Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PurposeThe RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter.MethodsARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting.ResultsARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation.ConclusionsThe ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated

Mapping of heterologous expressed sequence tags as an alternative to microarrays for study of defense responses in plants

BACKGROUND: Microarray technology helped to accumulate an immense pool of data on gene expression changes in response to different environmental factors. Yet, computer- generated gene profiling using expressed sequence tags (EST) represents a valuable alternative to microarrays, which allows efficient discovery of homologous sequences in evolutionarily different species and comparison of gene sets on the whole genome scale. In this study, we used publicly available EST database derived from different plant species infected with a variety of pathogens, to generate an expression profile of homologous genes involved in defense response of a model organism, Arabidopsis thaliana. RESULTS: EST-driven prediction identified 4,935 genes (16% of the total Arabidopsis genome) which, according to the origin of EST sets, were associated with defense responses in the reference genome. Profiles of defense-related genes, obtained by mapping of heterologous EST, represent putative Arabidopsis homologs of the corresponding species. Comparison of these profiles in pairs and locating common genes allowed estimating similarity between defense-related gene sets of different plant species. To experimentally support computer data, we arbitrarily selected a number of transcription factor genes (TF) detected by EST mapping. Their expression levels were examined by real-time polymerase chain reaction during infection with yellow strain of Cucumber mosaic virus, a compatible virus systemically infecting Arabidopsis. We observed that 65% of the designated TF were upregulated in accordance with the EST-generated profile. CONCLUSION: We demonstrated that heterologous EST mapping may be efficiently used to reveal genes involved in host defense responses to pathogens. Upregulated genes identified in this study substantially overlap with those previously obtained by microarrays

Еffect of the essential oil of <i>Satureja montana</i> L. on the growth of cultures of conditionally pathogenic microorganisms

Introduction. Essential oils contain antimicrobial components that are highly active against a wide range of microorganisms. Essential oils are natural, environmentally safe, low-toxic substances with a minimal list of side effects; no antimicrobial resistance is formed to them. The aim of the research was to study the influence of the essential oil of Satureja montana L., growing in the Crimea, on the growth of cultures of opportunistic microorganisms. Materials and methods. The short-term effect of savory oil on the growth of referenсе strains of microorganisms was studied in accordance with the European Standard for determining the rate of inactivation of microorganisms by the test substance (1997). To study the long-term effect of savory oil on clinical isolates of Staphylococcus aureus, we used the method of dilutions in a liquid medium, followed by measurement of the optical density of growth of the suspension culture biomass. The effect of savory oil on the formation of biofilms by clinical isolates of S. aureus was also studied. Results. Whole savory oil and its dilutions of 1 : 10 and 1 : 100 with short-term action (1060 min) completely suppressed the growth of referenсе strains of bacteria; growth of the referenсе strain Candida albicans CCM 885 was inhibited only by whole oil and a 1 : 10 dilution, while a 1 : 100 dilution had a bacteriostatic effect. Dilutions of essential oil 1 : 100 and 1 : 1000 had a pronounced antibacterial effect on the suspension culture of clinical isolates of S. aureus. Savory oil also inhibited biofilm formation by 11 isolates S. aureus. Conclusion. The essential oil of Satureja montana L. exhibits a pronounced antimicrobial effect against referenсе strains of S. aureus ATCC 25923, Escherichia coli ATCC 25922 and fungi C. albicans CCM 885. The antibacterial effect of this essential oil on clinical isolates of S. aureus allows us to offer it as a component of combined preparations for the treatment of infections caused by antibiotic-resistant strains of staphylococcus

Types of Void Space in the Bazhenov Reservoir Rocks

The deposits of the Bazhenov formation are a unique reservoir of unconventional oil reserves in Western Siberia. They contain both solid organic matter (kerogen) and liquid light oil. The successful development of these hydrocarbons is largely determined by the adequacy of the void space models. The aim of the study is to identify the types of void space in the sediments of the Bazhenov formation and to identify the distribution patterns across the section of the researched wells. The void space was studied by electron and optical microscopy, and the mineral composition of the rocks was determined by X-ray diffraction analysis. The deposits of the Bazhenov productive formation in the territory of Western Siberia are represented by a wide complex of lithotypes, including various kinds of silicites, carbonate, clay rocks, and mixtites. The reservoir space in the reservoir rocks of the Bazhenov formation is a complex and hierarchically subordinated system, which includes voids and fractures of various sizes, configurations, and genesis. The void space of the Bazhenov formation is characterized by a fairly high degree of spatial heterogeneity, which is controlled by lithological, facies, and tectonic factors, as well as the direction of catagenetic processes

Gene expression profiling in viable but nonculturable (VBNC) cells of Pseudomonas syringae pv syringae

Pseudomonas syringae infects diverse crop plants and comprises at least 50 different pathovar strains with different host ranges. More information on the physiological and molecular effects of the host inhibitory environment on the pathogen is needed to develop resistant cultivars. Recently, we reported an in vitro model system that mimics the redox pulse associated with the oxidative burst in plant cells inoculated with Pseudomonas syringae pv. syringae. Using this system, we demonstrated that oxidation of acetosyringone, a major extracellular phenolic compound induced in some plants in response to bacteria, rendered Pseudomonas syringae. pv. syringae to a viable but nonculturable (VBNC) state. Here we performed a large scale transcriptome profiling of P.s.pv. syringae in the VBNC state induced by acetosyringone treatment and identified bacterial genes and pathways presumably associated with this condition. The findings offer insight into what events occur when bacterial pathogens are first encountered and host defense responses are triggered. The acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance. We believe that this is the first work on global gene expression profiling of VBNC cells in plant pathogenic bacteria

Down-regulated differentially expressed genes (DEGs) in alfalfa plants from cultivar ZG9830.

<p><b>(A)</b> Venn diagram depicting a number of unique and common down-regulated DEGs between susceptible and resistant plants 24 hours after inoculation. <b>(B)</b> Functional categorization of the unique genes down-regulated genes in susceptible plants.</p

Volatile Evolution of Long Non-Coding RNA Repertoire in Retinal Pigment Epithelium: Insights from Comparison of Bovine and Human RNA Expression Profiles

Currently, several long non-coding RNAs (lncRNAs) (TUG1, MALAT1, MEG3 and others) have been discovered to regulate normal visual function and may potentially contribute to dysfunction of the retina. We decided to extend these analyses of lncRNA genes to the retinal pigment epithelium (RPE) to determine whether there is conservation of RPE-expressed lncRNA between human and bovine genomes. We reconstructed bovine RPE lncRNAs based on genome-guided assembly. Next, we predicted homologous human transcripts based on whole genome alignment. We found a small set of conserved lncRNAs that could be involved in signature RPE functions that are conserved across mammals. However, the fraction of conserved lncRNAs in the overall pool of lncRNA found in RPE appeared to be very small (less than 5%), perhaps reflecting a fast and flexible adaptation of the mammalian eye to various environmental conditions

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding