Probing the debromination of the flame retardant decabromodiphenyl ether in sediments of a boreal lake.

After decades of use of polybrominated diphenyl ethers (PBDEs) as flame retardants, a large reservoir of these toxins has accumulated in ecosystems worldwide. The present study used an innovative approach to examine whether the fully brominated PBDE decabromodiphenyl ether (decaBDE) degrades to more toxic congeners in aquatic environments. The authors incubated intact sediment microcosms with high-purity [(13)C]decaBDE in a remote boreal lake to assess its debromination under ambient conditions. Although the addition of [(13)C]decaBDE increased total PBDE concentrations in sediment more than 10-fold, the relative amount of [(13)C]decaBDE in sediment did not change significantly over a 1-mo incubation. However, observation of small quantities of lower-brominated [(13)C]BDEs lent support to the hypothesis that decaBDE is slowly debrominated. The authors observed a significant increase in octaBDEs and nonaBDEs in profundal, but not littoral, sediment over 30 d. A second experiment in which sediment was incubated under different light and oxygen regimes yielded a surprising result-oxygen significantly stimulated the formation of octaBDEs and nonaBDEs. The authors also conducted a large-scale in situ enclosure experiment in which they followed the fate of experimentally added decaBDE in sediment over 26 mo, but that study yielded little evidence of decaBDE debromination. Overall, the authors suggest that the debromination of decaBDE occurs very slowly, if at all, in natural sediment of boreal lakes, in contrast to the rapid degradation kinetics reported by most laboratory-based studies, which are usually conducted by dissolving decaBDE in organic solvents. The findings reinforce the need for field studies on contaminant fate to inform environmental policy decisions.

Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry.

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.

Mass spectrometry-based absolute protein quantification: PSAQ™ strategy makes use of "noncanonical" proteotypic peptides.

Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.

Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.

Production and use of stable isotope-labeled proteins for absolute quantitative proteomics.

In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein's concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used, are generally added to samples after protein isolation and digestion. Thus, if protein enrichment is necessary, they do not allow correction for protein losses that may occur during sample pre-fractionation, nor do they allow the tryptic digestion yield to be taken into account. To reduce these limitations we have developed the PSAQ (Protein Standard Absolute Quantification) strategy using full-length stable isotope-labeled proteins as quantification standards. These standards and the target proteins share identical biochemical properties. This allows standards to be spiked into samples at an early stage of the analytical process. Thanks to this possibility, the PSAQ method provides highly accurate quantification results, including for samples requiring extensive biochemical pre-fractionation. In this chapter, we describe the production of full-length stable isotope-labeled proteins (PSAQ standards) using cell-free expression devices. The purification and quality control of protein standards, crucial for good-quality and accurate measurements, are also detailed. Finally, application of the PSAQ method to a typical protein quantification assay is presented.

[A critical review of the organizational engineering of Québec's mental health action plan]. / Critique de l'ingénierie organisationnelle du Plan d'action en santé mentale 2005-2010.

The Plan d'action en santé mentale 2005-2010 commands a substantial reform of mental health services organization. In order to achieve this, the Plan draws upon a set of ideas that appear somewhat unsubstantiated in regards to the sciences of organization. This article examines a few of these ideas. The managerial rhetoric of the Plan is anchored in an organizational and mechanistic archetype known for its inadequacy in the central mission of organizations of complex human services such as those concerning mental health. The mechanist rationality adopted marginalizes the real source of the value of mental health services, of practitioners and their social institutions. It participates in a movement that renders organizations always bigger, more abstract and impersonal steered from a distance by superficial indicators. There are good reasons to believe that this approach of organizational engineering will accentuate the eroding of the individual and collective capacities of delivering services of great value.

Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples.

To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.

Isotope dilution strategies for absolute quantitative proteomics.

The development of mass spectrometry (MS)-based methodologies for high-throughput protein identification has generated a concomitant need for protein quantification. Numerous MS-based relative quantification methodologies have been dedicated to the extensive comparison of multiple proteomes. On the other hand, absolute quantification methodologies, which allow the determination of protein concentrations in biological samples, are generally restricted to defined sets of proteins. Depending on the selected analytical procedure, absolute quantification approaches can provide accurate and precise estimations. These analytical performances are crucial for specific applications such as the evaluation of clinical biomarker candidates. According to bioanalytical guidelines, accurate analytical processes require internal standards and quality controls. Regarding MS-based analysis of small molecules, isotope dilution has been recognized as the reference method for internal standardization. However, protein quantification methodologies which rely on the isotope dilution principle have been implemented in the proteomic field only recently. In these approaches, the sample is spiked with defined amounts of isotope-labeled analogue(s) of specific proteolytic peptide(s) (AQUA and QconCAT strategies) or protein(s) (PSAQ strategy). In this review, we present a critical overview of these isotope dilution methodologies.

Innovative application of mass spectrometry for the characterization of staphylococcal enterotoxins involved in food poisoning outbreaks.

Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.

Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks.

Staphylococcal enterotoxins are major causing agents of food-borne diseases. Their detection in food remnants for risk assessment or food poisoning outbreaks investigation suffers from a lack in comprehensive immunological tools. In this study, we demonstrate that the combination of immunocapture and Protein Standard Absolute Quantification (PSAQ) strategy, which uses isotope-labeled enterotoxins as internal standards for MS-based analysis, is powerful to specifically identify and quantify these contaminating agents in food matrices. This approach is believed to significantly improve the elucidation of staphylococcal food poisoning outbreaks.

Isotope-labeled protein standards: toward absolute quantitative proteomics.

Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. enzyme-linked immunosorbent assay), MS is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (Protein Standard Absolute Quantification) that uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our Protein Standard Absolute Quantification methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.

Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis.

Spontaneous calcium activity can be detected in embryonic mouse cortical slices as fluorescence intensity variations, in the presence of a fluorescent calcium indicator. Current methods to detect and quantify these variations depend heavily on experimenters whose judgement may interfere with measurement. In the present work, we developed new software called CalSignal for automatic detection and tracking of cellular bodies and quantification of spontaneous calcium activity on time-series of confocal fluorescence images. Analysis of 28 neocortical slices revealed that 21.0% of detected cells displayed peaks of fluorescence corresponding to spontaneous activity, with a mean frequency of one peak per 4 min. This activity was blocked in the absence of extracellular calcium but was not modified after depletion of calcium stores with thapsigargin or blockade of voltage-gated calcium channels with Ni2+. Further, statistical analysis of calcium activity revealed concomitant activation of distant cells in 24 slices, and the existence of a significant network of synchrony based on such coactivations in 17 slices out of 28. These networks enclosed 84.3% of active cells, scattered throughout the neocortical wall (mean distance between cellular bodies, 111.7 microm). Finally, it was possible to identify specific cells which were synchronously active with more neighbouring cells than others. The identity of these nodal cells remains to be investigated to fully comprehend the role of spontaneous calcium activity, before synaptogenesis, in shaping cortical neurogenesis.

Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane.

Maurocalcine (MCa) is a 33-amino-acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. External application of MCa to cultured myotubes is known to produce Ca2+ release from intracellular stores. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long lasting channel openings in a mode of smaller conductance. Here we investigated the way MCa proceeds to cross biological membranes to reach its target. A biotinylated derivative of MCa was produced (MCa(b)) and complexed with a fluorescent indicator (streptavidine-cyanine 3) to follow the cell penetration of the toxin. The toxin complex efficiently penetrated into various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called cell-penetrating peptides. Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1-2 min), making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.

Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.

P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

Early expression of sodium channel transcripts and sodium current by cajal-retzius cells in the preplate of the embryonic mouse neocortex.

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Na(v)1.3 (SCN3) Na channel isoform.

Two EPR-detectable [4Fe-4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus.

NADH:ubiquinone oxidoreductases (Complex I) contain a subunit, TYKY in the bovine enzyme and NuoI in the enzyme from Rhodobacter capsulatus, which is assumed to bind two [4Fe-4S] clusters because it contains two sets of conserved cysteine motifs similar to those found in the 2[4Fe-4S] ferredoxins. It was recently shown that the TYKY subunit is not an ordinary 2[4Fe-4S] ferredoxin, but has a unique amino acid sequence, which is only found in NAD(P)H:quinone oxidoreductases and certain membrane-bound [NiFe]-hydrogenases expected to be involved in redox-linked proton translocation [FEBS Lett. 485 (2000) 1]. We have generated a set of R. capsulatus mutants in which five out of the eight conserved cysteine residues in NuoI were replaced by other amino acids. The resulting mutants fell into three categories with virtually no, intermediate or quite normal Complex I activities. EPR-spectroscopic analysis of the membranes of the C67S and C106S mutants, two mutants belonging to the second and third group, respectively, showed a specific 50% decrease of the EPR signal attributed to cluster N2. It is concluded that the NuoI (TYKY) subunit binds two clusters N2, called N2a and N2b, which exhibit very similar spectral features when analyzed by X-band EPR spectroscopy.

[Is merging organizations the solution to service integration?]. / L'intégration des services passe-t-elle par des fusions d'établissements?

Organizational models inherited from the time when institutionalizing patients was common practice are now obsolete. Service coordination, undergone earlier within a same institution, is now governed by professionnals linked to various organizations and different settings. One of the difficulties of integration of these services is that contributing organizations often continue to operate individually, consequently going against the pursued goal. The solution most often proposed to counter this compartimentalization consists in reinforcing integration of organizations, which will in time favor integration of various services. One of the ways to realize this organizational integration is to reduce the number of existing organizations, by merging for example, a certain number of them. It is this path of hierarchical organization that the Québec health care system has taken during the 1990. Other paths that of networking have also been experienced during the same period. To judge of the relevance of these options, the authors propose a reversal of the dominating perspective according to which service integration necessarily pass through installing mecanisms of organizational integration. Withought minimizing the importance of such mecanisms, the authors put forth that integration of services commands first and foremost collaboration between professional health workers. Through this angle, the relevance of an organizational integration model depends on the impact it will have on the capacity and willingness of health workers to work in a concerted way. The authors submit that the fact of giving to a sole authority the govern of existing organizations is not the panacea some would like to believe.