Hafnium (IV) oxide obtained by atomic layer deposition (ALD) technology promotes early osteogenesis via activation of Runx2-OPN-mir21A axis while inhibits osteoclasts activity.

BACKGROUND: Due to increasing aging of population prevalence of age-related disorders including osteoporosis is rapidly growing. Due to health and economic impact of the disease, there is an urgent need to develop techniques supporting bone metabolism and bone regeneration after fracture. Due to imbalance between bone forming and bone resorbing cells, the healing process of osteoporotic bone is problematic and prolonged. Thus searching for agents able to restore the homeostasis between these cells is strongly desirable. RESULTS: In the present study, using ALD technology, we obtained homogeneous, amorphous layer of hafnium (IV) oxide (HfO2). Considering the specific growth rate (1.9Å/cycle) for the selected process at the temperature of 90 °C, we performed the 100 nm deposition process, which was confirmed by measuring film thickness using reflectometry. Then biological properties of the layer were investigated with pre-osteoblast (MC3T3), pre-osteoclasts (4B12) and macrophages (RAW 264.7) using immunofluorescence and RT-qPCR. We have shown, that HfO2 (i) enhance osteogenesis, (ii) reduce osteoclastogenesis (iii) do not elicit immune response and (iv) exert anti-inflammatory effects. CONCLUSION: HfO2 layer can be applied to cover the surface of metallic biomaterials in order to enhance the healing process of osteoporotic bone fracture.

Equine herpesvirus type 1 (EHV-1)-induced rearrangements of actin filaments in productively infected primary murine neurons.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell culture in vitro. Immunofluorescent labeling and confocal microscopy revealed the formation of long, thin projections in neurons infected with the Jan-E strain, which was probably associated with enhanced intracellular spread of the virus. The EHV-1 Rac-H strain caused disruption of the microfilaments system and general depolymerization of actin, but treatment of neurons with cytochalasin D or latrunculin A resulted in limitation of viral replication. It can therefore be assumed that actin filaments are required only at the early stages of infection. Our results allow us to suggest that the actin cytoskeleton participates in EHV-1 infection of primary murine neurons but is not essential, and that other components of the cytoskeleton and/or cellular mechanisms may be also involved during EHV-1 infection.

Bone marrow-origin stem/progenitor cells in the mammary gland of heifers.

The aim of the study was to estimate the size of bone marrow-origin stem/progenitor population in 2-year old nonpregnant Holstein-Friesian heifers. Quantitative and qualitative analysis was done using scanning cytometry and confocal microscopy of mammary tissue slices labelled with the combination of two markers: Sca-1 (marker of stem-progenitor cells) and CD45 (marker of hematopoietic cells). The average (+/- SEM) percentage of Sca-1POS CD45 POS cells was 0.89 +/- 0.21. They were localized mainly outside of mammary ducts, in the stroma and sometimes intraluminally. Our results indicate that the subpopulation of Sca-1POS cells bearing CD45 antigen may enrich the niche of mammary stem/progenitor cells from the bone marrow and participate in the growth of the mammary gland in post-pubertal heifers.

Influence of mutation in cj0183 and cj0588 genes for colonization abilities of Campylobacter jejuni in Caco-2 cells using confocal laser scanning microscope.

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with homology to virulence factors found in other bacteria. Previous studies showed that single mutation in the cj0183 gene does not affect adhesion of C. jejuni to the Caco-2 cell line whereas protein encoded by cj0588 is involved in adherence to the Caco-2 cells. In the presented study differences in invasion index were observed between mutants in both genes and single mutation of cj0588 in 81116 and 81-176 C. jejuni strains This fact indicates that Cj0183 protein might play some role in invasion of bacteria into host cells.

Apoptotic and necrotic changes in cultured murine neurons infected with equid herpesvirus 1.

Equid herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae, is a neurotropic virus, that causes latent infections in the nervous system of the natural host. All alphaherpesviruses have developed sophisticated strategies to interfere with the host cell apoptotic mechanisms, but the ability of EHV-1 to induce apoptosis in neurons has not been determined yet. In this study, apoptotic and necrotic changes in cultured murine neurons were methods identifying key stages of apoptosis. These methods have demonstrated characteristic apoptosis features, like DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage in the infected cells. It seems likely that apoptosis was the predominant way of cell death in EHV-1-infected murine neurons. However, we showed also that during acute EHV-1 infection the majority of infected neurons remained unchanged and survived for more than eight weeks in culture, suggesting some protective mechanisms induced by the virus. Furthermore, it was shown that infection of neurons with EHV-1 has no significant influence on the level of the caspase 3, 7, and 8. We speculate that the control of apoptosis may be the key mechanism regulating the balance between productive and latent infection at the site of virus persistence.

Opposite effects of two different strains of equine herpesvirus 1 infection on cytoskeleton composition in equine dermal ED and African green monkey kidney Vero cell lines: application of scanning cytometry and confocal-microscopy-based image analysis in a quantitative study.

Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that alpha-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and alpha-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.

ZnTe-ZnO core-shell radial heterostructures grown by the combination of molecular beam epitaxy and atomic layer deposition.

ZnTe-ZnO core-shell radial heterostructures were grown using a new method of combining molecular beam epitaxy (MBE) and atomic layer deposition (ALD). Zinc telluride nanowires (core) were grown on a GaAs substrate using gold catalyzed vapor-liquid-solid mechanism. An atomic layer deposition technique using diethyl zinc and deionized water as precursors was applied for zinc oxide shell formation. The core-shell ZnTe-ZnO heterostructures thus obtained were characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction and photoluminescence measurements.

Optically detected magnetic resonance in bismuth-doped silica glass.

We report on the observation of optically detected magnetic resonance (ODMR) in bismuth-doped silica glass. To explain the results of the experiment we adopted the intramolecular charge transfer model for Bi(5+)O(n)(2-) molecules in the frame of a semiempirical molecular orbitals approach. The results of our calculations are in good agreement with observed features of luminescence and ODMR experiments.

Combination of hydroxyapatite islets with Ti3P surface layer produced on titanium alloy for bone implants.

This study is concerned with the properties and bioactivity and biocompatibility of hydroxyapatite islets deposited on a new composite layer Ti3P+Ti2Ni type produced by a duplex method on Ti6Al4V titanium alloy. The microstructure and chemical composition of a produced surface layers and hydroxyapatite coating were investigated using scanning electron microscope equipped with EDS. Their bioactivity were examined in simulated body fluid and analyzed with XPS. Dissolution of hydroxyapatite was tested in culture medium during 12 days of incubation. Biocompatibility was investigated in osteoblast Saos2 line culture in contact with the tested material. Cell proliferation and activity were determined by the MTT test and measurement of alkaline phosphatase activity, respectively. Cell distribution was analyzed under a confocal microscope. The produced surface layers have a diffusion character with fine-grained structure and about 4 microm thick external zone of Ti3P. The experiments revealed higher bioactivity and biocompatibility of the Ti3P in comparison with reference titanium alloy. Hydroxyapatite islets were 0.8 mm in diameter and about 300 nm thick. They partially dissolved during the experiment what lead to formation on Ti3P between hydroxyapatite islets a precipitate containing Ca and P. Biocompatibility analyzed under confocal microscope in range of cell adhesion with osteoblast cells of Saos2 line revealed initial the highest osteoblast adhesion on Ti3P between hydroxyapatite islets and increasing on hydroxyapatite during following days. Cell were characterized by high proliferation and ALP activity. Therefore, the high bioactivity and biocompatibility of Ti3P and profitable hydroxyapatite properties make this composite layer promising for increasing implant fixation in vivo.

Wheat (Triticum aestivum) seedlings secrete proteases from the roots and, after protein addition, grow well on medium without inorganic nitrogen.

This paper reports on the role of proteases secreted by roots in nitrogen capture by plants. The study was conducted on aseptically cultivated wheat seedlings (Triticum aestivum cv. Tacher) obtained from embryos isolated from grains. Seedlings were cultivated for 21 days on deionised water, Murashige Skoog medium (MS), MS without inorganic nitrogen (IN), and MS without IN, in which IN was replaced by casein (0.01%, 0.1% or 1%). Comparison of seedlings grown on these media showed that casein entirely compensated for the lack of inorganic nitrogen in the medium. Shoots and roots of seedlings cultivated on MS medium with this protein had higher fresh weight than those cultivated on MS medium without casein. The increase in fresh weight of seedlings was correlated with casein concentration and proteolytic activity in the medium. In conclusion, wheat that uses proteases secreted by the roots can directly utilise proteins in the medium as a source of nitrogen without prior digestion by microbial proteases and without protein mineralisation. These results suggest the important role of organic nitrogen fertilisers in increasing wheat yield.

Mechanisms involved in the development of the small intestine mucosal layer in postnatal piglets.

The use of complementary visualization and measurement techniques allowed accurate description and quantification of changes in the intestinal mucosal architecture and provided a comprehensive outlook on the dynamics of remodelling and maturation processes of the mucosal layer taking place in the small intestine of piglets from birth to weaning. The aim of the study was to examine the early postnatal development of the small intestine in pigs. Three techniques were used: scanning electron microscopy (measurements of villus density and shape, height of enterocytes and microvilli, cell exfoliation, and location of extrusion zones), optical microscopy (cross section, measurement of structures: villus length and width; crypt depth; mucosal thickness), and confocal microscopy (cell localization, apoptosis, exfoliation and migration). The postnatal development of the mucosal layer of the small intestine was reflected in changes in the density, length, width, and shape of villi, crypt depth, replacement of enterocyte population, and arrangement. The presence of deep transverse furrows on villus corpus and vacuolated fetal-type enterocytes in the mucosal layer of the small intestine, which are able to engulf large amounts of colostrum shortly after birth, appears to play an important role in the observed phenomenon of straightening of the villus height and increasing of the villus diameter shortly after birth. We hypothesized that the intestinal mucosal layer is compressed before birth and ready to unfold within a short time after birth.

Molecular mechanism of programmed cell death in the gut epithelium of neonatal piglets.

In the intestinal mucosa of pig, calf and rat neonates, we observed the cells die in the packets which suggests involvement of some paracrine factors. The death signal was transferred via tissue continuum as well as across the gut lumen, and the involvement of TGF-beta1 and TNFalpha was demonstrated. Present study aimed to clarify the molecular mechanisms of programmed cell death in the mucosa of the small intestine of pig neonates. Groups (packets) of cells and the neighboring cells underwent apoptosis, and expressed an enhanced TGF-RII. In the dying cells the death signal promoted via TGF-RII was associated with enhanced expression of active caspase 8, TGF-beta1, TNFalpha and Bid. Quantitative study showed that high expression of TGF-beta1 was positively correlated with expression of BID and negatively with BCL-2, illustrating the transmission of signal from TGF-RII through SMAD cascade and RunX protein. We hypothesize that TGF-beta1 sensitizes the enterocytes for TNFalpha signaling and both cytokines control the apoptosis process in the gut epithelium. Intensive mitosis triggers many errors in DNA replication, and the role of p53 is to detect them and promote either repair or apoptosis. During first days of live all damaged cells were directed towards apoptosis while at day 7 at least some of them were repaired. Autophagy, the second form of programmed cell death, was recognized by its key marker MAP I LC3. Our data showed the colocalization of MAP I LC3 with active caspase 3 thus suggesting a coexistence between these two forms of cell death, at least in the early postnatal life.

The effect of supplementing sow with bioactive substances on neonatal small intestinal epithelium.

Development of the small intestinal epithelium in early postnatal period has a significant influence on pig's survival rate and further productivity. The aim of this research was to verify whether the diet supplementation of pregnant and lactating sow with a blend of bioactive substances (flax seed, rapeseed, linden inflorescence, taurine, L-carnitine and tocopherol acetate) had an effect on the development of intestinal epithelium in their offspring. The doses of bioactive substances were calculated to meet the demands for optimal supply of the pig fetuses and newborns. Pig neonates from two groups of sows, control and supplemented, were sacrificed at the day 1, 2, 4, 7 and 14 of life. The samples taken from mid-jejunum were evaluated for mitosis (Ki67), apoptosis (active caspase 3), autophagy (MAP I LC3), and DNA damage (p53). Increase of mitotic index was noticed at day 1, 4 and 7 for supplemented group when compared to the control. Reduction of apoptotic index was observed at day 2 as compared to control. A tendency toward elevated autophagy was observed during the first 2-4 postnatal days in both groups. p53 expression was significantly lower in supplemented group as compared to control. Overall, the mitosis to programmed cell death ratio was increased and the maturation of epithelial cells quickened. We suppose that the supplementation of pregnant and lactating sow diet with bioactive substances enhanced maturation of the small intestinal epithelium in their offspring during the early postnatal period.

Effect of butyrate infusion into the rumen on butyrate flow to the duodenum, selected gene expression in the duodenum epithelium, and nutrient digestion in sheep.

The aim of the study was to determine the effect of butyrate infusion into the rumen on butyrate flow to the duodenum, expression of short-chain fatty acid (SCFA) transporters (monocarboxylate transporter-1, -2, and -4) and receptors (G protein coupled receptor-41 and -43) in the duodenal epithelium and nutrient digestion in sheep. Eight wethers (39.0 ± 3.00 kg; mean ± SD) with ruminal and T-shape duodenal cannulas were allocated to 4 × 4 replicated Latin square design with each experimental period lasting for 21 d (12 d of adaptation and 9 d for data and sample collection). Experimental treatments were: 1) distilled water infusion into the rumen (CONT); 2) 15 g/d of butyric acid infusion into the rumen (BUT15); 3) 30 g/d of butyric acid infusion into the rumen (BUT30); and 4) 45 g/d of butyric acid infusion into the rumen (BUT45). The daily dose of butyrate was infused into the rumen via the rumen cannula, with 200 mL of solution of butyric acid and distilled water, at a constant rate (0.1389 mL/min) throughout the day using a peristaltic pump. Correspondingly, 200 mL/d of distilled water was infused into the rumen of CONT. The wethers were fed daily 900 g of chopped meadow hay and 200 g of concentrate in two equal meals at 0600 and 1800 h. Butyrate infusion into the rumen did not affect total SCFA concentration in the rumen fluid ( > 0.11). Molar proportion of butyrate in total SCFA linearly increased, and molar proportion of acetate and isovalerate linearly decreased ( ≤ 0.02) with an increasing amount of butyrate infused into the rumen. The molar proportion of butyrate in total SCFA in the duodenal digesta linearly increased ( < 0.01), and butyrate flow to duodenum tended to linearly increase ( = 0.06) with an increasing dose of exogenous butyrate delivered to the rumen. Butyrate infusion into the rumen did not affect ( ≥ 0.14) the mRNA expression of monocarboxylate transporter-2 and -4 and G protein coupled receptor-43 in the duodenal epithelium. The G protein coupled receptor-41 and monocarboxylate transporter-1 mRNA expression in the duodenal epithelium was very low in many of the analyzed samples. Digestibility of organic matter, neutral detergent fiber, and acid detergent fiber in the stomach (forestomach and abomasum) decreased for BUT15 and BUT30 and then increased for BUT45 (quadratic, ≤ 0.04); however, neither digestibility in the intestine nor total tract digestibility differed between treatments ( ≥ 0.10).

Titanium oxide thin films obtained with physical and chemical vapour deposition methods for optical biosensing purposes.

This work discusses an application of titanium oxide (TiOx) thin films deposited using physical (reactive magnetron sputtering, RMS) and chemical (atomic layer deposition, ALD) vapour deposition methods as a functional coating for label-free optical biosensors. The films were applied as a coating for two types of sensors based on the localised surface plasmon resonance (LSPR) of gold nanoparticles deposited on a glass plate and on a long-period grating (LPG) induced in an optical fibre. Optical and structural properties of the TiOx thin films were investigated and discussed. It has been found that deposition method has a significant influence on optical properties and composition of the films, but negligible impact on TiOx surface silanization effectiveness. A higher content of oxygen with lower Ti content in the ALD films leads to the formation of layers with higher refractive index and slightly higher extinction coefficient than for the RMS TiOx. Moreover, application of the TiOx film independently on deposition method enables not only for tuning of the spectral response of the investigated biosensors, but also in case of LSPR for enhancing the ability for biofunctionalization, i.e., TiOx film mechanically protects the nanoparticles and induces change in the biofunctionalization procedure to the one typical for oxides. TiOx coated LSPR and LPG sensors with refractive index sensitivity of close to 30 and 3400nm/RIU, respectively, were investigated. The ability for molecular recognition was evaluated with the well-known complex formation between avidin and biotin as a model system. The shift in resonance wavelength reached 3 and 13.2nm in case of LSPR and LPG sensors, respectively. Any modification in TiOx properties resulting from the biofunctionalization process can be also clearly detected.

Application of sulforaphane: histopathological study of intraportal transplanted pancreatic islets into livers of diabetic rats.

BACKGROUND: Islet cell transplantation is a promising method to restore insulin independence to patients with type 1 diabetes mellitus. A main problem in clinical islet transplantation is the fact that only a small percentage of allogeneic islet-transplanted type 1 diabetic patients can completely omit insulin injections after transplantation. One reason for the impaired survival of islet grafts is aberration of the function of islets due to toxic agents, including oxygen radicals and nitric oxide, which arise during warm or cold ischemic time. Therefore, in clinical islet transplantation, islets have been preserved with a mixture of antioxidants to reduce free radical-mediated damage of transplanted beta cells. Our aim was to examine hepatic tissue after metabolic normalization following intraportal islet transplantation after application of sulforaphane. MATERIALS AND METHODS: Islets were isolated from pancreata of WAG rats. Sulforaphane (24 mg/kg) was administered 24 hours before isolated islets were transplanted into the liver through the portal vein (1200 +/- 100 per rat). At 9 months after transplantation the animals were killed and liver tissue removed for morphological examination. RESULTS: This report indicated that the intrahepatic portal vein site was indeed an excellent locus for implantation of free pancreatic islets. The islet grafts developed rich vascularization derived from both venous and arterial sources. The islet cells maintained their structural and functional integrity after implantation. CONCLUSION: Our results showed that sulforaphane improved islet function in vivo, indicating that combination of a free radical scavenger and an antioxidant (sulforaphane) may be used to increase the effectiveness of islet transplantation.

Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

Changes in ATP level and iron-induced ultra-weak photon emission in bull spermatozoa, caused by membrane peroxidation during thermal stress.

ATP level, cell motility and viability, oxygen uptake, pyruvate kinase activity, and ultra-weak photon emission (UPE) induced by red-ox Fe(2+)-ascorbate cycling system were studied in fresh, in previously equilibrated in a glycerol diluent, and in cryopreserved bull spermatozoa, exposed to thermal stress by incubation of the cells at 44 degrees C. A sharp drop in motility and viability of fresh spermatozoa and even more so, of equilibrated and cryopreserved cells was accompanied by accumulation of ATP. When cell movement was totally inhibited, ATP utilization was decreased, while chemical energy continued to be produced by cell pyruvate kinase, one of the key glycolytic enzymes, which in spermatozoa is very active (6500 IU/g protein) and insensitive to feed-back inhibition by excess of ATP and L-cysteine. Accumulation of ATP during incubation at 44 degrees C in 0.9% NaCl was accompanied by rapid decrease in oxygen consumption by fresh spermatozoa and an increase in Fe(2+)-ascorbate induced UPE, followed by a sharp decrease in ATP level observed at the end of induced UPE measurement. The increase in photon emission due to lipid peroxidation was highly correlated with the increase in cell ATP level caused by thermal stress.

Spectra of the formaldehyde-induced ultraweak luminescence from yeast cells.

An increase in the intensity and distinct spectral changes of ultraweak luminescence from the yeast Saccharomyces cerevisiae were measured when the metabolism of cells was drastically altered. A small emission peak and a red emission band 680-850 nm appeared when air-dried cells were imbibed in water. Lethal concentrations of HCHO (0.01%-10%) elicited a 2500 fold increase of the emission intensity and distinct spectral alterations. A transient 500-580 nm emission appeared in the initial phase of interaction. Then a gradually increasing long-lasting red emission band centered around 620 nm predominated in the total spectral range covering 470-850 nm. These emissions were not correlated with minor changes in fluorescence emission and excitation spectra originating from tryptophan, flavins, and unidentified emitters.

Impact of yttria stabilization on Tb3+ intra-shell luminescence efficiency in zirconium dioxide nanopowders.

This paper reports the observation of Tb(3+) 4f-4f emission gain in ZrO2 nanocrystals stabilized by Y2O3 as the amount of stabilizer increases from 0% to 10% mol. The nanocrystals were obtained via microwave solvothermal technology. The photoluminescence properties of as-grown samples are investigated. The possibility of biological applications of the material is tested on living organisms (mice). The result indicates the potential use of the studied material as a luminescent nanomarker.