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Introduction: The prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level. Methods: We studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE). Results: TG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes. Conclusion: Here we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems.

Autoantibodies targeting the lung tissue were identified in severe COVID-19 patients in this retrospective study. Fifty-three percent of 104 patients developed anti-pulmonary antibodies, the majority of which were IgM class, suggesting that they developed upon infection with SARS-CoV-2. Anti-pulmonary antibodies correlated with worse pulmonary function and a higher risk of multiorgan failure that was further aggravated if 3 or more autoantibody clones were simultaneously present (multi-producers). Multi-producer patients were older than the patients with less or no autoantibodies. One of the identified autoantibodies (targeting a pulmonary protein of ~ 50 kDa) associated with worse clinical outcomes, including mortality. In summary, severe COVID-19 is associated with the development of lung-specific autoantibodies, which may worsen the clinical outcome. Tissue proteome-wide tests, such as the ones applied here, can be used to detect autoimmunity in the post-COVID state to identify the cause of symptoms and to reveal a new target for treatment.

Age represents a major risk factor in heart failure (HF). However, the mechanisms linking ageing and HF are not clear. We aimed to identify the functional, morphological and transcriptomic changes that could be attributed to cardiac ageing in a model of slowly progressing HF in Tgαq*44 mice in reference to the cardiac ageing process in FVB mice. In FVB mice, ageing resulted in the impairment of diastolic cardiac function and in basal coronary flow (CF), perivascular and interstitial fibrosis without changes in the cardiac activity of angiotensin-converting enzyme (ACE) or aldosterone plasma concentration. In Tgαq*44 mice, HF progression was featured by the impairment of systolic and diastolic cardiac function and in basal CF that was associated with a distinct rearrangement of the capillary architecture, pronounced perivascular and interstitial fibrosis, progressive activation of cardiac ACE and systemic angiotensin-aldosterone-dependent pathways. Interestingly, cardiac ageing genes and processes were represented in Tgαq*44 mice not only in late but also in early phases of HF, as evidenced by cardiac transcriptome analysis. Thirty-four genes and 8 biological processes, identified as being ageing related, occurred early and persisted along HF progression in Tgαq*44 mice and were mostly associated with extracellular matrix remodelling and fibrosis compatible with perivascular fibrosis resulting in coronary microvascular dysfunction (CMD) in Tgαq*44 mice. In conclusion, accelerated and persistent cardiac ageing contributes to the pathophysiology of chronic HF in Tgαq*44 mice. In particular, prominent perivascular fibrosis of microcirculation resulting in CMD represents an accelerated cardiac ageing phenotype that requires targeted treatment in chronic HF.

Artificial intelligence cardiac MRI measurements were validated and used to assess future patient mortality.

Severe cases of COVID-19 are characterized by an inflammatory burst, which is accompanied by multiorgan failure. The elderly population has higher risk for severe or fatal outcome for COVID-19. Inflammatory mediators facilitate the immune system to combat viral infection by producing antibodies against viral antigens. Several studies reported that the pro-inflammatory state and tissue damage in COVID-19 also promotes autoimmunity by autoantibody generation. We hypothesized that a subset of these autoantibodies targets cardiac antigens. Here we aimed to detect anti-cardiac autoantibodies in severe COVID-19 patients during hospitalization. For this purpose, 104 COVID-19 patients were recruited, while 40 heart failure patients with dilated cardiomyopathy and 20 patients with severe aortic stenosis served as controls. Patients were tested for anti-cardiac autoantibodies, using human heart homogenate as a bait. Follow-up samples were available in 29 COVID-19 patients. Anti-cardiac autoantibodies were detected in 68% (71 out of 104) of severe COVID-19 patients. Overall, 39% of COVID-19 patients had anti-cardiac IgG autoantibodies, while 51% had anti-cardiac autoantibodies of IgM isotype. Both IgG and IgM anti-cardiac autoantibodies were observed in 22% of cases, and multiple cardiac antigens were targeted in 38% of COVID-19 patients. These anti-cardiac autoantibodies targeted a diverse set of myocardial proteins, without apparent selectivity. As controls, heart failure patients (with dilated cardiomyopathy) had similar occurrence of IgG (45%, p= 0.57) autoantibodies, while significantly lower occurrence of IgM autoantibodies (30%, p= 0.03). Patients with advanced aortic stenosis had significantly lower number of both IgG (11%, p= 0.03) and IgM (10%, p< 0.01) type anti-cardiac autoantibodies than that in COVID-19 patients. Furthermore, we detected changes in the anti-cardiac autoantibody profile in 7 COVID-19 patients during hospital treatment. Surprisingly, the presence of these anti-cardiac autoantibodies did not affect the clinical outcome and the prevalence of the autoantibodies did not differ between the elderly (over 65 years) and the patients younger than 65 years of age. Our results demonstrate that the majority of hospitalized COVID-19 patients produce novel anti-cardiac IgM autoantibodies. COVID-19 also reactivates resident IgG autoantibodies. These autoantibodies may promote autoimmune reactions, which can complicate post-COVID recuperation, contributing to post-acute sequelae of COVID-19 (long COVID).

The case presented shows the clinical signs, diagnosis and surgical management of idiopathic bile duct cysts in a bearded dragon (Pogona vitticeps). A 12-year-old female bearded dragon presented with constipation resulting from the consumption of sand. A soft mass about 4 cm wide could be palpated in the mid-coelom after the substrate passed. The animal started eating, but soon started showing signs of kyphosis. Ultrasound, radiography and computed tomography examinations revealed that the mass was a cyst 4 cm in diameter. Diagnostic laparotomy was performed and the large cyst and affected liver tissue were removed with a partial hepatectomy. Cytology of the cyst was unremarkable, but histopathological examination showed the lesions to be bile duct cysts. No sign of malignancy or inflammation could be seen.

Angiotensin-converting enzyme 2 (ACE2) is essential for SARS-CoV-2 cellular entry. Here we studied the effects of common comorbidities in severe COVID-19 on ACE2 expression. ACE2 levels (by enzyme activity and ELISA measurements) were determined in human serum, heart and lung samples from patients with hypertension (n= 540), heart transplantation (289) and thoracic surgery (n= 49). Healthy individuals (n= 46) represented the controls. Serum ACE2 activity was increased in hypertensive subjects (132%) and substantially elevated in end-stage heart failure patients (689%) and showed a strong negative correlation with the left ventricular ejection fraction. Serum ACE2 activity was higher in male (147%), overweight (122%), obese (126%) and elderly (115%) hypertensive patients. Primary lung cancer resulted in higher circulating ACE2 activity, without affecting ACE2 levels in the surrounding lung tissue. Male sex resulted in elevated serum ACE2 activities in patients with heart transplantation or thoracic surgery (146% and 150%, respectively). Left ventricular (tissular) ACE2 activity was unaffected by sex and was lower in overweight (67%), obese (62%) and older (73%) patients with end-stage heart failure. There was no correlation between serum and tissular (left ventricular or lung) ACE2 activities. Neither serum nor tissue (left ventricle or lung) ACE2 levels were affected by RAS inhibitory medications. Abandoning of ACEi treatment (non-compliance) resulted in elevated blood pressure without effects on circulating ACE2 activities. ACE2 levels associate with the severity of cardiovascular diseases, suggestive for a role of ACE2 in the pathomechanisms of cardiovascular diseases and providing a potential explanation for the higher mortality of COVID-19 among cardiovascular patients. Abandoning RAS inhibitory medication worsens the cardiovascular status without affecting circulating or tissue ACE2 levels.

The environment of a city is influenced by global and local climate changes, pollution load from transport, industry and local heat sources. Green spaces as part of the urban green infrastructure fulfil multiple ecosystem services and improve the environmental and residential quality of a city. The level of positive effects of green spaces depends on their area, distribution within the city and the proportion of trees. The aim of the paper was to evaluate land cover structure in a selected segment of the housing estate Klokočina in Nitra, Slovakia. The evaluation focused on the share of biologically active and inactive surfaces, as well as the parameters of woody plant structure in green spaces. Green areas account for 58.70%, while built-up areas, parking lots and roads cover in total 41.30%. Biologically inactive anthropogenic horizontal and of building envelope areas represent 67.30%. Biologically active green areas represent 32.70%. The share of areas with tree crown overlap on horizontal areas is 20.82%, other areas are paved surfaces or grassland. We calculated the indices of the quality of green spaces from the individual categories of areas: proportional green area index (PGAI), effective green area index (EGAI) and tree shade index (TSI). The tree species composition consists of 36 deciduous and 11 coniferous tree species, and 20 shrub species. We evaluated the biometric traits of trees as the tree height, crown width, stem girth and tree vitality class (TVC). The correlations between tree traits were statistically tested with a significant result. In the group of biologically active areas of greenery, we propose to increase the proportion of trees, to establish more natural lawns and xerophyte flower beds. In the category of inactive areas, we propose the reconstruction of parking lots to grass paved surfaces.

ObjectiveWe assessed the diagnostic and prognostic implications of early cardiac magnetic resonance (CMR), CMR-based deformation imaging and conventional risk factors in patients with troponin-positive acute chest pain and non-obstructed coronary arteries.MethodsIn total, 255 patients presenting between 2009 and 2019 with troponin-positive acute chest pain and non-obstructed coronary arteries who underwent CMR in ≤7 days were followed for a clinical endpoint of all-cause mortality. Cine movies, T2-weighted and late gadolinium-enhanced images were evaluated to establish a diagnosis of the underlying heart disease. Further CMR analysis, including left ventricular strain, was carried out.ResultsCMR (performed at a mean of 2.7 days) provided the diagnosis in 86% of patients (54% myocarditis, 22% myocardial infarction (MI) and 10% Takotsubo syndrome and myocardial contusion (n=1)). The 4-year mortality for a diagnosis of MI, myocarditis, Takotsubo and normal CMR patients was 10.2%, 1.6%, 27.3% and 0%, respectively. We found a strong association between CMR diagnosis and mortality (log-rank: 24, p<0.0001). Takotsubo and MI as the diagnosis, age, hypertension, diabetes, female sex, ejection fraction, stroke volume index and most of the investigated strain parameters were univariate predictors of mortality; however, in the multivariate analysis, only hypertension and circumferential mechanical dispersion measured by strain analysis were independent predictors of mortality.ConclusionsCMR performed in the early phase establishes the proper diagnosis in patients with troponin-positive acute chest pain and non-obstructed coronary arteries and provides additional prognostic factors. This may indicate that CMR could play an additional role in risk stratification in this patient population.

Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) plays an important role in the maintenance of healthy cognitive function. Strong evidence demonstrates that age-related cerebromicrovascular endothelial dysfunction and consequential impairment of NVC responses contribute importantly to cognitive decline. Recent studies demonstrate that NAD+availability decreases with age in the vasculature and that supplemental NAD+precursors can ameliorate cerebrovascular dysfunction, rescuing NVC responses and improving cognitive performance in aged mice. The mechanisms underlying the age-related decline in [NAD+] in cells of the neurovascular unit are likely multifaceted and may include increased utilization of NAD+by activated poly (ADP-ribose) polymerase (PARP-1). The present study was designed to test the hypothesis that inhibition of PARP-1 activity may confer protective effects on neurovascular function in aging, similar to the recently demonstrated protective effects of treatment with the NAD+ precursor nicotinamide mononucleotide (NMN). To test this hypothesis, 24-month-old C57BL/6 mice were treated with PJ-34, a potent PARP inhibitor, for 2 weeks. NVC was assessed by measuring CBF responses (laser speckle contrast imaging) in the somatosensory whisker barrel cortex evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. Treatment with PJ-34 improved NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory. PJ-34 treatment also improved endothelium-dependent acetylcholine-induced relaxation of aorta rings. Thus, PARP-1 activation, likely by decreasing NAD+availability, contributes to age-related endothelial dysfunction and neurovascular uncoupling, exacerbating cognitive decline. The cerebromicrovascular protective effects of pharmacological inhibition of PARP-1 highlight the preventive and therapeutic potential of treatments that restore NAD+ homeostasis as effective interventions in patients at risk for vascular cognitive impairment (VCI).

ABSTRACTA popular gantry-type placement machine includes several interconnected, autonomously operating component placement modules and the machine was designed so as to able to use different kinds of placement heads and vacuum nozzles in the modules, which can be easily changed. Although this increases the flexibility of the production line, the reconfiguration phases of the modules may be unproductive and one should keep them to a minimum. In addition, the production times can be shortened by balancing the workloads of the machine modules. Here, a two-step optimisation method for the machine reconfiguration and workload balancing in the case of multiple Printed Circuit Borad (PCB) batches of different sizes and PCB types is presented. The objective is to minimise the total production time, and keep the machine configuration the same for all batches. The proposed algorithm is iterative and it applies integer programming for the workload balancing along with an evolutionary algorithm that searches for the best machine configuration. In experiments, for single PCB types the proposed algorithm obtained optimal or near optimal solutions. For multiple PCB types the solutions favour the PCB types that have a bigger production time due to greater batch sizes, but the total production time is still close to optimal.

The presence of the “mistletoe sign” on cardiac MR and coronary CT angiographic images is probably rare but might be a characteristic manifestation of retroperitoneal fibrosis.

Using ‘Urban Atlas’ as a data source, the authors present and critically discuss in this paper the application of figure-ground plans in combination with complex land-use maps as a tool for spatial analysis of urban agriculture in European cities and their multifunctional green infrastructure. The selected cities and metropolitan areas (including Dublin, Ruhr Metropolis, Geneva and Sofia) represent different regions in Europe from the Northwest to the Southeast. Urban fabric, agriculture and non-agricultural open spaces have been analysed and compared as the main land-use components. Agricultural open spaces include arable land with annual crops and permanent crops, such as vineyards, fruit trees and olive groves; pastures; as well as complex and mixed cultivation patterns. The results reveal the scale and land-use diversity of metropolitan regions and different spatial patterns of urban agriculture at the regional level and in central urban areas.

Case hardened, double-walled train wheel, produced in the foundry of Abraham Ganz in 1867 and used for long time on the railroad tracks, was studied and evaluated by modern methods of materials sciences. The investigations indicated the presence of 0.059m% antimony (Sb), distributed uniformly in the material of the wheel. This quantity is several times higher than the average Sb amounts (&lt;0.01m%) of the other components get into the castings. Sb was detected even in the several mm thick crust; except in the outermost surface layer with &lt; 1 µm thickness. Based on the 20th century comprehensive studies, the antimony content, detected in the wheel, was found to be ideal. In grey iron it is a powerful pearlite stabilizer which has favourable effects on its mechanical properties and serviceability.The investigation of the surface structure proved that the excellent hardness (~600 HV) at the crust surface was due to the “nano-composite” structure formed from the pearlite as a consequence of severe plastic deformation during service. This was also promoted by the high amount (4.09m%) of carbon present in the casting. The microstructure refinement during operation most probably contributes to the long service lifetime of the wheels.The secret of the Abraham Ganz’s train wheels is inherently present – besides the novel construction and production technology (case hardening) – in the above mentioned composition and the structure of the material.

DNA double-strand breaks (DSBs) are induced by SPO11 during meiosis to initiate recombination-mediated pairing and synapsis of homologous chromosomes. Germline genome integrity requires spatiotemporal control of DSB formation, which involves the proteinaceous chromosome axis along the core of each meiotic chromosome. In particular, a component of unsynapsed axes, HORMAD1, promotes DSB formation in unsynapsed regions where DSB formation must occur to ensure completion of synapsis. Despite its importance, the underlying mechanism has remained elusive. We identify CCDC36 as a direct interactor of HORMAD1 (IHO1) that is essential for DSB formation. Underpinning this function, IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes that are predicted activators of DSB formation. HORMAD1 is needed for robust recruitment of IHO1 to unsynapsed axes and efficient formation and/or stabilization of these recombinosomes. Thus, we propose that HORMAD1–IHO1 interaction provides a mechanism for the selective promotion of DSB formation along unsynapsed chromosome axes.

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.

Basal forebrain (BF) plays an important role in the regulation of cortical activation. Somatostatin (SOM) is present both in local neurons as well as in fibers in the BF. In previous studies, SOM axons were found to innervate corticopetal cholinergic cells and SOM was found to presynaptically modulate GABA and glutamate release onto cholinergic neurons in the BF. However, no systematic analysis is available about the EEG effects of SOM or its analog, octreotide (OCTR) injected directly into the BF. In the present experiments, EEG changes were examined following an OCTR injection (0.5 microliter, 500 nmol) into the BF areas containing several choline acetyl transferase-immunoreactive neurons of urethane-anaesthetized rats. Fronto-occipital EEG was recorded on both sides and relative EEG power was calculated in the delta (0–3 Hz), theta (3–9 Hz), alpha (9–16 Hz) and beta (16–48 Hz) frequency bands. OCTR injected to the BF failed to induce significant EEG changes and did not affect tail pinch-evoked cortical activation. Lack of effect may be attributed to the urethane anaesthesia as well as to the possible complex interactions between SOM and BF cholinergic and GABAergic neurons.

Aims:The goal of this study was to investigate the importance of the vascular angiotensin convertase enzyme (ACE) in coronary artery bypass graft surgery (CABG) patients. Methods:Vascular tissue (distal saphenous vein [n= 163] and/or radial artery [n= 120] segments) and blood samples were collected from CABG patients (n= 81). We studied (i) the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contractions; (ii) vascular and plasma ACE concentrations; and (iii) ACE genotype of the patients enrolled. Results:The ratio of the potencies (EC50) of AngII and AngI was significantly lower in radial artery compared to the saphenous vein (0.17 ± 0.03 nM and 0.51 ± 0.14 nM, respectively, P= 0.003), suggesting a 3‐fold more effective AngI conversion in saphenous vein samples. Angiotensin constrictions were inhibited with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous vein compared to radial artery (9.7 ± 1.0 ng/mg and 5.3 ± 0.7 ng/mg, respectively, P= 0.01). Serum but no tissue ACE concentration was determined by ACE insertion/deletion polymorphism. Accordingly, no relation was found between serum and tissue ACE expression. Conclusion:ACE‐inhibitor therapy targeting tissue located ACE may be beneficial to patients with saphenous vein grafts after CABG surgery.

The effect of the boron in steels is widely known as an element enhancing hardenability, but its role can also be related to the enamelability of steels. While the improvement of the hardenability is due to the solute boron, in the case of enameling grade steels the boron should be kept in form of precipitates 1. The hydrogen accumulated during processing and enameling diffuses to the metal/enamel interface and lead to the formation of the so called fish-scale failure. To prevent this it is needed an adequate type, number and dimension of precipitates which act as hydrogen traps hinder the free diffusion of the hydrogen in steel. The paper deals with the possibilities of investigation by scanning electron microscopy of the boron containing precipitates in the low carbon Al-killed steels apt for two side enamelling.

Hydroxyapatite (HAP) is the main inorganic component of mammal bone and teeth. It is one of the few bioactive materials, which helps cell growing. The objective of this study was to fabricate hydroxyapatite-biopolimer nanofiber mats. To achieve this goal, two different suspensions were used. Approximately 1 μm diameter size fibers with large HAP agglomerates were fabricated using acetone and acetic acid. Fibers with a larger diameter size and homogenous distributed HAP particles were produced applying acetone and isopropanol. During the experiment the effect of the processing parameters: applied voltage, diameter of needle, the distance of the needle tip and the collector, flow rate was analyzed.

The excellent gas sensing properties of the tungsten oxides have been manifested first of all in nanostructure and 1D, and 2D open structured forms. For optimal performance the sensing layer substrates should be of large specific surface. In this paper we report on electrospinning – a candidate for fabrication of large specific surface tungsten oxide nanofibers. Fibrous tissues doped with tungstic acid hydrate (H2WO4.H2O) and tungsten oxide one third hydrate (WO3.1/3H2O) has been created and characterized by X-ray diffraction, scanning electron microscope and energy dispersive spectroscopy in order to learn about the changes the materials suffer during the process.

Hydroxyapatite (HAp) was successfully produced from recycled eggshell, seashell and phosphoric acid by using two different type of milling method (attrition milling and ball milling). According to the analysis, the attrition milling resulted nanosize HAp even after milling, while the ball milling process provided HAp only after a 400oC, 2 h long heat treatment. The grain size in both cases were approximately preserved during the heat treatment. The effect of temperature on stoichiometry, morphology and crystallinity of HAp powders were investigated. The structures of the HAp were characterized by X-ray diffraction and Scanning Electron Microsopy.

Studies of oblique deposited nanostructured SiOx films by ellipsometric and IR spectroscopies are presented, as additional information is obtained by scanning electron microscopy (SEM). The films were deposited onto Si substrates under a 75° incidence vapor by vacuum evaporation of SiO and were annealed in Ar at 950oC. The thickness and composition of the films were estimated from the ellipsometric data analysis applying the Bruggeman effective approximation theory. Three-layer optical model described satisfactory the annealed film structure and verified the formation of nanocrystalline Si clusters. SEM micrographs showed that evaporated films consisted of silicon oxide pillars separated by air space and tilted at an angle of ~57o to Si substrate surface. The thin silica pillars were most probably free from Si-SiO2 interface leading to absence of strong absorption on LO vibrations in the IR spectrum. The estimated porosity factor was ~62 %. By annealing, the film oxidized to SiO2 but it remained columnar with a porosity factor of ~47 %.

The wetting angle (Θ) of molten Ag (Zn, Cu, Ga) drops on graphite substrate is investigated. A texture formation was found in the substrate/drop interphase region after solidification. The enrichment (segregation) of Ag atoms in the interface layer was observed in the Ag-Cu alloy, and unexpected mixing between graphite substrate and the Ag-Ga melt was also detected in the solidified structure.

The rat's prefrontal cortex plays a role in integration of feeding-related information. In this study, we investigated the neuronal activity changes of medial prefrontal cortex during licking of sucrose solution by freely moving rats. We found two different types of excitatory and two different types of inhibitory single neuron responses time locked to the beginning of the licking clusters. Changes in firing rates occurred either within 2 s before the first lick of the licking cluster or during the licking cluster. These observations suggest that neuronal responses in the medial prefrontal cortex may represent anticipation and consummation of liquid food reward.

Protective, chromate substitute thin layers on roughened galvanized surfaces produced at OCAS (Arcelor, Belgium) were characterized and compared using Scanning Electron Microscopy (SEM+EDS), Atomic Force Microscopy (AFM), Nanoindentation and X-ray Photoemission Spectroscopy (XPS). EDX maps, line scans and point analyses obtained at various places of the surfaces have shown differences between the CVD and silane nanolayers in the matter of thickness distribution and composition. At cross-section specimens the thickness of the layers could be shown. The hardness differences caused by layer thickness variations are hard to follow by nanoindentation as the penetration depth of the indenter is much larger than the thickness of the coatings. XPS measurements can distinguish between the chemical states of silicon in CVD and silane coatings.

Hexagonal (h-) WO3 was prepared through heating hexagonal ammonium tungsten bronze (HATB), (NH4)0.07(NH3)0.04(H2O)0.09WO2.95. By adjusting the heating temperature and atmosphere of HATB, we could control the oxidation state of tungsten atoms and the residual NH3/NH4 + content in h-WO3. The as-produced h-WO3 nanoparticles with different composition were tested as gas sensors and the effect of composition on gas sensing properties was studied. Our results showed that oxidized h-WO3 had the best sensitivity to H2S.

The transient receptor potential type V1 channel (vanilloid receptor 1, TRPV1) is a Ca(2+)-permeable nonspecific cation channel activated by various painful stimuli including ischemia. We hypothesized that TRPV1 is expressed in the arterioles and is involved in the regulation of microvascular tone. We found that TRPV1 stimulation by capsaicin (intra-arterial administration) of the isolated, perfused right hind limb of the rat increased vascular resistance (by 98 +/- 21 mm Hg at 10 mug) in association with decreased skeletal muscle perfusion and elevation of skin perfusion (detected by dual-channel laser Doppler flowmetry). Denervation of the hind limb did not affect capsaicin-evoked changes in vascular resistance and tissue perfusion in the hind limb but reduced the elevation of perfusion in the skin. In isolated, pressurized skeletal (musculus gracilis) muscle arterioles (diameter, 147 +/- 35 mum), capsaicin had biphasic effects: at lower concentrations, capsaicin (up to 10 nM) evoked dilations (maximum, 32 +/- 13%), whereas higher concentrations (0.1-1 muM) elicited substantial constrictions (maximum, 66 +/- 7%). Endothelium removal or inhibition of nitric-oxide synthase abolished capsaicin-induced dilations but did not affect arteriolar constriction. Expression of TRPV1 was detected by reverse transcriptase-polymerase chain reaction in the aorta and in cultured rat aortic vascular smooth muscle cells (A7r5). Immunohistochemistry revealed expression primarily in the smooth muscle layers of the gracilis arteriole. These data demonstrate the functional expression of TRPV1 in vascular smooth muscle cells mediating vasoconstriction of the resistance arteries. Because of the dual effects of TRPV1 stimulation on the arteriolar diameter (dilation in skin, constriction in skeletal muscle), we propose that TRPV1 ligands represent drug candidates for tissue-specific modulation of blood distribution.

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single‐strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin‐ and dye labeled reaction products are immobilized on microbeads and the homo‐ and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero‐ from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single‐strand discontinuities can also be detected, by combining nick‐translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single‐strand lesions formed at hypersensitive sites in response to apoptotic stimuli. © 2008 International Society for Analytical Cytology

Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t1/2 of 30 ± 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (Ki = 6400 ± 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodo-resiniferatoxin (5-iodo-RTX) displayed a Ki of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.

Abstract Calpain-1 is a ubiquitous intracellular Ca2+-activated protease, which has been implicated in the pathogenesis of reversible myocardial depression (i.e. myocardial stunning) that follows ischemia and reperfusion via myofibrillar protein degradation. However, the target proteins of this degradative process in the human myocardium have not yet been identified. In order to compare the levels of Calpain-1 susceptibility within a set of human myofibrillar proteins (titin, α-fodrin, desmin, troponin T (cTnT), troponin I (cTnI) and α-actinin), crude left ventricular tissue homogenates were incubated for 0.5, 15, 30, 60 or 120 min in the presence of Calpain-1 (1 U or 5 U). Differences in the kinetics and extents of protein degradation were subsequently evaluated by using silver-stained SDS-polyacrylamide gels and Western immunoblot analyses. These assays revealed myofibrillar proteins with high (titin and α-fodrin), moderate (desmin and cTnT), or low (cTnI and α-actinin) relative Calpain-1 susceptibilities. The level of phosphorylation of cTnI did not explain its relatively low Calpain-1 susceptibility. Moreover, the molecular mass distributions of the truncated α-fodrin, desmin and cTnI fragments resulting from Ca2+-dependent autoproteolysis exhibited marked similarities with those of their Calpain-1-clipped products. These in vitro results shed light on a number of structural (titin, α-fodrin, desmin and α-actinin) and regulatory (cTnT and cTnI) proteins within the contractile apparatus as potential targets of Calpain-1. Their degradation may contribute to the development of postischemic stunning in the human myocardium. (Mol Cell Biochem 278: 1–8, 2005)

The aim of this study was to find if nanocrystal layers obtained by well-established nanotechnology are able to induce deposition of hydroxyapatite [Ca10(PO4)6(OH)2]. It is known that nanosized objects and porous structures influence biological events and they may be used to create biologically integrated multifunctional devices including biomaterials and sensors. In this work, sequential physical vapour deposition of CdSe and SiO, or SiOx film was used to modify glass substrates. To study the ability of the nanostructured surfaces to induce hydroxyapatite deposition, samples were immersed in a simulated body fluid and simultaneously irradiated with a scanning laser beam for a few minutes. This resulted in a porous sponge-like non homogeneous hydroxyapatite layer, consisting of networks of aggregates of nano dimensions on the modified surfaces. Analysis showed higher Ca and P contents in the stripes of the laser-substrate interaction, which indicated the influence of the laser energy. The method of laser-liquid-solid interaction used has led to a synergistic effect due to the simultaneous use of the nanostructured substrate, aqueous solution and laser energy.

The mitotic cell cycle can be described as an alternation between two states. During mitosis, MPF (mitosis promoting factor) is high and keeps inactive its numerous molecular antagonists. In interphase, MPF is inactivated, and the antagonists prevail. The transition between the two states is ensured by 'helper' molecules that favour one state over the other. It has long been assumed that active MPF (a dimer of cyclin B and cyclin-dependent kinase 1) induces exit from mitosis by activating APC:Cdc20, a ubiquitin ligase responsible for cyclin B degradation. The molecular details have not been fully worked out yet, but recent results show that MPF and the ubiquitin ligase are not involved in a simple negative feedback loop. While it is proven that MPF activates APC, new data suggest that MPF inhibits Cdc20, i.e., that MPF and Cdc20 are antagonists. We introduce this new idea into a published model for cell cycle regulation in Xenopus laevis, and study its dynamical behavior. We show that the new wiring permits oscillations with a simpler and smaller network than previously envisaged and that the antagonism between MPF and Cdc20 suggests a new interpretation of the spindle checkpoint.

The mechanism by which mutations of the cardiac troponin I (cTnI) gene evoke familial hypertrophic cardiomyopathy (fHCM) is unknown. In this investigation the potential effects of three fHCM-related cTnI mutations on Calpain-1-induced cTnI degradation were tested, and a study was made of whether additional conformational changes due to troponin complex formation and protein kinase A-induced phosphorylation affect the intensity of cTnI proteolysis. Purified recombinant wild-type cTnI and three of its fHCM-related missense mutants (R145G, G203S and K206Q), alone or in the troponin complex (i.e. together with troponin C and troponin T), in the non-phosphorylated or protein kinase A-bisphosphorylated forms were proteolyzed in vitroin the presence of Calpain-1 (0.05–2.5U) at 30°C. Following incubation with Calpain-1 for 0.5, 30, 60 or 120 min, the extent of protein degradation was evaluated through the use of Western immunoblotting and densitometry. The results indicated that both the wild-type and the mutant cTnI molecules were susceptible to Calpain-1. However, the degradation of the cTnI molecules in the troponin complex was less intense than that of the non-complexed forms. Moreover, phosphorylation by protein kinase A conferred effective protection against cTnI proteolysis. The data suggested that mutations in the central inhibitory domain (R145G) and in the C-terminal region (G203S and K206Q) of cTnI do not affect its Calpain-1-mediated degradation, or the phosphorylation-induced protection against proteolysis.

The effect of phosphorylation in the N‐terminal region of myosin phosphatase target subunit 1 (MYPT1) on the interactions with protein phosphatase 1 catalytic subunit (PP1c) and with phosphorylated 20 kDa myosin light chain (P‐MLC20) was studied. Protein kinase C (PKC) phosphorylated threonine‐34 (1 mol/mol), the residue preceding the consensus PP1c‐binding motif (35KVKF38) in MYPT11–38, but this did not affect binding of the peptide to PP1c. PKC incorporated 2 mol Piinto MYPT11–296suggesting a second site of phosphorylation within the ankyrin repeats (residues 40–296). This phosphorylation diminished the stimulatory effect of MYPT11–296on the P‐MLC20 phosphatase activity of PP1c. Binding of PP1c or P‐MLC20 to phosphorylated MYPT11–296was also attenuated. It is concluded that phosphorylation of MYPT1 by PKC may therefore result in altered dephosphorylation of myosin.

The effect of phosphorylation in the N-terminal region of myosin phosphatase target subunit 1 (MYPT1) on the interactions with protein phosphatase 1 catalytic subunit (PP1c) and with phosphorylated 20 kDa myosin light chain (P-MLC20) was studied. Protein kinase C (PKC) phosphorylated threonine-34 (1 mol/mol), the residue preceding the consensus PP1c-binding motif (35KVKF38) in MYPT11–38, but this did not affect binding of the peptide to PP1c. PKC incorporated 2 mol Piinto MYPT11–296suggesting a second site of phosphorylation within the ankyrin repeats (residues 40–296). This phosphorylation diminished the stimulatory effect of MYPT11–296on the P-MLC20 phosphatase activity of PP1c. Binding of PP1c or P-MLC20 to phosphorylated MYPT11–296was also attenuated. It is concluded that phosphorylation of MYPT1 by PKC may therefore result in altered dephosphorylation of myosin.

The eukaryotic cell division cycle consists of two characteristic states: G1, when replication origins of chromosomes are in a pre-replicative state, and S/G2/M, when they are in a post-replicative state (Nasmyth, 1995). Using straightforward biochemical kinetics, we show that these two states can be created by antagonistic interactions between cyclin-dependent kinases (Cdk) and their foes: the cyclin-degradation machinery (APC) and a stoichiometric inhibitor (CKI). Irreversible transitions between these two self-maintaining steady states drive progress through the cell cycle: at “Start” a cell leaves the G1 state and commences chromosome replication, and at “Finish” the cell separates the products of replication to the incipient daughter cells and re-enters G1. We propose that a protein-phosphatase, by up-regulating the APC and by stabilizing the CKI, plays an essential role at Finish. The phosphatase acts in parallel pathways; hence, cells can leave mitosis in the absence of cyclin degradation or in the absence of the CKI.

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