Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A,B, and D

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.     

Low-pressure discharge induced by microwave radiation with a stochastically jumping phase

Results are presented from experimental studies on the unique beam-plasma generator of microwave radiation with a stochastically jumping phase (MWRSJP). To interpret the experimental results, a computer code was developed that allows one to simulate the process of gas ionization by electrons heated in the MWRSJP field and the behavior of plasma particles in such a field. The conditions for ignition and maintenance of a microwave discharge in air by MWRSJP are found both experimentally and theoretically, and the pressure range in which the power required for discharge ignition and maintenance is minimum are determined.     

Fluorescent probes for examination of spermatozoa in cryoprotective mediums

By fluorescent spectroscopy and microscopy methods the possibility of fluorescent probes DSM, E-176, 3-DAB and FME application for study of cryoprotective agents' influence on the dog spermatozoa are investigated. It is established that FME and 3-DAD dyes are suitable for the posed problem solving, and the DSM and E-176 probes have restrictions owing to enough strong fluorescence from cryoprotectant solutions. It is shown that the fluorescent probes investigated influence the cells motility to different degree. The perspectives of investigated dyes application for study of cryoprotective agent' finfluence on spermatozoa are considered.     

Atomic force microscopy study of peptides homologous to beta-domain of alpha-lactalbumins

Symmetrical peptide GYDTQAIVENNESTEYG (WT, Wild Type) identical to 35-51 aminoacid residues of human alpha-lactalbumin (HLA) and peptide GYDTQTVVNNNGHTDYG (ID, IDeal symmetry) homologous to beta-domain of mammalian alpha-lactalbumins can form amyloid-like fibrils in conditions required for fibrillogenesis of HLA. The latter peptide can also form fibrils in deionized water. Fibrils formed by these peptides can cause forming of HLA amyloid-like aggregates in physiological conditions. These results provide an evidence for presence of amyloidogenic determinant in beta-domain of alpha-lactalbumin. Thus, symmetry in the primary structure may play the role in fibrillogenesis of proteins.     

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents

Molecular Biology - Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic...     

Variability of nisin-synthesizing wild strain Lactococcus lactis subsp. lactis 119 under the effect of ultraviolet light]

To increase the nisin synthesizing activity of the natural strain Lactococcus lactis subsp. lactis 119 isolated from sour milk, UV irradiation in different doses was used followed by isolation of productive clones. The highest mutation effect was observed with the dose of 76,000 erg/mm2, when 11% of the cells increased their productivity by 12.8% at the minimum survival rate. Two-step UV irradiation and adaptive selection on the nisin-contaning medium provided isolation of a strain with the activity 42.6% higher than that of the initial strain (3850 IU/ml). Natural and UV-induced variability of the strain by the nisin synthesis, growth rate, carbohydrate consumption and sensitivity to antibiotics of various groups were studied.