Недавно была обнаружена ассоциация полиморфизмов гена SIRT1, кодирующего сиртуин-1 (фермент, обладающий деацетилазной или АДФ-рибозилтрансферазной активностью, в частности, деацетилирующий транскрипционные факторы, вовлеченные в регуляцию стресса), с тревогой и депрессией, однако его роль в механизмах развития депрессии остается малоизученной. В работе проведен анализ фармакологических эффектов ресвератрола, (активатор SIRT1 – 40 мг/кг, 4 недели, внутрибрюшинно) и никотинамида (ингибитора SIRT1 – 500 мг/кг, 4 недели, внутрибрюшинно) на проявления депрессивно-подобного поведения у мышей, вызванные хроническим непредсказуемым стрессом (ХНС). Ресвератрол снижал двигательную активность в тесте «Открытое поле» вне зависимости от ХНС, не влиял на социальное поведение и продолжительность неподвижности в тесте «Принудительное плавание» («поведенческое отчаяние»). В то время как никотинамид оказывал антидепрессивное действие в тесте «Принудительное плавание», не влияя на двигательную активность, но подавлял социальную мотивацию у животных, подверженных ХНС. Эффекты никотинамида — фармакологического блокатора SIRT1 оказались более выражены в условиях хронического стресса, чем влияние активатора SIRT1, ресвератрола. Актуальны дальнейшие исследования роли SIRT1 в молекулярно-генетических депрессогенных механизмах, вызванных стрессом.

Activating mutations of EGFR are associated with sensitivity of non-small cell lung cancer (NSCLC ) to tyrosine kinase inhibitors (TKI). Liquid biopsy using circulating cell-free tumor DNA (cfDNA) is proposed in cases when formalin fixed paraffin embedded (FFPE) tumor tissue is not available and for monitoring of EGFR status. In the study we evaluated new qPC R assay for EGFR mutations in plasma cfDNA. Sensitivity of the assay was 1 % of the mutant allele for L858R, L861Q, S768I mutations and deletions in exon 19, and 5 % of the mutant allele for G719X or T790M mutations Before surgery, mutation was detected in plasma of 4 out of 7 patients (57 %) with mutant EGFR in FFPE tumor tissue. Mutations found in cfDNA completely matched those found in tumor tissue in 2 cases. In one case with G719X and S768I mutations in FFPE tissue, only S768I was found in cfDNA. In another case, T790M was detected in plasma in addition to L858R that was present in tumor tissue. No EGFR mutations were detected in plasma DNA from 12 healthy volunteers and 13 cases of NSCLC with wt EGFR suggesting 100 % specificity of the assay. Liquid biopsy detected EGFR mutations in cfDNA in 8 of 16 cases of NSCLC with mutant EGFR being under therapy with TKI. Among them, 7 cases had mutations in liquid biopsy that matched those in tumor tissue and another case had T790M in addition to L858R. In 3 cases increased mutant allele frequency was detected 212 months before clinical progression.

Aim of the study. Aim of the study was to estimate the occurrence of pathogenic mutations in the BRCA1 gene in Russian breast cancer patients.Material and methods. Complete coding sequence of the BRCA1 gene of 445 early onset breast cancer patients (under 40 years) from Novosibirsk region (Russia) were analyzed by targeted Next Generation Sequencing (NGS) using Ion Torrent platform. Results. Forty (9%) carriers of various pathogenic mutations were revealed. Thirty five (7,9%) patients carried 5382insC mutation, described earlier as a founder mutation for Slavic population. Five (1.1%) patients carried various pathogenic mutations, namely C61G, 462delCC, E143X, 4153delA, and IVS18+1G>T. Besides, 29 genetic variants with no clinical significance or with unknown clinical significance were detected in BRCA1 gene among 445 early onset breast cancer patients. Conclusions. Data on the frequency of genetic variations in the BRCA1 gene among early onset breast cancer patients in the Novosibirsk Region (Russia) were obtained. Proportion of the 5382insC mutation is 87.5% of all pathogenic mutations in the BRCA1 gene found in patients.

Activating mutations of EGFR are associated with sensitivity of non-small cell lung cancer (Nsclc) to tyrosine kinase inhibitors (tKI). liquid biopsy using circulating cell-free tumor dNA (cfdNA) is proposed in cases when formalin fixed paraffin embedded (FFpE) tumor tissue is not available and for monitoring of EGFR status. In the study we evaluated new qpcR assay for EGFR mutations in plasma cfdNA. sensitivity of the assay was 1 % of the mutant allele for l858R, l861Q, s768I mutations and deletions in exon 19, and 5 % of the mutant allele for g719X or t790M mutations Before surgery, mutation was detected in plasma of 4 out of 7 patients (57 %) with mutant EGFR in FFpE tumor tissue. Mutations found in cfdNA completely matched those found in tumor tissue in 2 cases. In one case with g719X and s768I mutations in FFpE tissue, only s768I was found in cfdNA. In another case, t790M was detected in plasma in addition to l858R that was present in tumor tissue. No EGFR mutations were detected in plasma dNA from 12 healthy volunteers and 13 cases of Nsclc with wt EGFR suggesting 100 % specificity of the assay. liquid biopsy detected EGFR mutations in cfdNA in 8 of 16 cases of Nsclc with mutant EGFR being under therapy with tKI. Among them, 7 cases had mutations in liquid biopsy that matched those in tumor tissue and another case had t790M in addition to l858R. In 3 cases increased mutant allele frequency was detected 212 months before clinical progression. key words: liquid biopsy, qpcR, egfR, mutation, lung cancer. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Activating somatic mutations within the tyrosine kinase (TK) domain in the epidermal growth factor receptor (EGFR) protein are associated with the sensitivity of tumors to EGFRTK inhibitors [1]. Most of these mutations (~90 %) occur as either in-frame microdeletions that affect codons 746–750 in exon 19 (19del), or a missense mutation (L858R) in exon 21 of EGFR. In addition, rare activating mutations (L861Q, G719S/A/C, S768I and others) make up 10–15 % of cases with mutant EGFR. Besides, T790M mutation occurs in ~50 % of cases of acquired resistance to the first generation EGFR TK inhibitors (TKI). Tumors with T790M mutation are sensitive to therapy with osimertinib, which is the third generation TKI [2]. The European Society for Medical Oncology (ESMO) and American Society of Clinical Oncology (ASCO) recommend an EGFR mutation testing for selection of NSCLC patients who could benefit from anti-EGFR therapy [3, 4]. DNA from formalin fixed paraffin embedded (FFPE) tumor tissue is usually used to test mutations. In cases when tumor tissue is not available, the liquid biopsy using blood plasma that contains circulating cell-free tumor DNA (cfDNA) from tumor cells can be tested for clinically actionable mutations [5]. Major challenges of liquid biopsy are low concentration of cfDNA in plasma, and low frequency of mutant allele in cfDNA due to high background of DNA from normal cells. The assays for liquid biopsy are based on different methods including quantitative PCR (qPCR), digital PCR (dPCR), next generation sequencing (NGS) [6]. NGS can test a broad range of mutations with high sensitivity but the assay is expensive, technically demanding and has turnaround time of 12 weeks. Tests based on qPCR and dPCR detect only hot spot mutations but provide fast turnaround time of several days. dPCR has a high sensitivity and can detect mutations with mutant allele frequency (MAF) below 0,1 %. However, the high analytical sensitivity of dPCR often cannot be fully explored due to not sufficient concentration of cfDNA in plasma. The sensitivity of qPCR can be 1 % MAF. Importantly, equipment and reagents for qPCR are commonly available. The aim of our study was to evaluate liquid biopsy for EGFR mutations using new test based on qPCR. We used limited multiplex PCR (mPCR) of regions in 1821 exons of EGFR with hot spots for mutations

PCR clamping/wild-type blocking PCR with non-extendable locked nucleic acid (LNA) oligonucleotides is used for sensitive detection of somatic mutations in tumors. Various versions of the technique use different DNA polymerases and LNA oligonucleotides with and without additional phosphorothioate modifications. Here we studied requirements for successful PCR clamping with LNA oligonucleotides and Taq DNA polymerase for analysis of mutations in KRAS and BRAF genes by means of real-time PCR and Sanger sequencing. We found that addition of phosphorothioate linkages at the 5’-end of LNA oligonucleotide to protect from 5’- exonuclease activity of Taq DNA polymerase did not improve clamping. For most target sequences, efficient clamping was observed at melting temperature of LNA oligonucleotide 20‑25°C above annealing/extension temperature of the PCR with a 2-step protocol. Under such conditions, simple and sensitive detection of mutations in KRAS and BRAF genes was feasible using real-time PCR with TaqMan probes or Sanger sequencing.

The BRCA1 5382insC and CHEK2 1100 delC mutations were analyzed in 3850 of non-selected breast cancer patients residing in Novosibirsk region, Russia. One hundred seventy probands, BRCA1 5382insC or CHEK2 1100delC mutation carriers, were found. The study demonstrated that more than 80 % of probands informed relatives about their hereditary or familial cancer risk. Fifty-nine BRCA1 5382insC and CHEK2 1100 delC mutation carriers were found among 144 proband’s relatives. Mutation carriers or their relatives were interviewed two years after starting the project. At least 18 % of the mutation carriers reported primary tumor or recurrence of the tumor 2 years after starting the project.

Mutations in KRAS and BRAF genes in 80 colorectal cancer (CRC) samples from Russian patients were tested using two methods: 1) allele-specific real-time PCR (as-rt PCR) and 2) wild-type blocking PCR with Sanger sequencing (WTBS). Material and methods. Sections of fresh frozen or formalin-fixed paraffin embedded tumor tissue from 80 patients were used in the study. Tumor tissue content was determined on H&E stained sections. Samples were first tested by as-rtPCR for common mutations of the KRAS gene (G12C, G12S, G12R, G12V, G12D, G12A, G13D) and mutations BRAFV600E. After that samples were evaluated in PCR with oligonucleotide blocking amplification of wild-type DNA for enrichment with mutant allele followed by Sanger sequencing of the PCR DNA (WTBS method). Results. In 5 (6.3 %) cases samples had low tumor tissue content (<20 %). In reconstruction experiments both methods detected 1–5 % mutant allele. Mutations of KRAS and BRAF genes were found in 37 (46 %) and 3 (3.8 %) of the clinical cases, respectively. Classification in to wild-type and mutant samples by both methods was in agreement in 79 (98.8 %) cases. A single case with rare mutation KRASG13R was detected by WTBS, but was missed by as-rtPCR since this mutation is not included in the test. Of note, KRAS mutations were detected by both tests in two cases with low tumor content. Two cases were found with multiple mutations: one with KRASG12V and G13D, and one with KRASG13D and BRAFV600E. Conclusion. The frequency of mutations in CRC was 46 % for mutations in the KRAS gene, and 3.8 % for the BRAF. We showed 98.8% agreement in KRAS mutation detection by sensitive Sanger sequencing and as-rt PCR. The data on the frequencies of mutations are in agreement with studies in other countries. This in the first study to discover CRC case with multiple mutations KRASG13D and BRAFV600E.

Меланома кожи (МК) является этиологически гетерогенным заболеванием, развитие которого связано с воздействием как средовых, так и генетических факторов. С использованием сегрегационного анализа и сравнительной геномной гибридизации были выделены наследственные онкологические синдромы, на фоне которых развивается МК, и картированы гены, вовлеченные в наследственный канцерогенез МК. В молекулярный патогенез спорадической МК вовлечены онкогены и гены-супрессоры, входящие в состав различных сигнальных каскадов. Гиперактивация сигнального пути RAS-RAF-MEK-MAPK наблюдается до 90% случаев МК человека, при этом соматические мутации в гене BRAF являются определяющими в 50-70% случаев МК.

The article explores the issues related to the legal regime of dividends in joint-stock companies: legal regulation, the procedure of calculation and payment. The authors point out the problems that need special attention in the study of civil and corporate law.

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.

The introduction of foreign peptides into alkaline phosphatase that have a minimal influence on the enzymatic activity of a protein is described in the work. Calculated based on the methods of molecular dynamics the longest surface loops of alkaline phosphatase, which are not adjacent to both active center of a dimeric enzyme and contact surface between its monomers, were used as sites of the introduction of foreign peptides. The length of loops was estimated by several methods. Two loops were used to genetically engineer the introduction of peptides. Experiments demonstrated that the introduction of several peptides after the Ala218 residue of alkaline phosphatase insignificantly influences on the enzyme activity. According to experimental data, the selection of the loop for introducing the foreign peptide can be determined using the methods of molecular dynamics based on calculating the mobility of dihedral angles. In order to create macromolecules with new features by introducing foreign polypeptides in enzymatically active proteins, it is possible in practice to use an estimation of the loop length by means of the methods of molecular dynamics.

Frequencies of the 5382insC mutation in the BRCA1 gene and the 1100delC mutation in the CHEK2 gene were compared in the group of breast cancer patients and the large-scale sample, consisting of 7920 DNA specimens from healthy residents of the city of Novosibirsk. Higher frequencies of these mutations in the patient group compared to the control sample (1.95% versus 0.25% for BRCA1 5382insC, and 1.78% versus 0.40% for CHEK2 1100delC) were observed, pointing to their association with susceptibility to breast cancer (OR = 7.86; 95% CI 3.51–17.30 and OR =4.46: 95% CI 2.04–9.49, respectively).

The paper presents the description of the experiments in line with the rational concept for the "safe" insertion of guest polypeptides into the alkaline phosphatase with the minimal influence of the inserts on the enzymatic activity of the protein. Several approaches are described in the paper for the surface loop length estimation and two loops were used as the sites for guest peptides introduction by gene engineering technique. The experiments clearly demonstrate that insertions of several peptides after Ala218 of alkaline phosphatase (the site was selected by loop length analysis) do not block the activity of the enzyme. According the experimental data, the selection of the loops for the guest peptides insertion can be defined by the mobility of backbone dihedral angles during molecular dynamics simulation of alkaline phosphatase. The paper demonstrates the possibility to use in practice the estimation of loop length based on the mobility of backbone dihedral angles during molecular dynamics simulation. Indeed, it looks that the proteins with new features can be constructed by the introduction of new polypeptides into the enzymatically active proteins.