Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

Self-potential investigations of a gravel bar in a restored river corridor

Self-potentials (SP) are sensitive to water fluxes and concentration gradients in both saturated and unsaturated geological media, but quantitative interpretations of SP field data may often be hindered by the superposition of different source contributions and time-varying electrode potentials. Self-potential mapping and close to two months of SP monitoring on a gravel bar were performed to investigate the origins of SP signals at a restored river section of the Thur River in northeastern Switzerland. The SP mapping and subsequent inversion of the data indicate that the SP sources are mainly located in the upper few meters in regions of soil cover rather than bare gravel. Wavelet analyses of the time-series indicate a strong, but non-linear influence of water table and water content variations, as well as rainfall intensity on the recorded SP signals. Modeling of the SP response with respect to an increase in the water table elevation and precipitation indicate that the distribution of soil properties in the vadose zone has a very strong influence. We conclude that the observed SP responses on the gravel bar are more complicated than previously proposed semi-empiric relationships between SP signals and hydraulic head or the thickness of the vadose zone. We suggest that future SP monitoring in restored river corridors should either focus on quantifying vadose zone processes by installing vertical profiles of closely spaced SP electrodes or by installing the electrodes within the river to avoid signals arising from vadose zone processes and time-varying electrochemical conditions in the vicinity of the electrodes.

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Synchronous and baroceptor-sensitive oscillations in skin microcirculation: evidence for central autonomic control.

To determine whether skin blood flow is local or takes part in general regulatory mechanisms, we recorded laser-Doppler flowmetry (LDF; left and right index fingers), blood pressure, muscle sympathetic nerve activity (MSNA), R-R interval, and respiration in 10 healthy volunteers and 3 subjects after sympathectomy. We evaluated 1) the synchronism of LDF fluctuations in two index fingers, 2) the relationship with autonomically mediated fluctuations in other signals, and 3) the LDF ability to respond to arterial baroreflex stimulation (by neck suction at frequencies from 0.02 to 0.20 Hz), using spectral analysis (autoregressive uni- and bivariate, time-variant algorithms). Synchronous LDF fluctuations were observed in the index fingers of healthy subjects but not in sympathectomized patients. LDF fluctuations were coherent with those obtained for blood pressure, MSNA, and R-R interval. LDF fluctuations were leading blood pressure in the low-frequency (LF; 0.1 Hz) band and lagging in the respiratory, high-frequency (HF; approximately 0.25 Hz) band, suggesting passive "downstream" transmission only for HF and "upstream" transmission for LF from the microvessels. LDF fluctuations were responsive to sinusoidal neck suction up to 0.1 Hz, indicating response to sympathetic modulation. Skin blood flow thus reflects modifications determined by autonomic activity, detectable by frequency analysis of spontaneous fluctuations.

The influence of drugs used in cardiac anaesthesia and critical care on multiple electrode aggregometry: an in-vitro volunteer study.

BACKGROUND: Multiple electrode aggregometry (MEA) is a point-of-care test evaluating platelet function and the efficacy of platelet inhibitors. In MEA, electrical impedance of whole blood is measured after addition of a platelet activator. Reduced impedance implies platelet dysfunction or the presence of platelet inhibitors. MEA plays an increasingly important role in the management of perioperative platelet dysfunction. In vitro, midazolam, propofol, lidocaine and magnesium have known antiplatelet effects and these may interfere with MEA interpretation. OBJECTIVE: To evaluate the extent to which MEA is modified in the presence of these drugs. DESIGN: An in-vitro study using blood collected from healthy volunteers. SETTING: Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 2010 to 2011. PATIENTS: Twenty healthy volunteers. INTERVENTION: Measurement of baseline MEA was using four activators: arachidonic acid, ADP, TRAP-6 and collagen. The study drugs were then added in three increasing, clinically relevant concentrations. MAIN OUTCOME MEASURE: MEA was compared with baseline for each study drug. RESULTS: Midazolam, propofol and lidocaine showed no effect on MEA at any concentration. Magnesium at 2.5 mmol l had a significant effect on the ADP and TRAP tests (31 ± 13 and 96 ± 39 AU, versus 73 ± 21 and 133 ± 28 AU at baseline, respectively), and a less pronounced effect at 1 mmol l on the ADP test (39 ± 0 AU). CONCLUSION: Midazolam, propofol and lidocaine do not interfere with MEA measurement. In patients treated with high to normal doses of magnesium, MEA results for ADP and TRAP-tests should be interpreted with caution. TRIAL REGISTRATION: Clinicaltrials.gov (no. NCT01454427).

Nuclear scaffold proteins are differently sensitive to stabilizing treatment by heat or Cu++.

The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.

Weight-bearing bones are more sensitive to physical exercise in boys than in girls during pre- and early puberty: a cross-sectional study.

We carried out a cross-section study of the sex-specific relationship between bone mineral content and physical activity at sites with different loading in pre- and early pubertal girls and boys. There was significant sensitivity of bone mineral content of the hip to physical exercise in boys, but not in girls. BACKGROUND: Since little is known whether there are sex differences in sensitivity of bone to loading, we investigated sex differences in the cross-sectional association between measures of physical activity (PA) and bone mass and size in pre- and early pubertal children of both sexes. METHODS: We measured bone mineral content/density (BMC/BMD) and fat-free mass (FFM) in 269 6- to 13-year-old children from randomly selected schools by dual-energy X-ray absorptiometry. Physical activity (PA) was measured by accelerometers and lower extremity strength by a jump-and-reach test. RESULTS: Boys (n = 128) had higher hip and total body BMC and BMD, higher FFM, higher muscle strength and were more physically active than girls (n = 141). Total hip BMC was positively associated with time spent in total and vigorous PA in boys (r = 0.20-0.33, p < 0.01), but not in girls (r = 0.02-0.04, p = ns), even after adjusting for FFM and strength. While boys and girls in the lowest tertile of vigorous PA (22 min/day) did not differ in hip BMC (15.62 vs 15.52 g), boys in the highest tertile (72 min/day) had significantly higher values than the corresponding girls (16.84 vs 15.71 g, p < 0.05). CONCLUSIONS: Sex differences in BMC during pre- and early puberty may be related to a different sensitivity of bone to physical loading, irrespective of muscle mass.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2.

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

A filtering method to correct time-lapse 3D ERT data and improve imaging of natural aquifer dynamics

We have developed a processing methodology that allows crosshole ERT (electrical resistivity tomography) monitoring data to be used to derive temporal fluctuations of groundwater electrical resistivity and thereby characterize the dynamics of groundwater in a gravel aquifer as it is infiltrated by river water. Temporal variations of the raw ERT apparent-resistivity data were mainly sensitive to the resistivity (salinity), temperature and height of the groundwater, with the relative contributions of these effects depending on the time and the electrode configuration. To resolve the changes in groundwater resistivity, we first expressed fluctuations of temperature-detrended apparent-resistivity data as linear superpositions of (i) time series of riverwater-resistivity variations convolved with suitable filter functions and (ii) linear and quadratic representations of river-water-height variations multiplied by appropriate sensitivity factors; river-water height was determined to be a reliable proxy for groundwater height. Individual filter functions and sensitivity factors were obtained for each electrode configuration via deconvolution using a one month calibration period and then the predicted contributions related to changes in water height were removed prior to inversion of the temperature-detrended apparent-resistivity data. Applications of the filter functions and sensitivity factors accurately predicted the apparent-resistivity variations (the correlation coefficient was 0.98). Furthermore, the filtered ERT monitoring data and resultant time-lapse resistivity models correlated closely with independently measured groundwater electrical resistivity monitoring data and only weakly with the groundwater-height fluctuations. The inversion results based on the filtered ERT data also showed significantly less inversion artefacts than the raw data inversions. We observed resistivity increases of up to 10% and the arrival time peaks in the time-lapse resistivity models matched those in the groundwater resistivity monitoring data.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected. OBJECTIVES: To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients. STUDY DESIGN: Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method. RESULTS: This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load. CONCLUSION: The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.

High b-value diffusion-weighted imaging: A sensitive method to reveal white matter differences in schizophrenia.

Over the last 10 years, diffusion-weighted imaging (DWI) has become an important tool to investigate white matter (WM) anomalies in schizophrenia. Despite technological improvement and the exponential use of this technique, discrepancies remain and little is known about optimal parameters to apply for diffusion weighting during image acquisition. Specifically, high b-value diffusion-weighted imaging known to be more sensitive to slow diffusion is not widely used, even though subtle myelin alterations as thought to happen in schizophrenia are likely to affect slow-diffusing protons. Schizophrenia patients and healthy controls were scanned with a high b-value (4000s/mm(2)) protocol. Apparent diffusion coefficient (ADC) measures turned out to be very sensitive in detecting differences between schizophrenia patients and healthy volunteers even in a relatively small sample. We speculate that this is related to the sensitivity of high b-value imaging to the slow-diffusing compartment believed to reflect mainly the intra-axonal and myelin bound water pool. We also compared these results to a low b-value imaging experiment performed on the same population in the same scanning session. Even though the acquisition protocols are not strictly comparable, we noticed important differences in sensitivities in the favor of high b-value imaging, warranting further exploration.

Electrochemical As(III) whole-cell based biochip sensor.

The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.