INTRODUCTION: Puumala virus (family Hantaviridae, genus Orthohantavirus) is distributed in most regions of the European part of Russia. However, information about its genetic variants circulating on the territory of the Central Federal District is extremely scarce. MATERIALS AND METHODS: Rodents' tissue samples were tested after reverse transcription by PCR for the presence of hantaviral RNA. The amplified fragments of the L segment were sequenced by the Sanger method. For two samples, sequences of all three segments were obtained using the NGS method. Phylogenetic trees were built in the MEGA-X software. RESULTS: Puumala virus was found in six samples. Based on the phylogenetic analysis of sequences of three segments, the obtained genetic variants belong to the sublineage previously designated as W-RUS. CONCLUSION: A genetic variant of the Puumala virus, belonging to the subline W-RUS, circulates on the territory of the Volokolamsk district of Moscow region.
Orthohantavirus , Puumala virus , Animals , Puumala virus/genetics , Phylogeny , Orthohantavirus/genetics , Moscow/epidemiology , Arvicolinae
Polyepitope DNA vaccine inducing T-cell-mediated immune response against cancer-specific antigens is a promising tool for selective elimination of tumor cells. Breast cancer-specific polyepitope DNA vaccine was designed using TEpredict and PolyCTLDesigner software on the basis of immunogenic peptides of HER2 and Mammaglobin-1 (Mam) tumor antigens. LPS-free preparations of plasmid DNA encoding polyepitope T-cell antigen and full-length copies of HER2 and Mam antigens were obtained. TaqMan-PCR systems for evaluation of the expression of immunogens in cells were created. The protocol of vaccine DNA delivery into dendritic cells was optimized. Expression of the target immunogens in dendritic cells derived from human peripheral blood mononuclear fraction after transfection with plasmid DNA preparations is demonstrated.
Breast Neoplasms/immunology , Cancer Vaccines/immunology , Dendritic Cells/immunology , Mammaglobin A/immunology , Receptor, ErbB-2/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/immunology , Breast Neoplasms/prevention & control , Cell Line, Tumor , HEK293 Cells , Humans , Immunotherapy/methods , Interleukin-10/biosynthesis , Interleukin-6/biosynthesis , Polymerase Chain Reaction
The major methods of microRNA extraction from different biological fluids (particularly, serum and plasma), approaches to the analysis of microRNA concentration and composition, normalization methods used in data analysis are outlined in the review. The advantages and disadvantages of the described methodological approaches are being highlighted. Special attention is given to microRNAs, circulating in blood, which could be used as the markers for minimally invasive lung cancer diagnostics, prediction of antitumor treatment efficiency and disease prognosis. Prospects and limitations arising from the evaluation of clinical significance of microRNAs as the potential tumor markers, and emerging as roles of various microRNAs in the pathogenesis of lung cancer become known, are discussed.
Biomarkers, Tumor/blood , Lung Neoplasms/genetics , MicroRNAs/genetics , RNA, Neoplasm/genetics , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/blood , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , MicroRNAs/blood , Neoplastic Cells, Circulating/pathology , Prognosis , RNA, Neoplasm/blood
Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.
Chromosomes, Human , Cytogenetics/methods , Cytosine/metabolism , DNA Probes , DNA Restriction Enzymes/metabolism , In Situ Hybridization, Fluorescence/methods , Plasmids/genetics , Base Sequence , Chromosomes, Human/chemistry , Chromosomes, Human/genetics , Cytosine/chemistry , DNA/chemistry , DNA/genetics , DNA Primers/chemistry , DNA Primers/genetics , DNA Primers/metabolism , DNA Probes/chemistry , DNA Probes/genetics , DNA Restriction Enzymes/genetics , Gene Library , Genome, Human , Humans , Metaphase , Molecular Sequence Data , Plasmids/chemistry , Plasmids/metabolism , Polymerase Chain Reaction/methods , RNA, Ribosomal, 28S/chemistry , RNA, Ribosomal, 28S/metabolism , Repetitive Sequences, Nucleic Acid
The composition and kinetics of accumulation of extracellular DNA in cultures of primary human endotheliocytes, cervical adenocarcinoma, and mycoplasma-infected cervical adenocarcinoma cells were studied. The content of DNA bound to cell surface did not change during culturing. The concentration of extracellular DNA in culture medium increased during the lag phase and at the beginning of the exponential growth phase, which probably attests to active secretion of DNA by cells. Spontaneous extracellular DNA synthesis was observed only in cell culture infected with mycoplasma.
DNA/analysis , Mycoplasma/isolation & purification , Cell Line, Transformed , Electrophoresis, Agar Gel , HeLa Cells , Humans
