The Level of LINE-1 mRNA Is Increased in Extracellular Circulating Plasma RNA in Patients with Colorectal Cancer.

We performed a comparative quantitative analysis of LINE-1 mRNA levels in extracellular total plasma RNA in patients with colon cancer and practically healthy donors. Quantitative multiplex PCR with reverse transcription was used to assess the level of LINE-1 and 18S rRNA mRNA in extracellular total plasma RNA. The median of LINE-1 mRNA values in colon cancer patients (4.95) was significantly higher than in healthy donors (2.3) (p=0.037). It was shown for the first time that the level of LINE-1 mRNA in total RNA from blood plasma can be determined in the format of a liquid biopsy and serve as a new potential non-invasive marker of colon cancer.

Comparison of polymerase chain reaction and flow cytometry for measuring telomere length of human leukocytes.

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.

[Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis].

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Spectrum of TP53 Mutations in BRCA1/2 Associated High-Grade Serous Ovarian Cancer.

Objective: Mutations in TP53 lead to loss of function (LOF) or gain of function (GOF) of the corresponding protein p53 and produce a different effect on the tumor. Our goal was to determine the spectrum of somatic TP53 variants in BRCA1/2 associated high-grade serous ovarian cancer (HGSOC). Methods: The population under study comprised of HGSOCs with pathogenic variants in BRCA1 (n = 78) or BRCA2 (n = 21). Only chemo-naive and platinum-sensitive patients were included in this study. The case group of the IARC database (n = 1249) with HGSOC not stratified by BRCA status was used as a reference. A custom NGS panel was used for sequencing TP53 and mutational hot-spots of other genes, and p53 expression was evaluated by immunohistochemistry for 68 cases of HGSOCs. Results: Somatic TP53 variants (95) or inhibition of wild-type p53 expression (3) were observed in 98 cases. The sample with normal p53 had CDKNA1 variants. The frequency of truncating variants was significantly higher than in the reference cohort (30.3 vs. 21.0%, p = 0.01). Most of the samples (41/68) demonstrated low (or absent) expression of p53, and 17 samples overexpressed p53. LOH was typical for TP53 nonsense variants (14/15). In total, 68/95 samples were LOH positive and showed LOH in all tumorous cells, thus indicating the driver effect of TP53 mutations. Three specimens had KRAS, BAX, APC, and CTNNB1 subclones variants. Conclusion: High frequency of TP53 truncating variants, the low expression of mutant p53, and low incidence of oncogene mutations show potential GOF properties of p53 to be poorly represented in BRCA1/2 associated HGSOC.

Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products.

We developed a protocol for detection of mutations in the pncA gene associated with M. tuberculosis resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZAR DNA isolates carrying mutations in the pncA gene, 3 PZAR isolates without mutations in the pncA gene, and 20 PZAS isolates without mutations in the pncA gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the pncA gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy - 96.55% vs. 93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.

Mitochondrial DNA analysis of ancient sheep from Altai.

A comparative analysis of the genetic diversity of ancient and modern sheep can shed light on the origin of these animals and their distribution as well as help to evaluate the role of humans at each formation stage of different sheep breeds. Here we isolated ancient DNA and performed sequencing of the mitochondrial DNA D-loop from 17 sheep bone remains (~4000-1000 years old) found in the archaeological complexes in the south of Altai (Western Siberia). The length of the sequences obtained ranged between 318 and 586 bp. The haplotype diversity and nucleotide diversity were 0.801 ± 0.081 and 0.0096 ± 0.0014 respectively. The average number of nucleotide differences was ~3.1. Nucleotide sequence analysis revealed that 15 specimens were nested within previously described A,B,C,D and E lineages and that two specimens had a basal position relative to the rest of the analyzed samples. A relatively high diversity of sheep haplotypes, including the presence of two basal haplotypes, indicates that the Altai region may have been a transport route of human migration. Further ancient DNA analysis of other specimens and deeper genome sequencing of samples with novel haplotypes is needed to better understand the demographic history of sheep in Southern Siberia.

Antagonistic Activity of Intestinal Lactobacteria from Domestic Fowl against Clinical Isolates of Salmonella enterica.

Strain parameters of Salmonellas and Lactobacteria affecting their antagonistic activity were studied using an original method. Real-time PCR was applied for measuring the decrease in the concentration of field isolates of Salmonellas isolated from chickens in the presence of field isolates of Lactobacteria also isolated from the gastrointestinal tract of chickens and broiler chickens. Dispersion analysis showed that the probability of suppression of Salmonella growth by Lactobacteria is determined by strain parameters of both Lactobacteria and Salmonellas (p<0.001). However, strain parameters of Lactobacteria were found to be more important that Salmonella strain parameters during co-culturing.

DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin's Inhibitory Effect.

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30 kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-70 °C, and both were inactivated via heating at 95 °C for 15 min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.

Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.

Magnetofection of human somatic cells with magnetite and cobalt ferrospinel nanoparticles.

Superparamagnetic nanoparticles varying by their chemical composition and synthesis method were used to transfer DNA into somatic cells under the influence of constant magnetic field (method of magnetofection). Magnetite particles obtained by mechanochemical synthesis ensured higher expression of the marker gene GFP (evaluated by fluorescence intensity of the cell lysate) then particles of ferric oxide obtained by chemical co-precipitation and cobalt ferrospinel particles obtained by the mechanochemical method.

Tissue-specific expression of splice variants of human IL-4 and IL-6 gene mRNA.

The spectrum of alternatively spliced IL-4 and IL-6 gene mRNA was studied in peripheral blood mononuclears from healthy donors and in human fetal tissues. It was found that the expression of alternatively spliced IL-4 and IL-6 gene mRNA in fetal tissues is tissue specific and that hemopoiesis- and immunopoiesis-related tissues differ by the amount of IL-4 and IL-4δ2 mRNA. An mRNA variant IL-4alt3 carrying partial exon 3 deletion was for the first time identified in human mononuclear cells.

[Biodegradation of polyhydroxyalkanoates by soil microbiocoenoses of different structures and detection of microorganisms-destructors].

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbiocoenoses of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA destructors under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium. and Zygosporium.

Highest prevalence of the Mycobacterium tuberculosis Beijing genotype isolates in patients newly diagnosed with tuberculosis in the Novosibirsk oblast, Russian Federation.

In order to assess the genetic diversity of Mycobacterium tuberculosis in the Novosibirsk Region and determine profiles of resistance, 106 M. tuberculosis isolates were analysed. Fifty (47 %) isolates were identified using variable number tandem repeat typing as being in the Beijing family, of which eight (16 %) were type M2 isolates with the genetic profile 233325153533424 and eight (16 %) were type M11 isolates with the genetic profile 233325173533424, both of which are widespread in Russia. Mutations associated with resistance to isoniazid and rifampicin were identified. Of 48 isolates with resistance to isoniazid, 42 (87.5 %) contained a Ser(315)→Thr substitution in the katG gene and one contained a T→A substitution at position -34 of the promoter region of the ahpC gene. Of 31 isolates with resistance to rifampicin, 19 (61 %) each contained a mutation (TCG→TTG) at codon 531 of the rpoB gene. Two isolates each contained a mutation (GAC→GTC) at codon 516 of the rpoB gene and two others each contained a substitution at codon 526 of the rpoB gene, leading to a His→Asn substitution in one case and a His→Asp substitution in another case. One isolate contained a mutation (CTG→CCG) at codon 533 of the rpoB gene. An association between the Beijing genotype and multidrug resistance was demonstrated (R = 0.2, P = 0.032). However, it was interesting to note that a significant proportion (46 %) of isolates were sensitive to all drugs tested.

[Features of interaction of monoclonal antibody B2 with polycyclicaromatic hydrocarbons and peptide-mimotope of benzo[a]pyrene].

To define of possible mechanism of monoclonal antibody B2 interaction with haptens and peptide-mimotope of benzo[a]pyrene Fab-fragment structure was modeled. Antibody active centre structure was defined using docking with a number of different polyclic aromatic hydrocarbons. It was shown that active center of monoclonal antibody B2 composed of two binding pockets. Correlation was revealed between experimental and calculated data on relative free energies of monoclonal antibody B2 binding with different ligands. We found that synthetic peptide-mimotope of benzo[a]pyrene weakly competeswith benzo[a]pyrene conjugate in monoclonal antibody B2 binding. Immunization of mice with peptide-mimotope conjugate did not revealed antibodies to benzo[a]pyrene. To define structural features of peptide-mimotope of benzo[a]pyrene preliminary model of peptide-mimotope in the frame of pIII protein was calculated. It was shown that tryptophan located inside of peptide can be exhibited on a surface and accessible to antibody. Obtained modeling results could be applied for following optimizations ofbenzo[a]pyrene peptide-mimotope and active center of monoclonal antibody B2.

Selection of naturally occurring autoantibodies to interleukin-18 from phage display library.

Four unique phage single-chain variable fragments (scFvs) to recombinant human interleukin 18 (IL-18) has been selected from a naïve combinatorial library of human scFvs. Binding of unique phage antibodies with IL-18 was tested by ELISA and Western-blotting. No cross reactivity with tumor necrosis factor α, interferons α and γ was shown for the selected antibodies. The gene segments encoding V(D)J regions of selected antibodies exhibited a high degree of homology to germline genes, therefore we suggest that the selected scFv's belong to repertoire of naïve autoantibodies.

[New approach to synthesis of peptide immunomimetic of benzo[a]pyrene].

A novel approach to discovery of the peptide with an internal immunological image of carcinogen is suggested in this work. The hybridomas producing monoclonal antibodies (mAb) B2 against benzo[a]pyrene and benz[a]antracene were prepared. Polyclonal Ab against benzo[a]pyrene (Bp) and benz[a]antracene (Ba), antracene, chrysene, and pyrene were also prepared. We identified the related peptide-presenting phage specificity binding to mAT B2 and polyclonal Ab against Bp from 12 large random peptide libraries using phage display method. ICR mice were immunized with specific binding of positive phage clone for studying its immunogenicity. The Bp antibodies were found in the blood serum. All positive phage clones carried the sequence. Thus, the unique peptide with an internal immunological image of Bp may be the new candidate for anticarcinogenic vaccine.

[Cloning and expression of the gene of chymotrypsin-like protease of human kallikrein-7 in Escherichia coli and isolation of recombinant protein].

A sample of the human cDNA was used to amplify the segment encoding biosynthesis of chymotrypsin-like protease of kallikrein-7 and its cloning into the expressing plasmid pET23a(+). Biosynthesis of KLK-7 in transformed E. coil BL21(DE3) cells was accompanied by formation of insoluble inclusion bodies. The recombinant KLK-7 was extracted from the inclusion bodies using 7 M urea in the presence of 2-mercaptoethanol. The extracted recombinant KLK-7 was purified using methods of metal-chelate and ion-exchange chromatography, converted into a soluble form, and used for preparing monospecific antiserum.

Alternative splicing of interleukin-6 mRNA in mice.

Expression of mRNA for interleukin-6, interleukin-6Delta3, and interleukin-6Delta5 was detected in placental tissue (second and third trimesters of pregnancy) and spleen of mice immunized with sheep erythrocytes in high dose. We hypothesize that translation of mRNA yields proteins capable of binding to individual subunits of the interleukin-6 receptor and possessing effector functions.

[KatG Ser3 15Thr mutation as the main reason of isoniazid resistance in Mycobacterium tuberculosis isolated in the Novosibirsk and Kemerovo Regions]. / Mutatsiia Ser315Thr gena katG--osnovnaia prichina ustoichivosti k izoniazidu u izoliatov Mycobacterium tuberculosis, rasprostranennykh v Novosibirskoi i Kemerovskoi oblastiakh.

KatG Ser3 15Thr mutation is one of the main reasons of resistance to isoniazid in Mycobacterium tuberculosis isolates. The frequency rate of the above mutation among isoniazid-resistant isolates made 94% in Novosibirsk Region and 93% in Kemerovo Region. The use of PCR-restriction fragment length polymorphism (PCR-RFLP) can be regarded as an adequate method for rapid screening of isoniazid resistance in Mycobacterium tuberculosis isolated in the West-Siberian Region.

Alternative splicing of murine interleukin-4 mRNA.

We found an alternative form of mRNA with spliced second exon (IL-4delta2 mRNA) in mouse bone marrow and splenic cells. At rest, the amount of IL-4 mRNA markedly surpassed that of IL-4delta2 mRNA. Stimulation increased the content of both mRNA forms, but the alternative variant is accumulated more intensively and rapidly. We did not detect predominance of IL-4delta2 mRNA over full-length mRNA variant in the studied mouse tissues.