The investigation of post-thaw chilled storage and the applicability of large-scale cryopreservation in chub (Squalius cephalus) sperm.

Chub (reophillic cyprinids) is one of the most sensitive bioindicator fish of environmental changes following anthropogenic activities. The improvement of different biotechnological procedures could help support its conservation and strengthen the natural populations. The aim of this study was to compare the effects of two different hormonal agents (carp pituitary extract and Ovopel™) on various motility parameters (pMOT-%, DAP-µm, VCL µm s-1, VSL-µm s-1, LIN-%, ALH-µm, BCF-Hz) of fresh and cryopreserved/thawed sperm (stored at 4 °C for 6 h). Additionally, we sought to develop a novel, large-scale cryopreservation method for chub sperm, assessing freezing methods (Styrofoam box and a controlled-rate freezer) and different containers (0.5, 5 mL straw and 4 mL cryotube) for sperm cryopreservation. The results of this study indicated no difference between the carp pituitary extract and Ovopel treated groups in either the fresh or frozen/thawed sperm (at 0, 3, 6, hour post thawing, P = 0.4351). In contrast, the quality of the thawed chub sperm was negatively affected after 3 h chilled storage in both hormonal treatments (P = 0.0036, P < 0.0001). When assessing the motility parameters of the sperm between the 5 mL straw and 4 mL cryotube groups cryopreserved in a Styrofoam Box, no difference was observed (P = 0.103). Additionally, sperm loaded in 4 mL cryotubes showed no difference in motility when cryopreserved with either the Styrofoam box or controlled-rate freezer methods (P = 0.109). A similar hatching rate was observed in sperm preserved using the Styrofoam box (35 ± 7 %) and controlled rate freezer (25 ± 9 %) methods (P = 0.300). In a second fertilization trial, hatching rate was similar between control (72 ± 19 %) and cryopreserved (4 mL cryotube and Styrofoam box, 61 ± 5 %) groups. (P = 0.257). Based on our findings and its standard features (less species specific, precise dose calculation), Ovopel can be a good candidate for the stimulation of spermiation in chub sperm prior to cryopreservation. Furthermore, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.

Application of hydroxypropyl methylcellulose as a protective agent against magnesium stearate induced crystallization of amorphous itraconazole.

Itraconazole is a fungicide drug which has low bioavailability due to its poor water solubility. Amorphous solid dispersion (ASD) is a tool that has the potential to greatly increase the dissolution rate and extent of compounds. In this work, the dissolution of tablets containing the ASD of itraconazole with either hydroxypropyl methylcellulose (HPMC) or vinylpyrrolidone-vinyl acetate copolymer (PVPVA) was compared in order to find a formulation which can prevent the drug from the precipitation caused by magnesium stearate. Formulations containing the PVPVA-based ASD with HPMC included in various forms could reach 90% dissolution in 2 h, while HPMC-based ASDs could release 100% of the drug. However, HPMC-based ASD had remarkably poor grindability and low bulk density, which limited its processability and applicability. The latter issue could be resolved by roller compacting the ASD, which significantly increases the bulk density and the flowability of the powder blends used for tableting. This roller compaction step might be a base for the industrial application of HPMC-based, electrospun ASDs.

Note: 4-bounce neutron polarizer for reflectometry applications.

A neutron polarizer using four successive reflections on m = 2.5 supermirrors was built and installed at the GINA neutron reflectometer at the Budapest Neutron Centre. This simple setup exhibits 99.6% polarizing efficiency with 80% transmitted intensity of the selected polarization state. Due to the geometry, the higher harmonics in the incident beam are filtered out, while the optical axis of the beam remains intact for easy mounting and dismounting the device in an existing experimental setup.

Investigation of Deteriorated Dissolution of Amorphous Itraconazole: Description of Incompatibility with Magnesium Stearate and Possible Solutions.

Disadvantageous crystallization phenomenon of amorphous itraconazole (ITR) occurring in the course of dissolution process was investigated in this work. A perfectly amorphous form (solid dispersion) of the drug was generated by the electroblowing method (with vinylpyrrolidone-vinyl acetate copolymer), and the obtained fibers were formulated into tablets. Incomplete dissolution of the tablets was noticed under the circumstances of the standard dissolution test, after which a precipitated material could be filtered. The filtrate consisted of ITR and stearic acid since no magnesium content was detectable in it. In parallel with dissolution, ITR forms an insoluble associate, stabilized by hydrogen bonding, with stearic acid deriving from magnesium stearate. This is why dissolution curves do not have the plateaus at 100%. Two ways are viable to tackle this issue: change the lubricant (with sodium stearyl fumarate >95% dissolution can be accomplished) or alter the polymer in the solid dispersion to a type being able to form hydrogen bonds with ITR (e.g., hydroxypropyl methylcellulose). This work draws attention to one possible phenomenon that can lead to a deterioration of originally good dissolution of an amorphous solid dispersion.

Mortality and the relationship of somatic comorbidities to mortality in schizophrenia. A nationwide matched-cohort study.

AIM: We conducted a matched-cohort study to assess mortality in schizophrenia and the relationship of mortality with comorbid somatic conditions and suicide attempts. METHOD: A full-population register-based prospective matched-cohort study was performed including all eligible patients with schizophrenia in Hungary between 01/01/2005 and 31/12/2013. Control subjects were individually matched to patients with schizophrenia at a 5:1 ratio. The principal outcome measure was death due to any reason. A non-parametric approach was used for descriptive statistical purposes, the Kaplan-Meier model for survival analysis, and the Cox proportional-hazards regression model for inferential statistics. RESULTS: Patients with schizophrenia (n=65,169) had substantially higher risk of all-cause mortality than the control subjects (n=325,435) (RR=2.4; P<0.0001). Comorbidities and suicide attempts were associated with significantly increased mortality in both groups. As compared to the controls, 20-year old males with schizophrenia had a shorter life expectancy by 11.5years, and females by 13.7years; the analogous numbers for 45-year old schizophrenics were 8.1 and 9.6years, respectively. CONCLUSIONS: A significant mortality gap - mainly associated with somatic comorbidities - was detected between patients with schizophrenia and individually matched controls. Improved medical training to address the disparity in mortality, and many other factors including lack of resources, access to and model of medical care, lifestyle, medication side effects, smoking, stigma, need for early intervention and adequate health care organization could help to better address the physical health needs of patients with schizophrenia.

Food and water intake, body temperature and metabolic consequences of interleukin-1ß microinjection into the cingulate cortex of the rat.

In order to elucidate whether cytokine mechanisms of the cingulate cortex (cctx) are important in the central regulation of homeostasis, in the present study, feeding-metabolic effects of direct bilateral microinjection of interleukin-1ß (IL-1ß) into the cctx of the rat have been investigated. Short- (2h), medium (12h) and long-term (24h) food and water intakes and body temperature were measured after the intracerebral administration of this primary cytokine or vehicle solution, with or without paracetamol pretreatment. The effect of IL-1ß on the blood glucose level of animals was examined in glucose tolerance test (GTT), and concentrations of relevant plasma metabolites (total cholesterol, HDL, LDH, triglycerides, uric acid) were additionally also determined following the above microinjections. In contrast to causing no major alteration in the food and water intakes, the cytokine treatment evoked significant increase in the body temperature of the rats. Prostaglandin-mediated mechanisms were shown to have important role in the mode of this action of IL-1ß, since paracetamol pretreatment partially prevented the development of the above mentioned hyperthermia. In the GTT, no considerable difference was observed between the blood glucose levels of the cytokine treated and control animals. Following IL-1ß microinjection, however, significant decrease of HDL and total cholesterol was found. Our present findings indicate that elucidating the IL-1ß mediated homeostatic control mechanisms in the cingulate cortex may lead to the better understanding not only the regulatory entities of the healthy organism but also those found in obesity, diabetes mellitus and other worldwide rapidly spreading feeding-metabolic disorders.

Adipokines as atherothrombotic risk factors in obese subjects: Associations with haemostatic markers and common carotid wall thickness.

BACKGROUND AND AIMS: Some crucial associations between obesity-related altered adipokine levels and the main factors of atherosclerotic, atherothrombotic processes are not fully known. We analysed the relationships of classic adipokines, namely leptin, resistin, adiponectin, tumour necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) with the markers of platelet activation, including mean platelet volume (MPV), platelet surface/soluble P-selectin, platelet-derived microparticles (PMPs), the parameters of coagulation abnormalities and common carotid intima-media thickness (IMT) in obese patients with or without atherosclerotic comorbidities in comparison to age- and sex-matched controls. METHODS AND RESULTS: We enrolled 154 obese individuals, including 98 suffering from atherosclerotic concomitant conditions, 56 free of atherosclerotic comorbidities and 62 healthy controls. Plasma levels of leptin, resistin, adiponectin, TNF-α, IL-6, soluble P-selectin, and plasminogen activator inhibitor-1 antigen (PAI-1 ag) were analysed by ELISA. Platelet surface P-selectin and PMPs were measured by flow cytometry. IMT was detected by ultrasonography. Adipokines were closely associated with markers of platelet hyperactivity, hypercoagulability, hypofibrinolysis and IMT. Significant independent associations were found between leptin and platelet count (p < 0.0001), MPV (p = 0.019), PMPs (p < 0.0001), fibrinogen (p = 0.001), factor VIII (FVIII) activity (p = 0.035); adiponectin and PAI-1 ag (p = 0.035); resistin and soluble P-selectin (p = 0.002); TNF-α and PAI-1 ag (p < 0.0001); and IL-6 and fibrinogen (p = 0.011). Finally, leptin (p = 0.0005), adiponectin (p = 0.019), IL-6 (p = 0.001), MPV (p = 0.0003), PMP (p = 0.008), and FVIII activity (p = 0.043) were independent predictors of IMT. CONCLUSION: Overall, we suggest that in obese subjects altered adipokine levels play a key role in common carotid atherosclerosis both directly and through haemostatic parameters.

Plasma E-selectin levels can play a role in the development of diabetic retinopathy.

PURPOSE: Diabetic retinopathy is one of the leading causes of blindness. There are several risk factors, such as the duration of diabetes or glycemic control of the patient; however, several biochemical factors also alter the process. Our aim was to investigate the role of soluble E-selectin in the formation of diabetic retinopathy. PATIENTS AND METHODS: Fifty-seven patients (37 female and 20 male, aged 61.71 ± 12.31 years) and 14 healthy control subjects (ten female and four male, aged 63.06 ± 10.46 years) were enrolled in the study. We measured the soluble E-selectin level in the plasma of patients by ELISA. All patients underwent careful ophthalmological examination, including ophthalmoscopy and color fundus photography, while diabetic retinopathy grading was performed in line with the 2012 classification of the American Academy of Ophthalmology (AAO). RESULTS: The soluble E-selectin level was significantly higher in patients with diabetes compared to controls (32.95 ng/ml vs. 26.55 ng/ml, p = 0.03). Dividing patients into groups by the presence of retinopathy, the E-selectin level was also significantly higher in the retinopathy group (p < 0.05). When we examined diabetic patients by the severity of retinopathy (groups A, B, and C, by the guidelines of the AAO), however, we did not find any significant difference in soluble E-selectin levels, although it tended to be higher in group B. CONCLUSIONS: An elevated E-selectin level can play a role in the development of diabetic retinopathy, but it does not seem to alter disease severity. However, glycemic control and the reduction of cardiovascular risk factors may also alter the level of E-selectin that might play a role in the prevention of diabetic retinopathy.

Impaired glucose tolerance after streptozotocin microinjection into the mediodorsal prefrontal cortex of the rat.

The mediodorsal prefrontal cortex (mdPFC) is a key structure of the central glucose-monitoring (GM) neural network. Previous studies indicate that intracerebral streptozotocin (STZ) microinjection-induced destruction of local chemosensory neurons results in feeding and metabolic alterations. The present experiments aimed to examine whether STZ microinjection into the mdPFC causes metabolic deficits. To do so, glucose tolerance test (GTT) and measurements of plasma metabolites were performed in STZ-treated or control rats. Intraperitoneal D-glucose load was delivered 20 min or 4 weeks following the intracerebral microinjection of STZ or saline (acute or subacute GTT, respectively). The STZ-treated rats displayed acute glucose intolerance: at the 120th min of the test, blood glucose level of these rats was significantly higher than that of the ones in the control group. When determining the plasma level of various metabolites, 30 min following the intracerebral STZ or saline microinjection, the triglyceride concentration of the STZ-treated rats was found to be reduced compared with that of the control rats. The GM neurons of the mdPFC are suggested to be involved in the organization of complex metabolic processes by which these chemosensory cells contribute to adaptive control mechanisms of the maintenance of homeostasis.

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.

Direct comparison of kilohertz- and megahertz-repetition-rate femtosecond damage threshold.

We performed femtosecond laser-induced damage threshold (fs LIDT) measurements with substantially different repetition rate Ti:sapphire laser systems: a 1 kHz regenerative amplifier and a 4.3 MHz long-cavity oscillator. All other pulse parameters are kept the same. Comparative measurements of a dielectric high reflector, a chirped mirror, and metallic mirrors show at least a factor of 2.7 lower fs LIDT at megahertz repetition rates. We attribute this to thermally assisted damage mechanisms supported by complex heat transfer simulations.

Use of virulence determinants and seropathotypes to distinguish high- and low-risk Escherichia coli O157 and non-O157 isolates from Europe.

The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

GINA--a polarized neutron reflectometer at the Budapest Neutron Centre.

The setup, capabilities, and operation parameters of the neutron reflectometer GINA, the recently installed "Grazing Incidence Neutron Apparatus" at the Budapest Neutron Centre, are introduced. GINA, a dance-floor-type, constant-energy, angle-dispersive reflectometer is equipped with a 2D position-sensitive detector to study specular and off-specular scattering. Wavelength options between 3.2 and 5.7 Å are available for unpolarized and polarized neutrons. Spin polarization and analysis are achieved by magnetized transmission supermirrors and radio-frequency adiabatic spin flippers. As a result of vertical focusing by a five-element pyrolytic graphite monochromator, the reflected intensity from a 20 × 20 mm(2) sample has been doubled. GINA is dedicated to studies of magnetic films and heterostructures, but unpolarized options for non-magnetic films, membranes, and other surfaces are also provided. Shortly after its startup, reflectivity values as low as 3 × 10(-5) have been measured by the instrument. The instrument capabilities are demonstrated by a non-polarized and a polarized reflectivity experiment on a Si wafer and on a magnetic film of [(62)Ni/(nat)Ni](5) isotope-periodic layer composition. The facility is now open for the international user community. Its further development is underway establishing new sample environment options and spin analysis of off-specularly scattered radiation as well as further decreasing the background.