Two-dimensional reversed-phase liquid chromatography coupled to MALDI TOF/TOF mass spectrometry : an approach for shotgun proteome analysis

Large-scale proteome analysis of complex biological mixtures requires high resolving power separations, high-throughput mass spectrometry and accurate detection, and bioinformatics tools. In the present work LC-MALDI TOF/TOF mass spectrometry was combined for the first time with a two-dimensional separation based on reversed-phase chromatography at high and low pH for the analysis of the cytosolic proteome of Corynebacterium glutamicum. The proteome coverage achieved by this approach, 55%, is the highest reported up to day for this bacterium. A total of 1644 proteins including single-peptide based identifications were identified. The sample was also analysed by other conventional methods in the proteome analysis. The classical 2D-PAGE approach presents more limitations and delivered 166 different proteins including enzymes of the main metabolic pathways. Complementary results were found at peptide level for the LC-MALDI and 2D-PAGE approaches. Fractions collected during the first dimension at high pH were analysed by LC-MALDI MS and compared with the analysis carried out by LC-ESI-IT MS, which identified 745 proteins [1]. Further comparison with results found in literature confirmed that the two-dimensional reversed phase combination with LC-MALDI MS/MS is a promising tool in the proteome analysis. The MALDI based approach showed higher sensitivity than other approaches and was able to identify proteins over a larger dynamic range, based on the codon adaptation indexes of the proteins.An die Large-Scale Proteomanalyse von komplexen biologischen Mischungen sind eine Reihe von Anforderungen gestellt. Unter Anderem sind Trennungen mit hohem Auflösungsvermögen, High-Throughput Massenspektrometrie, präzise Massenbestimmung und Bioinformatik-Tools erforderlich. Gegenstand der vorliegenden Arbeit ist die Evaluierung der Kombination aus LC-MALDI-TOF/TOF Massenspektrometrie mit einer zwei-dimensionalen Trennung basierend auf Reverse-Phase-Chromatographie bei hohen und niedrigen pH-Werten. Des cytosolischen Proteoms von Corynebacterium glutamicum wurde mit der Methode analysiert. Dies ermöglichte die Indentifizierung von 55% des Proteoms von Corynebacterium glutamicum und ist damit der nach momentanem Kenntnisstand effizienteste Ansatz. Insgesamt konnten 1644 verschiedene Proteine identifiziert werden. Konventionelle Methoden der Proteomanalyse erreichten deutlich schlechtere Ergebnisse. So erzielte der klassische 2D-PAGE-Ansatz bei deutlich mehr Einschränkungen nur 166 Proteinidentifikationen inklusive wichtiger Enzyme der Stoffwechselwege. Komplementäre Ergebnisse konnten auf der Peptid-Ebene für LC-MALDI und 2D-PAGE Ansätze gefunden werden. Fraktionen der ersten Dimension bei hohem pH-Wert mittels LC-MALDI MS wurden analysiert und mit Ergebnissen aus LC-ESI-IT MS [1] Untersuchungen verglichen. Es hat sich gezeigt, dass der MALDI-basierte Ansatz höhere Sensitivität aufweist und in der Lage war Proteine über eine weitaus größere dynamische Reichweite bezogen auf den Codon Adaptation Index zu identifizieren

Mutual information between heart rate variability and respiration for emotion characterization

Objective: Interest in emotion recognition has increased in recent years as a useful tool for diagnosing psycho-neural illnesses. In this study, the auto-mutual and the cross-mutual information function, AMIF and CMIF respectively, are used for human emotion recognition. Approach: The AMIF technique was applied to heart rate variability (HRV) signals to study complex interdependencies, and the CMIF technique was considered to quantify the complex coupling between HRV and respiratory signals. Both algorithms were adapted to short-term RR time series. Traditional band pass filtering was applied to the RR series at low frequency (LF) and high frequency (HF) bands, and a respiration-based filter bandwidth was also investigated (). Both the AMIF and the CMIF algorithms were calculated with regard to different time scales as specific complexity measures. The ability of the parameters derived from the AMIF and the CMIF to discriminate emotions was evaluated on a database of video-induced emotion elicitation. Five elicited states i.e. relax (neutral), joy (positive valence), as well as fear, sadness and anger (negative valences) were considered. Main results: The results revealed that the AMIF applied to the RR time series filtered in the band was able to discriminate between the following: relax and joy and fear, joy and each negative valence conditions, and finally fear and sadness and anger, all with a statistical significance level p¿-value 0.05, sensitivity, specificity and accuracy higher than 70% and area under the receiver operating characteristic curve index AUC 0.70. Furthermore, the parameters derived from the AMIF and the CMIF allowed the low signal complexity presented during fear to be characterized in front of any of the studied elicited states. Significance: Based on these results, human emotion manifested in the HRV and respiratory signal responses could be characterized by means of the information-content complexityPeer ReviewedPostprint (author's final draft

Prognostic value of biomarkers after cardiopulmonary bypass in pediatrics: The prospective PANCAP study

Objective:To assess the usefulness of procalcitonin, pro-adrenomedullin and pro-atrial natriuretic peptide as predictors of need for mechanical ventilation and postoperative complications (need for inotropic support and bacterial infection) in critically ill pediatric patients after cardiopulmonary bypass. Design:A prospective, observational study Setting: Pediatric intensive care unit. Patients: Patients under 18 years old admitted after cardiopulmonary bypass. Measuraments and main results: Serum levels of procalcitonin, pro-adrenomedullin and pro-atrial natriuretic peptide were determined immediately after bypass and at 24-36 hours. Their values were correlated with the need for mechanical ventilation, inotropic support and bacterial infection. One hundred eleven patients were recruited. Septal defects (30.6%) and cardiac valve disease (17.1%) were the most frequent pathologies. 40.7% required mechanical ventilation, 94.6% inotropic support and 15.3% presented invasive bacterial infections. Pro-adrenomedullin and pro-atrial natriuretic peptide showed significant high values in patients needing mechanical ventilation. Cut-off values higher than 1.22 nmol/L and 215.3 pmol/L, respectively for each biomarker, may indicate need for mechanical ventilation with an AUC of 0.721 and 0.746 at admission and 0.738 and 0.753 at 24-36 hours, respectively but without statistical differences. Pro-adrenomedullin and procalcitonin showed statistically significant high values in patients with bacterial infections. Conclusions: After bypass, pro-adrenomedullin and pro-atrial natriuretic peptide are suitable biomarkers to predict the need for mechanical ventilation. Physicians should be alert if the values of these markers are high so as not to progress to early extubation. Procalcitonin is useful for predicting bacterial infection. This is a preliminary study and more clinical studies should be done to confirm the value of pro-adrenomedullin and pro-atrial natriuretic peptide as biomarkers after cardiopulmonary bypass

Patterns of sedentary behavior in overweight and moderately obese users of the Catalan primary-health care system

Background and objectives Prolonged sitting time (ST) has negative consequences on health. Changing this behavior is paramount in overweight/obese individuals because they are more sedentary than those with normal weight. The aim of the study was to establish the pattern of sedentary behavior and its relationship to health, socio-demographics, occupation, and education level in Catalan overweight/obese individuals. Methods A descriptive study was performed at 25 healthcare centers in Catalonia (Spain) with 464 overweight/moderately obese patients, aged25 to 65 years. Exclusion criteria were chronic diseases which contraindicated physical activity and language barriers. Face-to-face interviews were conducted to collect data on age, gender, educational level, social class, and marital status. Main outcome was ‘sitting time’ (collected by the Marshall questionnaire); chronic diseases and anthropometric measurements were registered. Results 464 patients, 58.4% women, mean age 51.9 years (SD 10.1), 76.1% married, 60% manual workers, and 48.7% had finished secondary education. Daily sitting time was 6.2 hours on working days (374 minutes/day, SD: 190), and about 6 hours on non-working ones (357 minutes/day, SD: 170). 50% of participants were sedentary 6 hours. The most frequent sedentary activities were: working/academic activities around 2 hours (128 minutes, SD: 183), followed by watching television, computer use, and commuting. Men sat longer than women (64 minutes more on working days and 54 minutes on non-working days), and individuals with office jobs (91 minutes), those with higher levels of education (42 minutes), and younger subjects (25 to 35 years) spent more time sitting. Conclusions In our study performed in overweight/moderately obese patients the mean sitting time was around 6 hours which was mainly spent doing work/academic activities and watching television. Men, office workers, individuals with higher education, and younger subjects had longer sitting time. Our results may help design interventions targeted at these sedentary patients to decrease sitting time.

Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases

Zweidimensionale Reversed-Phase-Flüssigchromatographie gekoppelt mit MALDI TOF/TOF Massenspektrometrie : eine Methode für die Shotgun-Proteomanalyse

Large-scale proteome analysis of complex biological mixtures requires high resolving power separations, high-throughput mass spectrometry and accurate detection, and bioinformatics tools. In the present work LC-MALDI TOF/TOF mass spectrometry was combined for the first time with a two-dimensional separation based on reversed-phase chromatography at high and low pH for the analysis of the cytosolic proteome of Corynebacterium glutamicum. The proteome coverage achieved by this approach, 55%, is the highest reported up to day for this bacterium. A total of 1644 proteins including single-peptide based identifications were identified. The sample was also analysed by other conventional methods in the proteome analysis. The classical 2D-PAGE approach presents more limitations and delivered 166 different proteins including enzymes of the main metabolic pathways. Complementary results were found at peptide level for the LC-MALDI and 2D-PAGE approaches. Fractions collected during the first dimension at high pH were analysed by LC-MALDI MS and compared with the analysis carried out by LC-ESI-IT MS, which identified 745 proteins [1]. Further comparison with results found in literature confirmed that the two-dimensional reversed phase combination with LC-MALDI MS/MS is a promising tool in the proteome analysis. The MALDI based approach showed higher sensitivity than other approaches and was able to identify proteins over a larger dynamic range, based on the codon adaptation indexes of the proteins.An die Large-Scale Proteomanalyse von komplexen biologischen Mischungen sind eine Reihe von Anforderungen gestellt. Unter Anderem sind Trennungen mit hohem Auflösungsvermögen, High-Throughput Massenspektrometrie, präzise Massenbestimmung und Bioinformatik-Tools erforderlich. Gegenstand der vorliegenden Arbeit ist die Evaluierung der Kombination aus LC-MALDI-TOF/TOF Massenspektrometrie mit einer zwei-dimensionalen Trennung basierend auf Reverse-Phase-Chromatographie bei hohen und niedrigen pH-Werten. Des cytosolischen Proteoms von Corynebacterium glutamicum wurde mit der Methode analysiert. Dies ermöglichte die Indentifizierung von 55% des Proteoms von Corynebacterium glutamicum und ist damit der nach momentanem Kenntnisstand effizienteste Ansatz. Insgesamt konnten 1644 verschiedene Proteine identifiziert werden. Konventionelle Methoden der Proteomanalyse erreichten deutlich schlechtere Ergebnisse. So erzielte der klassische 2D-PAGE-Ansatz bei deutlich mehr Einschränkungen nur 166 Proteinidentifikationen inklusive wichtiger Enzyme der Stoffwechselwege. Komplementäre Ergebnisse konnten auf der Peptid-Ebene für LC-MALDI und 2D-PAGE Ansätze gefunden werden. Fraktionen der ersten Dimension bei hohem pH-Wert mittels LC-MALDI MS wurden analysiert und mit Ergebnissen aus LC-ESI-IT MS [1] Untersuchungen verglichen. Es hat sich gezeigt, dass der MALDI-basierte Ansatz höhere Sensitivität aufweist und in der Lage war Proteine über eine weitaus größere dynamische Reichweite bezogen auf den Codon Adaptation Index zu identifizieren

Mutual information between heart rate variability and respiration for emotion characterization

Objective: Interest in emotion recognition has increased in recent years as a useful tool for diagnosing psycho-neural illnesses. In this study, the auto-mutual and the cross-mutual information function, AMIF and CMIF respectively, are used for human emotion recognition. Approach: The AMIF technique was applied to heart rate variability (HRV) signals to study complex interdependencies, and the CMIF technique was considered to quantify the complex coupling between HRV and respiratory signals. Both algorithms were adapted to short-term RR time series. Traditional band pass filtering was applied to the RR series at low frequency (LF) and high frequency (HF) bands, and a respiration-based filter bandwidth was also investigated (). Both the AMIF and the CMIF algorithms were calculated with regard to different time scales as specific complexity measures. The ability of the parameters derived from the AMIF and the CMIF to discriminate emotions was evaluated on a database of video-induced emotion elicitation. Five elicited states i.e. relax (neutral), joy (positive valence), as well as fear, sadness and anger (negative valences) were considered. Main results: The results revealed that the AMIF applied to the RR time series filtered in the band was able to discriminate between the following: relax and joy and fear, joy and each negative valence conditions, and finally fear and sadness and anger, all with a statistical significance level p¿-value 0.05, sensitivity, specificity and accuracy higher than 70% and area under the receiver operating characteristic curve index AUC 0.70. Furthermore, the parameters derived from the AMIF and the CMIF allowed the low signal complexity presented during fear to be characterized in front of any of the studied elicited states. Significance: Based on these results, human emotion manifested in the HRV and respiratory signal responses could be characterized by means of the information-content complexityPeer Reviewe

Ethical attitudes of intensive care paediatricians as regards patients with spinal muscular atrophy type 1

Introduction: Spinal muscular atrophy type 1 (SMA-1) is a progressive and fatal disease that leads to ethical problems for Paediatric professionals. Our objective was to determine the ethical options of Paediatric Intensive Care Unit (PICU) paediatricians as regards a child with SMA-1 and respiratory failure. Material and methods: A cross-sectional descriptive study was conducted using an anonymous questionnaire sent to PICUs in Spain (which can be accessed through the Spanish Society of Paediatric Critical Care web page). Results: Of the 124 responses analysed, 70% were from women, 51% younger than 40 years, 54% from a PICU with more than 10 beds, 69% with prior experience in such cases, and 53% with religious beliefs. In the last patient cared for, most paediatricians opted for non-invasive mechanical ventilation (NIV) and limitation of therapeutic effort (LET) in case of NW failure. Confronted with a future hypothetical case, half of paediatricians would opt for the same plan (NIV + LET), and 74% would support the family's decision, even in case of disagreement. Age, prior experience and sex were not related to the preferred options. Paediatricians with religious beliefs were less in favour of initial LET. Less than two-thirds (63%) scored the quality of life of a child with SMA-1 and invasive mechanical ventilation as very poor. Conclusions: Faced with child with SMA-1 and respiratory failure, most paediatricians are in favour of initiating NIV and LET when such support is insufficient, but they would accept the family's decision, even in case of disagreement. (C) 2015 AsociaciOn Espanola de Pediatria. Published by Elsevier Espana, S.L.U. All rights reserved