Demonstration of Single-Barium-Ion Sensitivity for Neutrinoless Double-Beta Decay Using Single-Molecule Fluorescence Imaging.

A new method to tag the barium daughter in the double-beta decay of ^{136}Xe is reported. Using the technique of single molecule fluorescent imaging (SMFI), individual barium dication (Ba^{++}) resolution at a transparent scanning surface is demonstrated. A single-step photobleach confirms the single ion interpretation. Individual ions are localized with superresolution (∼2 nm), and detected with a statistical significance of 12.9σ over backgrounds. This lays the foundation for a new and potentially background-free neutrinoless double-beta decay technology, based on SMFI coupled to high pressure xenon gas time projection chambers.

AEgIS at ELENA: outlook for physics with a pulsed cold antihydrogen beam.

The efficient production of cold antihydrogen atoms in particle traps at CERN's Antiproton Decelerator has opened up the possibility of performing direct measurements of the Earth's gravitational acceleration on purely antimatter bodies. The goal of the AEgIS collaboration is to measure the value of g for antimatter using a pulsed source of cold antihydrogen and a Moiré deflectometer/Talbot-Lau interferometer. The same antihydrogen beam is also very well suited to measuring precisely the ground-state hyperfine splitting of the anti-atom. The antihydrogen formation mechanism chosen by AEgIS is resonant charge exchange between cold antiprotons and Rydberg positronium. A series of technical developments regarding positrons and positronium (Ps formation in a dedicated room-temperature target, spectroscopy of the n=1-3 and n=3-15 transitions in Ps, Ps formation in a target at 10 K inside the 1 T magnetic field of the experiment) as well as antiprotons (high-efficiency trapping of [Formula: see text], radial compression to sub-millimetre radii of mixed [Formula: see text] plasmas in 1 T field, high-efficiency transfer of [Formula: see text] to the antihydrogen production trap using an in-flight launch and recapture procedure) were successfully implemented. Two further critical steps that are germane mainly to charge exchange formation of antihydrogen-cooling of antiprotons and formation of a beam of antihydrogen-are being addressed in parallel. The coming of ELENA will allow, in the very near future, the number of trappable antiprotons to be increased by more than a factor of 50. For the antihydrogen production scheme chosen by AEgIS, this will be reflected in a corresponding increase of produced antihydrogen atoms, leading to a significant reduction of measurement times and providing a path towards high-precision measurements.This article is part of the Theo Murphy meeting issue 'Antiproton physics in the ELENA era'.

[Doping, sport and addiction--any links?]. / Dopage, pratique sportive et addiction--quels liens?

Sport is widely encouraged as it is beneficial for health. However, high-performance sport is more and more associated to rather suspicious practices; doping is one of the best example. From a physician point of view, the use of doping agents is obviously a major concern because taking such products often induce serious adverse effects on health. The present manuscript aims to inform physicians about the most frequent doping practices. It also points out that intensive sport can generate an "addictive" behavior sharing with "common"addictions a loss of practice control, a lack of interest in other activities and even a sport's practice detrimental to athlete's health. Analysis of the doping issue needs to take this reality into account as some doping products display an established " addictive" effect.

USE OF MICRO-DAMS IN POTATO FURROWS TO REDUCE EROSION AND RUNOFF AND MINIMISE SURFACE WATER CONTAMINATION THROUGH PESTICIDES.

The use of micro-dams in potato furrows is an interesting technology to reduce erosion and runoff in hilly areas. These phenomena are major sources of surface water contamination by nutrients and plant protection products (Gillijns et al., 2005). In 2011 Bayer CropScience set up a trial in collaboration with the Walloon Agricultural Research Centre (CRA-W) and ULg-Gembloux Agro-Bio Tech in Huldenberg (Belgium) to demonstrate this technique in potatoes. Micro-dams create barriers between furrows in order to encourage rainwater to infiltrate in the soil rather than to run off. The results from the trial over this year confirm that the application of micro-dams is effective in reducing erosion and runoff significantly. The total loss of plant protection products (PPP) to surface water is dramatically reduced and also strongly depends on the physic-chemical characteristics of the active ingredients. In addition, the technique tends to produce a higher yield of potato tubers as an effect of an optimised utilisation of the available rainwater and nutrients.

Innovative methodology to transfer conventional GC-MS heroin profiling to UHPLC-MS/MS.

Nowadays, in forensic laboratories, heroin profiling is frequently carried out by gas chromatography coupled with mass spectrometry (GC-MS). This analytical technique is well established, provides good sensitivity and reproducibility, and allows the use of large databases. Despite those benefits, recently introduced analytical techniques, such as ultra-high-pressure liquid chromatography (UHPLC), could offer better chromatographic performance, which needs to be considered to increase the analysis throughput for heroin profiling. With the latter, chromatographic conditions were optimized through commercial modeling software and two atmospheric pressure ionization sources were evaluated. Data obtained from UHPLC-MS/MS were thus transferred, thanks to mathematical models to mimic GC-MS data. A calibration and a validation set of representative heroin samples were selected among the database to establish a transfer methodology and assess the models' abilities to transfer using principal component analysis and hierarchical classification analysis. These abilities were evaluated by computing the frequency of successful classification of UHPLC-MS/MS data among GC-MS database. Seven mathematical models were tested to adjust UHPLC-MS/MS data to GC-MS data. A simplified mathematical model was finally selected and offered a frequency of successful transfer equal to 95%.

The suppressor of cytokine signalling 2 (SOCS2) is a key repressor of insulin secretion.

AIMS/HYPOTHESIS: Suppressor of cytokine signalling (SOCS) proteins are powerful inhibitors of pathways involved in survival and function of pancreatic beta cells. Whereas SOCS1 and SOCS3 have been involved in immune and inflammatory processes, respectively, in beta cells, nothing is known about SOCS2 implication in the pancreas. METHODS: Transgenic (tg) mice were generated that constitutively produced SOCS2 in beta cells (betaSOCS2) to define whether this protein is implicated in beta cell functioning and/or survival. RESULTS: Constitutive production of SOCS2 in beta cells leads to hyperglycaemia and glucose intolerance. This phenotype is not a consequence of decreased beta cell mass or inhibition of insulin synthesis. However, insulin secretion to various secretagogues is profoundly altered in intact animals and isolated islets. Interestingly, constitutive SOCS2 production dampens the rise in cytosolic free calcium concentration induced by glucose, while glucose metabolism is unchanged. Moreover, tg islets have a depletion in endoplasmic reticulum Ca(2+) stores, suggesting that SOCS2 interferes with calcium fluxes. Finally, in betaSOCS2 mice proinsulin maturation is impaired, leading to an altered structure of insulin secretory granules and augmented levels of proinsulin. The latter is likely to be due to decreased production of prohormone convertase 1 (PC1/3), which plays a key role in proinsulin cleavage. CONCLUSIONS/INTERPRETATIONS: SOCS2 was shown to be a potent regulator of proinsulin processing and insulin secretion in beta cells. While its constitutive production is insufficient to induce overt diabetes in this mouse model, it causes glucose intolerance. Thus, increased SOCS2 production could be an important event predisposing to beta cell failure.

Constitutive expression of suppressor of cytokine signalling-3 in skeletal muscle leads to reduced mobility and overweight in mice.

AIMS/HYPOTHESIS: Due to their ability to regulate various signalling pathways (cytokines, hormones, growth factors), the suppressor of cytokine signalling (SOCS) proteins are thought to be promising therapeutic targets for metabolic and inflammatory disorders. Hence, their role in vivo has to be precisely determined. METHODS: We generated transgenic mice constitutively producing SOCS-3 in skeletal muscle to define whether the sole abundance of SOCS-3 is sufficient to induce metabolic disorders and whether SOCS-3 is implicated in physiological roles distinct from metabolism. RESULTS: We demonstrate here that chronic expression of SOCS-3 in skeletal muscle leads to overweight in mice and worsening of high-fat diet-induced systemic insulin resistance. Counter-intuitively, insulin sensitivity in muscle of transgenic mice appears to be unaltered. However, following constitutive SOCS-3 production, several genes had deregulated expression, among them other members of the SOCS family. This could maintain the insulin signal into skeletal muscle. Interestingly, we found that SOCS-3 interacts with calcineurin, which has been implicated in muscle contractility. In Socs-3 transgenic muscle, this leads to delocalisation of calcineurin to the fibre periphery. Relevant to this finding, Socs-3 transgenic animals had dilatation of the sarcoplasmic reticulum associated with swollen mitochondria and decreased voluntary activity. CONCLUSIONS/INTERPRETATION: Our results show that constitutive SOCS-3 production in skeletal muscle is not in itself sufficient to induce the establishment of metabolic disorders such as diabetes. In contrast, we reveal a novel role of SOCS-3, which appears to be important for muscle integrity and locomotor activity.

Allergic bronchial asthma due to Dermatophagoides pteronyssinus hypersensitivity can be efficiently treated by inoculation of allergen-antibody complexes.

Antigen-antibody complexes were made from allergens of the common house dust mite, Dermatophagoides pteronyssinus (Dpt) and an excess of purified autologous specific antibodies. These complexes have been used to treat Dpt-hypersensitive patients who suffered from chronic bronchial asthma. Clinical symptoms and medication intake were followed by filling in diary cards. Peak expiratory flow, measured four times a day, was also followed. Intradermal skin tests and bronchial challenge tests were performed with allergen together with an evaluation of nonspecific bronchial reactivity. Specific IgE and IgG antibodies were assayed after separation from the bulk of serum immunoglobulins by immunoadsorption. The study was carried out over two years according to a double-blind protocol. Intradermal inoculation of antigen-antibody complexes resulted in a marked reduction of both clinical and medication scores. No systemic side-effects were observed and only mild wheal and flare reactions were noted at the injection site. The treatment showed a drastic reduction of specific skin and bronchial reactivities with only marginal effects on nonspecific bronchial reactivity. Concentrations of specific IgE antibodies decreased significantly during the first weeks of treatment and remained at these lower values throughout the study. Specific IgG antibodies actually decreased in the majority of treated patients. The total amount of allergen used in this study was less than 1% of the amount currently used for conventional hyposensitization with the same allergen. These findings show that antigen-antibody complex inoculation is an efficient and safe means of treating allergic bronchial asthma and that the mechanism of action is likely to differ from conventional hyposensitization.

Optimization and validation of a fast supercritical fluid chromatography method for the quantitative determination of vitamin D3 and its related impurities.

In the uprising context of green analytical chemistry, Supercritical Fluid Chromatography (SFC) is often suggested as an alternative to Normal Phase Liquid Chromatography. Indeed, SFC provides fast, efficient and green separations. In this report, the quantitative performances of SFC were challenged on a real-life case study: the Quality Control (QC) of vitamin D3. A rapid and green SFC method was optimized thanks to the Design of Experiments-Design Space (DoE-DS) methodology. It provided robust and high quality separation of the compounds within a 2min timeframe, using a gradient of ethanol as co-solvent of the carbon dioxide. The analytical method was fully validated according to the total error approach, demonstrating the compliance of the method to the specifications of U.S. Pharmacopeia (USP: 97.0-103.0%) and European Pharmacopeia (EP: 97.0-102.0%) for an interval of [50-150%] of the target concentration. In order to allow quantification of impurities using vitamin D3 as an external standard in SFC-UV, correction factors were determined and verified during method validation. Thus, accurate quantification of impurities was demonstrated at the specified levels (0.1 and 1.0% of the main compound) for a 70.0-130.0% dosing range. This work demonstrates the validity of an SFC method for the QC of vitamin D3 raw material and its application to real samples. Therefore, it supports the switch to a greener and faster separative technique as an alternative to NPLC in the pharmaceutical industry.

Activation, but not inhibition, by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell.

The effect of glucose on the Ca2+-activated K+ permeability in pancreatic islet cells was investigated by measuring the rate of 86Rb efflux, 45Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in 86Rb outflow, 45Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased 86Rb and 45Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in 86Rb and 45Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca2+ of the Ca2+-sensitive modality of K+ extrusion. On the contrary, as a result of its effect upon Ca2+ handling, glucose stimulates the Ca2+-activated K+ permeability.

Ca2+ entry through L-type voltage-sensitive Ca2+ channels stimulates the release of human chorionic gonadotrophin and placental lactogen by placental explants.

The release of human chorionic gonadotrophin (hCG) and placental lactogen (hPL) by human placental explants can be stimulated by Ca2+ entry. The aim of the present study was to characterize the modality of Ca2+ entry in the presence of high extracellular K+ concentration ([K+]o). A rise in [K+]o from 5 to > or = 50 mM induced a rapid and marked increase in the release of hCG and hPL from human term placental explants. The stimulatory effects of an excess [K+]o on the release of hCG and hPL were blocked in the absence of extracellular Ca2+ or in the presence of 0.5 mM Co2+. The presence of 50 microM methoxyverapamil, 20 microM nifedipine or 40 microM Cd2+ in the medium inhibited the stimulatory effects of [K+]o addition. Lastly, 40 microM Ni2+ failed to affect the increases in hCG and hPL releases elicited by [K+]o addition. Our data clearly show that a rise in [K+]o stimulates the release of hCG and hPL from placental explants. These secretory effects can be viewed as resulting from a Ca2+ entry through voltage-sensitive Ca2+ channels of the L-type.

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells.

The removal of extracellular HCO3- together with a decrease in pCO2, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of 45Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca2+ and abolished at low extracellular Na+ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca2+. The removal of HCO3- and decrease in the pCO2 also reduced the magnitude of both the secondary rise in 45Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca2+ from islet cells as mediated by Na+ -Ca2+ countertransport and the inflow of Ca2+ by gated Ca2+ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.

Effect of extracellular sodium removal upon 86Rb outflow from pancreatic islet cells.

The present study was undertaken to characterize the effect of extracellular Na+ removal on 86Rb outflow from perifused rat pancreatic islets. Complete Na+ omission inhibited 86Rb outflow whether the islets were perifused in the presence or in the absence of extracellular Ca2+. Ouabain (1 mM) did not reduce the inhibitory effect of Na+ deprivation, whilst diphenylhydantoin (72.9 microM) mimicked the Na+-removal-induced fall in 86Rb outflow. Glucose (16.7 mM) lost its capacity to inhibit 86Rb outflow when the perifusate was deprived of extracellular Na+. These results indicate that Na+ omission reproduces the inhibitory effect of glucose on 86Rb outflow. The reduction in 86Rb outflow recorded after Na+ deprivation could be mediated by an intracellular acidification and/or a decrease in the intracellular Na+ activity. It is tempting to speculate that the capacity of glucose to reduce the B-cell Na+ content may participate in the process by which the sugar decreases K+ permeability.

Sodium nitroprusside inhibits glucose-induced insulin release by activating ATP-sensitive K+ channels.

Sodium nitroprusside (SNP) has been reported to be a potent stimulator of cGMP formation in different tissues, including pancreatic islets. The present study aimed at comparing the effects of sodium nitroprusside and dibutyryl cGMP on 86Rb outflow, 45Ca outflow, short-term 45Ca uptake, cytosolic Ca2+ concentration and insulin release from rat pancreatic islet cells. The data indicate that cGMP potentiates whilst SNP inhibits the glucose-induced insulin release. This inhibitory effect appears to be mediated by the activation of ATP-sensitive K+ channels leading to a decrease in Ca2+ influx and subsequent reduction in cytosolic free Ca2+ concentration. Whatever the exact mechanism(s) underlying the capacity of sodium nitroprusside to enhance the K+ permeability of the B-cell membrane, the drug appears to be an inadequate pharmacological tool to characterize the involvement of cGMP in the insulin secretory process. The experimental results also suggest that cGMP potentiates glucose-induced insulin release without affecting ionic movements.

Na+-H+ exchange in the process of glucose-induced insulin release from the pancreatic B-cell. Effects of amiloride on 86Rb, 45Ca fluxes and insulin release.

The effect of amiloride, an inhibitor of Na+-H+ exchange, on intracellular pH (pHi), 86Rb outflow, 45Ca outflow and insulin release from pancreatic rat islets was examined. In the 0.1-1 mM range, amiloride transiently reduced pHi of glucose-deprived islets and allowed glucose to induce a sustained decrease in pHi of the islet cells. Amiloride reproduced the effect of glucose to decrease 86Rb and 45Ca outflow. In the presence of glucose (5.6 mM or more), amiloride (100 microM) acted synergistically with the sugar to reduce K+ outflow, and to stimulate 40Ca inflow and insulin release from perifused islets. These results add strong support to the view that the generation of protons through the metabolism of glucose represents an important step in the process of glucose-induced release. The stimulation by glucose of Na+-H+ exchange apparently masks and even overcomes the glucose-induced decrease in pHi otherwise expected from the increase in catabolic fluxes.

Regulation of calcium fluxes in rat pancreatic islets. Quinine mimics the dual effect of glucose on calcium movements.

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on 45Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces 45Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 microM quinine stimulated 45Ca net uptake, 45Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on 45Ca net uptake, 45Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate 45Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in 45Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na+ concentration. At a low concentration (5 microM), quinine, although reducing 86Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit 45Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in 45Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced 45Ca efflux from perifused islets. It is concluded that quinine, by reducing K+ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K+ conductance.

Modalities of gliclazide-induced Ca2+ influx into the pancreatic B-cell.

The hypoglycemic sulfonylurea gliclazide stimulated 45Ca efflux and insulin release from prelabeled and perifused pancreatic rat islets, whether in the absence or presence of glucose (8.3 mM). The gliclazide-induced increase in 45Ca efflux is thought to reflect a stimulation of 40Ca influx into islet cells; it is suppressed in the absence of extracellular Ca2+ or in the presence of the organic CA2/-antagonist verapamil. In the absence of glucose, the ED50 for the inhibitory action of verapamil on gliclazide-stimulated 45Ca efflux was almost identical to that found when the process of 40Ca-45Ca exchange was stimulated by an increase in the extracellular concentration of K+, a procedure that leads to the depolarization of the plasma membrane. In the presence of glucose, however, the cationic response to gliclazide displayed an increased sensitivity toward the inhibitory action of verapamil. It is proposed that hypoglycemic sulfonylureas facilitate Ca2+ inflow into islet cells mainly through voltage-sensitive Ca2+ channels. In the presence of glucose, however, the stimulant action of hypoglycemic sulfonylureas upon Ca2+ entry into islet cells may entail a modality of Ca2+ transport not identical to that evoked by depolarization of the plasma membrane.

The stimulus-secretion coupling of amino acid-induced insulin release. Secretory and oxidative response of pancreatic islets to L-asparagine.

L-Asparagine (2-10 mM) failed to affect insulin secretion from rat pancreatic islets incubated in the absence of exogenous nutrient or presence of D-glucose, but caused a dose-related and progressive enhancement of insulin release evoked by L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylate, or 2-ketoisocaproate. The secretory response to the combination of L-asparagine and L-leucine was augmented by theophylline and inhibited in the absence of extracellular Ca2+ or presence of either menadione or methylamine. L-Asparagine augmented leucine-stimulated 45Ca net uptake. The ATP content, rate of O2 uptake, and malate/pyruvate ratio were not significantly different in islets exposed to L-leucine alone or to both L-asparagine and L-leucine, respectively. In the sole presence of L-asparagine, however, the malate/oxalacetate ratio was decreased and the malate/pyruvate ratio increased, relative to basal values. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine might be due to an accelerated generation rate of cytosolic NADPH, rather than to any sizable increase in either islet respiration or steady-state cytosolic NADPH/NADP+ ratio.

Mechanisms of sulfonylurea-induced insulin release.

The mechanisms responsible for the stimulation of insulin release from the pancreatic beta-cell by hypoglycemic sulfonylureas are reviewed herein. One hypothesis postulates that these agents act, at the level of the plasma membrane, by causing the closure of a class of K+ channels characterized by their sensitivity to ATP. This may lead to depolarization of the plasma membrane, gating of voltage-sensitive Ca2+ channels, increase in cytosolic Ca2+ activity, and activation of the effector system for insulin release. However, it is not evident that the closure of ATP-sensitive K+ channels accounts for effects of sulfonylureas such as inhibition of K+ inflow into the islet cells, increase in their Na+ content, or even stimulation of Ca2+ inflow and insulin release at physiological or higher concentrations of D-glucose.

Towards a full integration of optimization and validation phases: An analytical-quality-by-design approach.

When using an analytical method, defining an analytical target profile (ATP) focused on quantitative performance represents a key input, and this will drive the method development process. In this context, two case studies were selected in order to demonstrate the potential of a quality-by-design (QbD) strategy when applied to two specific phases of the method lifecycle: the pre-validation study and the validation step. The first case study focused on the improvement of a liquid chromatography (LC) coupled to mass spectrometry (MS) stability-indicating method by the means of the QbD concept. The design of experiments (DoE) conducted during the optimization step (i.e. determination of the qualitative design space (DS)) was performed a posteriori. Additional experiments were performed in order to simultaneously conduct the pre-validation study to assist in defining the DoE to be conducted during the formal validation step. This predicted protocol was compared to the one used during the formal validation. A second case study based on the LC/MS-MS determination of glucosamine and galactosamine in human plasma was considered in order to illustrate an innovative strategy allowing the QbD methodology to be incorporated during the validation phase. An operational space, defined by the qualitative DS, was considered during the validation process rather than a specific set of working conditions as conventionally performed. Results of all the validation parameters conventionally studied were compared to those obtained with this innovative approach for glucosamine and galactosamine. Using this strategy, qualitative and quantitative information were obtained. Consequently, an analyst using this approach would be able to select with great confidence several working conditions within the operational space rather than a given condition for the routine use of the method. This innovative strategy combines both a learning process and a thorough assessment of the risk involved.