Development of a Test System to Detect the Omicron Variant of SARS-CoV-2 and the Frequency of Its Detection in Patients.

We developed a new test system to detect the omicron variant of SARS-CoV-2 using allele-specific reverse transcription PCR and estimated the frequency of its detection in patients living in the Novosibirsk Region. Clinical samples were divided into 3 groups: samples collected from December 1 to December 30, 2021 (group 1; n=66), from December 30, 2021 to January 10, 2022 (group 2; n=20), and from January 11 to January 22, 2022 (group 3; n=101). Based on the identification of 5 mutations specific to SARS-CoV-2 (B.1.1.529), two systems of oligonucleotide primers and probes were developed for detecting this coronavirus genotype in clinical samples. Limit of detection (LOD95) was 4×103 genome equivalents per 1 ml of clinical sample for the first test system and 2×103 for the for the second test system. The omicron variant of SARS-CoV-2 was absent in group 1 of studied samples, but was detected in 20% (4/20) of group 2 samples and 88% of group 2 samples collected within less than 2 weeks of January 2022. Using developed test system, we showed that in less than 2 weeks the omicron variant has become dominant in patients, which confirms previously published data on its exceptional contagiousness.

Design of Polyepitope DNA Vaccine against Breast Carcinoma Cells and Analysis of Its Expression in Dendritic Cells.

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.

[Circulating microRNAs in lung cancer: prospects for diagnostics, prognosis and prediction of antitumor treatment efficiency].

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.

MIRA analysis of RARß2 gene methylation in DNA circulating in the blood in lung cancer.

Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARß2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein.

RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofluorometry and electrophoresis. Experiments on RLS(40) tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specific small interfering RNA (siRNA). These findings suggest that active cyclophosphamide metabolite can be a substrate for P-glycoprotein.

[Molecular-genetic markers in lung cancer diagnostics].

The major approaches to different lung cancer marker development are outlined in the review, including genetic, epigenetic, protein, transcryptomic, proteomic, metabolic, and miRNA markers. As far as epigenetic changes are among the earliest events in malignant transformation, methylated markers are thoroughly discussed. Special attention is given to minimally invasive tumor markers, which could be detected in easily accessible biological fluids, because they can be useful for screening and early diagnostics of cancer (before its clinical manifestation) as well as for verification of standard methods of diagnostics. Extracellular nucleic acids, circulating in blood (cirNA), are highlighted as the potential source of material for the early lung cancer diagnostics, prediction of antitumor treatment efficiency, post-treatment monitoring and disease prognosis.

[The peculiarities of rat tissue reactions to intraperitoneal implants made out of biodegradable polyhydroxyalkanoates].

The reaction of rat tissues to intraperitoneal placement of implants made out of biodegradable polyhydroxyalkanoates (PHA), was studied at different time points after the operation by the methods of light microscopy. It was found that after intraperitoneal PHA implant placement, the active adhesive process started leading to formation of fibrous adhesions between PHA implants and intestinal loops. When PHA implants were used in the form of films, they became surrounded by a thick fibrous capsule. As a result of PHA implant placement in the form of ultrathin fibers, the extensive foreign body granulomas with perifocal inflammation and sclerosis of surrounding tissues were formed. In these granulomas the polymer fragmentation and phagocytosis by macrophages with formation of giant cells of foreign body occured. It is concluded that PHA implants are not biodegradable and induce tissue reactions similar to those caused by other foreign bodies.

[Assay of methylated gene RARbeta2 in circulating DNA of blood from patients with lung cancer as a potential prognostic marker].

Blood-based methylated DNA gene RARbeta2 in circulating plasma (cir DNA) and one associated with blood cell surface were assayed in patients with non small cell lung cancer before and after combined treatment. The levels in both appeared to be significantly higher than in healthy subjects. Enhanced levels prior to treatment were associated with greater advancement of the disease and unfavorable prognosis (overall survival). After two courses of neoadjuvant therapy plus surgery methylation indices fell down to match those in healthy subjects. Our data may be instrumental in working out additional criteria to be used in diagnosis, prognosis and follow-up of patients with non small cell lung cancer.

Taxonomic composition and biodiversity of the gut microbiome from patients with irritable bowel syndrome, ulcerative colitis, and asthma.

To date, the association of an imbalance of the intestinal microbiota with various human diseases, including both diseases of the gastrointestinal tract and disorders of the immune system, has been shown. However, despite the huge amount of accumulated data, many key questions still remain unanswered. Given limited data on the composition of the gut microbiota in patients with ulcerative colitis (UC) and irritable bowel syndrome (IBS) from different parts of Siberia, as well as the lack of data on the gut microbiota of patients with bronchial asthma (BA), the aim of the study was to assess the biodiversity of the gut microbiota of patients with IBS, UC and BA in comparison with those of healthy volunteers (HV). In this study, a comparative assessment of the biodiversity and taxonomic structure of gut microbiome was conducted based on the sequencing of 16S rRNA genes obtained from fecal samples of patients with IBS, UC, BA and volunteers. Sequences of the Firmicutes and Bacteroidetes types dominated in all samples studied. The third most common in all samples were sequences of the Proteobacteria type, which contains pathogenic and opportunistic bacteria. Sequences of the Actinobacteria type were, on average, the fourth most common. The results showed the presence of dysbiosis in the samples from patients compared to the sample from HVs. The ratio of Firmicutes/Bacteroidetes was lower in the IBS and UC samples than in HV and higher the BA samples. In the samples from patients with intestinal diseases (IBS and UC), an increase in the proportion of sequences of the Bacteroidetes type and a decrease in the proportion of sequences of the Clostridia class, as well as the Ruminococcaceae, but not Erysipelotrichaceae family, were found. The IBS, UC, and BA samples had signif icantly more Proteobacteria sequences, including Methylobacterium, Sphingomonas, Parasutterella, Halomonas, Vibrio, as well as Escherichia spp. and Shigella spp. In the gut microbiota of adults with BA, a decrease in the proportion of Roseburia, Lachnospira, Veillonella sequences was detected, but the share of Faecalibacterium and Lactobacillus sequences was the same as in healthy individuals. A signif icant increase in the proportion of Halomonas and Vibrio sequences in the gut microbiota in patients with BA has been described for the f irst time.

Controlled Fragmentation of Urinary Stones as a Method of Preventing Inflammatory Infections in the Treatment of Urolithiasis (Experience in Successful Clinical Use).

The introduction of technologically advanced methods of lithotripsy into medical practice changes the nature of postoperative complications. Among them, the main complications are inflammatory infections. This largely determines the search for new, improved methods of stone fragmentation avoiding small stone fragments and dissemination of the pelvicalyceal system of the kidney with stone-associated infection. The authors have developed a method for controlled stone fragmentation using a continuous-wave diode laser with a hot-spot effect at the optical fiber end. The aim of the study was to evaluate the efficacy of controlled urinary stone fragmentation using a continuous-wave diode laser with a highly heated distal end of the optical fiber light guide as a method of preventing inflammatory infections in clinical practice. MATERIALS AND METHODS: We analyzed 1666 case histories of urolithiasis patients who underwent percutaneous nephrolithotripsy/ nephrolithoextraction and contact ureterolithotripsy/ureteroextraction, we also performed a prospective analysis of complications based on the Clavien-Dindo classification in 90 patients who underwent fine fragmentation of stones with various lithotripters: ultrasonic, pneumatic, and holmium laser. The method of controlled stone fragmentation by a diode laser with a hot-spot effect was tested on postoperative samples of 26 renal calculi. For the first time in clinical practice, this method was tested in the bladder cavity (n=10). RESULTS: In the percutaneous nephrolithotripsy group, postoperative infectious and inflammatory complications occurred in 34.1% of cases, in the percutaneous nephrolithoextraction group - in 24.6%, in the contact ureterolithotripsy group - in 7.8%, in the ureterolithoextraction group - in 2.5%. The analysis made it possible to identify factors promoting the development of infectious and inflammatory complications. For the first time in clinical practice, there were successfully performed ten operations of stone fragmentation using a continuous-wave diode laser with a hot-spot effect. Controlled coarse fragmentation of stones providing the possibility to reduce the number of infectious and inflammatory complications was performed in the bladder as a model for testing the method. CONCLUSION: The method of laser-induced controlled coarse fragmentation of stones with a hot-spot effect, developed and tested in clinical practice, is promising for the prevention of infectious and inflammatory complications in patients with potentially infected stones since their fine fragmentation and, consequently, spread of stone-associated toxins and microflora within the urinary system is avoided.

[Cholesterol-modified anti-MDR1 small interfering RNA: uptake and biological activity].

Small interfering RNAs (siRNA) are considered to be potent agents for specific gene silencing, but troubles in delivery of siRNA into cells limit their biomedical application. An accumulation of siRNA coupled with cholesterol residue at the 5'-end of the "passenger" strand (chol-siPHK) was investigated in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfectant levels of both unmodified and chol-siRNAs were very low, whereas transfectant substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency for the chol-siRNA being higher than that for the corresponding siRNA. Multiple drug resistance phenotype reversing activity of anti-MDR1-siRNAs targeted to the 557-577 region of MDR1 gene mRNA was investigated in KB-8-5 cancer cells. The chol-siRNA induced cancer cells' death in the presence of vinblastine doses tolerated before more effectively than the conventional siRNA did.

[Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference].

In the present study we have applied the siRNA approach for substantial reduction of AML1-ETO and RUNX1 (K83N) expression, which are frequently found in the leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5'-untranslated region of mRNA for the mutant RUNX1 (K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of the oncogenes following the introduction of shRNAs into cells.

[An RNA sequence determines the speed of its splitting by artificial ribonucleases].

Phosphodiester bonds in RNA situated between similar nucleotides but in different sequences (context) were split under the action of artificial and natural ribonucleases with different speeds, and the reason for this phenomenon has not yet been fully revealed. In this study, the influence of one-nucleotide substitution on the sensitivity to splitting of the phosphodiester bonds in linear and structured RNA with homologous sequences is studied for the first time. It is indicated that the introduction of one-nucleotide substitution in the RNA sequence significantly (up to 10 times) changes the speed of the splitting of the bonds that are separated from the substitution point not only by 1-3, but also 6-8 nucleotides, by artificial ribonucleases. The observed regularities may be explained by the fact that the introduction of a one-nucleotide substitution significantly changes the stacking interactions and the net of hydrogen bonds in the RNA molecule. The applied value of this study consists of the ability of using low-molecular artificial ribonucleases with the aim of choosing the region of the binding of the oligonucleotide in the construction of a conjugate for the site-directed cutting of RNA, because the choice of a phosphodiester bond (motif) easily subjected to splitting significantly determines the effectiveness of artificial ribonucleases of directed action.

[Application of a two-layer dynamic self-regulating alloplasty in treatment of patients for giant postoperative abdominal hernia].

A new technique of two-layer dynamical abdominal wall alloplasty was applied in 2005-2008 in 10 patients, ageing (55.3 +/- 12.6) yrs old at average, for giant postoperative abdominal hernia, based on a G. Valenti principle of dynamical self-regulating prosthesis and the method of temporary closure of operation wound, according to Wittmann Patch procedure. It was proved, that introduction of original technique of a two-layer dynamical self-regulating abdominal wall alloplasty permits to escape postoperatively the abdominal compartment syndrome development.

[Preperitoneal alloplasty of inguinal canal].

The results of treatment of 221 patients for inguinal hernia, including preperitoneal hernioplasty in 117 and hernioplasty according to Lichtenstein method in 104 of them, were analyzed. After the operation the testis shells watering had occurred in 6 patients, the postoperative cicatrix infiltrate--in 6, operative wound hematoma--in 4. The hernia recurrence had occurred, while fol-lowing patients up from 6 months till 4 years, in one patient only after operation, performed according to Lichtenstein method. In patients, to whom preperitoneal plasty of inguinal canal was per-formed, a specific complications, associated with the operation performance, were absent. Preperitoneal plasty secures a safe strengthening of hernial gates. The net implant fixation in preperi-toneal space permits to escape the feeling of a "foreign body" pres-ence in inguinal area of the patients.

A New MicroRNA Cluster Involved in the Reprogramming to a Pluripotent State.

Reprogramming of somatic cells to a pluripotent state is a complex, multistage process that is regulated by many factors. Among these factors, non-coding RNAs and microRNAs (miRNAs) have been intensively studied in recent years. MiRNAs play an important role in many processes, particularly in cell reprogramming. In this study, we investigated the reprogramming of rat fibroblasts with a deleted locus encoding a cluster comprising 14 miRNAs (from miR-743a to miR-465). The deletion of this locus was demonstrated to decrease significantly the efficiency of the cell reprogramming. In addition, the cells produced by the reprogramming differed from rat embryonic and induced pluripotent stem cells, which was an indication that reprogramming in these cells had not been completed. We suggest that this miRNA cluster or some of its members are involved in regulating the reprogramming of rat cells to a pluripotent state.

[Circulating deoxyribonucleic acids in blood and their using in medical diagnostics].

Circulating nucleic acids were discovered more then 30 years ago and were intensively investigated for the last ten years. The manuscript review the data regarding sources of circulating deoxyribonucleic acids in blood, factors influence on their circulation, like blood nucleases, and excretion of DNA from blood. Application of circulating DNA in practical medicine is also discussed.

[Extraembryonic endoderm stem cell lines from common voles of the genus Microtus].

Twenty-eight independent extraembryonic endoderm (XEN) stem cell lines have been obtained from morula and blastocyst cells of common voles. Most cell lines form very few cell-cell contacts when growing and morphologically correspond to the XEN that were earlier described in mice. In addition, XEN cell lines with atypical morphology forming colonies have been obtained for the first time. Both types of XEN lines rapidly proliferate, retain their morphology and karyotype during more than 25 passages in cell culture, and express genes characteristic of XEN. One of two X chromosomes in XEN lines with karyotype XX has been shown to be inactive and associated with the Xist gene transcript. It has been demonstrated that the paternal X chromosome is inactive.