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Aim. To evaluate an impact of different S6K1 isoforms expression in MCF-7 cells on initiation of Epithelial to Mesenchymal Transition (EMT). Methods. Immunocytochemical analysis, Real-Time PCR, Western blot. Results. We have demonstrated that unbalanced expression of p60, p70 and p85-S6K1 isoforms in MCF-7, namely downregulation of p70 and p85, and basal p60 expression, mediated by the CRISPR-Cas9 gene editing resulted in altered morphology and increased cell motility. Such changes were associated with the expression of genes whose products are involved in the regulation of cell motility, interaction with the extracellular matrix and loss of cellular adhesion demonstrating their increased potential for invasion and metastatic activity. qPCR analysis confirmed increased expression of a whole spectrum of genes associated with mesenchymal characteristics and loss of epithelial specific markers. Additionally, we observed a complete repression of the estrogen receptor 1 (ESR1) expression and downregulation of HER2/NEU (ERBB2). Conclusions. Our data demonstrate for the first time the implication of S6K1 isoforms in the regulation of EMT in MCF-7 cells by triggering for the concomitant onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type and has a strong negative impact on ESR1 expression. At the tumor level that can mean breast tumor transition to a more aggressive most probably triple negative molecular subtype. [ABSTRACT FROM AUTHOR]

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Autoantibodies, which are produced against tumor-associated antigens, are potential tumor markers and attract a growing interest for cancer detection, differential diagnostics and prognosis., Objective: To evaluate the diagnostic significance of 40 antigens identified by immunoscreening of cDNA libraries from thyroid and colon cancers by allogenic screening with different tumor types patients' sera., Method: Plaque-spot serological assay., Results: Increased frequency of antibody response in sera of cancer patients compared with that of healthy donors was shown toward 14 antigens, 8 of which (CG016, BTN3A3, FKBP4, XRCC4, TSGA2, ACTR1A, FXYD3 and CTSH) have revealed exclusively cancer-related serological profile., Conclusion: Allogenic screening of 40 SEREX-antigens with sera from cancer patients and healthy donors allowed us to reveal 14 antigens with potential diagnostic significance. These antigens and their cognate autoantibodies could be considered as valuable targets for further analysis as potential cancer biomarkers.

Host cells use metabolic signaling through the LXRα nuclear receptor to defend against Listeria monocytogenes infection. 25-Hydroxycholesterol is a natural ligand of LXRs that is produced by the enzyme cholesterol 25-hydroxylase (CH25H). We found that expression of Ch25h is upregulated following L. monocytogenes infection in a beta interferon (IFN-β)-dependent fashion. Moreover, increased Ch25h expression promotes survival of L. monocytogenes-infected cells and increases sensitivity of the host to infection. We determined that expression of Cd5l, a prosurvival gene, is controlled by CH25H. In addition, we found that CD5L inhibits activation of caspase-1, promoting survival of infected macrophages. Our results reveal a mechanism by which an intracellular pathogen can prolong survival of infected cells, thus providing itself with a protected environment in which to replicate.

Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-gamma)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-gamma or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

Genetic makeup of the host plays a significant role in the course and outcome of infection. Inbred strains of mice display a wide range of sensitivities to Listeria monocytogenes infection and thus serve as a good model for analysis of the effect of genetic polymorphism. The outcome of L. monocytogenes infection in mice is influenced by the ability of this bacterium to induce expression of interferon beta mRNA, encoded in mouse by the Ifnb1 (interferon beta 1, fibroblast) gene. Mouse strains that lack components of the IFN beta signaling pathway are substantially more resistant to infection. We found that macrophages from the ByJ substrain of the common C57BL/6 inbred strain of mice are impaired in their ability to induce Ifnb1 expression in response to bacterial and viral infections. We mapped the locus that controls differential expression of Ifnb1 to a region on Chromosome 7 that includes interferon regulatory factor 3 (Irf3), which encodes a transcription factor responsible for early induction of Ifnb1 expression. In C57BL/6ByJ mice, Irf3 mRNA was inefficiently spliced, with a significant proportion of the transcripts retaining intron 5. Analysis of the Irf3 locus identified a single base-pair polymorphism and revealed that intron 5 of Irf3 is spliced by the atypical U12-type spliceosome. We found that the polymorphism disrupts a U12-type branchpoint and has a profound effect on the efficiency of splicing of Irf3. We demonstrate that a naturally occurring change in the splicing control element has a dramatic effect on the resistance to L. monocytogenes infection. Thus, the C57BL/6ByJ mouse strain serves as an example of how a mammalian host can counter bacterial virulence strategies by introducing subtle alteration of noncoding sequences., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The identification and characterization of genes either induced or repressed in human brain tumors are important in understanding the mechanism of tumor initiation and progression, stages of malignancy, in developing new approaches to the diagnosis and treatment of tumors. In this paper, differential hybridization of arrayed human fetal brain and postnatal brain and postnatal brain cDNA libraries revealed differences in the rate of hybridization signals with total cDNA probes of the human brain and glioblastoma multiforme for more than 150 cDNA clones. Sixteen nucleotide sequences with changed contents in tumors were identified by repeated differential hybridization of the cDNA clones selected by primary screening with the same total cDNA probes of the human brain and glioblastoma multiforme and by Northern-hybridization of RNA samples from the human and glial tumors. The results of an analysis of the increased expression of the gene encoding apolipoprotein E. DNA-binding protein B, mitochondrial (16S and 12S) and cytoplasmic 28S rRNAs, Alu-containing transcripts, inactivation of cullin 1 gene and potential tumor suppressor gene TSC-22 are described.

Two identical strains of tobacco type TVM have been isolated in the region with 137Cs nuclear contamination with density of 12.6 Cu/km2 recombinant plasmids (pTVM9, pTVM9,5) containing cDNA of complete provirus and C-end sequence of cDNA of specific capsid protein from one of isolated viruses have been obtained. The capsule proteins of isolated strains have the higher 19.5 +/- 1.9 kDa molecular weight than standard TVM strain (17.5 kDa) as to SDS-PAAG electrophoresis data. No differences in distribution of fragments immunoactive to control antiserum have been found when using immunoblot analysis of capsid proteins of isolates and standard strain treated by tripsin. Sequencing analysis of cDNA pTVM9,5 has revealed non-conservative amino acid replacement of serine by tyrosine in position 149 for homologous region of capsid protein of standard TVM strain, which allows to suppose the mediated effect of specific ecological situation on the appearance of such replacement.

Considerable decrease of expression of some "brain-specific" genes obtained in previous experiments was detected by means of differential hybridization of gridded human fetal brain cDNA library with total cDNA probes, synthetized on mRNAs isolated from the normal brain cells and several tumor cells. In contrast, for few genes expressed on low level in human fetal brain the evident increase of expression level in human brain tumour cells was found. The nucleotide sequences of some of these genes and previously isolated genes which have different specificity of expression were determined.

The major liver and brain transcripts having different specificity of expression were isolated and characterized by differential hybridization of human fetal liver and human fetal brain cDNA libraries with total cDNA probes from different human tissues. The sequencing and analysis of nucleotide sequences indicated that the most isolated cDNA clones have contained unknown genes.