[Application of GLAD-PCR analysis for the methylation sites detection in the regulatory areas of tumor-suppressor genes ELMo1 and EsR1 in colorectal cancer].

Aberrant methylation of regulation regions of tumorsuppressor genes is showed for many cancer diseases. In course of this modification an enzyme DNMT3 methylates RCGY sites in CpG-islands of regulation regions producing R(5mC)GY sites. Earlier we developed GLAD-PCR assay to determine R(5mC)GY site in a definite position of human genome. In this work we have applied GLAD-PCR assay to determine R(5mC)GY sites in regulation regions of ESR1 and ELMO1 tumor-suppressor genes. We have studied a fragment of first exon of ELMO1 gene and a part of ESR1 promoter region in DNA preparations from malignant cell line SW837 and colorectal tumor samples. We have checked four sites in each region and found two highly methylated sites: GCGC in first exon of ELMO1 gene and GCGT in promoter region of ESR1 gene. Site GCGT is weakly methylated in healthy tissues and more methylated in the most of colorectal samples. Site GCGC is not methylated in healthy tissues and significantly methylated in 60% of colorectal samples. A possibility to use GLAD-PCR assay for cancer diagnostics is discussed.

Coronaviruses (Coronaviridae) of bats in the northern Caucasus and south of western Siberia.

INTRODUCTION: Bats are natural reservoirs of coronaviruses (Coronaviridae), which have caused three outbreaks of human disease SARS, MERS and COVID-19 or SARS-2 over the past decade. The purpose of the work is to study the diversity of coronaviruses among bats inhabiting the foothills and mountainous areas of the Republics of Dagestan, Altai and the Kemerovo region. MATERIALS AND METHODS: Samples of bat oral swabs and feces were tested for the presence of coronavirus RNA by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It has been shown that the greater horseshoe bats (Rhinolophus ferrumequinum), inhabiting the Republic of Dagestan, are carriers of two different coronaviruses. One of the two coronaviruses is a member of the Sarbecovius subgenus of the Betacoronavirus genus, which includes the causative agents of SARS and COVID-19. The second coronavirus is assigned to the Decacovirus subgenus of the Alphacoronavirus genus and is most similar to viruses identified among Rhinolophus spp. from European and Middle Eastern countries. In the Altai Republic and Kemerovo region, coronaviruses belonging to the genus Alphacoronavirus, subgenus Pedacovirus, were found in the smooth-nosed bats: Ikonnikov`s bat (Myotis ikonnikovi) and the eastern bat (Myotis petax). The virus from the Altai Republic from M. ikonnikovi is close to viruses from Japan and Korea, as well as viruses from Myotis spp. from European countries. The virus from the Kemerovo region from M. petax groups with coronaviruses from Myotis spp. from Asian countries and is significantly different from coronaviruses previously discovered in the same natural host.

GLAD-PCR Assay of R(5mC)GY Sites in the Regulatory Region of Tumor-Suppressor Genes Associated with Gastric Cancer.

At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.

[Application of GLAD-PCR Assay for Study on DNA Methylation in Regulatory Regions of Some Tumor-Suppressor Genes in Lung Cancer].

Hypermethylation of the gene regulatory regions are common for many cancer diseases. In this work we applied GLAD-PCR assay for identificating of the aberrantly methylated RCGY sites in the regulatory regions of some downregulated genes in tissue samples of lung cancer (LC). This list includes EFEMP1, EPHA5, HOXA5, HOXA9, LHX1, MYF6, NID2, OTX1, PAX9, RARB, RASSF1A, RXRG, SIX6, SKOR1 and TERT genes. The results of DNA samples from 40 cancer and 25 normal lung tissues showed a good diagnostic potential of selected RCGY sites in regulatory regions of MYF6, SIX6, RXRG, LHX1, RASSF1A and TERT genes with relatively high sensitivity (80.0 %) and specificity (88.0 %) of LC detection in tumor DNA.

[Functional polymorphism of p53 and CCR5 genes in the long-lived of the Siberian region]. / Funktsional'nyi polimorfizm genov p53 i CCR5 u dolgozhitelei Sibirskogo regiona.

The P53 protein is a key regulator of modified-cell apoptosis. The functional oligonucleotide polymorphism of the p53 gene causes the substitution of arginine (Arg) for praline (Pro) in the codon 72. A reduced apoptotic activity of p53 and, as a consequence, development of oncology pathology is associated with the above polymorphism. CCR5 is a compound transmembrane receptor-protein, which apart from chemokines, binds with some molecules and is a coreceptor for HIV-1. 32 bp deletion within the CCR5 encoding region results in the loss of the protein's receptor function. It has been demonstrated that the transmission of the "external" (in respect to cell) stimulus, via the CCR5 system, induces expression of the p53 gene and initiates apoptosis. Allele variants and p53 and CCR5 genotypes (separately and in combinations) were investigated, within the present case study, for 131 long-livers from Novosibirsk and Tyumen Regions. A trend was detected towards accumulation of the p53 Pro alleles in association with the CCR5del32 allele in the study group, which, as the authors believe, can enhance the genome resistance to variable factors that cut the life span.