Molecular Genetics, Microbiology and Virology

When we use cell cultures in research, we rarely think about the story behind them, which can be intriguing, providing insight, and sometimes tragic. In the 1950s, the culture of HeLa cells unexpectedly became well known scientifically and became one of the most famous cell cultures. These cells were taken from a woman named Henrietta Lacks, who had cervical cancer and died shortly afterward, and the HeLa cell line proved to be an essential tool for several generations of scientists around the world in developing new treatments and biomedical research. These cells have become unique due to their immortality, endless division, easy cultivation, and adaptation to conservation conditions. At the same time, HeLa cells remain a simplified imitation of the human body.

The epidemic of cholera in Siberia and the Russian Far East during the seventh pandemic was recorded as sporadic cases of introduction of the infection into the territory or severe local outbreaks associated with the infection’s importation in the 1970s and 1990s. The genetic diversity and relationship of 83 V. cholerae O1 El Tor strains isolated from the region with epidemic complications were studied on the basis of the multilocus analysis of the variable number tandem repeats at five loci: VcA, VcB, VcC, VcD, and VcG. The VcB locus was characterized by the maximum polymorphism in the sample of the studied strains, while the VcC locus displayed the minimum polymorphism. Cluster analysis revealed the differentiation of V. cholerae strains during epidemic complications into seven clusters depending on the structure of the pathogenic determinants (typical and atypical El Tor vibrios), as well as on the time and direction of the pathogen importation. In some outbreaks in the Far East in 1999, the heterogeneity of the etiological agent populations was shown (five genotypes were detected among the outbreak strains from Yuzhno-Sakhalinsk, and six genotypes were determined among the strains from Primorskii krai). At the same time, the predominant MLVA profiles and their single locus variants were identified. The absence of a strict connection between the territory and the genotypes of V. cholerae outbreak strains (one strain from Yuzhno-Sakhalinsk showed the identity of the allele profile with the isolates from Vladivostok), as well as no correspondence of the V. cholerae genotype to a specific infection focus, was shown. According to the data of the MLVA typing, the V. cholerae O1 El Tor strains isolated from the Siberian and Far Eastern regions (Russia) with epidemic complications were characterized by the heterogeneity of their genetic organization, which agrees with the period and direction of their importation to the territory. During sporadic outbreaks of cholera, the clonality of the etiological agent was revealed. Closely related subclones, the emergence of which could result from the presence of the causative agent in the environment or passaging through a susceptible organism, were identified.

One of the most popular nonviral delivery systems for gene therapy constructs are carriers based on polyethylenimine (PEI) DNA complexes. A number of disadvantages associated with the lack of targeted delivery and increased cytotoxicity are overcome by adding auxiliary molecules to the complexes. An example of this is chondroitin sulfate (CS). The purpose of this work was to assess the effect of CS on the transfection properties of DNA–PEI complexes under different conditions of their preparation and transfection protocols. All complexes were prepared in solutions with high and low ionic strength. Transfection of C26 cells was performed according to two protocols differing in the presence of serum in the medium. The portion of transfected cells, transgene expression level, and cell viability were the main parameters of assessing the transfection efficiency. In binary DNA–PEI complexes prepared in different salt conditions, using different transfection protocols, the difference in the portion of transfected cells reached ten times. Addition of CS improved this transfection efficiency indicator up to 6.5 times, while the maximum difference in this indicator for the corresponding ternary complexes was reduced to 2.5 times. Changes in the proportion of CS in the composition of the complexes had an insignificant effect on their transfection properties. In the case of complexes prepared in high ionic strength solutions, the order of CS addition was also important. The best results of transfection efficiency were achieved with ternary complexes prepared in low ionic strength solutions, using a serum-free protocol, while these indicators were comparable with the data for Lipofectamine 2000. The addition of chondroitin sulfate improves the transfection properties of DNA–PEI complexes and makes them less dependent on the methods of preparation and transfection.

Recombinant adenoviruses are widely used for delivery and expression of transgenes for therapeutic purposes and in fundamental research. AdEasy™ adenoviral vector system allows for rapid and efficient generation of replication-deficient adenoviruses. One important crucial step in producing recombinant adenoviruses is the generation of a recombinant adenoviral plasmid in E. coli BJ5183 cells via homologous recombination between the transgene encoding shuttle vector and the pAdEasy-1 plasmid, which carries a significant part of human adenovirus serotype 5 (Ad5) genome. In many published protocols, the necessity of electroporation for the transformation of the shuttle vector and pAdEasy-1 into E. coli cells is emphasized, thus making necessary the availability of the appropriate instruments and consumables. Here, we have proposed an optimized protocol for the generation of recombinant adenoviruses with the use of chemically competent E. coli cells only, without the use of electroporation. We have demonstrated the efficient transformation of linearized nonpurified shuttle vector into chemically competent E. coli BJ5183-pAdEasy-1 cells allowing identification of clones with correct recombinant adenoviral plasmids. Using the optimized protocol, we have generated four functional replication-deficient adenoviruses expressing target proteins in HEK293 cells. The protocol that has been suggested provides a significantly simplified and cheaper method for the generation of recombinant adenoviruses due to lack of requirement to use instruments and consumables for electroporation.

The method of application laser transfer of silver and copper nanoparticles for the first time has been shown to be effective against biofilms formed on a solid substrate. It has been experimentally confirmed that this effect is not associated with the influence of the laser itself. The proposed method allows one to increase the locality, availability, and efficiency of biofilm destruction due to the bactericidal effect of metal nanoparticles with a slight direct laser effect on the biofilm.

Endemic goiter is one of the most common endocrine diseases in Russian Federation. There is a strong need for new methods to detect and treat this disease. Analysis of nuclear abnormalities in buccal epithelial cells can be used to estimate overall exposure of patients to various endo- and exogenic factors associated with a disorder. We performed an association study of nuclear abnormalities in buccal epithelial cells and as well polymorphisms of genes encoding superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) in patients with endemic goiter. We observed an increased frequency of nuclear abnormalities related to early stages of apoptosis and necrosis in buccal epithelial cells of patients with endemic goiter when compared to cells from healthy individuals. Nuclear abnormalities in patients with endemic goiter are more common in heterozygous carriers of minor alleles of SOD1 G7958A and SOD2 58T>C polymorphisms when compared to homozygous individuals. Furthermore the presence of minor allele of SOD2 60C>T polymorphism in individuals with endemic goiter decreases the frequency of nuclear abnormalities. The results of this study could be used for the further development of effective and low-cost methods of endemic goiter diagnostics.

Objectives of the present study were to evaluate the antibacterial activity of the two types of inorganic magnesium hydroxide [Mg(OH)₂] and calcium carbonate [CaCO₃] nanoparticles (NPs) on the growth of three Gram-negative bacteria, e.g., Escherichia coli, Pseudomonas aeruginosa and Serratia marcescens and three Gram-positive bacteria, e.g., Streptococcus pyogenes, Staphylococcus aureus and Streptococcus bovis. The synthesis of these NPs was done by a microwave hydrothermal method. The structures and sizes of synthesized nanoparticles were investigated using X-ray diffractometer. Antimicrobial susceptibility of different NPs was determined at 20, 50 and 100 mg/mL by the agar-well diffusion method, growth reduction at the aqueous solution and time-kill assay. The antimicrobial effects across NPs and bacterial species were shown to be dose-dependent. The results of the different experiments indicated that smaller NP sizes have higher antibacterial effects. M29 [Mg(OH)₂-29] nanoparticles followed by silver (Ag) and C1 (CaCO₃-1) showed the highest influence on bacterial growth rates, while similar ability to kill bacteria across treatment time. In addition, Gram-negative bacteria were more affected in terms of the inhibition zone and reduction of growth rates after 24 h as well as in terms of the prolonged treatment of NPs up to 36 h due to the influence of different nanoparticles. We recommend to search the chance of further using M29 and C1 in medicine and industry.