[Analysis of the Complete Tomato Aspermy Virus Genomes Suggests Reassortment in Russian Isolates from Chrysanthemum].

Tomato aspermy virus (TAV, genus Cucumovirus from the family Bromoviridae) is one of the most common and harmful chrysanthemum viruses, causing severe flower distortion, size reduction, and color breaking. Metatranscriptome sequencing of chrysanthemum plants of the Ribonette and Golden Standard cultivars from the collection of the Nikita Botanical Garden (Yalta, Republic of Crimea) generated TAV-related RNA reads. The complete genomes of two Russian isolates of the virus were assembled from the reads. This is the first report of full-length TAV genomes from Russia. Typically of cucumoviruses, the segmented TAV genome is represented by three single-stranded positive-sense linear RNA molecules of 3412 (RNA1), 3097 (RNA2) and 2219 (RNA3) nucleotides. Five open reading frames (ORF) have been identified that encode replicase (ORF1), RNA-dependent RNA polymerase (ORF2a), silencing suppressor protein (OFR2b), movement protein (OFR3a) and the coat protein (ORF3b). The identity of TAV genomes from the two chrysanthemum cultivars was 99.8% for all three viral RNAs; with other TAV isolates from GenBank it was 97.5-99.7% (RNA1), 93.8-99.8% (RNA2), and 89.3-99.3% (RNA3). Phylogenetic analysis showed that RNA1 and RNA3 of the Russian isolates were assigned to heterogeneous groups of TAV isolates found on various plant species in different regions of the world. At the same time, RNA2 clearly clustered with tomato isolates SKO20ST2 from Slovenia and PV-0220 from Bulgaria and, to a lesser extent, with the Iranian isolate Ker.Mah.P from petunia and the Chinese isolate Henan from chrysanthemum. The incongruence of phylogenetic trees reconstructed from different genome segments suggests pseudo-recombination (reassortment) in the Russian TAV isolates.

[Novel Genes Associated with the Development of Carotid Paragangliomas].

Carotid paragangliomas (CPGLs) are rare neuroendocrine tumors of the head and neck. "Germline" and somatic mutations in a number of genes were shown to be associated with the development of CPGLs; however, molecular mechanisms of the tumor pathogenesis have not been fully understood. In the work, we have used whole exome sequencing data of 52 CPGLs obtained earlier. Using MutSigCV, the search for genes with high mutation rate was performed. Thirty four genes (MADCAM1, SARM1, ZFPM1, CTDSP2, DSPP, POTED, ANP32B, FRG2B, BAGE3, CCDC89, ACOT2, KRTAP10-1, ATXN1, GXYLT1, MUC2, AQP7, TMPRSS13, KRTAP4-3, PRR21, PSPH, PLBD1, ZNF595, IGSF3, PRR16, FAM157A, KCNJ12, HYDIN, IGFBP2, KIAA1671, DISC1, MUC6, XKR3, HRNR, and MUC4) potentially associated with the CPGL initiation and progression were revealed. The involvement of these genes in the pathogenesis of CPGLs was first shown, and possible mechanisms of their participation in that were discussed.

[Identification of Fusion Transcripts in Leukеmic Cells by Whole-Transcriptome Sequencing].

Genetic aberrations in leukemia often lead to the formation of expressed chimeric genes, which should be assessed for proper diagnosis and therapy. Modern methods of molecular diagnostic mainly allow to identify already known fusion genes. RNAseq is an efficient tool for identification of rare and novel chimeric transcripts. Here we present the results of the whole transcriptome analysis of bone marrow samples from five patients with acute myeloblastic leukemia and one, with myelodysplastic syndrome. The whole-transcriptome analysis was performed using Illumina/Solexa approach. We found rare or unknown chimeric transcripts including ETV6-MDS1, MN1-ETV6, OAZ1-PTMA, and MLLT10-GRIA4. Each of these transcripts was confirmed by RT-PCR and Sanger sequencing.

[Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of HK2 Expression in Colorectal Cancer].

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from "The Cancer Genome Atlas" (TCGA) and five microRNA-mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.

[CAX3 Gene is Involved in Flax Response to High Soil Acidity and Aluminum Exposure].

Understanding the molecular mechanisms of plant response to unfavorable conditions is necessary for the effective selection of tolerant genotypes. Earlier, using high-throughput transcriptome sequencing of flax plants after exposure to aluminum ions (Al^(3+)) and high soil acidity, we detected stress-induced alteration in the expression of several genes, including CAX3, which encodes Ca^(2+)/H^(+)-exchanger involved in calcium ion transport. Here we describe CAX3 mRNA levels in flax cultivars either tolerant (Hermes and TMP1919) or sensitive (Lira and Orshanskiy) to Al^(3+). Stress-induced increased expression of CAX3 was detected only in aluminum-tolerant flax cultivars. The product of CAX3 gene may participate in flax response to high soil acidity and high Al^(3+) concentration through Ca^(2+)-mediated intracellular regulation.

[Transcription Factor SAP30 Is Involved in the Activation of NETO2 Gene Expression in Clear Cell Renal Cell Carcinoma].

Clear cell renal cell carcinoma (ccRCC) is a common oncourological disease with a high mortality level. The incidence of this type of cancer is constantly increasing, while molecular mechanisms involved in the disease initiation and progression remain far from being fully understood. A problem of the search for novel markers is crucial for improvement of diagnosis and therapy of ccRCC. We have previously found that the disease is characterized by increased expression of the NETO2 gene. In the present study, we showed that isoform 1 (NM_018092.4) makes the main contribution to the upregulation of this gene. Using original CrossHub software, "The Cancer Genome Atlas" (TCGA) project data were analyzed to identify possible mechanisms of NETO2 gene activation in ccRCC. The absence of significant contribution of methylation to the increase of mRNA level of the gene was observed. At the same time, a number of genes encoding transcription factors, which could potentially regulate the expression of NETO2 in ccRCC, were identified. Three such genes (MYCBP, JMY, and SAP30) were selected for the further analysis of their mRNA levels in a set of ccRCC samples with quantitative PCR. We showed a significant increase in mRNA level of one of the examined genes, SAP30, and revealed its positive correlation with NETO2 gene expression. Thus, upregulation of NETO2 gene is first stipulated by the isoform 1 (NM_018092.4), and the probable mechanism of its activation is associated with the increased expression of SAP30 transcription factor.

[Suppression of NR0B2 gene in Clear Cell Renal Cell Carcinoma Is Associated with Hypermethylation of Its Promoter].

Clear cell renal cell carcinoma (ccRCC) is a common urologic malignancy. Understanding of the transcriptional regulation of oncogenes and tumor suppressor genes involved is critical for the development of the treatments for renal tumors. Using ccRCC subdivision of the TCGA dataset, we identified NR0B2 encoding orphan nuclear receptor as a tumor suppressor candidate in renal tissue. In independent cohort of primary renal tumors, quantitative PCR experiments confirmed significant suppression of NR0B2 mRNA in 86% of ccRCC samples studied. In 80% of these cases, we detected the hypermethylation of the NR0B2 pro-moter region. These results suggest that NR0B2 is a tumor suppressor gene in ccRCC, and that the hypermethylation of promoter region is the main mechanism of its downregulation.

[The role of microRNA in abiotic stress response in plants].

Regulation of gene expression via microRNA is the key mechanism of response to biotic and abiotic stresses in plants. There are a lot of experimental data on the biological function of microRNAs in response to different stresses in various plant species. This review contains up-to-date information on molecular mechanisms of microRNA action in plants in response to abiotic stresses, including drought, salinity, mineral nutrient deficiency or imbalance.

[Molecular genetic mechanisms of drug resistance in prostate cancer].

The major problem in prostate cancer treatment is the development of drug resistance and especially important, cross-resistance. The mechanisms of drug resistance, which are divided into ligand-dependent (requiring the presence of androgens in the cell) and independent (not requiring the presence of androgens) are reviewed. The mechanisms are mainly represented with mutations of the androgen receptor and expression of aberrant constitutively active splice variants, as well as up-regulation of genes involved in androgens synthesis.

[Downregulation of OGDHL expression is associated with promoter hypermethylation in colorectal cancer].

Cell metabolic reprogramming is one of the cancer hallmarks. Glycolysis activation, along with suppression of oxidative phosphorylation and, to a lower extent, the TCA cycle, occurs in the majority of malignant tumors. A bioinformatics search for the glucose metabolism genes that are differentially expressed in colorectal cancer (CC) was performed using the data of The Cancer Genome Atlas (TCGA) Project. OGDHL for an oxoglutarate dehydrogenase complex subunit, which is involved in the TCA cycle and is indirectly responsible for the induction of apoptosis, was identified as one of the most promising candidates. A quantitative PCR analysis showed, on average, an eightfold downregulation of OGDHL in 50% (15/30) of CC samples. Based on the TCGA data, promoter hypermethylation was assumed to be a major mechanism of OGDHL inactivation. Bisulfite sequencing identified the OGDHL promoter region (+327 ... +767 relative to the transcription start site) that is often methylated in CC samples with downregulated ODGHL expression (80%, 8/10) and is possibly crucial for gene inactivation. Thus, frequent and significant OGDHL downregulation due to hypermethylation of a specific promoter region was demonstrated for CC. The OGDHL promoter methylation pattern was assumed to provide a marker for differential diagnosis of CIMP+ (CpG island methylator phenotype) tumors, which display dense hypermethylation of the promoter region in many genes.

[Evaluation of Gene Expression of Hexokinases in Colorectal Cancer with the Use of Bioinformatics Methods].

One of the hallmarks of cancer is the change of energy metabolism, mainly activation of glycolysis that occurs even at early stages of tumorigenesis. The glycolysis activation can be caused by overexpression of hexokinases, primarily HK1 and HK2. Colorectal cancer, which takes the third place in the cancer morbidity and mortality rates worldwide, is believed to be accompanied with overexpression of HK2, which is .considered a marker of poor prognosis. With the use of the developed CrossHub tool, we performed the analysis of the Cancer Genome Atlas RNA-Sequencing data, which, on the contrary, revealed the prevalence of the down-regulation of HK2 gene and only slight expression alterations in HK1 gene. The Cancer Genome Atlas is the largest resource in the field of molecular oncology that accumulated genomic, transcriptomic and methylomic data for thousands of sample of more than 20 cancers. The transcriptome analysis data for colorectal cancer (283 tumor samples and 41 matched normal samples) were in accord with the results of further qPCR expression level evaluation. Up-regulation of HK1 and HK2 genes was observed only in a part of samples: 12% for HK1 and 30% for HK2. At the same time, the HK2 mRNA level decrease was shown in 50% of cases. Correlation analysis revealed the consistency in HK1 and HK2 expression alterations (Spearman's rank correlation coefficient r(s) = 0.43, p < 0.01), that could be explained by common deregulation mechanisms of these genes in colorectal tumors. The HK3 expression level was significantly increased in 60% of samples. Most likely, just hexokinase 3 contributes significantly to the activation of glycolysis in colorectal cancer.

A new reliable reference gene UBA52 for quantitative real-time polymerase chain reaction studies in pyloric cecal tissues of the starfish Asterias rubens.

The starfish Asterias rubens is one of the most abundant echinoderm species in the White, Barents, North, and Baltic Seas. This species is an important component of marine ecosystems and a model object for certain biological studies, in particular those requiring quantitative estimation of gene expression. As a rule, expression at the transcriptional level is estimated by real-time qPCR using the ΔΔCt method, which allows the comparison of the copy number of target gene transcripts in samples with unknown mRNA/cDNA concentration. Application of this method requires normalization of the results relative to genes with stable expression levels (reference genes). The identification of reference genes is still a challenging task since data of this kind are missing for certain taxa, whereas the use of "standard" endogenous control genes without additional tests might lead to erroneous conclusions. We performed a preliminary analysis of the expression of many housekeeping genes in the pyloric ceca of A. rubens by high-throughput sequencing under normal and heat shock conditions. For one of them, the ubiquitin gene UBA52, low variation of expression (not greater than 2-fold) was shown using real-time qPCR. Tissues of pyloric ceca of normal adults and underyearlings and of adults after heat shock were used. The data obtained suggest that the UBA52 gene may be used as reference for normalization of gene expression at the mRNA level in the starfish A. rubens and probably in closely related species.

[Preparation stimulating hair growth possibly acts by inhibiting hair follicle ageing experiments on mice and transcriptome analysis].

Preparation stimulating hair growth (PSHG) was studied on mice of various strains (Balb/c, CBA, C57BI/6, and outbred). It was shown that a long-term (44 months) application of PSHG does not reliably affect the appearance of young healthy mice but does induce increase in the hair follicle size. No adverse consequences of the PSHG application were observed. Naturally occurring propagating regenerative hair waves peculiar to mice were preserved. In older mice (more than 2 years) with signs of alopecia, application of PSHG caused an overgrowing of bald patches within two months. Transcriptome analysis of the PSHG effect performed in fibroblast cell culture showed that PSHG stimulates processes of tissue development and remodeling. These observations together with previous findings showing that PSHG stimulates autophagy and induces death of cells subjected to oxidative stress may suggest that the mechanism of the PSHG effect involves stimulation of regeneration of skin and its derivatives owing to more efficient elimination of senescent and damaged follicle cells.

Modification of human fibroblast properties in microtransplant.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell "rejuvenation" in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 µ) filaments, which excludes large old cells.

[Differential expression of genes that encode glycolysis enzymes in kidney and lung cancer in humans].

Glycolysis is a main catabolic pathway of glucose metabolism, accompanied by ATP synthesis. More than 30 enzymes are involved in glycolysis, and genes that encode them can be considered housekeeping genes due to the high conservatism and evolutionary antiquity of the process. We studied the expression of these genes in kidney papillary cancer and planocellular lung cancer via the bioinformatic analysis of transcriptome database and method of quantitative real time PCR. Quantitative analysis of mRNA level demonstrated that only a part ofgenes that encode glycolysis enzymes maintain relatively stable mRNA level, including the HK1, ADPGK, GPI, PGK1, and PKM2 genes in kidney papillary cancer and the ADPGK, ALDOA, GAPDH, PGK1, BPGM, ENO1, and PKM2 genes in planocellular lung cancer. The frequent increase in the mRNA expression of PFKP, ALDOA, and GAPDH genes in kidney cancer, as well as the GPI gene in lung cancer, were detected for the first time by real time PCR. For other genes, their differential expression was demonstrated; the cases of both a decrease and increase in the mRNA level were detected. Thus, several genes that can be used as control genes in transcriptome analysis by real time PCR in kidney and lung cancer, as well as a number of differentially expressed genes that can be potential oncomarkers, were identified.

[Increase in NETO2 gene expression is a potential molecular genetic marker in renal and lung cancers].

Multiple changes in the genome, transcriptome, and proteome are frequent in cancer cells. A search for molecular markers based on DNA, mRNA, or proteins is a main method to develop early specific diagnostics for cancer. While universal markers are still unavailable, similar trends are known for the expression patterns of particular genes in certain epithelial tumors. A bioinformatic screening of transcriptomic databases identified the NETO2 gene as a new potential promising marker of renal cancer. A substantial increase in NETO2 mRNA level was detected in 90% clear-cell renal cell carcinomas, 70% of non-small cell lung cancers, and 50% of papillary renal cancers by real-time PCR. The NETO2 mRNA level was increased to a lesser extent in cervical carcinoma and colon cancer and tended to decrease in cancer of the stomach. The NETO2 gene, which codes for a membrane glycoprotein with an unclear function, was assumed to provide a new promising marker for early diagnosis in renal cancer and non-small cell lung cancer.

Interplay between RNA interference and heat shock response systems in Drosophila melanogaster.

The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. Here, we investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster We demonstrated that pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. It was also shown that deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR.

Genome-wide mapping of hot spots of DNA double-strand breaks in human cells as a tool for epigenetic studies and cancer genomics.

Hot spots of DNA double-strand breaks (DSBs) are associated with coordinated expression of genes in chromosomal domains (Tchurikov et al., 2011 [1]; 2013). These 50-150-kb DNA domains (denoted "forum domains") can be visualized by separation of undigested chromosomal DNA in pulsed-field agarose gels (Tchurikov et al., 1988; 1992) and used for genome-wide mapping of the DSBs that produce them. Recently, we described nine hot spots of DSBs in human rDNA genes and observed that, in rDNA units, the hot spots coincide with CTCF binding sites and H3K4me3 marks (Tchurikov et al., 2014), suggesting a role for DSBs in active transcription. Here we have used Illumina sequencing to map DSBs in chromosomes of human HEK293T cells, and describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE53811 and associated with the study published in DNA Research (Kravatsky et al., 2015). Our data indicate that H3K4me3 marks often coincide with hot spots of DSBs in HEK293T cells and that the mapping of these hot spots is important for cancer genomic studies.