Improvement of the performances of a commercial hand-held laser-induced breakdown spectroscopy instrument for steel analysis using multiple artificial neural networks.

In this article, we present a study on the optimization of the analytical performance of a commercial hand-held laser-induced breakdown spectroscopy instrument for steel analysis. We show how the performances of the instrument can be substantially improved using a non-linear calibration approach based on a set of Artificial Neural Networks (ANNs), one optimized for the determination of the major elements of the alloy, and the others specialized for the analysis of minor components. Tests of the instrument on steel samples used for instrument internal calibration demonstrate a comparable accuracy with the results of the ANNs, while the latter are considerably more accurate when unknown samples, not used for calibration/training, are tested.

Epstein-Barr Virus-Infected Plasma Cells Infiltrate Erosive Oral Lichen Planus.

Epstein-Barr virus (EBV), in addition to its transforming properties, contributes to the pathogenesis of several inflammatory diseases. Here, we investigated its involvement in oral lichen planus (OLP), a common autoimmune-like disease of unknown etiopathogenesis that can display a malignant potential. EBV-infected cells (EBV+ cells) were sought in a large series of clinically representative OLPs ( n = 99) through in situ hybridization to detect small noncoding EBV-encoded RNAs. Overall, our results demonstrated that EBV was commonly found in OLP (74%), with significantly higher frequency (83%) in the erosive form than in the reticular/keratinized type mild form (58%). Strikingly, many erosive OLPs were massively infiltrated by large numbers of EBV+ cells, which could represent a large part of the inflammatory infiltrate. Moreover, the number of EBV+ cells in each OLP section significantly correlated with local inflammatory parameters (OLP activity, infiltrate depth, infiltrate density), suggesting a direct relationship between EBV infection and inflammatory status. Finally, we characterized the nature of the infiltrated EBV+ cells by performing detailed immunohistochemistry profiles ( n = 21). Surprisingly, nearly all EBV+ cells detected in OLP lesions were CD138+ plasma cells (PCs) and more rarely CD20+ B cells. The presence of EBV+ PCs in erosive OLP was associated with profound changes in cytokine expression profile; notably, the expression of key inflammatory factors, such as IL1-ß and IL8, were specifically increased in OLP heavily infiltrated with EBV+ PCs. Moreover, electron microscopy-based experiments showed that EBV+ PCs actively produced EBV viral particles, suggesting possible amplification of EBV infection within the lesion. Our study thus brings conclusive evidence showing that OLP is commonly infiltrated with EBV+ PCs, adding a further puzzling element to OLP pathogenesis, given that PCs are now considered to be major regulatory immune cells involved in several autoimmune diseases (ClinicalTrials.gov NCT02276573).

The possible role of chromosome X variability in hypertensive familiarity.

Familiarity participates in the pathogenesis of hypertension, although only recently, whole genome studies have proposed regions of the human genome possibly involved in the transmission of the hypertensive phenotype. Although studies have mainly focused on autosome, hitherto the influence of sex on familial transmission of hypertension has not been considered. We analysed the database of the Campania Salute Network of Hypertension center of the Federico II University Hospital of Naples (Italy), using dichotomous variables for paternal and maternal familiarity and gender (male and female) of 12 504 hypertensive patients (6868 males and 5636 females) and 6352 controls (3484 males and 2868 females), totaling 18 856 subjects. In the hypertensive group, familiarity was present in 75% of cases with odds of 3.77 and in only 26% of the normotensives with odds of 0.94. The odds ratio (OR) indicated that familiarity increases the risk of developing hypertension by 2.91 (95% confidence interval (CI)=2.67-3.17, P<0.001) times. Additionally, maternal familiarity was 37% (OR=3.01, 95% CI=2.66-3.41, P<0.001), paternal familiarity was 21% (OR=2.31, 95% CI=2.01-2.68, P<0.001) and the double familiarity was 17% (OR=3.45, 95% CI=2.87-4.01, P<0.001), thus suggesting a plausible association between maternal familiarity and development of hypertension; this finding was observed both in male and in female patients, although the phenomenon was larger in males. Given the dominance of maternal transmission in males, by genome-wide analysis of the X chromosome, we found two regions that were differently distributed in male hypertensives with maternal hypertension. Our data highlight the importance of genetic variants in the X chromosome to the maternal transmission of the hypertensive phenotype.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions.

Comparison between CT and MR in perfusion imaging assessment of high-grade gliomas.

PURPOSE: The aim of our study was to compare the relative cerebral blood volume (CBV) values obtained by first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) T2-weighted magnetic resonance (MR) and perfusion computed tomography (P-CT) imaging in high-grade gliomas (HGG) in the same patient population. MATERIALS AND METHODS: Sixteen patients with histologically proven HGG underwent P-CT and DSC-MR brain imaging. P-CT studies were obtained using a four-row multislice CT scanner and postprocessed with a commercial software package based on a deconvolution-based technique. DSC-MR images were obtained at 1.5 T with a first-pass dynamic susceptibility contrast-enhanced T2-weighted sequence. P-CT and DSC-MR images were obtained within 4 days of each another, always before surgery. Maximum CBV ratios normalised with contralateral white matter (rCBV) were calculated. Statistical analysis was performed with the classical parametric statistic procedure. RESULTS: A linear correlation between maximum rCBV values obtained with P-CT and DSC-MR imaging was evident. The best linear model is CT=slope×MR+error and provides a highly significant estimate of the slope equal to 1.08. Thus CT results can be predicted from MR values. Therefore, it is also possible to predict MR results from CT values by estimating the linear model MR=slope×CT+error. DSC-MR imaging gave lower rCBV average values (4.92±1.52) compared with P-CT (5.56±1.55). CONCLUSIONS: In our population of patients, P-CT and DSC-MR imaging showed proportional results in rCBV assessment of HGGs, and thus both modalities may be used interchangeably in HGG of the brain.

Induction of different types of cell death after normothermic liver ischemia-reperfusion.

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular autophagy, apoptosis, and necrosis. The aim of this study was to investigate these three types of cell death in normothermic liver I-R in rats. A segmental normothermic ischemia of the liver was induced for 120 minutes. Liver autophagy was evaluated by transmission electron microscopy and LC3 (Light Chain 3) immunohistochemical studies. Liver apoptosis was assessed by FLIVO (FLuorescence in vIVO) and TUNEL (TdT-mediated dUTP nick end labeling) assays. Liver necrosis was determined by optical microscopic examination. Autophagy was increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Fluorescence microscopy showed in situ caspase-3 and -7 specific activity to be increased in ischemic liver lobes after 6 hours of reperfusion, compared with nonischemic lobes. Quantitative analysis of apoptotic cells evaluated by the TUNEL method showed a clearly significant increase in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Necrotic cell death was significantly increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes (P < .005). In conclusion, 120 minutes normothermic liver I-R resulted in increased autophagic, apoptotic and necrotic cell death.

Water and trehalose: how much do they interact with each other?

The observation made by early naturalists that some organisms could tolerate extreme environmental condisions and "enjoy the advantage of real resurrection after death" [ Spallanzani , M. Opuscules de Physique Animale et Vegetale 1776 (translated from Italian by Senebier , J. Opuscules de Physique Animale et Vegetale 1787 , 2 , 203 - 285 )] stimulated research that still continues to this day. Cryptobiosis, the ability of an organism to tolerate adverse environments, such as dehydration and low temperatures, still represents an unsolved and fascinating problem. It has been shown that many sugars play an important role as bioprotectant agents, and among the best performers is the disaccharide trehalose. The current hypothesis links the efficiency of its protective role to strong modifications of the tetrahedral arrangement of water molecules in the sugar hydration shell, with trehalose forming many hydrogen bonds with the solvent. Here, we show, by means of state-of-the-art neutron diffraction experiments combined with EPSR simulations, that trehalose solvation induces very minor modifications of the water structure. Moreover, the number of water molecules hydrogen-bonded to the sugar is surprisingly small.

Dielectric relaxations in ribose and deoxyribose supercooled water solutions.

The relaxation dynamic of ribose and deoxyribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of 150-250 K. Two relaxation processes are observed for all the hydration levels; the slower (process I) is related to the relaxation of the whole solution whereas the faster one (process II) is associated with the reorientation of water molecules in the mixture. As for other polymeric water solutions, dielectric data for process II indicate the existence of a critical water concentration above which water mobility is less restricted. According to these results, attenuated total reflectance Fourier transform infrared spectroscopy measurements of the same sugar solutions showed an increment in the intensity of the OH stretching sub-band close to 3200 cm(-1) as water content increases.

Solvation of KSCN in water.

The results of a neutron diffraction experiment performed on aqueous solutions of KSCN are analyzed and discussed in comparison with similar data for KCl and KOH solutions. The effect of the different ions on the structure of water and the differences and similarities among the ion solvation shells are discussed in detail. In particular it is shown that the effect of KSCN on the structure of water is visible as a shift and a broadening of the second peak of the oxygen-oxygen radial distribution function, which corresponds to a lower number of interstitial water molecules, compared to pure water. The hydration shell of the cation is similar to that found in the case of KCl solutions, and more interestingly the hydration shell of SCN- is asymmetric, with water molecules H-bonded to the N site and weakly correlated to the S site. These results provide a reasonable microscopic description of the mechanism for the high efficiency of thiocyanate in crystallizing basic proteins.

Quantum behavior of water protons in protein hydration shell.

Quantum effects on the water proton dynamics over the surface of a hydrated protein are measured by means of broadband dielectric spectroscopy and deep inelastic neutron scattering. Dielectric spectroscopy indicates a reduced energy barrier for a hydrogenated protein sample compared to a deuterated one, along with a large and temperature-dependent isotopic ratio, in good agreement with theoretical studies. Recent deep inelastic neutron scattering data have been reanalyzed, and now show that the momentum distribution of water protons reflects a characteristic delocalization at ambient temperatures. These experimental findings might have far-reaching implications for enzymatic catalysis involving proton transfer processes, as in the case of the lysozyme protein studied in this report.

Proton momentum distribution in a protein hydration shell.

The momentum distribution of protons in the hydration shell of a globular protein has been measured through deep inelastic neutron scattering at 180 and 290 K, below and above the crossover temperature Tc=1.23Tg, where Tg=219 K is the glass transition temperature. It is found that the mean kinetic energy of the water hydrogens shows no temperature dependence, but the measurements are accurate enough to indicate a sensible change of momentum distribution and effective potential felt by protons, compatible with the transition from a single to a double potential well. This could support the presence of tunneling effects even at room temperature, playing an important role in biological function.

Glassy behavior of a percolative water-protein system.

We show that is possible to look at the glass transition as a percolation transition in phase space. This study has been carried out on a hydrated globular enzyme for which the thermodynamic transition and the percolative transition could have a functional significance. The approach adopted is based on the identity of roles played respectively by the glass transition temperature T(o) and the critical hydration threshold h(c) for the percolation of protons on the surface and through the protein, given that dynamical arrest is observed at temperatures and hydration below T(o) and h(c). Theoretical predictions for temperature dependence of the nonexponentiality parameter, beta(KWW) , appearing in the KWW relaxation function, indicate that at high temperatures, beta(KWW) remains insensitive to temperature changes, whereas in the vicinity of the glass transition, beta(KWW) is linearly increasing with temperature. The low temperature limit of beta(KWW) is about 1/3 and its temperature-independent behavior starts at 1.23 T(g): both predictions have been verified in the present study.

Pre- and postsynaptic modulation of glycinergic and gabaergic transmission by muscarinic receptors on rat hypoglossal motoneurons in vitro.

The motor output of hypoglossal motoneurons to tongue muscles takes place in concert with the respiratory rhythm and is determined by the balance between excitatory glutamatergic transmission and inhibitory transmission mediated by glycine or GABA. The relative contribution by these transmitters is a phasic phenomenon modulated by other transmitters. We examined how metabotropic muscarinic receptors, widely expressed in the brainstem where they excite cranial motor nuclei, might influence synaptic activity mediated by GABA or glycine. For this purpose, using thin slices of the neonatal rat brainstem, we recorded (under whole-cell patch clamp) glycinergic or GABAergic responses from visually identified hypoglossal motoneurons after pharmacological block of glutamatergic transmission. Muscarine inhibited spontaneous and electrically induced events mediated by GABA or glycine. The amplitude of glycinergic miniature inhibitory postsynaptic currents was slightly reduced by muscarine, while GABAergic miniature inhibitory postsynaptic currents were unaffected. Motoneuron currents induced by focally applied GABA and glycine were depressed by muscarine with stronger reduction in glycine-mediated responses. Histochemical observations indicated the presence of M1, M2 and M5 subtypes of muscarinic receptors in the neonatal hypoglossal nucleus. These results suggest that muscarine potently depressed inhibitory neurotransmission on brainstem motoneurons, and that this action was exerted via preterminal and extrasynaptic receptors. Since the large reduction in inhibitory neurotransmission may contribute to overall excitation of brainstem motoneurons by muscarinic receptors, these data might help to understand the central components of action of antimuscarinic agents in preanesthetic medication or against motion sickness.

Ca increase in secretory granules of stimulated mast cells.

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.

Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium?

It is generally accepted that Ca is essentially involved in regulated secretion, but the role of this cation, as well as others such as Na, is not well understood. An illustrative example occurs in neurohypophysial secretion, where an experimentally induced increase in the cytosolic concentration of Na+ can induce continuous neuropeptide release. In contrast, an increase in cytosolic Ca2+ will have only a transient stimulatory effect. The secretion-promoting targets for Ca2+ are not known; they may be cytosolic, as is usually assumed, but they may also be intravesicular, especially in view of evidence that Ca-rich secretory vesicles are preferentially secreted. In the present work, we have investigated the movements of these cations into and out of secretory vesicles during stimulus-secretion coupling. Isolated rat neurohypophysial nerve endings were stimulated by potassium (55 mM) depolarization, and at 6 min (peak secretion) and 20 min after the onset of stimulation, the elemental content of individual secretory vesicles was measured by quantitative x-ray microanalysis. A depolarization-induced transient increase in intravesicular Na+ concentration was found to coincide with the onset of secretion. Moreover, only a predicted small fraction of peripheral vesicles-presumably the docked ones-were Na+-loaded. The low sulfur concentration of Na+-rich vesicles most likely resulted from vesicle swelling. The results suggest that high intravesicular Na+ concentrations in docked vesicles, occurring by Na+/Ca2+ exchange or by transient fusion pore opening, is a proximal event in exocytosis.

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens.

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.