[Evaluation of the influence of culturing on the intensity of biofilm formation by Klebsiella pneumoniae strains.] / Влияние условий культивирования на интенсивность биоплёнокообразования штаммами Klebsiella pneumoniaе.

Due to the prevalence of biofilm infections caused by Klebsiella pneumoniae, in laboratory diagnostic practice it has a great importance to obtain a standard model of Klebsiella biofilm for evaluating the bactericidal effect and effectiveness of antimicrobial drugs. Describes the method of Klebsiella biofilms formation in vitro. The intensity of biofilm formation was evaluated by the ability of bacteria to bind the crystal violet. The degree of film formation was measured by optical density. The presence of an intercellular matrix was confirmed by staining of Congo-red solution followed by light microscopy. The effect of exogenous and endogenous factors on biofilm formation by K. pneumoniae strains was investigated. The influence of the nutrient composition, the age of the culture («daily¼, «weekly¼), the presence of oxygen and the temperature conditions were studied. The nutrient composition of the medium significantly influenced on biofilm formation of K. pneumoniae: DMEM stimulated biofilm formation in most strains in vitro compared to TSB. The age of the culture (daily, weekly) did not significantly affect the biofilm formation of Klebsiella. At the same time, the temperature of culturing and the presence of oxygen can both stimulate and inhibit biofilm formation, depending on the strain under study. Most strains of Klebsiella better form a biofilm under aerobic conditions at 37º C.

[Magnesium and vitamin B2 status of children with bronchial asthma and obesity].

Insufficiency or deficiency of some micronutrients may be additional modifying factors that influence the pathogenesis of the disease and the effectiveness of standard pharmacotherapy. The aim of the study - to evaluate the level of magnesium and vitamin B2 in blood serum of patients with bronchial asthma and obesity in order to develop methods for individual correction of deficiency. Material and methods. The study included 51 children aged 12-17 years. The first group included 23 patients (12 girls and 11 boys) with asthma with comorbidities (obesity), and the second group consisted of 28 children (10 girls and 18 boys) with obesity. The concentration of magnesium in blood serum was determined by a colorimetric method without deproteinization, and vitamin B2 - by an immunological microbiological method. Results and discussion. When analyzing the concentration of magnesium in blood serum of the examined children, it was found that in patients with bronchial asthma and obesity, a reduced content of this mineral was observed in 15 (65.2%) patients. The average magnesium concentration was 0.66±0.02 mmol/l at a rate of 0.7-1.2 mmol/l. A statistically significant decrease in the magnesium level in children suffering from asthma and obesity was noted, compared with the level in children with obesity (0.66 [0.57; 0.73] vs 0.71 [0.67; 0.73] mmol/l, р<0.05). Low serum magnesium levels in obese patients were detected more rarely (р<0.05) - only in 6 (21.4%) children, mostly in patients with grade III obesity. The remaining 22 (78.6%) children had magnesium level within the normal range. Patients with low serum magnesium levels showed increased irritability, sleep disturbance, loss of memory and concentration. Vitamin B2 levels in all examined children were within the normal range (137-370 ng/ml). Conclusion. The results indicate a decrease in the concentration of magnesium and normal levels of vitamin B2 in serum in patients with bronchial asthma and obesity. Low serum magnesium levels were observed in children with low bronchial asthma control. To increase the effectiveness of therapy and control the symptoms of bronchial asthma, especially when combined with obesity, correction of the accompanying magnesium deficiency is necessary.

[Evaluation of anti-candida activity of metabolites of enterococcal clinical isolates.]

When studying the effect of the metabolic products of clinical isolates of enterococci on the viability of Candida albicans, it was found that metabolites of all tested strains of Enterococcus faecium, E. faecalis had a fungistatic effect. At the same time a reliable fungicidal effect is a strain-specific feature. It is better to use the method of delayed antagonism on double-layer agar to assess the antifungal effect of enterococcal metabolism products.

Cowpox in a human, Russia, 2015.

We investigated the first laboratory-confirmed human case of cowpox virus infection in Russia since 1991. Phylogenetic studies of haemagglutinin, TNF-α receptor-like protein and thymidine kinase regions showed significant differences with known orthopoxviruses, including unique amino-acid substitutions and deletions. The described cowpox virus strain, taking into account differences, is genetically closely related to strains isolated years ago in the same geographical region (European part of Russia and Finland), which suggests circulation of viral strains with common origin in wild rodents without spread over long distances and appearance in other parts of the world.

[Two cases of hydrophobia in the Republic of Tatarstan: in vivo and postmortem laboratory diagnosis].

The results of rabies in vivo and postmortem laboratory detection in two cases registered in the Republic of Tatarstan are reported: a victim bitten by a wolf in 2002 and another one bitten by a stray dog on Goa Island, India, in 2013. In the patient bitten by a wolf cornea imprints studies using the method of fluorescent antibodies (MFA) showed rabies-positive result 6 days before the patient's death. The results were confirmed by postmortem examination of different parts of the brain and salivary glands using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. In the patient bitten by a stray dog the rabies virus specific antigen was detected by eye cornea studies using the MFA method and saliva studies using the ELISA. The rabies virus genome was also isolated from saliva and tear fluid using nested reverse-transcription polymerase chain reaction (RT-PCR) 9 days before the patient's death. The in viva studies results were consistent with the postmortem study of different parts of the brain using the MFA, enzyme-linked immunosorbent assay (ELISA), optical microscopy, and bioassay methods. All the infection-positive results of both in viva and postmortem studies were consistent with the clinical studies, i.e. rabies diagnosis was confirmed. The analysis of the rabies virus gene G fragment nucleotide sequence of 238 nd length showed a slight difference between the studied isolates (2 rabies) and the RABV AY9563I9 (1.68%), difference by 10.5% from the Vnukovo-32 vaccine strains and by 10.9% from the SAD B19 rabies strain, respectively (rabies viruses of 1st genotype). It was also significantly different from the lissaviruses of 2,4,5, and 6 genotypes (21 .0-32.7%). The obtained results indicate phylogenetic closeness of the studied isolates (2 rabies) with the RABV AY956319 rabies virus strain belonging to the 1st genotype.

Genetic evidence for a novel familial Alzheimer's disease locus on chromosome 14.

Familial Alzheimer's disease (FAD) has been shown to be genetically heterogeneous, with a very small proportion of early onset pedigrees being associated with mutations in the amyloid precursor protein (APP) gene on chromosome 21, and some late onset pedigrees showing associations with markers on chromosome 19. We now provide evidence for a major early onset FAD locus on the long arm of chromosome 14 near the markers D14S43 and D14S53 (multipoint lod score z = 23.4) and suggest that the inheritance of FAD may be more complex than had initially been suspected.

Markers of transmembrane and energy exchange in cells of terminal placental villi in spontaneous and induced pregnancy.

We performed a comparative morphological study of the placentas in spontaneous and induced pregnancy. Immunohistochemical analysis revealed pronounced decrease in the expression of markers of transmembrane (α-SNAP 23, annexin 3) and energy (ferritin light chain, ATP5J) in placental terminal villi.

Peroxisome proliferator-activated receptors of trophoblast cells in miscarriage.

Comparative histological study of uterine curettage specimens in missed abortion in women with spontaneous and induced pregnancy and progressing spontaneous pregnancy revealed similar changes in the endometrium and placental villi. Immunohistochemical assay showed enhanced expression of PPARγ and increased proliferation index (Ki-67) in cells of the villous and extravillous trophoblasts in missed abortion.

The N-terminal domain of c-Myc associates with alpha-tubulin and microtubules in vivo and in vitro.

The polymerization of alpha- and beta-tubulin into microtubules results in a complex network of microfibrils that have important structural and functional roles in all eukaryotic cells. In addition, microtubules can interact with a diverse family of polypeptides which are believed to directly promote the assembly of microtubules and to modulate their functional activity. We have demonstrated that the c-Myc oncoprotein interacts in vivo and in vitro with alpha-tubulin and with polymerized microtubules and have defined the binding site to the N-terminal region within the transactivation domain of c-Myc. In addition, we have shown that c-Myc colocalizes with microtubules and remains tightly bound to the microtubule network after detergent extraction of intact cells. These findings suggest a potential role for Myc-tubulin interaction in vivo.

Effect of central hypervolemia on respiratory function.

Effects of central hypervolemia on respiratory function and compensatory capabilities of the respiratory system were studied in the anesthetized, vagally intact or vagotomized rats. Central hypervolemia was induced by a head-down tilt on -30 degree rotation. The tilt evoked an elevation of central venous pressure (from -2+/-0.4 cmH2O to 3.9+/-0.8 cmH2O). At 30 min after tilting, airway resistance and negative intrathoracic pressure (indirect measure of respiratory effort) significantly increased, whereas inspiratory flow, tidal volume, and minute ventilation decreased. Load compensatory response was strongly weakened. The tilt-induced esophageal pressure augmentation was suppressed by transection of the vagal nerves. In vagotomized animals inspiratory swings of the intrathoracic pressure increased barely to 116+/-15%, whereas they increased to 216+/-17% of control in vagally intact animals (P<0.05). We conclude that central hypervolemia increases mechanical loading and weakens compensatory capabilities of the respiratory system. Vagal afferents have a part in the realization of the respiratory response to central hypervolemia.

Prefrontal control of respiration.

The aim of the present study was to test the hypothesis that different parts of the prefrontal cortex could be involved in respiratory control. This hypothesis was tested by examining the changes in respiratory pattern produced by low intensity electrical stimulation of the insular and infralimbic cortex. The experiments were performed on the anaesthetised rats. It was found that the sites capable of inducing different kinds of respiratory responses were localized to the insular and infralimbic cortices. Microstimulation of the anterior insular cortex produced responses which manifested themselves in a decrease of inspiratory flow and tidal volume, but the respiratory time remained stable. Responsive sites within caudal insular and infralimbic cortex produced other alterations in breathing which included an increase of inspiratory flow, and decreases of tidal volume and respiratory time. These observations support the hypothesis proposed and localize distinct patterns of respiratory responses to different parts of the prefrontal cortex in the rat.

Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker.

The aminoglycoside 3'-phosphotransferase type VIII (APHVIII) encoding gene (aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7-8 mt+ for paromomycin resistance (PmR) at a frequency (1.3-1.9) x 10(-7), probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The PmR phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.

c-Raf kinase binds to N-terminal domain of c-Myc.

We have demonstrated that the 50 N-terminal amino acids of c-Myc bind a kinase activity, which phosphorylates Myc in vitro predominantly on Thr8. We also have shown that c-Raf, a widely known Ser/Thr kinase, involved in the Ras signaling pathway, binds to the same portion of c-Myc in vitro. In addition we were able to precipitate native c-Myc/Raf complex from various cell lysates. Physical interaction of Myc and Raf may potentially be a part of their well-known functional cooperation.

Incorporation of monoclonal antibodies in living rat pheochromocytoma PC12 cells. Evidence for the intracellular formation of immune complex between the incorporated antibody and a target protein.

PC12 cells permeabilized with a low concentration of digitonin (5 microM) under controlled conditions were loaded with monoclonal antibodies (MoAb) against the regulatory subunit type II (RII) of cAMP-dependent protein kinase. After digitonin removal from the nutrient medium (DMEM) the loaded cells repaired within 20-30 min and recontinued growth. The inserted MoAb stayed in the repaired cells at least for several hours. MoAb inhibiting the cAMP binding activity of neural RII [Grozdova et al. (1992) Biochem. Int. 27, 811-822; Sveshnikova et al. (1996) Biochem. Int. 39, 1063-1070] were shown to bind the target antigen inside the cells and influence the properties of intracellular protein kinases.

Human c-myc gene contains a regulatory site similar to consensus of interferon response sequence (IRS).

Expression of c-myc proto oncogene is regulated by multiple mechanisms. Here, we report that the consensus of the regulatory region of interferon-dependent genes, GGAAAN1-3 GAAA, was found after computer search in the 5'-terminal flank of human c-myc gene in position (-76:-67). In vitro transcription of c-myc gene fragments showed that the consensus region competes with oligonucleotide GGGAAAATGAAACT for binding to specific protein(s). This oligonucleotide was shown to bind selectively the interferon-dependent positive transcription factor. Transcription of c-myc fragments lacking 5'-terminal region up to positions -101 or +71 was initiated at two sites located in the first intron. These sites did not coincide with P1 in vivo RNA cap-site. Binding of the protein factor(s) to the regulatory region of c-myc gene -76:-67 blocked the in vitro transcription initiated in the first intron.

Mass effect and death from severe acute stroke.

BACKGROUND: In severe acute stroke, the degree of midline cerebral displacement is related to level of consciousness but not to survival. Early identification of patients at high risk of death from mass effect would assist patient management decisions. METHODS: We measured lesion volume, horizontal pineal displacement (PD), and horizontal septum pellucidum displacement (SD) on axial CT of consecutive patients with severe (Canadian Neurological Scale score < or = 5) acute hemispheric stroke. We correlated CT measurements with the probability of 14-day survival. RESULTS: Forty-six (39%) of 118 patients died within 14 days and 72 (61%) died within 1 year following stroke. Crude risk factors for 14-day mortality were as follows: lesion volume > or = 400 ml, SD > or = 9 mm, PD > or = 4 mm, intraventricular hemorrhage, and coma on admission. Only SD (p = 0.001) and coma on admission (p = 0.019) remained significant in multivariate analysis, but PD was highly correlated with SD (r = 0.82). PD of > or = 4 mm on a scan performed within 48 hours of stroke onset identified patients with a low probability of 14-day survival (0.16; CI 0 to 0.32) with a specificity of 89% and a sensitivity of 46%. CONCLUSIONS: The degree of horizontal midline cerebral displacement correlates with the likelihood of death following stroke. Patients with > or = 4 mm PD on CT performed within 48 hours of stroke onset are at high risk for early death.

Analysis of the c-FOS gene on chromosome 14 and the promoter of the amyloid precursor protein gene in familial Alzheimer's disease.

The c-FOS gene product, a putative transacting transcriptional regulator of the amyloid precursor protein (APP) gene, is a candidate locus for the familial Alzheimer's disease (FAD) mutation on chromosome 14 (FAD14). In light of this functional relationship, we investigated the nucleotide sequence and segregation of c-FOS and the nucleotide sequence of the 5' APP promoter. Single-stranded conformational polymorphisms (SSCPs) in the c-FOS gene revealed that c-FOS closely cosegregates with the FAD14 gene but does not show allelic association with FAD. A conservative third-position T-->C mutation was demonstrated in exon 2 (codon 84) of c-FOS, and a C-->G substitution was detected at -209 bp in the 5' promoter of APP. Neither were unique to FAD and are unlikely to be pathogenic or secondary modifiers of the FAD phenotype. We conclude that the c-FOS open reading frame is probably not the site of the FAD14 locus, but we cannot exclude the existence of modifier loci on chromosome 21.

Tryptophanyl-tRNA synthetase as a human autoantigen.

Autoantibodies to highly purified tryptophanyl-tRNA synthetase, consisting of two approximately 60-kDa subunits (6.1.1.2, TrpRS), were detected in some sera of donors and patients with various diagnosis using the newly developed 125I-TrpRS-radiodot, 125I-TrpRS-radioblot, ELISA and Western immunoblotting. The percentage of positive sera appears to be dependent upon the method of sera testing. The autoimmune sera recognized both the native and denatured TrpRS forms. The binding of the human serum to the 60-kDa band of tissue extract was demonstrable by the 125I-TrpRS-blot as well as Western blot techniques. The possible role of infections in the induction of anti-TrpRS antibodies and maintenance of the autoimmune response is discussed.

Mutation of the gene for the human lysosomal serine protease cathepsin G is not the cause of aberrant APP processing in familial Alzheimer disease.

Recent genetic linkage studies have implicated a gene on chromosome 14 in the pathogenesis of FAD. The identity of this gene remains unknown but it has been speculated that it may be involved in the cellular processing of the amyloid precursor protein (APP). We have analyzed the nucleotide sequence of the entire open reading frame of the cathepsin G gene located on chromosome 14q. No mutations were observed, suggesting that defects in this lysosomal protease are not responsible for aberrant accumulation of proteolytic products of APP in FAD brain tissue.

The clinical value of methods to measure carotid stenosis.

BACKGROUND: The North American (NASCET) and European (ECST) carotid surgery trials have shown a surgical benefit for symptomatic stenosis greater than 70%. The Asymptomatic Carotid Artery Surgery (ACAS) trial have shown some benefit for the stenosis greater than 60%. Although the NASCET/ACAS angiographic methods were similar, these are discrepant from ECST and have technical limitations inherent to measurement of the distal internal carotid artery (ICA) or guessing the ICA bulb diameter. METHODS: Consecutive carotid angiograms were analyzed to verify the relationships between proximal and distal aspects of the common carotid artery (CCA) and ICA bulb. We then compared the NASCET and ECST methods and, two new techniques, the Common Carotid (CC) and Carotid Stenosis Index (CSI). The CC method is based on a direct comparison of the residual lumen to the distal CCA diameter adjacent to the bulb. The CSI is based on the known relationship between the proximal CCA and ICA (1.2 x CCA diameter = proximal CCA diameter). The normal ICA bulb diameter can therefore be calculated from direct measurement of the CCA. RESULTS: 125 consecutive carotid angiograms were evaluated (250 arteries). Technical applicability of NASCET was 89%, ECST 95%, CC/CSI 99%. The CCA/ICA diameter ratios were established: 1.23 +/- 0.23 (ICA bulb/distal CCA), and 1.27 +/- 0.2 (ICA bulb/proximal CCA). The CCA is enlarged at its distal end that such the distal CCA/proximal CCA ratio is 1.04 +/- 0.12. The CC and CSI methods were statistically different in 8 of 10 groups when these methods were compared per decile stenosis (p < 0.04). However, CC and CSI methods disagreed in classifying patients into mild (0-29%), moderate (30-69%), and severe (70-99%) only in 3%, 5%, and 8% of cases. Linear regression analysis shows excellent correlation between the methods (CC = 15.7 + 0.82 x CSI, r2 = 0.92). Lumen asymmetry is most common with mild-to-moderate stenoses which may affect accuracy and reproducibility of measurements. CONCLUSIONS: We have confirmed previous data on the relationships between the components of the carotid artery. Of the different angiographic techniques, CSI is the most reliable validated method of measuring carotid stenosis, and is proposed as a bridge between results of carotid surgery trials, and to validate noninvasive modalities against angiography.