Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphology.

One of the most productive strategies for finding the functions of proteins is to study the consequences of loss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system, it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

[Structure of Neuroglobin from Cold-Water Sponge Halisarca dujardinii].

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

[Rational strategy for studying microbiome of the ocular surface of people using hard contact lenses by method of 16S rRNA gene metabarcoding]. / Ratsional'naya strategiya izucheniya mikrobioma glaznoi poverkhnosti pol'zovatelei zhestkikh kontaktnykh linz metodom metabarkodinga po genu 16S rRNK.

The study is based on the hypothesis that high taxonomic diversity of bacteria detectable on the eye surface by molecular genetic methods is attributed to the high level of its contamination by skin microflora. Such contamination would make it problematic to identify the fractions of real ocular surface microbiome, which remains behind the one-percent cut-off threshold adopted in the metagenomic analysis. Hard contact lenses for long-wearing act as a physical filter preventing DNA contamination from random microorganisms, and at the same time providing adhesion to the living cells of bacteria and fungi. To confirm this assumption, a detailed analysis of references was carried out, supplemented by original laboratory research. MATERIAL AND METHODS: The analysis included 16 hard contact lenses obtained from 11 patients with impaired refraction (myopia). Additionally, conjunctival mucosa scrapings were collected from 42 patients. Samples were cross-analyzed by 16S rRNA gene sequencing using 454 GS Junior (Ion Torrent) and Illumina MiSeq platforms. RESULTS: Results obtained by the Illumina platform (analysis of the V3-V4 variable region of the 16S rRNA gene) showed better convergence with the data of culture tests reported in the literature. The major microorganism groups found were: Acinetobacter (39%), Gluconacetobacter (10.8%), Propionibacterium (9.3%), Corynebacterium (9.3%), Staphylococcus (7.2%), Streptococcus (7%), Pseudomonas (4.1%), Micrococcus (3.3%), Yersinia (3%), Chondromyces (2.4%), Serratia (2.3%), and Bacillus (2.1%). Analysis of the samples obtained directly from the mucosa revealed dominance of typical skin-associated microorganisms. CONCLUSION: The present study proposes a contamination-reduction algorithm for microbiological testing of the ocular surface using hard contact lenses for prolonged wearing as a carrier for microbial DNA.

[Determination of EGFR gene somatic mutations in tissues and plasma of patients with advanced non-small cell lung cancer]. / Opredelenie somaticheskikh mutatsii v gene EGFR v tkaniakh i plazme bol'nykh nemelkokletochnym rakom legkogo.

The presence of activating mutations in the EGFR gene influences cell proliferation, angiogenesis, and increases metastatic ability; it has a significant impact on the choice of medical therapy of non-small cell lung cancer (NSCLC). The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests. The aim of this study was to design a real-time PCR-based diagnostic kit for fast and cheap of EGFR mutations testing in paraffin blocks and plasma, and kit validation using samples from patients with NSCLC, and also comparative estimation of diagnostic features of real-time PCR with wild type blocking and digital PCR for mutation testing in blood plasma. The study included 156 patients with various types of adenocarcinoma differentiation. It was designed a simple and efficient real-time PCR-based method of detecting L858R activating mutation and del19 deletion in the EGFR gene for DNA isolated from paraffin blocks. Kit for EGFR mutations was validated using 411 samples of paraffin blocks. The proposed system showed high efficiency for DNA testing from paraffin blocks: a concordance with results of testing with therascreen® EGFR RGQ PCR Kit ("Qiagen", Germany) was 100%. It has been shown the possibility of using this test system for the detection of mutations in plasma.

[Stress response genes expression analysis of barley Hordeum vulgare under space flight environment].

Transcriptome of barley Hordeum vulgare grown aboard International Space Station (ISS) was analyzed by means of microarray. It was revealed 500 genes with mRNA level, changed more than two folds in space environment. Among them are genes encoding stress response proteins, videlicet Heat Shock Proteins (HSP), Pathogenesis-Related Proteins (PR) and Antioxidant Proteins. Further analysis of these genes by real time PCR showed enhanced transcription level of Reactive oxygen Species (ROS) scavenging genes. The mRNA level of superoxide dismutase (sod) was 6 folds higher in space environment when compare to Earth conditions. Glutamyl transferase gene expression was enhanced 24 times in space. Transcription of catalase gene (cat) was increased 18 times and of ascorbate peroxidase was increased 3 times in space in comparison with ground control. For the first time it was shown that space flight environment may induce oxidative stress in plants.

Tide-associated biological rhythms of some White Sea littoral invertebrates.

We report the results from two years of laboratory observations of the tide-associated rhythms of activity of White Sea intertidal invertebrates, Mya arenaria (Bivalvia) and Gammarus finmarchicus (Amphipoda). The tidal associated activity of these invertebrates could not be estimate as a clear circatidal clock. Gammarus activity could be phase shifted by a 0.5 h exposure to turbulent water twice a day for 2-3 days. Mya's rhythm could be changed by a single drainage of aquariums lasting about 15 min. This kind of timing system may be a relatively primitive evolution feature.

[Assessing quality in the conducting of seminars for the advanced training of dentists]. / Otsenka kachestva provedeniia seminarov po povysheniiu kvalifikatsii vrachei-stomatologov.

Regular analysis of questionnaires distributed among students of self-supporting workshops carried out at the Training Center is aimed at elucidating the students' opinions about organization of workshops in order to optimize it and at collection of marketing information on the training and medical activities of the firm. The majority (90%) of respondents gave a positive answer to the question: "Will you advise your colleagues to go to workshops at the Training Center?" Exhibitions of materials offered by the firm and training of students to use these materials demonstrate the advantages thereof. Analysis of questionnaires showed that just a little part of dentists know the true price of their work and that the major part of the market of dental service is oriented, judging by the proposed costs, mainly at the solvent population.

[Fund for social development of regional health care coalitions in the conditions of collective bargaining]. / Fond sotsial'nogo razvitiia territorial'nogo meditsinskogo ob''edineniia v usloviiakh kollektivnogo podriada.

The introduction of economic incentives, the utilization of a fund for social development of institutions considerably increased the intensification of labour, improved health care indicators and the accessibility of health care for the population. The expedient form of work appeared to be collective bargaining which considerably expanded the rights of the leader and collective. ++ Interdepartmental cost-accountable relations with the future introduction of ++intra-structural cost-accountable relations is a prospective form of health services administration and management.

[Economic effectiveness of the introduction of a new method of conservative treatment of chalazion]. / Ekonomicheskaia éffektivnost' vnedreniia novogo sposoba konservativnogo lecheniia khalaziona.

The authors analyze the economic efficacy of a new method for chalazion therapy: corticosteroid injections deep into the pathologic focus. Such therapy may be carried out on an outpatient basis, the patients do not have to cease work for it. The authors emphasize high socioeconomic significance of introducing this method into clinical practice.

[Comparative effectiveness of the sanatorium treatment of patients with bronchopulmonary diseases in the Leningrad health resort zone and at the climatotherapy sanatoria of Yalta and Kislovodsk]. / Sravnitel'naia éffektivnost' sanatornogo lecheniia bol'nykh s bronkholegochnymi zabolevaniiami v usloviiakh Leningradskoi kurortnoi zony i v klimaticheskikh sanatoriiakh Ialty i Kislovodska.

Upon the comparison for Leningrad citizens, CNPD sanatorium treatment in climatic conditions of Leningrad Region was inferior in noway to that in southern climates of Yalta and Kislovodsk resorts as regards to relevant therapeutic response.