Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis

The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid (OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as model species. Shellfish matrix loading limits for the PPIA have been established, according to the shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48). Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs. The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Diagnosis of tuberculosis infection using interferon-γ-based assays

Interferon-γ-based assays, collectively known as IFN-γ release assays (IGRAs), have emerged as a reliable alternative to the old tuberculin skin test (TST) for the immunodiagnosis of tuberculosis (TB) infection. The 2 commercially available tests, the enzyme-linked immunosorbent assay (ELISA), QuantiFERON-TB Gold Intube (QFT-IT), and the enzyme-linked immunospot assay (ELISPOT), T-SPOT.TB, are more accurate than TST for the diagnosis of TB, since they are highly specific and correlate better with the existence of risk factors for the infection. According to the available data, T-SPOT.TB obtains a higher number of positive results than QFT-IT, while its specificity seems to be lower. Although the sensitivity of the IFN-γ -based assays may be impaired to some extent by cellular immunosuppression and extreme ages of life, they perform better than TST in these situations. Data from longitudinal studies suggest that IFN-γ-based tests are better predictors of subsequent development of active TB than TST; however this prognostic value has not been consistently demonstrated. This review focuses on the clinical use of the IFN-γ -based tests in different risk TB groups, and notes the main limitations and areas for future development.

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Multifunctionality and Diversity in Bacterial Biofilms

Bacteria are highly diverse and drive a bulk of ecosystem processes. Analysis of relationships between diversity and single specific ecosystem processes neglects the possibility that different species perform multiple functions at the same time. The degradation of dissolved organic carbon (DOC) followed by respiration is a key bacterial function that is modulated by the availability of DOC and the capability to produce extracellular enzymes. In freshwater ecosystems, biofilms are metabolic hotspots and major sites of DOC degradation. We manipulated the diversity of biofilm forming communities which were fed with DOC differing in availability. We characterized community composition using molecular fingerprinting (T-RFLP) and measured functioning as oxygen consumption rates, the conversion of DOC in the medium, bacterial abundance and the activities of five specific enzymes. Based on assays of the extracellular enzyme activity, we calculated how the likelihood of sustaining multiple functions was affected by reduced diversity. Carbon source and biofilm age were strong drivers of community functioning, and we demonstrate how the likelihood of sustaining multifunctionality decreases with decreasing diversity

Stable complexes involving acetylcholinesterase and amyloid-ß-peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

Prediction of enzyme function by combining sequence similarity and protein interactions

Background: A number of studies have used protein interaction data alone for protein function prediction. Here, we introduce a computational approach for annotation of enzymes, based on the observation that similar protein sequences are more likely to perform the same function if they share similar interacting partners. Results: The method has been tested against the PSI-BLAST program using a set of 3,890 protein sequences from which interaction data was available. For protein sequences that align with at least 40% sequence identity to a known enzyme, the specificity of our method in predicting the first three EC digits increased from 80% to 90% at 80% coverage when compared to PSI-BLAST. Conclusion: Our method can also be used in proteins for which homologous sequences with known interacting partners can be detected. Thus, our method could increase 10% the specificity of genome-wide enzyme predictions based on sequence matching by PSI-BLAST alone.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

Degradation of pheromone and plant volatile components by a same Odorant-Degrading Enzyme in the cotton leafworm, Spodoptera littoralis

Background: Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis , a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis

Introduction: Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA. Methods: Samples from 307 blood donors and patients with RA (322), psoriatic arthritis (133), systemic lupus erythematosus (119), and hepatitis C infection (84) were assayed by using CFFCP- and CCP2-based tests. Autoantibodies also were analyzed at baseline and during a 2-year follow-up in 98 early RA patients to determine their prognostic value. Results: With cutoffs giving 98% specificity for RA versus blood donors, the sensitivity was 72.1% for CFFCP1, 78.0% for CFFCP2, 71.4% for CFFCP3, and 73.9% for CCP2, with positive predictive values greater than 97% in all cases. CFFCP sensitivity in RA increased to 80.4% without losing specificity when positivity was considered as any positive anti-CFFCP status. Specificity of the three CFFCP tests versus other rheumatic populations was high (> 90%) and similar to those for the CCP2. In early RA, CFFCP1 best identified patients with a poor radiographic outcome. Radiographic progression was faster in the small subgroup of CCP2-negative and CFFCP1-positive patients than in those negative for both autoantibodies. CFFCP antibodies decreased after 1 year, but without any correlation with changes in disease activity. Conclusions: CFFCP-based assays are highly sensitive and specific for RA. Early RA patients with anti-CFFCP1 antibodies, including CCP2-negative patients, show greater radiographic progression.

Interferon-gamma release assays for the diagnosis of tuberculosis and tuberculosis infection in HIV-infected adults: a systematic review and meta-analysis.

Background: Despite the widespread use of interferon-gamma release assays (IGRAs), their role in diagnosing tuberculosis and targeting preventive therapy in HIV-infected patients remains unclear. We conducted a comprehensive systematic review to contribute to the evidence-based practice in HIV-infected people. Methodology/Principal Findings: We searched MEDLINE, Cochrane, and Biomedicine databases to identify articles published between January 2005 and July 2011 that assessed QuantiFERON H -TB Gold In-Tube (QFT-GIT) and T-SPOT H .TB (T-SPOT.TB) in HIV-infected adults. We assessed their accuracy for the diagnosis of tuberculosis and incident active tuberculosis, and the proportion of indeterminate results. The search identified 38 evaluable studies covering a total of 6514 HIV-infected participants. The pooled sensitivity and specificity for tuberculosis were 61% and 72% for QFT-GIT, and 65% and 70% for T-SPOT.TB. The cumulative incidence of subsequent active tuberculosis was 8.3% for QFT-GIT and 10% for T-SPOT.TB in patients tested positive (one study each), and 0% for QFT-GIT (two studies) and T-SPOT.TB (one study) respectively in those tested negative. Pooled indeterminate rates were 8.2% for QFT-GIT and 5.9% for T-SPOT.TB. Rates were higher in high burden settings (12.0% for QFT-GIT and 7.7% for T-SPOT.TB) than in low-intermediate burden settings (3.9% for QFT-GIT and 4.3% for T-SPOT.TB). They were also higher in patients with CD4 + T-cell count, 200 (11.6% for QFT-GIT and 11.4% for T-SPOT.TB) than in those with CD4 + T-cell count $ 200 (3.1% for QFT-GIT and 7.9% for T-SPOT.TB). Conclusions/Significance: IGRAs have suboptimal accuracy for confirming or ruling out active tuberculosis disease in HIV-infected adults. While their predictive value for incident active tuberculosis is modest, a negative QFT-GIT implies a very low short- to medium-term risk. Identifying the factors associated with indeterminate results will help to optimize the use of IGRAs in clinical practice, particularly in resource-limited countries with a high prevalence of HIV-coinfection.

Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

Background: Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results: We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA) representation. The optimization task is posed as a nonconvex nonlinear programming (NLP) problem that is solved by an outer- approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP) master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion: Our results show the utility of the suggested approach for investigating the evolution of adaptive responses to environmental changes. The proposed method can be used in other important applications such as the evaluation of parameter changes that are compatible with health and disease states.

Metabolomic assays of amoxicillin, cephapirin and ceftiofur in chicken muscle. Application to treated chicken samples by liquid chromatography quadrupole time-of-flight mass spectrometry

The aim of this study was to identity metabolites and transformation products (TPs) in chicken muscle from amoxicillin (AMX), cephapirin (PIR) and ceftiofur (TIO), which are antibiotics of the β-lactam family. Liquid chromatography coupled to quadrupole time-of-flight (QqTOF) mass spectrometry was utilized due to its high resolution, high mass accuracy and MS/MS capacity for elemental composition determination and structural elucidation. Amoxicilloic acid (AMA) and amoxicillin diketopiperazine (DKP) were found as transformation products from AMX. Desacetylcephapirin (DAC) was detected as a metabolite of PIR. Desfuroylceftiofur (DFC) and its conjugated compound with cysteine (DFC-S-Cys) were detected as a result of TIO in contact with chicken muscle tissue. The metabolites and transformation products were also monitored during the in vivo AMX treatment and slaughtering period. It was found that two days were enough to eliminate AMX and associated metabolites/transformation products after the end of administration.

Hepatocyte nuclear factor 4alpha transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5′-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.