Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification.

Background: Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis. Patients and methods: We developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients' outcomes. Results: Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016). Conclusions: Comprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.

Lipoxygenase Inhibitors Nordihydroguaiaretic Acid and Fungus Lecanicillum lecanii Extract Induce Death of Lymphoid Leukemia Cells.

We studied the effect of lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and fungus Lecanicillum lecanii extract on lymphatic leukemia P388 cells. The cells grown in the abdominal cavity of DBA2 mice for 7 days were transferred into a nutrient medium. The effect of lipoxygenase inhibitors was evaluated by changes in cell number, trypan blue staining, nucleus damage, and changes in cell distribution by DNA content after 22-h incubation. NDGA and fungus extract induced apoptotic death of lymphatic leukemia cells, which was seen from nucleus damage and reduced DNA content in cells. IC50 for NDGA and fungus extract was 0.66 and 5.5 µg/ml, respectively.

[Apoptotic endonuclease EndoG induces alternative splicing of telomerase catalytic subunit hTERT and death of tumor cells]. / Éndonukleaza EndoG indutsiruet al'ternativnyi splaising kataliticheskoi sub"edinitsy telomerazy hTERT i gibel' opukholevykh kletok.

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.

[Screening of Organisms Producing Inhibitors of 15-Lipoxygenase Among Micromycetes.]

The effects of extracts from the mycelium of Lecanicilium lecaniiNo.169, Beauveria fellina No.7 and Beauveria bassianaNo.15 on the activity of 15-lpoxygenase (15-LO) recovered from rat reticulocytes was investigated. The activity of 15-LO was determined by oxidation of linolic acid. The extract from the mycelium of the fungal complex was shown to inhibit 15-LO (IC50 of 12 mcg/ml). The inhibitory effect of the combined extract on 15-LO was due to the substances recovered from Lecanicilium lecanii No.169. The extract fractions responsible for the activity were determined and the compounds containing the fractions were identified. They proved to be 10 - 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol and genistein, a flavonoid from fraction 11. The possible role of the inhibitory effect of the compounds on 15-LO in the antiatherosclerotic activity of the fungal extract is discussed.

[Change of Tumor Cell Multiple Drug Resistance Inhibitory Activity of Oligomycins in Complexes with Lithium and Zinc].

Oligomycins and their complexes with lithium and zinc were shown to be less active vs. cyclosporin A in inhibition of transport proteins responsible for multiple drug resistance of lymphoid leukosis P388VR cells, while certain oligomycin complexes were tens or hundreds times more active than cyclosporin A by inhibition of transport proteins in another type of tumor cells, i.e. human larynx cancer Hep-2, that makes possible the use of the oligomycins complexes with lithium and zinc for inhibition of multiple drug resistance of certain tumor types.

[Determination of oligomycins hydrophobicity parameters].

The parameters of hydrophobicity of five oligomycins, i. e. A, B, C, F and SC-II were determined by HPLC. The location of the ascending hydrophobicity parameter was set: oligomycin B < oligomycin SC-II < oligomycin A < oligomycin F < oligomycin C.

Clinical characteristics of ovarian cancer classified by BRCA1, BRCA2, and RAD51C status.

We evaluated homologous recombination deficient (HRD) phenotypes in epithelial ovarian cancer (EOC) considering BRCA1, BRCA2, and RAD51C in a large well-annotated patient set. We evaluated EOC patients for germline deleterious mutations (n = 899), somatic mutations (n = 279) and epigenetic alterations (n = 482) in these genes using NGS and genome-wide methylation arrays. Deleterious germline mutations were identified in 32 (3.6%) patients for BRCA1, in 28 (3.1%) for BRCA2 and in 26 (2.9%) for RAD51C. Ten somatically sequenced patients had deleterious alterations, six (2.1%) in BRCA1 and four (1.4%) in BRCA2. Fifty two patients (10.8%) had methylated BRCA1 or RAD51C. HRD patients with germline or somatic alterations in any gene were more likely to be high grade serous, have an earlier diagnosis age and have ovarian and/or breast cancer family history. The HRD phenotype was most common in high grade serous EOC. Identification of EOC patients with an HRD phenotype may help tailor specific therapies.

[Physico-chemical properties and structure of oligomycin SC-II, produced by Streptomyces virginiae 17].

Under the screening programme for antibiotics with antifungal and immunosuppressive activities, Streptomyces virginiae 17 producing an oligomycin complex was isolated. Separation of the complex by HPLC showed that it contained two components at a ratio of 8:2. The physico-chemical characteristics of the components were investigated. The structure of oligomycin was assessed by 13C NMR and 1H NMR.

[Streptomyces sp. 17, an organism producing oligomycin SC-iI (culture characteristics and antibiotic biological properties)].

Under the screening programme for organisms producing substances with hypolipidemic and antifungal activity Streptomyces sp. 17 was isolated. The taxonomic properties of the strain were investigated. Active compounds, i.e. oligomycin A and oligomycin SC-II were isolated from a complex biosynthetic product. Oligomycin A showed high antifungal activity whereas oligomycin SC-II had also moderate antibacterial activity.

[Influence of natural complex compounds with hipolipidemic activity on expression of bacteria virulence factors].

Natural complex compounds with hipolipidemic activity, having considerable inhibitory effect on expression of bacteria virulence factors were isolated. Inhibitory properties of the compounds with respect to pyocyanine and protease formation, as well as their influence on the quorum sensing mechanism in Chromobacterium violacium were shown.

[The influence of compound aITEL1296 on telomerase activity and the growth of cancer cells].

Telomerase is a ribonucleoprotein that synthesizes telomeric repeats and identified as a promising target for anticancer therapy. Here we describe a new compound aITEL1296 as a potent telomerase inhibitor. Its inhibitory activity was a bit higher (IC50 = 0,19 +/- 0,02 ng/ml) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. Besides telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5,0 +/- 0,2 ng/ml for most sensitive cell line LnCap) but not normal fibroblast cell line.

[Hyphomycetes as organisms producing cyclodepsipeptides].

The review deals with cyclodepsipeptides produced by hyphomycetes. The cyclodepsipeptide compounds are prospective new agents for the treatment of diseases of the infectious and pathophysiologic nature.

[Production of destruxins by coprotrophic Beauveria feline no. 7 strain].

Beauveria feline No. 7 strain was isolated from the shrewmouse wool and characterized by production of a complex of destruxins cyclodepsipeptides. The strain was shown to produce significant quatities of destruxin B and pseudodestruxin C. The destruxins were found to be able to inhibit formation of biofilms by Pseudomonas aeruginosa ATCC 27833.

[Influence of natural hypolipidemic compounds on biofilm formation by Pseudomonas strains].

Natural compounds showing considerable inhibitory effect on formation of biofilms by P. aeruginosa were selected. Synergism of the compounds with gentamicin with respect to both the inhibition of the biofilm formation and the gentamicin antibacterial effect was stated.

[Organisms producing hypolipidemic compounds with antioxidant activity].

Complex compounds produced by fungal cultures of Lecanicilium and Beauveria with both high hypolipidemic and antioxydant activities were screened. Two fractions of the hypolipipidemic compounds with antioxidant activity of 95 and 75% in a dose of 25 mcg/ml were isolated.

[Complex of cyclosporins produced by Tolypocladium inflatum isolated from soil sample from Buriatiia].

A micromycete culture was isolated from a soil sample of Buryatiya and identified as Tolypocladium inflatum No. 2. The culture was shown to produce a complex of cyclosporins of unusual component structure: the content of cyclosporin A (55-60%) was the same as that in the substances produced by the majority of the described cultures, the content of cyclosporin B was much higher (about 40%) and the content of cyclosporin C was relatively low (about 3%). An appreciable content of cyclosporin (Leu4)Cs (3%) proved to be of interest.