Death receptors (DRs) and the participants of DR mediated signaling are characterized by a large number of mRNA isoforms generated by alternative splicing. Due to their high labor intensity and high cost, conventional methods (RT PCR and RT PCR in real time) are ineffective when the simultaneous detection of a plurality of mRNA isoforms is needed. In this regard, the use of DNA biochips is has prospective appli cations in analyzing the expression of many genes simultaneously. In this paper, we suggest an optimal strat egy of probes selection aimed at detecting the maximum number of mRNA splice variants generated by major participants of DR signaling. The objects of the study were 185 genes that form 1134 mRNA isoforms. As a result, a biochip design was developed that enables the detection of 499 mRNA isoforms (44% of total mRNA splice variants). The proposed strategy combines a high degree of modularity, the use of modern high perfor mance computers, and broad opportunities for setting up the selection criteria in accordance with the objec tives of the study.

The CD38 gene encodes a membrane bound protein that takes part in cell adhesion and catalyzes the formation of cyclic ADP ribose. The presence of the full size and alternative forms of CD38 mRNA in tumor tissue specimens from patients with colorectal cancer and tumor cell lines was analyzed by RT PCR. It was shown that tumor specimens contained cells that constantly express CD38; full size CD38 mRNA was detected more frequently than its alternative form. CD38 mRNA was found in Colo 205, T 84, HCT15, and HCT116 cells, but not in Caco 2 or SW 620 cells. CD38 expression was observed in all cases of stage I colo rectal cancer; at stages II, III, and IV, it was significantly less frequent. The frequency of CD38 mRNA detec tion did not depend on the localization or grade of the tumor, or the presence of metastases. An analysis using methylation sensitive restriction endonucleases showed that CpG dinucleotides were methylated at the bind ing sites of the Sp1 transcription factor and retinoic acid receptor in all tumor specimens studied, regardless of the presence or absence of CD38 mRNA. The obtained data suggest that CD38 expression in tumor cells of colorectal cancer patients is heterogeneous.

In Russia, of the whole variety of EBV- and HHV6-etiology diseases, only infectious mononucleosis is subject to official statistical reporting, which significantly complicates an objective assessment of their etiological structure, incidence rate, characteristics of the development of the epidemic process. Currently, information on the genetic heterogeneity of EBV, even at the level of the main types (EBV1 and EBV2), as well as HHV6A and HHV6B, their prevalence and clinical significance are limited mainly by foreign data. In Russia, there are isolated publications devoted to this issue. At the same time, the study of the circulation of genetic types (variants) and the use of this information in the implementation of epidemiological surveillance of some other infections have already become routine practice. One of the key issues is the level of development of laboratory support for molecular genetic monitoring. The purpose of this work was to improve the methodological base of differential detection of HHV6A/B and the main types of EBV. The material for the study was peripheral blood leukocytes of children aged 1-15 years with acute infectious mononucleosis (n = 50) and without clinical symptoms of this disease (n = 29). The detection and quantification of EBV DNA and HHV6 DNA was performed using real-time PCR. For differential determination of EBV1/EBV2 and HHV6A /HHV6B, an optimized one-round PCR with electrophoretic detection of amplification products in an agarose gel was used. According to the results of our own research, the frequency of detection of  EBV DNA and HHV6 DNA in acute infectious mononucleosis was 74% and 72%, and in the control group - 35% and 74%, respectively. It was found that among the examined children of the Nizhny Novgorod region, EBV1 and HHV6B prevail in the viral population, which is consistent with existing views about their geographical distribution in the adjacent territories. EBV2 was found in a single sample in the control group only. HHV6A was not detected in any of the studied groups. The methodological approach optimized in this work makes it possible to separately detect HHV6A/HHV6B and the main types of EBV according to a single laboratory protocol, and in combination with an additional stage of DNA concentration increases the diagnostic sensitivity of PCR analysis, minimizes the proportion of discordant and false negative results. Such an integrated approach can be applied for diagnostic, epidemiological and research purposes.

Transforming growth factor β 1 (TGFB1) likely contributes to the pathogenesis of Epstein-Barr virus (EBV)-mediated cancer. A microarray containing 59 probes for detecting mRNA of members of TGFB1-associated pathways was developed. mRNA expression of TGFB1 receptors and members of connected pathways were examined in peripheral blood leukocytes of patients during acute EBV infection and after recovery. TGFB1 and TGFBR2 mRNA expression was increased in patients with EBV infection. Similarly, mRNA expression of protein kinase C (PRKCB), MAP3K7, PDLIM7, and other members of TGFB1 and NF-κB signaling pathways increased. A shift of mRNA transcript variant expression of some key members (TGFBR2, PRKCB, and NFKBIB) of involved signaling pathways was detected. After the patients' recovery, most of the altered mRNA expression has been normalized. We speculate that in patients with EBV infection, members of TGFB1-associated pathways contribute to the suppression of proapoptotic and induction of pro-survival factors in leukocytes. The modulation of TGFB1-associated pathways may be considered as a potential risk factor in the development of EBV-associated tumors in patients with acute EBV infection.

Epstein—Barr virus (EBV) is one of the etiological agents causing infectious mononucleosis. A severe form of the disease can result in developing serious complications, which risk might also depend on the state of patient’s immune system. To date, no specific tests for assessing a risk of developing severe disease form are available. Our study was aimed at identifying molecular genetic markers of severe EBV-infectious mononucleosis (EBV-IM) in immunocompetent peripheral blood cells. Expression of 483 genes and gene transcripts regulating apoptosis, proliferation and differentiation of immunocompetent cells was measured in the peripheral blood leukocytes from patients with severe and moderate EBV-IM as well as apparently healthy donors. A DNA-microarray designed by us and subsequent data processing were carried out by using custom-made “MiDA” software. To identify markers of a severe form of the pathology, expression of each gene and transcript was compared in EBV-IM patients and apparently healthy donors. For each gene and transcript, the level of expression fold change and significance for binary classification were determined. Genes and transcripts, characterized by the maximum values of two determined parameters while comparing patients with severe infection and healthy donors, as well as patients with severe and moderate EBV-IM forms, were selected as markers of severe EBV-IM. Genes and transcripts with differed expression in patients with moderate EBV-IM and healthy donors, were excluded from the list of markers. In addition, sex- and age-linked markers with differed expression were excluded as well. The markers for severe EBV-IM consisted of apoptosis regulators (BCL2L11, BIRC3 genes and XIAP.NM_001167 transcript) and splicing factors (CELF6 gene and SF1.NM_201995 transcript). Compared with donors and patients with a moderate form of the disease, a decreased expression of BCL2L11, BIRC3 genes, transcripts SF1.NM_201995 and XIAP.NM_001167, as well as increased expression of the CELF6 gene were detected in the blood of patients with severe EBV-IM. The functional role of identified molecular markers suggests that severe EBV-IM is characterized by suppressed mitochondrial and activated TRAF-dependent apoptosis pathways in immunocompetent cells. The expression pattern for select markers is specific for severe EBV-MI, not associated with patient sex and age. Thus, study data may be used to develop specific tools for assessing a risk of developing complications of EBV mononucleosis.