Getting Ready for Large-Scale Proteomics in Crop Plants

Plants are an indispensable cornerstone of sustainable global food supply. While immense progress has been made in decoding the genomes of crops in recent decades, the composition of their proteomes, the entirety of all expressed proteins of a species, is virtually unknown. In contrast to the model plant Arabidopsis thaliana, proteomic analyses of crop plants have often been hindered by the presence of extreme concentrations of secondary metabolites such as pigments, phenolic compounds, lipids, carbohydrates or terpenes. As a consequence, crop proteomic experiments have, thus far, required individually optimized protein extraction protocols to obtain samples of acceptable quality for downstream analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). In this article, we present a universal protein extraction protocol originally developed for gel-based experiments and combined it with an automated single-pot solid-phase-enhanced sample preparation (SP3) protocol on a liquid handling robot to prepare high-quality samples for proteomic analysis of crop plants. We also report an automated offline peptide separation protocol and optimized micro-LC-MS/MS conditions that enables the identification and quantification of ~10,000 proteins from plant tissue within 6 h of instrument time. We illustrate the utility of the workflow by analyzing the proteomes of mature tomato fruits to an unprecedented depth. The data demonstrate the robustness of the approach which we propose for use in upcoming large-scale projects that aim to map crop tissue proteomes

Triglycerides and glycated hemoglobin for screening insulin resistance in obese patients

International audienceOBJECTIVE: Assessment of insulin resistance (IR) is essential in non-diabetic patients with obesity. Thus study aims to identify the best determinants of IR and to propose an original approach for routine assessment of IR in obesity. DESIGN AND PATIENTS: All adult with obesity defined by a body mass index >=30kg/m2, evaluated in the Nutrition Department between January 2010 and January 2015 were included in this cross-sectional study. Patients with diabetes were excluded. IR was diagnosed according to the HOMA-IR. Based on a logistic regression, we determined a composite score of IR. We then tested the variables with a principal component analysis and a hierarchical clustering analysis. RESULTS: A total of 498 patients with obesity were included. IR was associated with grade III obesity (OR=2.6[1.6-4.4], p\textless0.001), HbA1c>=5.7% (OR=2.6[1.7-4.0], p\textless0.001), hypertriglyceridemia \textgreater1.7mmol/l (OR=3.0[2.0-4.5], p\textless0.001) and age (OR=0.98[0.96-0.99], p=0.002). Exploratory visual analysis using factor map and clustering analysis revealed that lipid and carbohydrates metabolism abnormalities were correlated with insulin resistance but not with excessive fat accumulation and low-grade inflammation. CONCLUSIONS: Our results highlight the interest of simple blood tests such as HbA1c and triglyceride determination, which associated with BMI, may be widely available tools for screening IR in obese patients

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aβ1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aβ deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease

Enzymatic creatinine assays allowestimation of glomerular filtration rate in stages 1 and 2 chronic kidney disease using CKD-EPI equation

The National Kidney Disease Education Program group demonstrated that MDRD equation is sensitive to creatinine measurement error, particularly at higher glomerular filtration rates. Thus, MDRD-based eGFR above 60 mL/min/1.73 m2 should not be reported numerically. However, little is known about the impact of analytical error on CKD-EPI-based estimates. This study aimed at assessing the impact of analytical characteristics (bias and imprecision) of 12 enzymatic and 4 compensated Jaffe previously characterized creatinine assays on MDRD and CKD-EPI eGFR. In a simulation study, the impact of analytical error was assessed on a hospital population of 24 084 patients. Ability using each assay to correctly classify patients according to chronic kidney disease (CKD) stages was evaluated. For eGFR between 60 and 90 mL/min/1.73 m2, both equations were sensitive to analytical error. Compensated Jaffe assays displayed high bias in this range and led to poorer sensitivity/specificity for classification according to CKD stages than enzymatic assays. As compared to MDRD equation, CKD-EPI equation decreases impact of analytical error in creatinine measurement above 90 mL/min/1.73 m2. Compensated Jaffe creatinine assays lead to important errors in eGFR and should be avoided. Accurate enzymatic assays allow estimation of eGFR until 90 mL/min/1.73 m2 with MDRD and 120 mL/min/1.73 m2 with CKD-EPI equation.Peer reviewe

Transcriptional Response of Zebrafish Embryos Exposed to Neurotoxic Compounds Reveals a Muscle Activity Dependent hspb11 Expression

Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sopfixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds

Biomarkers of metabolic and cardiovascular risks

Il existe des interactions profondes entre les fonctions rénales et cardiaques. Un dysfonctionnement de l'un ou l'autre de ces deux organes entraîne un dysfonctionnement du second. Ces interactions entre cœur et rein sont regroupées sous le terme de syndromes cardio-rénaux. Les biomarqueurs sont, par définition des indicateurs objectivement mesurés d'un processus biologique normal, d'un processus pathologique ou d'une réponse pharmacologique à une intervention thérapeutique. Les marqueurs biologiques dont très utiles à l'exploration des phénomènes pathologiques cardiaques et rénaux. En pratique clinique, la fonction rénale est quotidiennement estimée à partir de biomarqueurs de filtration glomérulaire, la créatinine et la cystatine C en premier lieu. Dans l'exploration des fonctions cardiaques, des évolutions importantes ont eu lieu ces dernières années. Au niveau analytique, des améliorations significatives des performances ont abouti à la mise sur le marché de méthodes dites hypersensibles de mesure de la troponine. De plus de nouveaux marqueurs explorant de nouveaux aspects physiopathologiques de la dysfonction cardiaque sont également intensément étudiés, tels que les marqueurs de fibrose (ST2 soluble, galectine-3). Après une revue de la bibliographie des biomarqueurs rénaux et cardiaques, ce travail s'attache à l'étude de l'optimisation de l'usage des biomarqueurs rénaux et cardiaques, tout d'abord par la maîtrise des procédés analytiques puis dans l'utilisation de ceux-ci en pratique clinique. Dans une première partie consacrée aux marqueurs rénaux, nous cherchons à optimiser l'utilisation des marqueurs permettant d'estimer au mieux le débit de filtration glomérulaire, meilleur index connu de la fonction rénale. En pratique clinique, le débit de filtration glomérulaire est estimé à partir de la créatinine. Au niveau analytique, nous montrons dans ce travail que les méthodes colorimétriques, basées sur la réaction de Jaffé, devraient désormais être abandonnées au profit des méthode enzymatiques. Ces résultats sont illustrés dans différentes populations de patients, une cohorte issue des bases de données hospitalières ainsi qu'une population de patients cirrhotiques. Chez ces derniers, la créatinine présente d'importantes limites en tant que marqueur de filtration glomérulaire, en particulier en raison d'une diminution de la masse musculaire. La cystatine C représente dans ce contexte une alternative intéressante puisque ce marqueur n'est pas dépendant de la masse musculaire. Des algorithmes utilisant la cystatine C, seule ou en association avec la créatinine ont récemment été proposés dans la littérature. Dans une seconde partie, nous nous intéressons aux marqueurs cardiaques. Les troponines cardiaques sont des protéines présentes au niveau de l'appareil contractile du cardiomyocyte, relarguées dans la circulation en cas de nécrose cellulaire. L'arrivée récente de méthodes capables de mesurer des concentrations circulantes dans une part importante de la population saine a obligé d'une part à un contrôle strict des procédés analytiques par les fabricant et d'autre part à une adaptation des cliniciens afin de tirer partie des nouvelles informations apportées par ces marqueurs dans différentes populations présentant des élévations chroniques (sujets âgés, insuffisants rénaux chroniques) ou aiguës (cinétiques post infarctus du myocarde). Enfin, une étude concernant le ST2 soluble, marqueur émergent de fibrose dans l'insuffisance cardiaque est également présentée. En conclusion, l'optimisation de l'usage des biomarqueurs s'oriente à l'heure actuelle vers des stratégies multimarqueurs, comme l'association créatinine et cystatine C dans l'estimation du débit de filtration glomérulaire ou le développement de scores pronostics associant troponine, peptides natriurétiques et ST2 soluble dans l'insuffisance cardiaque.Profound interactions exist between cardiac and renal functions. Acute or chronic dysfunction of an organ may induce acute or chronic dysfunction of the other. These complex interactions have been grouped under the term cardiorenal syndrome.A Biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biological markers are useful for the exploration of pathological renal and cardiac phenomenon. In clinical practice, renal function is estimated from glomerular filtration markers, mainly creatinine and cystatin C. Much progress has recently been made recently toward exploration of cardiac dysfunction. From an analytical point of vue, improvement in measurement of cardiac troponine led to the so-called hypersensitive cardiac troponin assays. Furthermore, new markers of cardiac dysfunction are under initensive inverstigation. These markers(soluble ST2, galectin-3) provide information regarding specific pathophysiological pathways, such as fibrosis.After a review of the litterature regarding cardiac and renal biomarkers, this work aims at optimize the interpretation of abovementioned biological markers.In the fist part, consacred to renal markers, this work tries to optimize the estimation of glomerular filtration rate, firstly regarding analytical process then in clinical practice. Glomerular filtration rate is in clincal practice derived from creatinine level. Analytically, our work indaicates that compensated Jaffe methods for the measurement of creatinine should be replaced with enzymatic ones, which are muche more performant. These conclusions have been drawn in different populations, from hospital databases and in cirrhotic patients. In this population , creatinine as a filtration marker suffers from important limitations, mainly beacause of the loss of muscle mass observed in these patients. Cystatin C is an alternative filtration marker whose level is independent of muscle mass. Some algorithms predicting glomerular filtration rate from cystatin C, sole or in association with creatinine have recently been proposed.The second part of this work is consacred to cardiac markers. Cardiac troponins , proteins which are part of the contractile apparatus, are released in the blood flow in case of necrosis. The recent improvement of analytical methods, enabling measurement of cardiac troponine levels in at leats 50% of a healthy reference population require a precise control of manufacturing process. Furthermore, hypersensitive troponins require from physician an exact interpretation in patients with chronic (elderly, chronic kidney disease patients) or acute (post myocardial infarction kinetics) elevation. A study regarding soluble ST2, a n emerging marker of cardiac fibrosis, is also presentedOptimization of the use of biomarkers move nowadays toward multimarkers strategies as illustrated by approaches combining cystatin C with creatinine for estimating glomerular filtration rate or the development of scores for predicting mortality risk in heart failure patients based on cardiac troponins, natriuretic peptides and soluble ST2

Biomarqueurs des risques cardiaque et métabolique

Profound interactions exist between cardiac and renal functions. Acute or chronic dysfunction of an organ may induce acute or chronic dysfunction of the other. These complex interactions have been grouped under the term cardiorenal syndrome.A Biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biological markers are useful for the exploration of pathological renal and cardiac phenomenon. In clinical practice, renal function is estimated from glomerular filtration markers, mainly creatinine and cystatin C. Much progress has recently been made recently toward exploration of cardiac dysfunction. From an analytical point of vue, improvement in measurement of cardiac troponine led to the so-called hypersensitive cardiac troponin assays. Furthermore, new markers of cardiac dysfunction are under initensive inverstigation. These markers(soluble ST2, galectin-3) provide information regarding specific pathophysiological pathways, such as fibrosis.After a review of the litterature regarding cardiac and renal biomarkers, this work aims at optimize the interpretation of abovementioned biological markers.In the fist part, consacred to renal markers, this work tries to optimize the estimation of glomerular filtration rate, firstly regarding analytical process then in clinical practice. Glomerular filtration rate is in clincal practice derived from creatinine level. Analytically, our work indaicates that compensated Jaffe methods for the measurement of creatinine should be replaced with enzymatic ones, which are muche more performant. These conclusions have been drawn in different populations, from hospital databases and in cirrhotic patients. In this population , creatinine as a filtration marker suffers from important limitations, mainly beacause of the loss of muscle mass observed in these patients. Cystatin C is an alternative filtration marker whose level is independent of muscle mass. Some algorithms predicting glomerular filtration rate from cystatin C, sole or in association with creatinine have recently been proposed.The second part of this work is consacred to cardiac markers. Cardiac troponins , proteins which are part of the contractile apparatus, are released in the blood flow in case of necrosis. The recent improvement of analytical methods, enabling measurement of cardiac troponine levels in at leats 50% of a healthy reference population require a precise control of manufacturing process. Furthermore, hypersensitive troponins require from physician an exact interpretation in patients with chronic (elderly, chronic kidney disease patients) or acute (post myocardial infarction kinetics) elevation. A study regarding soluble ST2, a n emerging marker of cardiac fibrosis, is also presentedOptimization of the use of biomarkers move nowadays toward multimarkers strategies as illustrated by approaches combining cystatin C with creatinine for estimating glomerular filtration rate or the development of scores for predicting mortality risk in heart failure patients based on cardiac troponins, natriuretic peptides and soluble ST2.Il existe des interactions profondes entre les fonctions rénales et cardiaques. Un dysfonctionnement de l'un ou l'autre de ces deux organes entraîne un dysfonctionnement du second. Ces interactions entre cœur et rein sont regroupées sous le terme de syndromes cardio-rénaux. Les biomarqueurs sont, par définition des indicateurs objectivement mesurés d'un processus biologique normal, d'un processus pathologique ou d'une réponse pharmacologique à une intervention thérapeutique. Les marqueurs biologiques dont très utiles à l'exploration des phénomènes pathologiques cardiaques et rénaux. En pratique clinique, la fonction rénale est quotidiennement estimée à partir de biomarqueurs de filtration glomérulaire, la créatinine et la cystatine C en premier lieu. Dans l'exploration des fonctions cardiaques, des évolutions importantes ont eu lieu ces dernières années. Au niveau analytique, des améliorations significatives des performances ont abouti à la mise sur le marché de méthodes dites hypersensibles de mesure de la troponine. De plus de nouveaux marqueurs explorant de nouveaux aspects physiopathologiques de la dysfonction cardiaque sont également intensément étudiés, tels que les marqueurs de fibrose (ST2 soluble, galectine-3). Après une revue de la bibliographie des biomarqueurs rénaux et cardiaques, ce travail s'attache à l'étude de l'optimisation de l'usage des biomarqueurs rénaux et cardiaques, tout d'abord par la maîtrise des procédés analytiques puis dans l'utilisation de ceux-ci en pratique clinique. Dans une première partie consacrée aux marqueurs rénaux, nous cherchons à optimiser l'utilisation des marqueurs permettant d'estimer au mieux le débit de filtration glomérulaire, meilleur index connu de la fonction rénale. En pratique clinique, le débit de filtration glomérulaire est estimé à partir de la créatinine. Au niveau analytique, nous montrons dans ce travail que les méthodes colorimétriques, basées sur la réaction de Jaffé, devraient désormais être abandonnées au profit des méthode enzymatiques. Ces résultats sont illustrés dans différentes populations de patients, une cohorte issue des bases de données hospitalières ainsi qu'une population de patients cirrhotiques. Chez ces derniers, la créatinine présente d'importantes limites en tant que marqueur de filtration glomérulaire, en particulier en raison d'une diminution de la masse musculaire. La cystatine C représente dans ce contexte une alternative intéressante puisque ce marqueur n'est pas dépendant de la masse musculaire. Des algorithmes utilisant la cystatine C, seule ou en association avec la créatinine ont récemment été proposés dans la littérature. Dans une seconde partie, nous nous intéressons aux marqueurs cardiaques. Les troponines cardiaques sont des protéines présentes au niveau de l'appareil contractile du cardiomyocyte, relarguées dans la circulation en cas de nécrose cellulaire. L'arrivée récente de méthodes capables de mesurer des concentrations circulantes dans une part importante de la population saine a obligé d'une part à un contrôle strict des procédés analytiques par les fabricant et d'autre part à une adaptation des cliniciens afin de tirer partie des nouvelles informations apportées par ces marqueurs dans différentes populations présentant des élévations chroniques (sujets âgés, insuffisants rénaux chroniques) ou aiguës (cinétiques post infarctus du myocarde). Enfin, une étude concernant le ST2 soluble, marqueur émergent de fibrose dans l'insuffisance cardiaque est également présentée. En conclusion, l'optimisation de l'usage des biomarqueurs s'oriente à l'heure actuelle vers des stratégies multimarqueurs, comme l'association créatinine et cystatine C dans l'estimation du débit de filtration glomérulaire ou le développement de scores pronostics associant troponine, peptides natriurétiques et ST2 soluble dans l'insuffisance cardiaque

Méthodes d évaluation de la fonction rénale et application chez le patient cirrhotique

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF