Method of Selection of Bacteria Antibiotic Resistance Genes Based on Clustering of Similar Nucleotide Sequences.

A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, ß-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.

[Metabolomic profiling in culture media of day-5 human embryos]. / Profilirovanie metabolitov v pitatel'nykh sredakh piatidnevnykh émbrionov cheloveka.

The aim of this study was to determine the changes of metabolomic profiles in embryonic culture media (ECM) for the evaluation of quality and implantation potential of human embryos. ECM (n=163) were collected on day 5 before transfer or cryopreservation. Some embryos were used in preimplantation genetic screening for detection of aneuploidy karyotypes. Samples were subdivided into groups according to embryo morphological classification (by Gardner), genetic analysis and implantation data. ECM were extracted with methanol, precipitates were separated by centrifugation and metabolite production of individual embryo was analysed by LC-MS (the positive ion mode). After peak detection and retention time alignment, data were analysed using the PCA algorithm. MS fingerprinting analysis of embryo culture medium showed significant differences between morphologically divided groups. Intragroup comparisons did not reveal differences between subclasses. Genetic screening of embryos revealed 33 aneuploid karyotypes. It was shown that chromosome number did not affect the metabolite profiles comparing with the normal group. The culture media of embryos that were positive or negative for successful implantation showed specific signatures that allowed to distinguish embryos with different outcomes.The characterization of ECMs by LC-MS may facilitate more accurate selection of the best embryo for the implantation, improving single-embryo transfer and thus eliminating the risk and undesirable effects of multiple pregnancies.

[Comparative results of preimplantation genetic screening by array comparative genomic hybridization and new-generation sequencing].

Aneuploidies as quantitative chromosome abnormalities are a main cause of failed development of morphologically normal embryos, implantation failures, and early reproductive losses. Preimplantation genetic screening (PGS) allows a preselection of embryos with a normal karyotype, thus increasing the implantation rate and reducing the frequency of early pregnancy loss after IVF. Modern PGS technologies are based on a genome-wide analysis of the embryo. The first pilot study in Russia was performed to assess the possibility of using semiconductor new-generation sequencing (NGS) as a PGS method. NGS data were collected for 38 biopsied embryos and compared with the data from array comparative genomic hybridization (array-CGH). The concordance between the NGS and array-CGH data was 94.8%. Two samples showed the karyotype 47,XXY by array-CGH and a normal karyotype by NGS. The discrepancies may be explained by loss of efficiency of array-CGH amplicon labeling.

[Twin reversed arterial perfusion syndrome in the monochorionic diamniotic placenta with the development of an acardiac fetus]. / Sindrom obratnoi arterial'noi perfuzii v monokhorial'noi diamnioticheskoi platsente s razvitiem ploda-akardiusa.

The article describes an acardiac fetus in a patient with monochorionic diamniotic twin pregnancy with reversed arterial perfusion syndrome at 30 weeks' gestation. It gives postmortem fetal computed tomographic and pathoanatomic data. Microarray of acardiac fetal tissues revealed that there was deletion of chromosome 19 - arr [hg19] 19p13.3q11 (260,911-23,005,001) x1; size, 23 Mbp; the signal level in about 30% of fetal tissue cells containing deletion.

Role of Collagen Gene Polymorphisms in the Structure of Early Gestation Loss.

Studies of collagen gene polymorphisms associated with predisposition to early recurrent miscarriages revealed significant differences in the distribution of COL1A1 C-1997A C>A (rs1107946) genotypes and alleles in the group of pregnant patients with early miscarriages in comparison with controls (normal pregnancy). Identification of COL1A1 C-1997A C>A (rs1107946) collagen gene polymorphisms at the stage of pregnancy planning will make it possible to form early miscarriage risk groups for more thorough preparation to gestation and optimization of follow up of this patient population.

Comparison of the expression of immunomodulatory factors in cultures of mesenchymal stromal cells from human extraembryonic tissues.

mRNA of genes (tgfß, il6, il10, il1ß, and vegf165) involved in cell proliferation, inflammatory, anti-inflammatory, and angiogenic processes were detected and quantified in cultures of mesenchymal stromal cells of different passages derived from human extraembryonic tissues (amniotic sac, umbilical cord, chorionic villi and trophoblast of the placenta). The concentrations of IL-10, IL-6, IL-1ß, and TGFß were measured.

Expression of immunomodulator gene mRNA in co-culture of mesenchymal stromal cells from the placenta and human mononuclear blood cells.

The expression of mRNA of cytokines and immunoregulatory molecules characterizing the interaction of mesenchymal stromal cells from chorionic villi of postpartum placenta and allogenic mononuclear blood cells was studied during 3-day co-culturing of these cells. The expression of foxp3, il2ra, and il10 mRNA in floating mononuclear cells increased from day 1 to 3 in co-culture, which can refl ect the process of induction of regulatory T cells in the lymphocyte population under the action of mesenchymal stromal cells.

[Immunogenetic predictors of a rapid virologic response to antiviral therapy in patients with chronic hepatitis C].

AIM: To determine the correlation between interleukin 28B (IL28B) gene polymorphism in patients with chronic hepatitis C (CHC), the presence or absence a rapid virologic response to antiviral therapy, and a number of immunological characteristics as a basis for a personalized approach to treating the patients. SUBJECTS AND METHODS: Seventeen CHC patients infected with hepatitis C virus genotype 1b were examined and underwent genetic testing for IL28B gene polymorphism for rs12979860 (CC, CT or TT genotypes) and rs8099917 (TT, TG or GG genotypes) using the modified method of adjacent samples, which revealed single nucleotide substitutions in the genes. Their immunological parameters were identified by a flow cytometry technique by taking into account whether a rapid virologic response had been achieved. RESULTS: The key phenomena of a rapid virologic response in the representatives of different IL28B genotypes are the nonspecific proliferative activity of blood natural killer cells before treatment, as well as the count of regulatory T cells before and 4 weeks after therapy start. CONCLUSION: To predict the efficiency of antiviral therapy for CHC, it is desirable to supplement genetic studies with immunological data.

Protamine and fertilin mRNA: potential biomarkers of assisted reproductive technology outcomes.

We studied the relationship between the levels of protamines 1 and 2 (PRM1 and PRM2) and fertilin-ß (ADAM-2) mRNA expression and outcomes of infertility treatment using assisted reproductive technologies was studied. Analysis of the relationships between the outcomes of in vitro fertilization and embryo transfer and profiles of the expression of seminal genes PRM1, PRM2, ADAM-2 mRNA, evaluated by reverse transcription quantitative PCR was carried out in 79 couples. Significant differences in the expression of seminal PRM1, PRM2, ADAM-2 mRNA were detected in couples with different outcomes of in vitro fertilization and embryo transfer. The levels of seminal gene expression are potential predictors of the efficiency of in vitro fertilization and embryo transfer.