Designing of Drug Delivery Systems to Improve the Antimicrobial Efficacy in the Periodontal Pocket Based on Biodegradable Polyesters.

Delivery systems for biologically active substances such as proanthocyanidins (PCANs), produced in the form of electrospun nonwoven through the electrospinning method, were designed using a polymeric blend of poly(L-lactide-co-glycolide) (PLGA)and poly[(R,S)-3-hydroxybutyrate] ((R,S)-PHB). The studies involved the structural and thermal characteristics of the developed electrospun three-dimensional fibre matrices unloaded and loaded with PCANs. In the next step, the hydrolytic degradation tests of these systems were performed. The release profile of PCANs from the electrospun nonwoven was determined with the aid of UV-VIS spectroscopy. Approximately 30% of the PCANs were released from the tested electrospun nonwoven during the initial 15-20 days of incubation. The chemical structure of water-soluble oligomers that were formed after the hydrolytic degradation of the developed delivery system was identified through electrospray ionization mass spectrometry. Oligomers of lactic acid and OLAGA oligocopolyester, as well as oligo-3-hydroxybutyrate terminated with hydroxyl and carboxyl end groups, were recognized as degradation products released into the water during the incubation time. It was also demonstrated that variations in the degradation rate of individual mat components influenced the degradation pattern and the number of formed oligomers. The obtained results suggest that the incorporation of proanthocyanidins into the system slowed down the hydrolytic degradation process of the poly(L-lactide-co-glycolide)/poly[(R,S)-3-hydroxybutyrate] three-dimensional fibre matrix. In addition, in vitro cytotoxicity and antimicrobial studies advocate the use of PCANs for biomedical applications with promising antimicrobial activity.

Extraordinary Multi-Organismal Interactions Involving Bacteriophages, Bacteria, Fungi, and Rotifers: Quadruple Microbial Trophic Network in Water Droplets.

Our observations of predatory fungi trapping rotifers in activated sludge and laboratory culture allowed us to discover a complicated trophic network that includes predatory fungi armed with bacteria and bacteriophages and the rotifers they prey on. Such a network seems to be common in various habitats, although it remains mostly unknown due to its microscopic size. In this study, we isolated and identified fungi and bacteria from activated sludge. We also noticed abundant, virus-like particles in the environment. The fungus developed absorptive hyphae within the prey. The bacteria showed the ability to enter and exit from the hyphae (e.g., from the traps into the caught prey). Our observations indicate that the bacteria and the fungus share nutrients obtained from the rotifer. To narrow the range of bacterial strains isolated from the mycelium, the effects of bacteria supernatants and lysed bacteria were studied. Bacteria isolated from the fungus were capable of immobilizing the rotifer. The strongest negative effect on rotifer mobility was shown by a mixture of Bacillus sp. and Stenotrophomonas maltophilia. The involvement of bacteriophages in rotifer hunting was demonstrated based on molecular analyses and was discussed. The described case seems to be an extraordinary quadruple microbiological puzzle that has not been described and is still far from being understood.

X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants.

Contents Summary 432 I. Introduction 433 II. Preparation of plant samples for X-ray micro-analysis 433 III. X-ray elemental mapping techniques 438 IV. X-ray data analysis 442 V. Case studies 443 VI. Conclusions 446 Acknowledgements 449 Author contributions 449 References 449 SUMMARY: Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X-ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X-rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton-induced X-ray emission) or X-rays (X-ray fluorescence microscopy). Elucidating the cellular and tissue-level distribution of metal(loid)s is inherently challenging and accurate X-ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X-ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.

Diverse action of repeated corticosterone treatment on synaptic transmission, neuronal plasticity, and morphology in superficial and deep layers of the rat motor cortex.

One of the adverse effects of prolonged stress in rats is impaired performance of skilled reaching and walking tasks. The mechanisms that lead to these abnormalities are incompletely understood. Therefore, we compared the effects of twice daily repeated corticosterone injections for 7 days on miniature excitatory postsynaptic currents (mEPSCs), as well as on synaptic plasticity and morphology of layers II/III and V pyramidal neurons of the primary motor cortex (M1) of male Wistar rats. Corticosterone treatment resulted in increased frequency, but not amplitude, of mEPSCs in layer II/III neurons accompanied by increased complexity of the apical part of their dendritic tree, with no changes in the density of dendritic spines. The frequency and amplitude of mEPSCs as well as the parameters characterizing the complexity of the dendritic tree were not changed in layer V cells; however, their dendritic spine density was increased. While corticosterone treatment resulted in an increase in the amplitude of field potentials evoked in intralaminar connections within layer II/III, it did not influence field responses in layer V intralaminar connections, as well as the extent of chemically induced layer V long-term potentiation (chemLTP) by the application of tetraethylammonium (TEA, 25 mM). However, chemLTP induction in layer II/III was impaired in slices prepared from corticosterone-treated animals. These data indicate that repeated 7-day administration of exogenous corticosterone induces structural and functional plasticity in the M1, which occurs mainly in layer II/III pyramidal neurons. These findings shed light on potential sites of action and mechanisms underlying stress-induced impairment of motor functions.

Short-term repeated corticosterone administration enhances glutamatergic but not GABAergic transmission in the rat motor cortex.

It has been demonstrated that stress impairs performance of skilled reaching and walking tasks in rats due to the action of glucocorticoids involved in the stress response. Skilled reaching and walking are controlled by the primary motor cortex (M1); however, it is not known whether stress-related impairments in skilled motor tasks are related to functional and/or structural alterations within the M1. We studied the effects of single and repeated injections of corticosterone (twice daily for 7 days) on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) recorded from layer II/III pyramidal neurons in ex vivo slices of the M1, prepared 2 days after the last administration of the hormone. We also measured the density of dendritic spines on pyramidal cells and the protein levels of selected subunits of AMPA, NMDA, and GABAA receptors after repeated corticosterone administration. Repeatedly administered corticosterone induced an increase in the frequency but not in the amplitude of sEPSCs, while a single administration had no effect on the recorded excitatory currents. The frequency and amplitude of sIPSCs as well as the excitability of pyramidal cells were changed neither after single nor after repeated corticosterone administration. Treatment with corticosterone for 7 days did not modify the density of dendritic spines on pyramidal neurons. Corticosterone influenced neither the protein levels of GluA1, GluA2, GluN1, GluN2A, and GluN2B subunits of glutamate receptors nor those of α1, ß2, and γ2 subunits of the GABAA receptor. The increase in sEPSCs frequency induced by repeated corticosterone administration faded out within 7 days. These data indicate that prolonged administration of exogenous corticosterone selectively and reversibly enhances glutamatergic, but not GABAergic transmission in the rat motor cortex. Our results suggest that corticosterone treatment results in an enhancement of spontaneous glutamate release from presynaptic terminals in the M1 and thereby uncovers a potential mechanism underlying stress-induced motor functions impairment.

Chromium distribution in shoots of macrophyte Callitriche cophocarpa Sendtn.

The aim of the study was the analysis of Cr distribution in shoots of the macrophyte Callitriche cophocarpa by means of two X-ray-based techniques: micro X-ray fluorescence (µXRF) and electron probe X-ray microanalysis (EPXMA). Plants were treated with 100 µM (5.2 mg l(-1)) chromium solutions for 7 days. Cr was introduced independently at two speciations as Cr(III) and Cr(VI), known for their diverse physicochemical properties and different influence on living organisms. A comparative analysis of Cr(III)-treated plants by EPXMA and µXRF demonstrated high deposition of Cr in epidermal glands/hairs localized on leaves and stems of the plant shoots. Cr in Cr(III)-treated plants was recorded solely in glands/hairs, and the element was not present in any other structures. On the other hand, Cr in Cr(VI)-treated group of plants was rather found in vascular bundles. Moreover, the concentration of Cr in Cr(VI)-treated plants was significantly lower than in plants incubated in Cr(III) solution. The results obtained in this work suggest differences in chromium uptake, transport and accumulation dependent on the oxidative state of the element.

Dietary acrylamide disrupts the functioning of the biological clock.

Acrylamide (ACR) is a known carcinogen and neurotoxin. It is chronically consumed in carbohydrate-rich snacks processed at high temperatures. This calls for systematic research into the effects of ACR intake, best performed in an experimental model capable of detecting symptoms of its neurotoxicity at both high and low doses. Here, we study the influence of 10 µg/g (corresponding to the concentrations found in food products) and, for comparison, 60, 80 and 110 µg/g dietary ACR, on the fruit fly Drosophila melanogaster. We show that chronic administration of ACR affects lifespan, activity level and, most importantly, the daily and circadian pattern of locomotor activity of Drosophila. ACR-treated flies show well-defined and concentration-dependent symptoms of ACR neurotoxicity; a reduced anticipation of upcoming changes in light conditions and increased arrhythmicity in constant darkness. The results suggest that the rhythm-generating neural circuits of their circadian oscillator (biological clock) are sensitive to ACR even at low concentrations if the exposure time is sufficiently long. This makes the behavioural readout of the clock, the rhythm of locomotor activity, a useful tool for studying the adverse effects of ACR and probably other compounds.

Investigating the Anticancer Potential of Zinc and Magnesium Alloys: From Base Materials to Nanocoated Titanium Implants.

In this work, we show the in vitro anticancer potential of surgical wires, obtained from zinc (ZnMg0.004) or magnesium (MgCa0.7) alloys by spatial technology comprising casting, extrusion, and final drawing processes. We also present the selective anticancer effects of applied soluble multilayer nanocoatings of zinc and magnesium onto titanium surfaces using the pulse laser deposition method. In the latter, the titanium samples were produced via 3D printing using the selective laser melting method and coated with various combinations of zinc and magnesium layers. For cytotoxicity studies, human dental pulp-derived stem cells (hDPSCs) and human osteosarcoma SaOS-2 cell line were used as representatives of healthy and cancer cells. Cells were examined against the 0.3-3.0 cm2/mL material extract ratios obtained from experimental and steel surgical wires, the latter being the current clinical industry standard. The MgCa0.7 alloy wires were approx. 1.5 times more toxic to cancer cells at all examined extract ratios vs. the extracts from steel surgical wires that exhibited comparable toxicity towards healthy and cancer cells. The ZnMg0.004 alloy wires displayed increased toxicity towards cancer cells with decreasing extract ratios. This was also reflected in the increased anticancer effectiveness, calculated based on the viability ratio of healthy cells to cancer cells, from 1.1 to 4.0 times. Healthy cell viability remained at 80-100%, whereas cancer cell survival fluctuated at 20-75%, depending on the extract ratio. Furthermore, the culture of normal or cancer cells on the surface of Zn/Mg-coated titanium allowed us to select combinations of specific coating layers that yielded a comparable anticancer effectiveness to that observed with the experimental wires that ranged between 2 and 3. Overall, this work not only demonstrates the substantial anticancer properties of the studied wires but also indicates that similar anticancer effects can be replicated with appropriate nanocoatings on titanium samples. We believe that this work lays the groundwork for the future potential development of the category of new implants endowed with anticancer properties.

Analysis of biologically-derived small particles--searching for geometry correction factors using Monte Carlo simulation.

A Monte Carlo simulation was used to determine geometry correction factors that increase accuracy of quantitative X-ray microanalysis of laterally semithick biological materials. A model composed of cellulose with homogeneously distributed biological elements and lateral dimensions between 0.5-25 µm was chosen. The specimen was exposed to 5, 10, and 15 keV electrons, the net intensities of characteristic X-rays registered for the elements, and presented as a function of the lateral dimensions of the model. This showed the double decay exponential function fitted the distribution of X-ray intensities in relation to the model size. The applicability of the function as a correction method was successfully tested for 30 specimens with varying composition and dimensions. The value of relative error decreased from ±60% to ±5% when the correction was applied. Moreover, the minimal lateral size of the material was defined, below which the correction is not required. The simulation also revealed that the difference of the weighted sum of Z²/A between the unknown and the standard could reach 25% without significant influence on the quantitative results. The correction method could be helpful for accurate assessment of elemental composition in biological or organic matrices, when their lateral dimensions are smaller than the distribution range.

The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster.

Accumulating evidence from mammalian studies suggests the dual-faced character of heme oxygenase (HO) in oxidative stress-dependent neurodegeneration. The present study aimed to investigate both neuroprotective and neurotoxic effects of heme oxygenase after the ho gene chronic overexpression or silencing in neurons of Drosophila melanogaster. Our results showed early deaths and behavioral defects after pan-neuronal ho overexpression, while survival and climbing in a strain with pan-neuronal ho silencing were similar over time with its parental controls. We also found that HO can be pro-apoptotic or anti-apoptotic under different conditions. In young (7-day-old) flies, both the cell death activator gene (hid) expression and the initiator caspase Dronc activity increased in heads of flies when ho expression was changed. In addition, various expression levels of ho produced cell-specific degeneration. Dopaminergic (DA) neurons and retina photoreceptors are particularly vulnerable to changes in ho expression. In older (30-day-old) flies, we did not detect any further increase in hid expression or enhanced degeneration, however, we still observed high activity of the initiator caspase. In addition, we used curcumin to further show the involvement of neuronal HO in the regulation of apoptosis. Under normal conditions, curcumin induced both the expression of ho and hid, which was reversed after exposure to high-temperature stress and when supplemented in flies with ho silencing. These results indicate that neuronal HO regulates apoptosis and this process depends on ho expression level, age of flies, and cell type.

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster.

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.

Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843).

The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak's erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 µm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 µm(2), respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes.

Early-life Stress Modifies the Reactivity of Neurons in the Ventral Tegmental Area and Lateral Hypothalamus to Acute Stress in Female Rats.

Early-life stress (ELS) has long-term consequences, including an increased risk for drug abuse and psychiatric disorders later in life, which is higher in women than in men. The consequences of ELS include heightened sensitivity to stressful events. Here, we hypothesized that ELS changes the stress sensitivity of dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) and orexin (OXA) neurons in the lateral hypothalamus (LH), that are crucial for the control of motivated behaviors. We combined morphological and immunohistochemical approaches to investigate the effect of maternal separation (MS), a rodent model of the ELS, on the expression of c-Fos protein in putative DAergic and non-DAergic VTA and LH OXA neurons, as well as on dendritic spine density and morphology in the VTA of adult female rats. We showed that MS increased basal and acute restraint stress-induced c-Fos expression in putative DAergic neurons, specifically in the dorsomedial VTA, an area implicated in responsiveness to aversive stimuli. Conversely, restraint-induced increase in c-Fos expression in non-DAergic dorsolateral VTA neurons was dampened by MS. Furthermore, an increase in spine head diameter of VTA neurons and a concurrent decrease in dendritic spine density in dorsal VTA were observed. We also showed that MS changed the stress sensitivity of OXA neurons selectively in the dorsomedial hypothalamus (DMH), which is implicated in arousal and the stress response. These findings show the long-lasting consequences of ELS and indicate the selective, regional sensitivity of structures involved in the control of arousal, motivational behaviors and the stress response to ELS.

A Biological Study of Composites Based on the Blends of Nanohydroxyapatite, Silk Fibroin and Chitosan.

In this work, the biological properties of three-dimensional scaffolds based on a blend of nanohydroxyapatite (nHA), silk fibroin (SF), and chitosan (CTS), were prepared using a lyophilization technique with various weight ratios: 10:45:45, 15:15:70, 15:70:15, 20:40:40, 40:30:30, and 70:15:15 nHA:SF:CTS, respectively. The basic 3D scaffolds were obtained from 5% (w/w) chitosan and 5% silk fibroin solutions and then nHA was added. The morphology and physicochemical properties of scaffolds were studied and compared. A biological test was performed to study the growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). It was found that the addition of chitosan increases the resistance properties and extends the degradation time of materials. In vitro studies with human mesenchymal stem cells found a high degree of biotolerance for the materials produced, especially for the 20:40:40 and 15:70:15 (nHa:SF:CTS) ratios. The presence of silk fibroin and the elongated shape of the pores positively influenced the differentiation of cells into osteogenic cells. By taking advantage of the differentiation/proliferation cues offered by individual components, the composites based on the nanohydroxyapatite, silk fibroin, and chitosan scaffold may be suitable for bone tissue engineering, and possibly offer an alternative to the widespread use of collagen materials.

Surface Modification of PHBV Fibrous Scaffold via Lithium Borohydride Reduction.

In this study, lithium borohydride (LiBH4) reduction was used to modify the surface chemistry of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) fibers. Although the most common reaction employed in the surface treatment of polyester materials is hydrolysis, it is not suitable for fiber modification of bacterial polyesters, which are highly resistant to this type of reaction. The use of LiBH4 allowed the formation of surface hydroxyl groups under very mild conditions, which was crucial for maintaining the fibers' integrity. The presence of these groups resulted in a noticeable improvement in the surface hydrophilicity of PHBV, as revealed by contact angle measurements. After the treatment with a LiBH4 solution, the electrospun PHBV fibrous mat had a significantly greater number of viable osteoblast-like cells (SaOS-2 cell line) than the untreated mat. Moreover, the results of the cell proliferation measurements correlated well with the observed cell morphology. The most flattened SaOS-2 cells were found on the surface that supported the best cell attachment. Most importantly, the results of our study indicated that the degree of surface modification could be controlled by changing the degradation time and concentration of the borohydride solution. This was of great importance since it allowed optimization of the surface properties to achieve the highest cell-proliferation capacity.

Beneficial effect of cigarette smoking cessation on fibrin clot properties.

To examine the associations between cigarette smoking and preferable clot properties. Plasma fibrin clots from 21 randomly selected current smokers (n = 7), former smokers (n = 7) and non-smokers (n = 7) were analyzed, using scanning electron microscopy (SEM). With the use of the turbidimetric clotting and lysis assay in plasma, the maximum absorbance (MaxAbs(C), MaxAbs(L)) was measured and lysis time (Lys(50%)) was calculated. Smoking cessation significantly influenced fibrin fiber branching and density. Median fiber diameter was not changed. Lys(50%) was the highest in current smokers and was reduced in former smokers to the non-smoker level (2120 ± 385 versus 1771 ± 122 and 1724 ± 272 s; P = 0.04). Smoking cessation improves fibrin clot architecture which results in the lesser resistance to lysis.

Gellan gum hydrogels cross-linked with carbodiimide stimulates vacuolation of human tooth-derived stem cells in vitro.

The natural polysaccharides are promising compounds for applications in regenerative medicine. Gellan gum (GG) is the bacteria-derived polysaccharide widely used in food industry. Simple modifications of its chemical properties make GG superior for the development of biocompatible hydrogels. Beside reversible cationic integration of GG chains, more efficient binding is accomplished with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). However, the side-products of polymer cross-linking might affect viability and differentiation of stem cells introduced into the hydrogels. We found that O-acylisourea (EDU) stimulates autophagy-based vacuolation in both periodontal ligament and dental pulp stem cells. 24-h treatment of cells with GG extracts cross-linked with 15 mM EDC developed large cytoplasmic vacuoles. Freshly prepared EDU (2-6 mM) but not 15 mM EDC solutions initiated vacuole development with concomitant reduction of cell viability/metabolism. Most of the vacuoles stained with acridine orange displayed highly acidic environment further confirmed by flow cytometric analysis. Western blot of the LC3 autophagy marker followed by a transmission electron microscopy indicated the process is autophagy-dependent. We propose that the high reactivity of EDU with intracellular components initiates autophagy, although the targets of EDU remain unknown. Nevertheless, a burst release of EDU from GG hydrogels might modulate negatively cellular processes and final effectiveness of tissue regeneration.

Early life stress-induced alterations in the activity and morphology of ventral tegmental area neurons in female rats.

Childhood maltreatment, which can take the form of physical or psychological abuse, is experienced by more than a quarter of all children. Early life stress has substantial and long-term consequences, including an increased risk of drug abuse and psychiatric disorders in adolescence and adulthood, and this risk is higher in women than in men. The neuronal mechanisms underlying the influence of early life adversities on brain functioning remain poorly understood; therefore, in the current study, we used maternal separation (MS), a rodent model of early-life neglect, to verify its influence on the properties of neurons in the ventral tegmental area (VTA), a brain area critically involved in reward and motivation processing. Using whole-cell patch-clamp recordings in brain slices from adolescent female Sprague-Dawley rats, we found an MS-induced increase in the excitability of putative dopaminergic (DAergic) neurons selectively in the medial part of the VTA. We also showed an enhancement of excitatory synaptic transmission in VTA putative DAergic neurons. MS-induced alterations in electrophysiology were accompanied by an increase in the diameter of dendritic spine heads on lateral VTA DAergic neurons, although the overall dendritic spine density remained unchanged. Finally, we reported MS-related increases in basal plasma ACTH and corticosterone levels. These results show the long-term consequences of early life stress and indicate the possible neuronal mechanisms of behavioral disturbances in individuals who experience early life neglect.

Impact of long-lasting spontaneous physical activity on bone morphogenetic protein 4 in the heart and tibia in murine model of heart failure.

Bone morphogenetic protein 4 (BMP4) plays an important role in bone remodeling and in heart failure pathogenesis. The aim of this study was to evaluate the effect of spontaneous physical activity on the expression of BMP4 in the heart and tibia of the transgenic (Tgαq*44) mice, representing a model of chronic heart failure. Tgαq*44 and wild-type FVB mice (WT) were randomly assigned either to sedentary or to trained groups undergoing 8 weeks of spontaneous wheel running. The BMP4 protein expression in heart and tibiae was evaluated using Western immunoblotting and the phosphorus and calcium in the tibiae was assessed using the X-ray microanalysis. BMP4 content in the hearts of the Tgαq*44-sedentary mice was by ~490% higher than in the WT-sedentary mice, whereas in tibiae the BMP4 content of the Tgαq*44-sedentary mice was similar to that in the WT-sedentary animals. Tgαq*44 mice revealed by ~28% poorer spontaneous physical activity than the WT mice. No effect of performed physical activity on the BMP4 content in the hearts of either in the Tgαq*44 or WT mice was observed. However, 8-week spontaneous wheel running resulted in a decrease in the BMP4 expression in tibiae (by ~43%) in the group of Tgαq*44 mice only, with no changes in their bone phosphorus and calcium contents. We have concluded that prolonged period of spontaneous physical exercise does not increase the risk of the progression of the BMP4-mediated pathological cardiac hypertrophy and does not affect bone mineral status in the chronic heart failure mice.

Symbiotic microbes of Saxifraga stellaris ssp. alpigena from the copper creek of Schwarzwand (Austrian Alps) enhance plant tolerance to copper.

Saxifraga stellaris var. alpigena grows as one of the very few higher plants in the copper rich area of the "blue creek" in Austria. Two endophytes were isolated from it: Mortierella sp. (fungus), and Stenotrophomonas maltophilia (bacterium). Both microbes were practically inseparable due to resistance of the bacteria to the antibiotics tested. On PDA media, the fungus showed higher tolerance to copper than the bacterium, which disappeared from both the media and the surface of the fungus in the presence of 150 µM of Cu. However, at this Cu concentration, the bacteria were still detectable inside the mycelium and reappeared on the outside when transferred to media of lower Cu concentration. Microscopic studies of in vitro cultivated plants showed that the fungus was present in both, the roots and shoots of the plant. The effects of endophytes on plant performance were assessed in rhizoboxes filled with Cu-rich substratum; plants inoculated with both microbes showed better growth, survival and photosynthesis performance than the non-inoculated controls. The results of this study prove the beneficial influence of the isolated endophytes on the Cu tolerance of S. stellaris, and indicate the ecological potential of applying microbial consortia to plants under extreme environmental conditions.