A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues, The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-mul amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C-18 (150X4.6 mm, 5 mum) column at 35 degreesC. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored. (C) 2001 Elsevier Science B.V. All rights reserved.

Phase sensitive pumping and coherence of superposition states

The continuous parametric pumping of a superconducting lossy QED cavity supporting a field prepared initially as a superposition of coherent states is discussed. In contrast to classical pumping, we verify that the phase sensitivity of the parametric pumping makes the asymptotic behaviour of the cavity field state strongly dependent on the phase theta of the coherent state \ alpha > = \ alpha \e(i theta)>. Here we consider theta = pi /4, -pi /4 and we analyse the evolution of the purity of the superposition states with the help of the linear entropy and fidelity functions. We also analyse the decoherence process quantitatively through the Wigner function, for both states, verifying that the decay is slightly modified when compared to the free decoherence case: for theta = -pi /4 the process is accelerated while for theta = pi /4 it is delayed.

An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia

1. An ATP-sensitive K+ (K-ATP) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY. 2. Under current clamp conditions in physiological solutions, leveromakalim (10 muM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage clamp conditions in symmetrical (140 mM) K+ solutions, hath application of levcromakalim evoked an inward current with a density of similar to8 pA pF(-1) at -50 mV and a slope conductance of similar to9 nS, which reversed close to the potassium equilibrium potential (E-K). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide. 4. Bath application of either glibenclamide (10 muM) or tolbutamide (100 muM) depolarized adult intracardiac neurones by 3-5 mV, suggesting that a K-ATP conductance is activated under resting conditions and contributes to the resting membrane potential. 5. Activation of a membrane current by levcromakalim leas concentration dependent, with an EC50 of 1.6 muM. Inhibition of the levcromakalim-activated current by glibenclamide leas also concentration dependent, with an IC50 of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P-O2 similar to 16 mmHg) also activated a membrane current. These currents exhibited similar I-P characteristics to the levcroinakalim-induced current and were inhibited by glibenclamide. 7. Activation of K-ATP channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and. cardiac function during ischaemia-reperfusion.

The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.

New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 mug diuron l(-1). Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 mug l(-1) defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 mug chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 mumol quanta m(-2) s(-1) with Phaeodactylum and thylakoids, respectively.

On learning context-free and context-sensitive languages

The long short-term memory (LSTM) is not the only neural network which learns a context sensitive language. Second-order sequential cascaded networks (SCNs) are able to induce means from a finite fragment of a context-sensitive language for processing strings outside the training set. The dynamical behavior of the SCN is qualitatively distinct from that observed in LSTM networks. Differences in performance and dynamics are discussed.

Does research into sensitive areas do harm? Experiences of research participation after a child's diagnosis with Ewing's sarcoma

Objective: To investigate family members' experiences of involvement in a previous study (conducted August 1995 to June 1997) following their child's diagnosis with Ewing's sarcoma. Design: Retrospective survey, conducted between 1 November and 30 November 1997, using a postal questionnaire. Participants: Eighty-one of 97 families who had previously completed an in-depth interview as part of a national case-control study of Ewing's sarcoma. Main outcome measures: Participants' views on how participation in the previous study had affected them and what motivated them to participate. Results: Most study participants indicated that taking part in the previous study had been a positive experience. Most (n = 79 [97.5%]) believed their involvement would benefit others and were glad to have participated, despite expecting and finding some parts of the interview to be painful. Parents whose child was still alive at the time of the interview recalled participation as more painful than those whose child had died before the interview. Parents who had completed the interview less than a year before our study recalled it as being more painful than those who had completed it more than a year before. Conclusions: That people suffering bereavement are generally eager to participate in research and may indeed find it a positive experience is useful information for members of ethics review boards and other gatekeepers, who frequently need to determine whether studies into sensitive areas should be approved. Such information may also help members of the community to make an informed decision regarding participation in such research.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Low temperature induced spikelet sterility in rice. I. Nitrogen fertilisation and sensitive reproductive period

Low temperature during panicle development in rice increases spikelet sterility. This effect is exacerbated by high rates of nitrogen (N) application in the field. Spikelet sterility induced by low temperature and N fertilisation was examined in glasshouse experiments to clarify the mechanisms involved. In two glasshouse experiments, 12-h periods of low (18/13degreesC) and high (28/23degreesC) day/night temperatures were imposed over periods of 5-7 days during panicle development, to determine the effects of low temperature and N fertilisation on spikelet sterility. In one experiment, 50% sunlight was imposed together with low temperature to investigate the additive effects of reduced solar radiation and low temperature. The effect of increased tillering due to N fertilisation was examined by a tiller removal treatment in the same experiment. Pollen grain number and spikelet sterility were recorded at heading and harvest, respectively. Although there was no significant effect of low temperature on spikelet sterility in the absence of applied N, low temperature greatly increased spikelet sterility as a result of a reduction in the number of engorged pollen grains per anther in the presence of applied N. Spikelet sterility was strongly correlated with the number of engorged pollen grains per anther. Low temperature during very early ( late stage of spikelet differentiation-pollen mother cell stage) and peak ( second meiotic division stage-early stage of extine formation) microspore development caused a severe reduction in engorged pollen production mainly as a result of reduced total pollen production. Unlike low temperature, the effect of shading was rather small. The increased tillering due to application of high rates of N, increased both spikelet number per plant and spikelet sterility under low temperature conditions. The removal of tillers as they appeared reduced the number of total spikelets per plant and maintained a large number of engorged pollen grains per anther which, in turn, reduced spikelet sterility. The number of engorged pollen grains per anther determined the numbers of intercepted and germinated pollen grains on the stigma. It is concluded that N increased tillering and spikelet number per plant and this, in turn, reduced the number of engorged pollen grains per anther, leading into increased spikelet sterility under low temperature condition.

Optimizing electrode positioning in 3-lead ECG chest devices

Although the 12-lead electrocardiogram has become an essential medical and research tool, many current and envisaged applications would benefit from simpler devices, using 3-lead ECG configuration. This is particularly true for Ambient Assisted Living (in a broad perspective). However, the chest anatomy of female patients, namely during pregnancy, can hamper the adequate placement of a 3-lead ECG device and, very often, electrodes are placed below the chest rather than at the precise thoracic landmarks. Thus, the aim of this study was to compare the effect of electrode positioning on the ECG signal of pregnant women and provide guidelines for device development. The effect of breast tissue on the ECG signal was investigated by relating breast size with the signal-to-noise ratio, root mean square and R-wave amplitude. Results show that the 3-lead ECG should be placed on the breast rather than under the breast and indicate positive correlation between breast size and signal-to-noise ratio.

Neural Electrode Array Based on Aluminum: Fabrication and Characterization

A unique neural electrode design is proposed with 3 mm long shafts made from an aluminum-based substrate. The electrode is composed by 100 individualized shafts in a 10 × 10 matrix, in which each aluminum shafts are precisely machined via dicing-saw cutting programs. The result is a bulk structure of aluminum with 65 ° angle sharp tips. Each electrode tip is covered by an iridium oxide thin film layer (ionic transducer) via pulsed sputtering, that provides a stable and a reversible behavior for recording/stimulation purposes, a 40 mC/cm2 charge capacity and a 145 Ω impedance in a wide frequency range of interest (10 Hz-100 kHz). Because of the non-biocompatibility issue that characterizes aluminum, an anodization process is performed that forms an aluminum oxide layer around the aluminum substrate. The result is a passivation layer fully biocompatible that furthermore, enhances the mechanical properties by increasing the robustness of the electrode. For a successful electrode insertion, a 1.1 N load is required. The resultant electrode is a feasible alternative to silicon-based electrode solutions, avoiding the complexity of its fabrication methods and limitations, and increasing the electrode performance.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Detection of FRET signals with a wavelength sensitive device based on a-SiC:H

Glucose sensing is an issue with great interest in medical and biological applications. One possible approach to glucose detection takes advantage of measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor within a protein which undergoes glucose-induced changes in conformation. This demands the detection of fluorescent signals in the visible spectrum. In this paper we analyzed the emission spectrum obtained from fluorescent labels attached to a protein which changes its conformation in the presence of glucose using a commercial spectrofluorometer. Different glucose nanosensors were used to measure the output spectra with fluorescent signals located at the cyan and yellow bands of the spectrum. A new device is presented based on multilayered a-SiC:H heterostructures to detect identical transient visible signals. The transducer consists of a p-i'(a-SiC:H)-n/p-i(a-Si:H)-n heterostructure optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet (400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. Results show that the device photocurrent signal measured under reverse bias and using appropriate steady state optical bias, allows the separate detection of the cyan and yellow fluorescence signals. (C) 2013 Elsevier B.V. All rights reserved.

On the use of IEEE 802.15.4/Zigbee for time-sensitive wireless sensor network applications

Mestrado em Engenharia Electrotécnica e de Computadores

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.