[DNA mapping in the capsid of giant bacteriophage phiEL (Caudovirales: Myoviridae: Elvirus) by analytical electron microscopy].

INTRODUCTION: Giant phiKZ-like bacteriophages have a unique protein formation inside the capsid, an inner body (IB) with supercoiled DNA molecule wrapped around it. Standard cryo-electron microscopy (cryo-EM) approaches do not allow to distinguish this structure from the surrounding nucleic acid of the phage. We previously developed an analytical approach to visualize protein-DNA complexes on Escherichia coli bacterial cell slices using the chemical element phosphorus as a marker. In the study presented, we adapted this technique for much smaller objects, namely the capsids of phiKZ-like bacteriophages. MATERIAL AND METHODS: Following electron microscopy techniques were used in the study: analytical (AEM) (electron energy loss spectroscopy, EELS), and cryo-EM (images of samples subjected to low and high dose of electron irradiation were compared). RESULTS: We studied DNA packaging inside the capsids of giant bacteriophages phiEL from the Myoviridae family that infect Pseudomonas aeruginosa. Phosphorus distribution maps were obtained, showing an asymmetrical arrangement of DNA inside the capsid. DISCUSSION: We developed and applied an IB imaging technique using a high angle dark-field detector (HAADF) and the STEM-EELS analytical approach. Phosphorus mapping by EELS and cryo-electron microscopy revealed a protein formation as IB within the phage phiEL capsid. The size of IB was estimated using theoretical calculations. CONCLUSION: The developed technique can be applied to study the distribution of phosphorus in other DNA- or RNA-containing viruses at relatively low concentrations of the element sought.

Bacteriophage's Dualism in Therapy.

Careful selection of bacteriophages for phage therapy is needed to avoid undesirable consequences. Different approaches to phage therapy are compared: from the use of multispecies industrially produced phage mixtures with wide range of antibacterial activity to the 'magistral phage' approach in which bacteriophages are selected for treating individual patients.

[NOVEL APPROACH TO COMPOSITION OF, BACTERIOPHAGE MIXTURES FOR ANTIBACTERIAL THERAPY].

AIM: Evaluate antibacterial activity of an experimental mixture of phages, belonging to several well-studied species. MATERIALS AND METHODS: The study was carried out using a group of 55 clinical Pseudomonas aeruginosa strains of various origins,- 4 mono-species mixtures of 32 virulent bacteriophages (species phiKZ-, phiKMV-, phiPBl-, PaP3-like phages) and 2 novel phages, phiMK (species PaK-P2) and phiPerm5. Activity of preparations from mono-species mixtures of bacteriophages ofvarious species were compared with activity of 3 commercial mixtures. Standard methods of study of bacteriophages were used: determination of lytic activity by seeding onto bacterial lawns of P. aeruginosa, restriction analysis of phage DNA for confirmation of their be- longing to certain species. RESULTS: Cumulative activity of 6 mono-species mixtures of virulent phages was shown to be similar to lytic activity of commercial therapeutic mixtures used against P. aeruginosa infections. 54 of 55 strains of clinical isolates of P: aeruginosa showed sensitivity to experimental mixtures composed of mono-species mixtures of bacteriophages. 53 strains were lysed by commercial preparations. Wherein the possibility of accidental inclusion of moderate -bacteriophages in the experimental mixture is excluded. CONCLUSION: A possibility of creation of highly active therapeutic antibacterial preparations against P. aeruginosa using mono-species mixtures of 6 species of lytic bacteriophages is shown Use of such a mixture in therapy of lung infections reduces the risk of emergence of bacterial strains with increased virulence and patho- genicity during prolonged administration.

[STRESS-PROTECTIVE EFFECT OF CYTOFLAVIN ON CHRONIC CEREBRAL ISCHEMIA IN RATS.]

Analysis of stress-releasing blood reactions (electrophoretic mobility of red blood cells, WBC count, and leukocyte ratio) in rats with experimental cerebral ischernia under the action of cytoflavin held showed that cytoflavin in combination with basic therapy leads to further activation of compensatory-adaptive reactions of the body and stimulated peripheral stress-limiting mechanisms.

[USING MEXICOR TO CORRECT THE FUNCTIONAL PARAMETERS OF RED BLOOD CELLS IN RATS WITH TRAUMATIC BRAIN INJURY MODEL].

The effect of mexicor on the functional characteristics of erythrocytes under conditions of post-traumatic period has been studied on rats with traumatic brain injury (TBI) model. TBI leads to an increase in the aggregation of erythrocytes by 143% (p = 0.003), increases the concentration of malonic dialdehyde (MDA) by 40% (p = 0.006), and decreases the electrophoretic mobility of erythrocytes by 22% (p = 0.005) relative to the levels of these indices in intact animals. The administration of mexicor (8.0 mg/day, injected intraperitoneally twice per day for 12 days) upon TBI led to a maximum reduction in the MDA concentration by 61% (p = 0.03) and decreased the aggregation of erythrocytes by 36% (p = 0.01), increased the electrophoretic mobility of erythrocytes by 20% (p = 0.003) on the 3rd day of posttraumatic period, and increased the concentrations of 2,3-diphosphoglycerate by 23% (p = 0.02) on the 7th day as compared to the positive control. The most pronounced changes under the action of mexicor were observed within 3 - 7 days after the onset of TBI. The obtained data show the efficacy of mexicor in the treatment of rats with TBI model, which is manifested by a 40% (p = 0.02) shorter period of recovery of the functional parameters of red blood cells in comparison to the positive control.

[Low doses of ionizing radiation-induced apoptotic nature of erythrocyte hemolysis].

It is established that exposure of the red blood cells of the rats to low doses of ionizing radiation (0.04; 0.08; 0.16; 0.25 and 0.33 mGr) leads to non-linear changes in the processes of lipid peroxidation in the membrane of erythrocytes, their electrophoretic mobility, osmotic resistance. In a dose range from 0.08 to 0.16 mGr with ionizing radiation apoptosis that determines a temporary slowing of the process of hemolysis and stabilization of erythrocytes that is confirmed by morphological changes in erythrocytes is most likely triggered.

Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa.

A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, which has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor.

[Electrophoretic mobility and activity Na,K-ATPase of erytheocytes in rats under stress].

In this paper, we study the relation between the electrophoretic mobility of erythrocytes (EPME) and the activity of Na,K-ATPase of erythrocytes under the action of different types of stress factors (immobilization, hypoxia, radiation exposure), as well as the introduction of stress implementing agents (adrenaline, cortisol, bee and toad venom) rats. Activity of Na,K-ATPase and the change of EPME washed erythrocytes were examined through 15, 120 min and 24 hours after exposure. Found that at the initial stage (15 min after a stress-exposure) decrease of EPME associated with increased enzyme activity and further increase of EPME takes place on the background of the decrease of activity of Na,K-ATPase in all types of alterations, except the actions toad venom and cortisol. Toad venom and cortisol led to reduced activity of Na,K-ATPase and increase of EPME.

[New temperate Pseudomonas aeruginosa phage, phi297: specific features of genome structure].

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.

[Properties of the new D3-like Pseudomonas aeruginosa bacteriophage phiPMG1: genome structure and prospects for the use in phage therapy].

Results of studying the novel virulent phage phiPMG1 active on Pseudomonas aeruginosa are presented. phiPMG1 was shown to exhibit detectable homology and resemblance in the total genome structure with temperate converting phage D3. Phage phiPMG1 differs from D3 in that it fails to stably lysogenize bacteria and can grow on strains carrying plasmids that cause growth inhibition of phage D3 and some other phages. This significantly diminishes the probability of horizontal gene transfer with phage phiPMG1 and suggests the possible employment of this phage in phage therapy. A comparison of genome structures in phages phiPMG1 and D3 demonstrated not only high homology of 65 genes, but also the presence in the phiPMG1 genome of 16 genes that were not recorded in the files of NCBI database. Apparently, the evolution of genomes in phages of this species is mostly associated with migrations into other species of bacteria and recombinations with phages of other species (for example, F116). Detailed structural analysis a genome region in which the essential nonhomology is exhibited between three D3-like phages (D3, phiPMG1, and PAJU2) revealed that the phiPMG1 genome supposedly is phylogenetically closer than the others to the genome of a hypothetical ancestor phage belonging to this species.

[Individual parameters of general low-frequency magnetic therapy as a possibility for improving the clinical efficacy of the combined treatment of patients with essential arterial hypertension].

A new approach is proposed for the choice of low-frequency magnetic therapy on an individual basis using the results of analysis of heart rhythm variability. The clinical efficiency of low-frequency magnetic therapy incorporated in the combined treatment of 65 patients aged between 25 and 45 years with essential arterial hypertension was estimated. The statistically significant positive effects of the treatment included normalization of blood pressure and characteristics of heart rhythm variability as well as resolution of clinical symptoms of vegetative dysregulation.

Complete genome sequence of the giant Pseudomonas phage Lu11.

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ϕRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.

[The diagnostics of adaptive reactions of blood on application the stress-modulating therapy in patients with brain chronic ischemia].

The article deals with the results of analysis of electrophoretic mobility of erythrocytes and leukogram in patients with dyscirculatory encephalopathy on different stages of disease on application therapy with inclusion of stress-modulating pharmaceuticals into course of treatment. It is established that the electrophoretic mobiliy of erythrocytes makes it possible to evaluate the adaptive indicators blood in patients with dyscirculatory encephalopathy. The consideration of these indicators makes feasible the substantiation of inclusion of stress-modulating therapy into complex treatment of patients with chronic cerebrovascular inefficiency.

[Bacteriophage phi297--the new species of temperate phages Pseudomonas aeruginosa with a mosaic genome: potential use in phagotherapy].

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi97 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable oflysing bacteria, while the wild-type phage induced lysogeny, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm'). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lystic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.

[Genome instability of Pseudomonas aeruginosa phages of the EL species: examination of virulent mutants].

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.

[TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages].

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysogenizing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

[Changes in the electrophoretic mobility of erythrocytes by the action of low-intensity pulse magnetic field].

It has been shown that the exposure of "intact" rats and human blood to low-intensity pulse magnetic field leads to similar changes in the electrophoretic mobility of erythrocytes, which may be connected with the modification of the spectrum of their membrane proteins.

Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain.

We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

[Adaptation reactions of rat blood exposed to low intensity electromagnetic radiation].

It is carried out research of action low-intensive electromagnetic radiations--low-intensive laser radiation and radiations of the highest frequency on normal animals and at modelling the stress-reaction, caused by introduction of adrenaline. Absence of effects of system of blood is noted at action low-intensive electromagnetic radiations on normal an organism and them correction action on alteration an organism, shown in restoration of the broken parameters--leukocyte the blood count, electrophoretic mobility of erythrocytes and phospholipide's structure of their membranes.

[Induction and migration of cryptic/defective Salmonella enterica prophages as a consequence of infection with lytic phages is an additional factor in stability of a coevolutionary vector].

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.