Photosensitizing effects and physicochemical properties of chlorophyll a derivatives with hydrophilic oligoethylene glycol fragments at the macrocycle periphery.

In this work, screening studies of the cytotoxic effect of chlorins with fragments of di-, tri-, and pentaethylene glycol at the macrocycle periphery in relation to HeLa, A549, and HT29 cells were performed. It is shown that, despite different hydrophobicity, all the compounds studied have a comparable photodynamic effect. The conjugate of chlorin e6 with pentaethylene glycol, which has the lowest tendency to association among the studied compounds with tropism for low density lipoproteins and the best characteristics of the formation of molecular complexes with Tween 80, has a significant difference in dark and photoinduced toxicity (ratio IC50(dark)/IC50(photo) approximately 2 orders of magnitude for all cell lines), which allows to hope for a sufficiently large "therapeutic window". A study of the interaction of this compound with HeLa cells shows that the substance penetrates the cell and, after red light irradiation induces ROS appearance inside the cell, associated, apparently, with the photogeneration of singlet oxygen. These data indicate that photoinduced toxic effects are caused by damage to intracellular structures as a result of oxidative stress. Programmed type of cell death characterized with caspase-3 induction is prevailing. So, the conjugate of chlorin e6 with pentaethylene glycol is a promising antitumor PS that can be successfully solubilized with Tween 80, which makes it suitable for further in vivo studies.

[Pharmacological prevention of fibrosis in dacryosurgery]. / Medikamentoznaya profilaktika fibrotizatsii v dakriokhirurgii.

Chronic inflammatory process in the lacrimal drainage system is the main etiological factor leading to dacryostenosis and consequent obliteration - partial and total nasolacrimal duct obstruction. Prevention of this process is an urgent problem in dacryology. Currently, there is very little research on the development and use of conservative methods for treating dacryostenosis using anti-inflammatory, as well as anti-fibrotic drugs. In this regard, the main method of treating lacrimal drainage obstruction is dacryocystorhinostomy. However, the problem of recurrence after this operation has not been resolved. The causes of recurrence can be cicatricial healing of dacryocystorhinostomy ostium, canalicular obstruction, formation of granulations and synechiae in its area. Surgical methods of recurrence prevention are associated with possible complications, and there is conflicting data on the feasibility of their use. Based on this, the development of pharmacological methods for the prevention of fibrosis in dacryology is promising, among which the antitumor antibiotic Mitomycin C is the most studied. However, there are no specific scientifically substantiated recommendations for the use of this drug, and the data on its effectiveness vary. This has prompted researchers to look for and study alternative anti-fibrotic agents, such as antitumor drugs, glucocorticoids, hyaluronic acid, small molecule, biological, immunological and genetically engineered drugs, as well as nanoparticles. This review presents the current data on the efficacy and prospects of the use of these drugs in dacryology.

[The main provisions of programs of medical and social rehabilitation of patients with occupational diseases of ent organs]. / Poryadok razrabotki i osnovnye razdely programmy reabilitatsii postradavshego na proizvodstve s professional'nymi otorinolaringologicheskimi zabolevaniyami.

The article discusses the main, modern provisions of the programs of medical and social rehabilitation of patients with occupational diseases of the ENT organs. The criteria of the degree of loss of professional ability to work in hearing loss and malignant neoplasms of the upper respiratory tract are presented. Rehabilitation programs and forms are developed taking into account the needs of the victim in medical, social and vocational rehabilitation, taking into account the potential capabilities and abilities of the victim to carry out professional, domestic and social activities.

[Forensic examination of air gun dart wounds]. / Sudebno-meditsinskaya otsenka ranenii drotikami, metaemymi iz dukhovykh ruzhei.

The objective was to study the mechanism of formation of experimental wounds of the human body dummy by 12 different types of common air gun darts. The impact of dart tips design features on macro- and microscopic morphological patterns of wounds was established; the description of further laboratory diagnostics of the wounds was provided.

[Visually impaired older adults as a target group of social rehabilitation: towards the issue of the peculiarities of lifestyle.]

The article presents materials of an empirical study of the peculiarities of the way of life of visually impaired older people in a social rehabilitation context. In comparison with older people without disabling visual impairment, the older blind and visually impaired, according to the results of the conducted questionnaire survey, are characterized in terms of their position in society, the preservation of the «inner core¼ of the personality, involvement in social and cultural life, the need for novelty and expansion of «spiritual and cultural spaces¼, creative activity and social and cultural communication.

Association between Disturbances in Polyunsaturated Fatty Acid Metabolism and Development of Oxidative Stress during Experimental Diabetes Mellitus.

We studied the effect of streptozotocin-induced diabetes mellitus on the level of glycemia and some other indices of lipid metabolism, including fatty acid metabolism and LPO intensity, during the development of diabetes mellitus in rats. Even at the early terms of diabetes development, hypertriglyceridemia and hypercholesterolemia were accompanied by changes in the blood content of fatty acid (at the expense of ω3 and ω6 fatty acids) that persisted throughout the observation period. Intensification of LPO against the background of suppressed activity of antioxidant enzymes and reduced level of ω3 fatty acids attested to the development of oxidative stress. These data attest to antioxidant property of ω3 fatty acids, which is seen from positive correlations between these fatty acids and activity of antioxidant enzymes.

Evaluation of the Efficiency of Lytic Mycobacteriophage D29 on the Model of M. tuberculosis-Infected Macrophage RAW 264 Cell Line.

Culture of mouse macrophages (RAW 264.7 ATCC strain) in wells of a 6-well plate was infected with M. tuberculosis in proportion of 15 mycobacteria per one macrophage and then treated with a lytic strain of mycobacteriophage D29. Antibacterial efficacy of mycobacteriophages was studied using D29 phage (activity 108 plaque-forming units/ml) previously purified by ion exchange chromatography. After single and double 24-h treatment, the lysed cultures of macrophages were inoculated onto Middlebrook 7H10 agar medium. The number of mycobacterial colonies in control and test wells (at least 3 wells in each group) was 300.178±12.500 and 36.0±5.4, respectively (p<0.01).

Enzyme-Substrate Reporters for Evaluation of Substrate Specificity of HIF Prolyl Hydroxylase Isoforms.

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.

MOLECULAR-GENETIC PROPERTIES OF VIBRIO CHOLERAE EL TOR STRAINS CIRCULATING IN AFRICA.

The review contains some brief information on cholera epidemics in Africa. Based on the results of the whole genome sequencing of 30 clinical strains isolated in Africa in different periods of the 7th cholera pandemic (1985-2012), extensive genetic diversity has been revealed. It is demonstrated that at present cholera epidemics in Africa are caused by new variants of the agent, which emerged in South- Eastern Asia in consequence of not only new genes acquisition, but also genome alterations of pandemicity and pathogenicity islands. SNP analysis of 53 strains circulating at different times in the territory of the continent, as well as isolated in South-Eastern Asia, has been carried out. Phylogenetic relations between the majority of the African and Asian strains have been established. In addition, strains were shown to exist that are, apparently, endemic to the African region. Identified genetic diversity of the strains with varying virulence and drug resistance points out the necessity of continuous molecular monitoring of the cholera agent in Africa.

[The application of lytic micro-bacteriophage В29 for accelerated phenotype detection of sensitivity mycobacteria of tuberculosis to anti-tuberculosis medications.]

In conditions of prevalence of medicine-resistant strains of mycobacteria of tuberculosis necessity in accelerated, including phenotype techniques of detection of sensitivity of mycobacteria to anti-microbial chemotherapeutic medications in clinical samples is an actual issue. The results of application of accelerated phenotype techniques of detection of sensitivity of clinical strains of mycobacteria of tuberculosis to anti-microbial chemotherapeutic medications on the basis application of lytic mycobacteriophage D29 are presented. The principle of technique is in evaluation of reproduction of mycobacteriophage in cells of mycobacteria of tuberculosis in presence of sensitive to them anti-bacterial medications. The reproduction of mycobacteriophage is evaluated by quantitative analysis of phage DNA in polymerase chain reaction in real-time. The study used 102 clinical strains of mycobacteria of tuberculosis obtained after primary cultivation or re-cultivation in tubes of MGIT system (Bactec). After positive results of growth of mycobacteria of tuberculosis were obtained, the samples were incubated during 48 hours in CO2 incubator in the presence of critical concentrations of 10 widely applied in case of treatment tuberculosis medicinal substances in liquid nutrient medium Middlebrook 7H9 enriched with components OADC, in format of 24 well cultural plate with volume of nutrient medium 1 ml per well. Whereupon, in plate wells deposited 2x103 plaque-forming units of mycobacteriophage D29. After 24 hours a qualitative detection of phage DNA was implemented with polymerase chain reaction in real-time using reagents phage D29 ("Syntol", Russia). The increasing of threshold level of fluorescence of Ct more than to 2 cycles in samples with antibiotic as compared with control testifies sensitivity of the analyzed strain of mycobacteria of tuberculosis to antibiotic. The level of coincidence made up to 91% in comparative study with inoculation in Lowenstein-Jensen nutrient medium. The level of coincidence made up to 96% in comparative study with Bactec test-system of limited number of strains with establishment of sensitivity for 10 medications. The data was confirmed concerning inverse relationship of value ∆Ct and minimal inhibiting concentration of medication. The supposed high efficiency of possible reagents' set on the basis of presented technique on cost/quality criterion.

Detection and quantification of epithelial progenitor cell populations in human healthy and IPF lungs.

BACKGROUND: In the human lung, epithelial progenitor cells in the airways give rise to the differentiated pseudostratified airway epithelium. In mice, emerging evidence confers a progenitor function to cytokeratin 5 (KRT5(+)) or cytokeratin 14 (KRT14(+))-positive basal cells of the airway epithelium. Little is known, however, about the distribution of progenitor subpopulations in the human lung, particularly about aberrant epithelial differentiation in lung disease, such as idiopathic pulmonary fibrosis (IPF). METHODS: Here, we used multi-color immunofluorescence analysis to detect and quantify the distribution of airway epithelial progenitor subpopulations in human lungs obtained from healthy donors or IPF patients. RESULTS: In lungs from both, healthy donors and IPF patients, we detected KRT5(+)KRT14(-), KRT5(-)KRT14(+) and KRT5(+)KRT14(+) populations in the proximal airways. KRT14(+) cells, however, were absent in the distal airways of healthy lungs. In IPF, we detected a dramatic increase in the amount of KRT5(+) cells and the emergence of a frequent KRT5(+)KRT14(+) epithelial population, in particular in distal airways and alveolar regions. While the KRT14(-) progenitor population exhibited signs of proper epithelial differentiation, as evidenced by co-staining with pro-SPC, aquaporin 5, CC10, or MUC5B, the KRT14(+) cell population did not co-stain with bronchial/alveolar differentiation markers in IPF. CONCLUSIONS: We provide, for the first time, a quantitative profile of the distribution of epithelial progenitor populations in human lungs. We show compelling evidence for dysregulation and aberrant differentiation of these populations in IPF.

BIORESORBABLE PROPERTIES OF CHITOSAN FIBERS IN ENDOMYSIUM AND PERIMYSIUM MUSCLE TISSUE

Scanning electron microscopy and histologic analysis were used in the comparative in vivo study of resorption of chitosan fibers implanted into endomysium and perimysium of a rat latissimus dorsi muscle. It was demonstrated that the mechanism and rate of chitosan fiber resorption depend on the position of fibers in muscular tissue. After implantation of chitosan fibers into endomysium (when chitosan was in direct contact with muscle fibers), the formation of cross-sectional cracks, fragmentation of implanted fibers and its partial resorption were observed in 14 days. Complete chitosan resorption in endomysium occurred after 30 days only. Chitosan fibers implanted into perimysium preserved integrity for 7 days, and fibrous tissue was formed around implants. After 45 days of exposure, no signs of chitosan fiber destruction were registered in this case. Biocompatibility of chitosan fibers proved by effective adhesion and proliferation of mesenchymal stem cells on their surface.

[Genome structure and origin of nontoxigenic strains of Vibrio cholerae of El Tor biovar with different epidemiological significance].

Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA ­ tcpA + strains vary considerably and can be clustered into two separate groups, the CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates and the CTXφ­RS1φ­VPI+VSP­ isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTXφ­RS1φ­VPI+VSP­ strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTXφ­RS1φ­VPI+VSP­ strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.

[ACTUAL PROBLEMS OF EPIDEMIOLOGIC CONTROL, LABORATORY DIAGNOSTICS AND PROPHYLAXIS OF CHOLERA IN RUSSIAN FEDERATION].

Main problems of system of epidemiologic control for cholera active in Russian Federation, as well as laboratory diagnostics and vaccine prophylaxis of this especially dangerous infection, that had emerged in the contemporary period of the ongoing 7th pandemic of cholera, are discussed. Features of the genome of natural strains of Vibrio cholerae of El Tor biovar, that possess a poten- tial epidemic threat, as well as problems, that have emerged during isolation of these strains from samples of water of surface water bodies during their monitoring, are also examined. The main direction of enhancement of the system of epidemiologic control for cholera consist in develop- ment of a new algorithm of differentiation of administrative territories of Russian Federation by types of epidemic manifestations, as well as optimization of monitoring of environment objects. Integration of modern highly informative technologies into practice, as well as development of new generation diagnostic preparations based on DNA-chips and immunechips is necessary to increase effectiveness of the conducted operative and retrospective diagnostics in the contemporary period. Creation of national cholera vaccine, ensuring simultaneous protection from cholera causative agents of both O1 and O139 serogroups, is also required.

[ISOLATION OF ANTIBIOTICS RESISTANCE GENES IN VIBRIO CHOLERAE O1 AND O139 SEROGROUP STRAINS].

AIM: Determination of sensitivity of V. cholerae O1 serogroup El Tor biovar and O139 serogroup strains to antibiotics and determination of the presence of antibiotics resistance genes in their genome. MATERIALS AND METHODS: The studies were carried out in 75 V. cholerae O1 and O139 serogroup strains. Sensitivity of cultures to antibiotics was determined by disc-diffusion method. DNA isolation was carried out in the presence of 6M guanidine thiocyanate. PCR was carried out in multi-channel amplificator Tercyc. RESULTS: A multiplex PCR was constructed, that includes 5 primer pairs for the detection of O1 and O139 serogroup resistance genes of vibrios to sulfame- thoxazolum, streptomycin B, trimethoprim, the presence of SXT element, an amplification program was developed. Using the developed PCR, V. cholerae O1 serogroup El Tor biovar strains with multiple drug resistance were established to be imported into Russia in 1993. The presence of SXT elements with genes of resistance to 4 antibiotics simultaneously was detected precisely in these strains, that belong to toxigenic genovariants of V. cholerae El Tor biovar. All the El Tor vibrio strains imported in the subsequent years were shown to stably preserve SXT element, this indicates its important role in biology of cholera vibrios. O139 serogroup strains with intact SXT element and having a deletion of the gene coding trimethoprim resistance were isolated. CONCLUSION: The data obtained may be used to establish molecular-genetic mechanisms of emergence of antibiotics resistant strains of cholera vibrio, construction of novel gene diagnostic test-systems and carrying out passportization of strains that are stored in the State collection of pathogenic bacteria.

[EVALUATION OF FUNCTIONAL FEATURES AND STRESS RESISTANCE OF ISOGENIC TOXIGENIC AND NON-TOXIGENIC BIOVAR EL TOR VIBRIO CHOLERAE STRAINS].

AIM: Comparative evaluation of functional features of toxigenic biovar El Tor Vibrio cholerae strains and their spontaneous non-toxigenic mutants and study of their resistance to saline and oxidative stress. MATERIALS AND METHODS: 8 biovar El Tor V. cholerae strains were studied: 4 clinical strains isolated in 1970 from patients in Astrakhan and 4 spontaneous non-toxigenic mutants of these strains that have lost cholera toxin genes as a result of residence in river water at the temperature of 25°C. Protein composition was determined in polyacrylamide gel electrophoresis by Laemmli U.K. Stress resistance of the strains was studied by adding H2O2 to 20 mM concentration and NaCl to 3 mM concentration to the cultural suspension. RESULTS: Loss of cholera toxin genes was shown to be accompanied by changes in the level of expression of 17 proteins including those that take part in energy metabolism, glucose transport, chemotaxis and purine bases. Moreover, non-toxigenic strains were established to be 5-15 times more resistant to saline and oxidative stress compared with toxigenic strains. CONCLUSION: Non-toxigenic V. cholerae mutants adopt better to stress factors, therefore the loss of cholera toxin gene in water environment could be one of the methods of adaptation of pathogenic bacteria to changes in the environment.

[EFFECT OF OSMOTIC AND OXIDATIVE STRESS ON STRAINS OF GENOVARIANTS OF VIBRIO CHOLERAE EL TOR BIOVAR].

AIM: Study the effect of osmotic and oxidative stress on survivability and changes in phenotypic and genetic properties of strains of genovariants of V. cholerae El Tor biovar. MATERIALS AND METHODS: 22 strains of V. cholerae El Tor biovar were used in the study. Phenotypic properties of strains were studied in LB medium with the addition of the appropriate ingredients. Surface structures of cells were studied using scanning probe microscope "Solver P47-PRO". PCR was carried out using specific primers in "Tercic" amplificator. RESULTS: After 60 minutes of incubation in 3 M solution of NaCl and after 6 minutes in 20 mM solution of hydrogen peroxide, the amount of surviving cells of genovariants was, respectively, 3.0 - 25.0 and 4.3 - 7.6 times higher than for typical strains. One of the mechanisms of increased resistance of genovariants to high concentrations of salt was associated with the production of an extra exopolysaccharide layer on the cell surface at earlier periods than in typical strains. Osmotic stress results in a reversible reduction of mobility in strains of genovariants of V. cholerae El Tor biovar. Osmotic and oxidative stress was revealed to result in a loss of a number of mobile genetic elements in strains of genovariants. CONCLUSION: Genovariants of V. cholerae El Tor biovar, that had caused cholera outbreaks in Russia in 1993 -2001, in contrast to typical strains, isolated in 1970 - 1990, are more resistant to th effect of osmotic and oxidative stress, that, probably, facilitates their higher survivability in both the environment and macroorganism.

[Genovariants of the cholera agent biovar El Tor: construction, molecular-genetic, and proteomic analysis].

Experimental modeling of origination of the virulent Vibrio cholerae El Tor genovariants is presented. It was demonstrated that the genovariants obtained did not differ from the natural genetically modified strains emerged in a natural population of the agent, either in phenotypical or genotypic properties. Using the PCR assay and sequencing techniques it was proved that the constructed genovariants carried a CTX(Class phi) prophage genome region with ctxBl gene of the V. cholerae classical biovar in the chromosome. It is shown that the prophage structure alterations lead to the increase in the toxigenicity and virulence in the genovariants compared to the typical strain-recipient. Moreover, as regards proteomics, changes in the expression of 26 proteins that perform various functions in the cell, such as metabolism, energy exchange, transportation, etc., were demonstrated. The data are indicative of the impact that a new DNA region in the genome of the genovariants has on the expression level of different house-keeping genes. The results obtained testify to the fact that one of the mechanisms of the genovariant emergence in the natural populations of the agent can be horizontal gene transfer.

[Microevolution of cholera agent in the modern period].

AIM: To carry out comparative molecular genetic analysis of highly pathogenic atypical Vibrio cholerae strains biovar El Tor, isolated in the territory of RF, in order to determine micro-evolutionary alterations of cholera agent in the modern period. MATERIALS AND METHODS: 38 clinical strains have been examined by means of polymerase chain reaction, sequencing and MLVA-analysis. The selected strains were isolated at different periods of time during cholera epidemic complications and differed between each other in virulence. RESULTS: It is demonstrated that new variants have emerged in the course of short-term microevolution. Their genome structure and function differ from those of all previously known strains. The genome alterations have been caused by point mutations in ctxB u tcpA genes associated with virulence and located in CTXΦ prophage and pathogenicity island VPI-1 respectively, as well as by the extended deletion in pandemicity island VSP-II. Presented is the dynamics of genome structure and function alterations in modern strains. CONCLUSION: The discovered genomic alterations in the new variants of the agent evolved in the process of microevolution are indicative of their epidemic potential enhancement and probability of virulence potentiation.

[Comparative resistance of typical and genetically altered Vibrio cholerae biovar El Tor strains to the effect of unfavorable environmental factors].

AIM: Carry out comparative analysis of survival of typical strains and genovariants of V. cholerae biovar El Tor imported in different years to the territory of Russian Federation, in the absence of nutrients and under the conditions of temperature stress. MATERIALS AND METHODS: 24 V. cholerae biovar El Tor strains isolated in 1970 - 2011 were studied, 8 of those were typical isolates and 16--genetically altered variants. Strain survival was studied in 0.9% sodium chloride solution and autoclaved river water at various temperature modes (5, 25, 37 and 42 degrees C). Protein composition and exopolysaccharide production were determined by electrophoresis method by U.K. Laemmli. RESULTS: Genovariants as well as typical strains were shown to be able to exist for a long time (up to 5 months) in the absence of nutrients at the temperature of 25 degrees C. However, unlike typical eltor vibrios, genovariants were more resistant to temperature stress. As a result of adaptation to high temperature (42 degrees C) biosynthesis of porin proteins of outer membrane OmpU and/or OmpT is increased in genovariant cells, and at lower temperatures (5 degrees C)--exopolysaccharide. CONCLUSION: V. cholerae biovar El Tor genovariants are able to adapt to temperature change better, that may facilitate their higher survival in the environment.