Lymphatic Dissemination in Prostate Cancer: Features of the Transcriptomic Profile and Prognostic Models.

Radical prostatectomy is the gold standard treatment for prostate cancer (PCa); however, it does not always completely cure PCa, and patients often experience a recurrence of the disease. In addition, the clinical and pathological parameters used to assess the prognosis and choose further tactics for treating a patient are insufficiently informative and need to be supplemented with new markers. In this study, we performed RNA-Seq of PCa tissue samples, aimed at identifying potential prognostic markers at the level of gene expression and miRNAs associated with one of the key signs of cancer aggressiveness-lymphatic dissemination. The relative expression of candidate markers was validated by quantitative PCR, including an independent sample of patients based on archival material. Statistically significant results, derived from an independent set of samples, were confirmed for miR-148a-3p and miR-615-3p, as well as for the CST2, OCLN, and PCAT4 genes. Considering the obtained validation data, we also analyzed the predictive value of models based on various combinations of identified markers using algorithms based on machine learning. The highest predictive potential was shown for the "CST2 + OCLN + pT" model (AUC = 0.863) based on the CatBoost Classifier algorithm.

Key FAD2, FAD3, and SAD Genes Involved in the Fatty Acid Synthesis in Flax Identified Based on Genomic and Transcriptomic Data.

FAD (fatty acid desaturase) and SAD (stearoyl-ACP desaturase) genes play key roles in the synthesis of fatty acids (FA) and determination of oil composition in flax (Linum usitatissimum L.). We searched for FAD and SAD genes in the most widely used flax genome of the variety CDC Bethune and three available long-read assembled flax genomes-YY5, 3896, and Atlant. We identified fifteen FAD2, six FAD3, and four SAD genes. Of all the identified genes, 24 were present in duplicated pairs. In most cases, two genes from a pair differed by a significant number of gene-specific SNPs (single nucleotide polymorphisms) or even InDels (insertions/deletions), except for FAD2a-1 and FAD2a-2, where only seven SNPs distinguished these genes. Errors were detected in the FAD2a-1, FAD2a-2, FAD3c-1, and FAD3d-2 sequences in the CDC Bethune genome assembly but not in the long-read genome assemblies. Expression analysis of the available transcriptomic data for different flax organs/tissues revealed that FAD2a-1, FAD2a-2, FAD3a, FAD3b, SAD3-1, and SAD3-2 were specifically expressed in embryos/seeds/capsules and could play a crucial role in the synthesis of FA in flax seeds. In contrast, FAD2b-1, FAD2b-2, SAD2-1, and SAD2-2 were highly expressed in all analyzed organs/tissues and could be involved in FA synthesis in whole flax plants. FAD2c-2, FAD2d-1, FAD3c-1, FAD3c-2, FAD3d-1, FAD3d-2, SAD3-1, and SAD3-2 showed differential expression under stress conditions-Fusarium oxysporum infection and drought. The obtained results are essential for research on molecular mechanisms of fatty acid synthesis, FAD and SAD editing, and marker-assisted and genomic selection for breeding flax varieties with a determined fatty acid composition of oil.

Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-Quality Genome.

High-quality genome sequences help to elucidate the genetic basis of numerous biological processes and track species evolution. For flax (Linum usitatissimum L.)-a multifunctional crop, high-quality assemblies from Oxford Nanopore Technologies (ONT) data were unavailable, largely due to the difficulty of isolating pure high-molecular-weight DNA. This article proposes a scheme for gaining a contiguous L. usitatissimum assembly using Nanopore data. We developed a protocol for flax nuclei isolation with subsequent DNA extraction, which allows obtaining about 5 µg of pure high-molecular-weight DNA from 0.5 g of leaves. Such an amount of material can be collected even from a single plant and yields more than 30 Gb of ONT data in two MinION runs. We performed a comparative analysis of different genome assemblers and polishers on the gained data and obtained the final 447.1-Mb assembly of L. usitatissimum line 3896 genome using the Canu-Racon (two iterations)-Medaka combination. The genome comprised 1695 contigs and had an N50 of 6.2 Mb and a completeness of 93.8% of BUSCOs from eudicots_odb10. Our study highlights the impact of the chosen genome construction strategy on the resulting assembly parameters and its eligibility for future genomic studies.

Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.

Immunohistochemistry and Mutation Analysis of SDHx Genes in Carotid Paragangliomas.

Carotid paragangliomas (CPGLs) are rare neuroendocrine tumors often associated with mutations in SDHx genes. The immunohistochemistry of succinate dehydrogenase (SDH) subunits has been considered a useful instrument for the prediction of SDHx mutations in paragangliomas/pheochromocytomas. We compared the mutation status of SDHx genes with the immunohistochemical (IHC) staining of SDH subunits in CPGLs. To identify pathogenic/likely pathogenic variants in SDHx genes, exome sequencing data analysis among 42 CPGL patients was performed. IHC staining of SDH subunits was carried out for all CPGLs studied. We encountered SDHx variants in 38% (16/42) of the cases in SDHx genes. IHC showed negative (5/15) or weak diffuse (10/15) SDHB staining in most tumors with variants in any of SDHx (94%, 15/16). In SDHA-mutated CPGL, SDHA expression was completely absent and weak diffuse SDHB staining was detected. Positive immunoreactivity for all SDH subunits was found in one case with a variant in SDHD. Notably, CPGL samples without variants in SDHx also demonstrated negative (2/11) or weak diffuse (9/11) SDHB staining (42%, 11/26). Obtained results indicate that SDH immunohistochemistry does not fully reflect the presence of mutations in the genes; diagnostic effectiveness of this method was 71%. However, given the high sensitivity of SDHB immunohistochemistry, it could be used for initial identifications of patients potentially carrying SDHx mutations for recommendation of genetic testing.

Characterization of repeated DNA sequences in genomes of blue-flowered flax.

BACKGROUND: Members of different sections of the genus Linum are characterized by wide variability in size, morphology and number of chromosomes in karyotypes. Since such variability is determined mainly by the amount and composition of repeated sequences, we conducted a comparative study of the repeatomes of species from four sections forming a clade of blue-flowered flax. Based on the results of high-throughput genome sequencing performed in this study as well as available WGS data, bioinformatic analyses of repeated sequences from 12 flax samples were carried out using a graph-based clustering method. RESULTS: It was found that the genomes of closely related species, which have a similar karyotype structure, are also similar in the repeatome composition. In contrast, the repeatomes of karyologically distinct species differed significantly, and no similar tandem-organized repeats have been identified in their genomes. At the same time, many common mobile element families have been identified in genomes of all species, among them, Athila Ty3/gypsy LTR retrotransposon was the most abundant. The 30-chromosome members of the sect. Linum (including the cultivated species L. usitatissimum) differed significantly from other studied species by a great number of satellite DNA families as well as their relative content in genomes. CONCLUSIONS: The evolution of studied flax species was accompanied by waves of amplification of satellite DNAs and LTR retrotransposons. The observed inverse correlation between the total contents of dispersed repeats and satellite DNAs allowed to suggest a relationship between both classes of repeating sequences. Significant interspecific differences in satellite DNA sets indicated a high rate of evolution of this genomic fraction. The phylogenetic relationships between the investigated flax species, obtained by comparison of the repeatomes, agreed with the results of previous molecular phylogenetic studies.

Flax (Linum usitatissimum L.) response to non-optimal soil acidity and zinc deficiency.

BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed production. Unfavorable environments, such as nutrient deficiency and non-optimal soil acidity, decrease the quantity and quality of yield. Cultivation of tolerant to stress varieties can significantly reduce the crop losses. Understanding the mechanisms of flax response to the stresses and identification of resistance gene candidates will help in breeding of improved cultivars. In the present work, the response of flax plants to increased pH level and zinc (Zn) deficiency was studied. RESULTS: We performed high-throughput transcriptome sequencing of two flax cultivars with diverse tolerance to increased pH level and Zn deficiency: Norlin (tolerant) and Mogilevsky (sensitive). Sixteen cDNA libraries were created from flax plants grown under control conditions, increased pH level, Zn deficiency, and both stresses simultaneously, and about 35 million reads were obtained for each experiment type. Unfavorable pH resulted in significantly stronger gene expression alterations compared to Zn deficiency. Ion homeostasis, oxidoreductase activity, cell wall, and response to stress Gene Ontology terms were the most affected by unfavorable pH and Zn deficiency both in tolerant and sensitive flax cultivars. Upregulation of genes encoding metal transporters was identified under increased pH level, Zn deficiency, and both stresses simultaneously. Under Zn deficiency, only in tolerant cultivar Norlin, we revealed the induction of several photosynthesis-related genes and, in this way, this tolerant genotype could overcome unfavorable effects of reduced Zn content. CONCLUSIONS: We identified genes with expression alterations in flax under non-optimal soil acidity and Zn deficiency based on high-throughput sequencing data. These genes are involved in diverse processes, including ion transport, cell wall biogenesis, and photosynthesis, and could play an important role in flax response to the studied stresses. Moreover, genes with distinct expression changes between examined tolerant and sensitive genotypes could determine the mechanisms of flax tolerance to non-optimal soil acidity and Zn deficiency.

Novel potential causative genes in carotid paragangliomas.

BACKGROUND: Carotid paragangliomas (CPGLs) are rare neuroendocrine tumors that arise from the paraganglion at the bifurcation of the carotid artery and are responsible for approximately 65% of all head and neck paragangliomas. CPGLs can occur sporadically or along with different hereditary tumor syndromes. Approximately 30 genes are known to be associated with CPGLs. However, the genetic basis behind the development of these tumors is not fully elucidated, and the molecular mechanisms underlying CPGL pathogenesis remain unclear. METHODS: Whole exome and transcriptome high-throughput sequencing of CPGLs was performed on an Illumina platform. Exome libraries were prepared using a Nextera Rapid Capture Exome Kit (Illumina) and were sequenced under 75 bp paired-end model. For cDNA library preparation, a TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) was used; transcriptome sequencing was carried out with 100 bp paired-end read length. Obtained data were analyzed using xseq which estimates the influence of mutations on gene expression profiles allowing to identify potential causative genes. RESULTS: We identified a total of 16 candidate genes (MYH15, CSP1, MYH3, PTGES3L, CSGALNACT2, NMD3, IFI44, GMCL1, LSP1, PPFIBP2, RBL2, MAGED1, CNIH3, STRA6, SLC6A13, and ATM) whose variants potentially influence their expression (cis-effect). The strongest cis-effect of loss-of-function variants was found in MYH15, CSP1, and MYH3, and several likely pathogenic variants in these genes associated with CPGLs were predicted. CONCLUSIONS: Using the xseq probabilistic model, three novel potential causative genes, namely MYH15, CSP1, and MYH3, were identified in carotid paragangliomas.

The CIMP-high phenotype is associated with energy metabolism alterations in colon adenocarcinoma.

BACKGROUND: CpG island methylator phenotype (CIMP) is found in 15-20% of malignant colorectal tumors and is characterized by strong CpG hypermethylation over the genome. The molecular mechanisms of this phenomenon are not still fully understood. The development of CIMP is followed by global gene expression alterations and metabolic changes. In particular, CIMP-low colon adenocarcinoma (COAD), predominantly corresponded to consensus molecular subtype 3 (CMS3, "Metabolic") subgroup according to COAD molecular classification, is associated with elevated expression of genes participating in metabolic pathways. METHODS: We performed bioinformatics analysis of RNA-Seq data from The Cancer Genome Atlas (TCGA) project for CIMP-high and non-CIMP COAD samples with DESeq2, clusterProfiler, and topGO R packages. Obtained results were validated on a set of fourteen COAD samples with matched morphologically normal tissues using quantitative PCR (qPCR). RESULTS: Upregulation of multiple genes involved in glycolysis and related processes (ENO2, PFKP, HK3, PKM, ENO1, HK2, PGAM1, GAPDH, ALDOA, GPI, TPI1, and HK1) was revealed in CIMP-high tumors compared to non-CIMP ones. Most remarkably, the expression of the PKLR gene, encoding for pyruvate kinase participating in gluconeogenesis, was decreased approximately 20-fold. Up to 8-fold decrease in the expression of OGDHL gene involved in tricarboxylic acid (TCA) cycle was observed in CIMP-high tumors. Using qPCR, we confirmed the increase (4-fold) in the ENO2 expression and decrease (2-fold) in the OGDHL mRNA level on a set of COAD samples. CONCLUSIONS: We demonstrated the association between CIMP-high status and the energy metabolism changes at the transcriptomic level in colorectal adenocarcinoma against the background of immune pathway activation. Differential methylation of at least nine CpG sites in OGDHL promoter region as well as decreased OGDHL mRNA level can potentially serve as an additional biomarker of the CIMP-high status in COAD.

HK3 overexpression associated with epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a common cancer worldwide. The main cause of death in CRC includes tumor progression and metastasis. At molecular level, these processes may be triggered by epithelial-mesenchymal transition (EMT) and necessitates specific alterations in cell metabolism. Although several EMT-related metabolic changes have been described in CRC, the mechanism is still poorly understood. RESULTS: Using CrossHub software, we analyzed RNA-Seq expression profile data of CRC derived from The Cancer Genome Atlas (TCGA) project. Correlation analysis between the change in the expression of genes involved in glycolysis and EMT was performed. We obtained the set of genes with significant correlation coefficients, which included 21 EMT-related genes and a single glycolytic gene, HK3. The mRNA level of these genes was measured in 78 paired colorectal cancer samples by quantitative polymerase chain reaction (qPCR). Upregulation of HK3 and deregulation of 11 genes (COL1A1, TWIST1, NFATC1, GLIPR2, SFPR1, FLNA, GREM1, SFRP2, ZEB2, SPP1, and RARRES1) involved in EMT were found. The results of correlation study showed that the expression of HK3 demonstrated a strong correlation with 7 of the 21 examined genes (ZEB2, GREM1, TGFB3, TGFB1, SNAI2, TWIST1, and COL1A1) in CRC. CONCLUSIONS: Upregulation of HK3 is associated with EMT in CRC and may be a crucial metabolic adaptation for rapid proliferation, survival, and metastases of CRC cells.

CrossHub: a tool for multi-way analysis of The Cancer Genome Atlas (TCGA) in the context of gene expression regulation mechanisms.

The contribution of different mechanisms to the regulation of gene expression varies for different tissues and tumors. Complementation of predicted mRNA-miRNA and gene-transcription factor (TF) relationships with the results of expression correlation analyses derived for specific tumor types outlines the interactions with functional impact in the current biomaterial. We developed CrossHub software, which enables two-way identification of most possible TF-gene interactions: on the basis of ENCODE ChIP-Seq binding evidence or Jaspar prediction and co-expression according to the data of The Cancer Genome Atlas (TCGA) project, the largest cancer omics resource. Similarly, CrossHub identifies mRNA-miRNA pairs with predicted or validated binding sites (TargetScan, mirSVR, PicTar, DIANA microT, miRTarBase) and strong negative expression correlations. We observed partial consistency between ChIP-Seq or miRNA target predictions and gene-TF/miRNA co-expression, demonstrating a link between these indicators. Additionally, CrossHub expression-methylation correlation analysis can be used to identify hypermethylated CpG sites or regions with the greatest potential impact on gene expression. Thus, CrossHub is capable of outlining molecular portraits of a specific gene and determining the three most common sources of expression regulation: promoter/enhancer methylation, miRNA interference and TF-mediated activation or repression. CrossHub generates formatted Excel workbooks with the detailed results. CrossHub is freely available athttps://sourceforge.net/projects/crosshub/.

Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes.

BACKGROUND: The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n = 30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes. RESULTS: High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH. Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n = 30) could originate either as the result of hybridization of two diploid species (2n = 16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n = 16) and a diploid ancestor of modern L. narbonense (2n = 14). CONCLUSIONS: High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.

Differential gene expression in response to Fusarium oxysporum infection in resistant and susceptible genotypes of flax (Linum usitatissimum L.).

BACKGROUND: Flax (Linum usitatissimum L.) is a crop plant used for fiber and oil production. Although potentially high-yielding flax varieties have been developed, environmental stresses markedly decrease flax production. Among biotic stresses, Fusarium oxysporum f. sp. lini is recognized as one of the most devastating flax pathogens. It causes wilt disease that is one of the major limiting factors for flax production worldwide. Breeding and cultivation of flax varieties resistant to F. oxysporum is the most effective method for controlling wilt disease. Although the mechanisms of flax response to Fusarium have been actively studied, data on the plant response to infection and resistance gene candidates are currently very limited. RESULTS: The transcriptomes of two resistant and two susceptible flax cultivars with respect to Fusarium wilt, as well as two resistant BC2F5 populations, which were grown under control conditions or inoculated with F. oxysporum, were sequenced using the Illumina platform. Genes showing changes in expression under F. oxysporum infection were identified in both resistant and susceptible flax genotypes. We observed the predominant overexpression of numerous genes that are involved in defense response. This was more pronounced in resistant cultivars. In susceptible cultivars, significant downregulation of genes involved in cell wall organization or biogenesis was observed in response to F. oxysporum. In the resistant genotypes, upregulation of genes related to NAD(P)H oxidase activity was detected. Upregulation of a number of genes, including that encoding beta-1,3-glucanase, was significantly greater in the cultivars and BC2F5 populations resistant to Fusarium wilt than in susceptible cultivars in response to F. oxysporum infection. CONCLUSIONS: Using high-throughput sequencing, we identified genes involved in the early defense response of L. usitatissimum against the fungus F. oxysporum. In response to F. oxysporum infection, we detected changes in the expression of pathogenesis-related protein-encoding genes and genes involved in ROS production or related to cell wall biogenesis. Furthermore, we identified genes that were upregulated specifically in flax genotypes resistant to Fusarium wilt. We suggest that the identified genes in resistant cultivars and BC2F5 populations showing induced expression in response to F. oxysporum infection are the most promising resistance gene candidates.

Upregulation of NETO2 gene in colorectal cancer.

BACKGROUND: Neuropilin and tolloid-like 2 (NETO2) is a single-pass transmembrane protein that has been shown primarily implicated in neuron-specific processes. Upregulation of NETO2 gene was also detected in several cancer types. In colorectal cancer (CRC), it was associated with tumor progression, invasion, and metastasis, and seems to be involved in epithelial-mesenchymal transition (EMT). However, the mechanism of NETO2 action is still poorly understood. RESULTS: We have revealed significant increase in the expression of NETO2 gene and deregulation of eight EMT-related genes in CRC. Four of them were upregulated (TWIST1, SNAIL1, LEF1, and FOXA2); the mRNA levels of other genes (FOXA1, BMP2, BMP5, and SMAD7) were decreased. Expression of NETO2 gene was weakly correlated with that of genes involved in the EMT process. CONCLUSIONS: We found considerable NETO2 upregulation, but no significant correlation between the expression of NETO2 and EMT-related genes in CRC. Thus, NETO2 may be involved in CRC progression, but is not directly associated with EMT.

Gene expression profiling of flax (Linum usitatissimum L.) under edaphic stress.

BACKGROUND: Cultivated flax (Linum usitatissimum L.) is widely used for production of textile, food, chemical and pharmaceutical products. However, various stresses decrease flax production. Search for genes, which are involved in stress response, is necessary for breeding of adaptive cultivars. Imbalanced concentration of nutrient elements in soil decrease flax yields and also results in heritable changes in some flax lines. The appearance of Linum Insertion Sequence 1 (LIS-1) is the most studied modification. However, LIS-1 function is still unclear. RESULTS: High-throughput sequencing of transcriptome of flax plants grown under normal (N), phosphate deficient (P), and nutrient excess (NPK) conditions was carried out using Illumina platform. The assembly of transcriptome was performed, and a total of 34924, 33797, and 33698 unique transcripts for N, P, and NPK sequencing libraries were identified, respectively. We have not revealed any LIS-1 derived mRNA in our sequencing data. The analysis of high-throughput sequencing data allowed us to identify genes with potentially differential expression under imbalanced nutrition. For further investigation with qPCR, 15 genes were chosen and their expression levels were evaluated in the extended sampling of 31 flax plants. Significant expression alterations were revealed for genes encoding WRKY and JAZ protein families under P and NPK conditions. Moreover, the alterations of WRKY family genes differed depending on LIS-1 presence in flax plant genome. Besides, we revealed slight and LIS-1 independent mRNA level changes of KRP2 and ING1 genes, which are adjacent to LIS-1, under nutrition stress. CONCLUSIONS: Differentially expressed genes were identified in flax plants, which were grown under phosphate deficiency and excess nutrition, on the basis of high-throughput sequencing and qPCR data. We showed that WRKY and JAS gene families participate in flax response to imbalanced nutrient content in soil. Besides, we have not identified any mRNA, which could be derived from LIS-1, in our transcriptome sequencing data. Expression of LIS-1 flanking genes, ING1 and KRP2, was suggested not to be nutrient stress-induced. Obtained results provide new insights into edaphic stress response in flax and the role of LIS-1 in these process.

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells.

BACKGROUND: The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the "Warburg effect" and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear. RESULTS: The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis. CONCLUSIONS: We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

ITS and 16S rDNA metagenomic dataset of different soils from flax fields.

Flax (Linum usitatissimum L.), one of the important and versatile crops, is used for the production of oil and fiber. To obtain high and stable yields of flax products, L. usitatissimum varieties should be cultivated under optimal conditions, including the composition of the soil microbiome. We evaluated the diversity of microorganisms in soils under conditions unfavorable for flax cultivation (suboptimal acidity or herbicide treatment) or infected with causative agents of harmful flax diseases (Septoria linicola, Colletotrichum lini, Melampsora lini, or Fusarium oxysporum f. sp. lini). For this purpose, twenty-two sod-podzolic soil samples were collected from flax fields and their metagenomes were analyzed using the regions of 16S ribosomal RNA gene (16S rDNA) and internal transcribed spacers (ITS) of the ribosomal RNA genes, which are used in phylogenetic studies of bacteria and fungi. Amplicons were sequenced on the Illumina MiSeq platform (reads of 300 + 300 bp). On average, we obtained 8,400 reads for ITS and 43,300 reads for 16S rDNA per sample. For identification of microorganisms in the soil samples, the Illumina reads were processed using DADA2. The raw data are deposited in the Sequence Read Archive under the BioProject accession number PRJNA956957. Tables listing the microorganisms identified in the soil samples are available in this article. The obtained dataset can be used to analyze the fungal and bacterial composition of flax field soils and their relationship to environmental conditions, including suboptimal soil acidity and infection with fungal pathogens. In addition, it can help to understand the influence of herbicide treatment on the microbial diversity of flax fields. Another useful application of our data is the ability to assess the suitability of the soil microbiome for flax cultivation.

Expression of FAD and SAD Genes in Developing Seeds of Flax Varieties under Different Growth Conditions.

Flax seed is one of the richest plant sources of linolenic acid (LIN) and also contains unsaturated linoleic acid (LIO) and oleic acid (OLE). Stearoyl-ACP desaturases (SADs) and fatty acid desaturases (FADs) play key roles in the synthesis of flax fatty acids (FAs). However, there is no holistic view of which genes from the SAD and FAD families and at which developmental stages have the highest expression levels in flax seeds, as well as the influence of genotype and growth conditions on the expression profiles of these genes. We sequenced flax seed transcriptomes at 3, 7, 14, 21, and 28 days after flowering (DAF) for ten flax varieties with different oil FA compositions grown under three temperature/watering conditions. The expression levels of 25 genes of the SAD, FAD2, and FAD3 families were evaluated. FAD3b, FAD3a, FAD2b-2, SAD3-1, SAD2-1, SAD2-2, SAD3-2, FAD2a-1, and FAD2a-2 had the highest expression levels, which changed significantly during seed development. These genes probably play a key role in FA synthesis in flax seeds. High temperature and insufficient watering shifted the maximum expression levels of FAD and SAD genes to earlier developmental stages, while the opposite trend was observed for low temperature and excessive watering. Differences in the FAD and SAD expression profiles under different growth conditions may affect the FA composition of linseed oil. Stop codons in the FAD3a gene, resulting in a reduced LIN content, decreased the level of FAD3a transcript. The obtained results provide new insights into the synthesis of linseed oil.

Genome of Linum usitatissimum convar. crepitans expands the view on the section Linum.

Sequencing whole plant genomes provides a solid foundation for applied and basic studies. Genome sequences of agricultural plants attract special attention, as they reveal information on the regulation of beneficial plant traits. Flax is a valuable crop cultivated for oil and fiber. Genome sequences of its representatives are rich sources of genetic information for the improvement of cultivated forms of the plant. In our work, we sequenced the first genome of flax with the dehiscence of capsules-Linum usitatissimum convar. сrepitans (Boenn.) Dumort-on the Oxford Nanopore Technologies (ONT) and Illumina platforms. We obtained 23 Gb of raw ONT data and 89 M of 150 + 150 paired-end Illumina reads and tested different tools for genome assembly and polishing. The genome assembly produced according to the Canu-Racon ×2-medaka-POLCA scheme had optimal contiguity and completeness: assembly length-412.6 Mb, N50-5.2 Mb, L50-28, and complete BUSCO-94.6% (64.0% duplicated, eudicots_odb10). The obtained high-quality genome assembly of L. usitatissimum convar. crepitans provides opportunities for further studies of evolution, domestication, and genome regulation in the section Linum.

Comparative Genomic Analysis of Colletotrichum lini Strains with Different Virulence on Flax.

Colletotrichum lini is a flax fungal pathogen. The genus comprises differently virulent strains, leading to significant yield losses. However, there were no attempts to investigate the molecular mechanisms of C. lini pathogenicity from high-quality genome assemblies until this study. In this work, we sequenced the genomes of three C. lini strains of high (#390-1), medium (#757), and low (#771) virulence. We obtained more than 100× genome coverage with Oxford Nanopore Technologies reads (N50 = 12.1, 6.1, 5.0 kb) and more than 50× genome coverage with Illumina data (150 + 150 bp). Several assembly strategies were tested. The final assemblies were obtained using the Canu-Racon ×2-Medaka-Polca scheme. The assembled genomes had a size of 54.0-55.3 Mb, 26-32 contigs, N50 values > 5 Mb, and BUSCO completeness > 96%. A comparative genomic analysis showed high similarity among mitochondrial and nuclear genomes. However, a rearrangement event and the loss of a 0.7 Mb contig were revealed. After genome annotation with Funannotate, secreting proteins were selected using SignalP, and candidate effectors were predicted among them using EffectorP. The analysis of the InterPro annotations of predicted effectors revealed unique protein categories in each strain. The assembled genomes and the conducted comparative analysis extend the knowledge of the genetic diversity of C. lini and form the basis for establishing the molecular mechanisms of its pathogenicity.