Introduction. Mycoplasma hominis is a bacterium belonging to the class Mollicutes . It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517–622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection. Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division. Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms. Methodology. LC–MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer. Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC–MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis. Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Comparison of primate genomic sequences has demonstrated that the intra-and interspecific genetic variation is provided by retroelements (REs). The human genome contains many thousands of polymorphic RE copies, which are regarded as a promising source of new generation molecular genetic markers. However, the absence of systematized data on the RE number, distribution, genomic context, and abundance in various human populations limits the use of RE insertion polymorphism. We designed the first bilingual (Russian/English) web resource on the known polymorphic REs discovered both by our team and other researchers. The database contains the information about the genomic location of each RE, its position relative to known and predicted genes, abundance in human populations, and other data. Our web portal (http://labcfg.ibch.ru/home.html) allows a search of the database with user-specified parameters. The database makes it possible to most comprehensively analyze the RE distribution in the human genome and to design molecular genetic markers for studies of human genome diversity and biomedical applications.

At present, bacteriophages are considered as an alternative to antibiotics in prevention and treatment of bacterial infections, in particular cholera.The aimof the work was to demonstrate a method to obtain synthetic bacteriophage againstVibrio cholerae. Vibriophage N4 was selected as a subject for the study.Materials and methods. The genome sequence of vibriophage N4 (38.5 kb) was taken from the NCBI GenBank database. The sequence was divided into gene blocks of 1500–2000 bp. The gene blocks, in turn, were split into oligonucleotides. Sequence partitioning was carried out using the BAC-browser software that we have developed. Oligonucleotides were chemically synthesized; gene blocks were assembled from them. After that, the complete genome of vibriophage N4 was synthesized from the obtained gene blocks. The assembly of the synthetic genome took place in two stages. At the first stage, gene block cassettes of 5–7 pieces with sizes ranging from 7 to 10.5 thousand bp were generated via homologous recombination in yeast. The resulting cassettes were then amplified and used forin vitroassembly using 5’-3’ exonuclease and thermostable DNA polymerase. The resulting preparation was used for electroporation ofV. choleraecells.Results and discussion. The synthetic genome of vibriophage N4 was delivered into theV. choleraestrain M818 O1 biovar El Tor using electroporation. As a result, the formation of lytic plaques on the lawn ofV. choleraewas observed. The range of technologies we have developed: software for assembly design, enzymes and buffers for the synthesis of gene blocks and their crosslinking by homologous recombinationin vitro, the method for producing large-sized assemblies in yeast can be used to obtain artificial bacteriophages with a rational genome design.

BACKGROUND: Mammalian cell cultures play a key role in the pharmaceutical industry, requiring constant monitoring of the cell conditions during fermentation. In addition to monitoring of the physical-chemical parameters, it is important to evaluate the transcription state of cells, for which gene expression analysis is used. Currently, quantitative reverse transcription polymerase chain reaction (qPCR) is the dominant method. However, the loop-mediated isothermal amplification (LAMP) technique also attracts attention due to its high specificity, sensitivity and reaction rate. LAMP is becoming a promising tool for rapid analysis of gene expression, especially under the conditions of limited biological material or a large volume of samples. AIM: Development of a technique for semi-quantifying the expression level of target genes in human cell culture Expi293 using LAMP. MATERIALS AND METHODS: For LAMP, a recombinant large fragment of Bacillus stearothermophilus DNA polymerase (Bst-pol) was obtained, purified, and optimal reaction conditions were determined. SYBR Green I and LUCS13 were used as intercalating dyes. The amplification parameters for different concentrations of the enzyme and the dye were analyzed. Standard SYBR Green I kits were used for qPCR. Both methods were compared when analyzing the expression of IGF1, FGF2 and EIF3i genes in cell lines with an increased expression level of these genes. RESULTS: It has been shown that using LUCS13 dye provides the classic S-shape of the signal accumulation curve in LAMP, while using SYBR Green I dye causes artifacts. The optimal concentration of Bst-pol was 40 ng/µl. When comparing the two methods, it was found that LAMP has greater sensitivity, allows determining gene expression with an accuracy comparable to qPCR, demonstrating a shorter reaction time (up to 35 minutes). CONCLUSION: Although qPCR remains the main method for assessing the level of gene expression, LAMP offers a number of advantages that make it an attractive alternative for various biotechnological purposes. Due to its high speed, ease of execution and accessibility, as well as high sensitivity and specificity, LAMP is a valuable technique for rapid analysis of gene expression during cell culture monitoring.