[To the question of genetic predisposition to the development of professional sensorineural hearing loss]. / K voprosu o geneticheskoi predraspolozhennosti k razvitiyu khronicheskoi neirosensornoi tugoukhosti.

Objective was to study single-nucleotide polymorphisms (SNP) in CAT, NCL, HSPA1L, PCDH15, and PON2 genes and their associations with hearing impairment among the people working among noise-exposed workers of the mashine-building plant (JSC «Krasmash¼, Krasnoyarsk, Eastern Siberia, Russia). MATERIALS AND METHODS: The 443 employees of Krasmash JSC, who have been working under conditions of increased noise for at least 1 year, were surveyed and examined. A hearing study was performed by speech and tonal audiometry. Tonal audiometry was carried out in accord with according to a standard method in the frequency range 125-8000 Hz. People with chronic hearing impairment, survivors of meningitis and family history of hearing impairment were excluded from the study. The allelic composition of the studied genes was determined in the remaining group of 288 workers (study group). Polymorphisms were detected using bioluminescent method, developed by the authors earlier. The study group comprised 122 people with hearing impairment (experimental group) and 166 people without impairment (control group). RESULTS: The genotyping results of on allelic variants rs494024 (CAT), rs7598759 (NCL), rs2227956 (HSPA1L), rs7095441 (PCDH15) and rs7785846 (PON2) showed that their frequencies in the study group did not differ and were comparable with those for the European population. No statistically significant differences were revealed in the distribution of the genotypes of the studied mutations between the experimental and control groups. Also no statistically significant associations we found between hearing impairment and availability of two or several SNPs, or these SNPs and clinical characteristics of the disease (degree of hearing impairment, tinnitus). In the group of workers with an experience of 5 to 16 years, an association was found for hearing impairment and SNP rs494024, as well as when it is combined with rs7598759. CONCLUSIONS: The associations between SNP rs7598759, rs2227956, and rs7095441 and hearing impairment were not found. In the group of workers with 5-16 year experience, this association was found for SNP rs494024, as well as when it is combined with rs7598759. Discovered associations require further study.

Artificial blastocyst collapse prior to vitrification significantly improves Na+/K+-ATPase-dependent post-warming blastocoel re-expansion kinetics without inducing endoplasmic reticulum stress gene expression in the mouse.

Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

[The genetic aspects of occupational hearing impairment]. / Geneticheskie aspekty professional'noi neirosensornoi tugoukhosti.

This article was designed to be the overview of the current literature publications concerning the identification of the genetic markers of susceptibility to the noise-induced loss of hearing. The analysis of these data has demonstrated that the major gene polymorphisms associated with the development of this pathological condition are localized in the genes encoding for the antioxidant systems, potassium homeostasis, and adhesion molecules as well as in the genes involved in intercellular coupling, the mechanisms underlying the cellular response to stress, activation and regulation of heat shock proteins, and signaling function of the immune system. It is concluded that the further investigations into the genetic aspects of the full-genome sequencing techniques and the search for genomic associations could greatly contribute to the development of personalized medicine and the reduction of risks of occupational noise-induced sensorineural impairment of hearing.

[ Bioluminescent assay to detect melanocortin-1 receptor (MC1R) polymorphisms (R160W, R151C, and D294H)].

Several polymorphisms in melanocortin-1 receptor (MC1R) gene are shown to have associations with melanoma risk. In particular, rs1805007, rs1805008, and rs1805009 mutations causing the corresponding R151C, R160W, and D294H changes and associated with the phenotype ("red-hair mutations") are connected with melanoma and non-melanoma skin cancer risks. The work describes the approach to detect these polymorphisms based on primer extension reaction with the following dual bioluminescent assay. Model plasmids with polymorphic MC1R fragments as well as several clinical DNA samples were tested using the developed technique. The results were in good correlation with those obtained by Sanger sequencing.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence.

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

[The detection of Leiden mutation using technique of enzymatic extension of allele-specific primer with double bioluminescent detection (PED-Biolum)].

The article deals with comparing technique of detection of Leiden mutation on the basis of PEXT-reaction with subsequent bioluminescent microanalysis of products with technique based on RT-PCR. The sampling for testing comprised 83 specimen of genome DNA including 35 specimens with known Leiden heterozygote mutation. The commercial kit "SNP-express-PB" (Litex) was used as a comparison test. It is demonstrated that proposed approach is a simple in its application, effective and relatively inexpensive technique of detection of Leiden one-nucleotide polymorphism in gene V of blood coagulation factor. The technique "PED-Biolum" has no differences in comparison with commercial technique RT-PCR concerning ability to detect mutant allele and matches it in parameters of economic effectiveness.

[Bioluminescent reporters to identify gene allelic variants].

The method of single nucleotide polymorphism identification based on primer extension reaction (PEXT) with the following bioluminescent solid-phase microassay was developed. The recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent luciferase Renilla muelleri were used as reporters. Factor V Leiden polymorphism 1691 G-->A (R506Q) of human F5 gene genotyping was used for investigation. Genomic DNA was amplified by PCR using primers, flanking polymorphic site of 140 base pairs. PCR products were used as a template for two PEXT reaction using two primers with 3'-end nucleotides, complementary either normal or mutant alleles. At complementarity of template and allelic-typical primer its extension with DNA-polymerase takes place. The products carried biotin due to availability ofbiotinylated dUTP in the reactions mixture. The assay was carried out using obelin-streptavidin chemical conjugates. Optimal PEXT-reaction conditions providing high reliability of SNP genotyping were found. A new approach to determine both alleles in one well was developed applying two bioluminescent reporters. Availability of the proposed approach was shown in the study of clinical DNA samples.

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

[Human single chain antibodies directed to tumor necrosis factor].

Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.

Analytical techniques to recognize inclusion complexes formation involving monoterpenes and cyclodextrins: A study case with (-) borneol, a food ingredient.

Monoterpenes are non-polar secondary metabolites widely used by industry due to their excellent therapeutic, food-ingredient and cosmetic properties. However, their low solubility in water limits their use. In this sense, cyclodextrins (CDs) have been widely used to solve these technological challenges. Thus, this study aims to use (-)-borneol as a monoterpene model to prepare inclusion complexes between ß-CD and hydroxypropyl-ß-CD (HP-ß-CD) through different ways and characterize them in order to choose the best inclusion method to improve physicochemical properties of monoterpenes. To achieve this goal, the samples were prepared by physical mixture (PM), paste complex (PA) and freeze-drying complex (FD) and then, extensively characterized by thermal analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy, size particle, X-ray diffraction and nuclear magnetic resonance. The physicochemical results showed that freeze-drying was more effective to form inclusion complexes between (-)-borneol with both CDs. This research highlights the importance of recognizing the best method to prepare inclusion complexes, including food additives as (-)-borneol, to achieve better results in food preparations.

New pectin-based hydrogel containing imiquimod-loaded polymeric nanocapsules for melanoma treatment.

We developed a pectin-based hydrogel containing nanocapsules as a new strategy for melanoma treatment. Our first objective was to evaluate the nanoencapsulation effect of imiquimod on melanoma. Imiquimod-loaded polymeric nanocapsules (NCimiq) showed significant time-dependent decrease in cell viability after treatment at 3 µmol L-1 (79% viable cells in 24 h and 55% in 72 h), which was not observed in cells treated with the solution of the drug (IMIQ) (99% viable cells in 24 h and 91% in 72 h). The second objective was to develop the hydrogel containing the drug-loaded nanocapsules (PEC-NCimiq). In vitro release study showed that 63% of imiquimod was released from the pectin-based hydrogel containing the drug (PEC-imiq) after 2 h, while 60% of the drug was released from PEC-NCimiq after 8 h. In the permeation study, 2.5 µg of imiquimod permeated the skin within 8 h after the initial contact of PEC-NCimiq, whereas only 2.1 µg of drug permeated after 12 h of contact when PEC-imiq was assayed. Pectin-based hydrogels enabled the drug penetration in all skin layers, especially the dermis (PEC-NCimiq = 6.8 µg and PEC-imiq = 4.3 µg). In the adhesion study, PEC-NCimiq showed the highest adhesiveness (42% removed from the skin) in comparison to PEC-imiq (71% removed from the skin). In conclusion, the nanoencapsulation provided a higher cytotoxic effect of imiquimod in SK-MEL-28, and the incorporation of the drug-loaded nanocapsules in pectin-based hydrogel showed higher adhesiveness and deeper penetration of the drug into the skin. Graphical abstract.

Venus: an upper limit on intrinsic magnetic dipole moment based on absence of a radiation belt.

On the basis of the absence of energetic electrons (E(e) 45 kiloelectron volts) and protons (E(p) 320 kiloelectron volts) associated with Venus to within a radial distance of 10,150 kilometers from the center of the planet and using a physical similitude argument and the observational and theoretical knowledge of the magnetosphere of Earth, we conclude that the intrinsic magnetic dipole moment of Venus is almost certainly less than 0.01 and probably less than 0.001 of that of Earth. Corresponding upper limits on the magnetic field at the equatorial surface of Venus are about 350 and 35 x 10(-5) gauss, respectively.

Plasma observations at venus with galileo.

Plasma measurements were obtained with the Galileo spacecraft during an approximately 3.5-hour interval in the vicinity of Venus on 10 February 1990. Several crossings of the bow shock in the local dawn sector were recorded before the spacecraft passed into the solar wind upstream from this planet. Although observations of ions of the solar wind and the postshock magnetosheath plasmas were not possible owing to the presence of a sunshade for thermal protection of the instrument, solar wind densities and bulk speeds were determined from the electron velocity distributions. A magnetic field-aligned distribution of hotter electrons or ;;strahl'' was also found in the solar wind. Ions streaming into the solar wind from the bow shock were detected. Electron heating at the bow shock,

Plasma observations at Io with the Galileo spacecraft.

Plasma measurements made during the flyby of Io on 7 December 1995 with the Galileo spacecraft plasma analyzers reveal that the spacecraft unexpectedly passed directly through the ionosphere of Io. The ionosphere is identified by a dense plasma that is at rest with respect to Io. This plasma is cool relative to those encountered outside the ionosphere. The composition of the ionospheric plasmas includes O++, O+ and S++, S+, and SO2+ ions. The plasma conditions at Io appear to account for the decrease in the magnetic field, without the need to assume that Io has a magnetized interior.

Preliminary results on the plasma environment of saturn from the pioneer 11 plasma analyzer experiment.

The Ames Research Center Pioneer 11 plasma analyzer experiment provided measurements of the solar wind interaction with Saturn and the character of the plasma environment within Saturn's magnetosphere. It is shown that Saturn has a detached bow shock wave and magnetopause quite similar to those at Earth and Jupiter. The scale size of the interaction region for Saturn is roughly one-third that at Jupiter, but Saturn's magnetosphere is equally responsive to changes in the solar wind dynamic pressure. Saturn's outer magnetosphere is inflated, as evidenced by the observation of large fluxes of corotating plasma. It is postulated that Saturn's magnetosphere may undergo a large expansion when the solar wind pressure is greatly diminished by the presence of Jupiter's extended magnetospheric tail when the two planets are approximately aligned along the same solar radial vector.

[Affine magnetic sorbents supported on coal ash microspheres for recombinant protein isolation].

The results of the development and utilization of an affine magnetic sorbent with Ni2+ ions immobilized on coal ash microspheres are reported. The applicability of the material in the isolation of Histag proteins is demonstrated by examples of the recombinant green fluorescent protein from Clytia gregarium and the Ca2+ regulated photoprotein obelin from Obelia longissima. The specific sorption capacity of the sorbent was 2-7 mg/cm3 for medium-size proteins (20-30 kDa). The particles are suitable for chromatography with the presence of chaotropic agents and EDTA. They are easy to manipulate as isolation of a target protein takes 30-35 min. On one hand, the elevated affinity of the sorbent to proteins rich in native histidines may result in a high degree of irreversible sorption; on the other hand, it allows isolation of such proteins without the introduction of artificial polyhistidine tracts.

Imiquimod-loaded nanocapsules improve cytotoxicity in cervical cancer cell line.

This paper proposes the development of imiquimod-loaded polymeric nanocapsules formulation for the treatment of cervical cancer. The mechanism of death involved in the reduction of the cell viability as well as the production of an inflammation marker (IL-6) after the treatment in cell line SiHa have been evaluated. The formulation has significantly decreased the viability of the cells in a time-dependent manner, after 24, 48 and 72 h. Additionally, results showed a cellular decrease of almost 80% of the cells after 72 h of treatment. The formulation induced death by apoptosis, necrosis, autophagy, and increased the percentage of SubG1subpopulation of SiHa cells after 72 h. After the same time-interval, the formulation significantly prevented the appearance of colonies, showing effectiveness against SiHa. Finally, the formulation stimulated SiHa cells to release IL-6. These findings open new possibilities for the development of aqueous nanosuspension containing imiquimod as a novel strategy for the treatment of cervical cancer.

[A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter].

The recombinant Ca2+-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of a fine-grained polymer or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with a biotin residue on the 3'-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide, was used as a DNA template. The probe in the hybridization complex was labeled by elongation of the chain using Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, ensures a high sensitivity of the assay (no less than 10(-15) mol of DNA template), and enables quantitative determination of the amount of DNA template in the tested sample.

Extended low-dose dexamethasone suppression test for diagnosis of atypical Cushing's syndrome in dogs.

The purpose of this study was to evaluate extension of the low-dose dexamethasone suppression (LDDS) test from 8 h to 12 h to detect possible hypercortisolemia associated with atypical hyperadrenocorticism (AHAC). Twelve client-owned dogs were enrolled in the study: 6 healthy dogs (group 1) and 6 dogs with suspected AHAC (group 2). Baseline EDTA plasma samples were collected for endogenous ACTH determination using an immunoradiometric assay. Serum samples were collected before and at 4, 8, 10, and 12 h post-administration of 0.01 mg/kg dexamethasone IV for cortisol concentration determination via chemiluminescent assay. Mean endogenous ACTH concentration did not differ between groups (group 1: 22.4 pg/mL, group 2: 20.0 pg/mL; P > 0.2). Mean baseline cortisol concentration also did not differ significantly between groups (group 1: 3.03 µg/dL, group 2: 4.95 µg/dL; P > 0.2) nor was there any difference in mean cortisol concentration between the groups at any other time point (P > 0.2). The cortisol concentration from 1 dog in group 2 suppressed to 0.7 µg/dL at 8 h but increased to 1.5 µg/dL at 10 h and 3.7 µg/dL at 12 h post-dexamethasone. Based on results of this study, use of an extended LDDS test could not differentiate between healthy dogs and dogs with AHAC. Diagnosis of AHAC should continue to be based on prior established criteria until new testing has been identified.