Evaluation du risque Brettanomyces dans le vignoble libanais et étude cinétique de la bioconversion de l'acide p-coumarique en 4-éthylphénol

Les altérations sensorielles des vins dues à la présence des levures du genre Brettanomyces se caractérisent par une augmentation de la teneur en phénols volatils tel que le 4-éthylphénol. Le premier objectif de ce travail était de faire un état des lieux sur le risque « éthylphénol » au Liban en s'intéressant à la présence d'un précurseur (acide p-coumarique), du microorganisme responsable (Brettanomyces) et du produit final (4-éthylphénol) dans les vins élaborés dans ce pays. Une forte hétérogénéité de concentrations en acide p-coumarique a été observée avec des valeurs variant de 0 à 31,4 mg.L-1. Des niveaux importants de 4-éthylphénols de l'ordre de 1,367 mg.L-1 ont été détectés sur certains vins. Un dépistage du contaminant microbien a permis de confirmer pour la première fois la présence de Brettanomyces au Liban, les proportions restant toutefois assez faibles (3 % des échantillons testés). Une étude génétique a caractérisé les souches retenues qui se sont montrées diverses au sein de l'espèce. Le travail a porté ensuite sur l'analyse cinétique des étapes réactionnelles constituant le processus enzymatique de la bioconversion des substrats acide p-coumarique et 4-vinlphénol en 4-éthylphénol pour 5 souches de Brettanomyces bruxellensis d'origines libanaises et françaises. La variabilité entre les souches s'est exprimée aux niveaux génétique et cinétique. Des profils hétérogènes de bioréaction ont été mis en évidence en fonction de la nature des souches. L'analyse du bilan-matière a révélé l'existence probable de phénomènes d'adsorption sur les parois des Brettanomyces qui sont souche-dépendants. La dernière partie a été consacrée à l'évaluation du lien entre quantité de biomasse et production de 4-éthylphénol ainsi qu'à l'influence de quelques paramètres environnementaux (pH, source d'ammonium et milieu de culture) sur la cinétique réactionnelle

Evaluation du risque Brettanomyces dans le vignoble libanais et étude cinétique de la bioconversion de l'acide p-coumarique en 4-éthylphénol

Les altérations sensorielles des vins dues à la présence des levures du genre Brettanomyces se caractérisent par une augmentation de la teneur en phénols volatils tel que le 4-éthylphénol. Le premier objectif de ce travail était de faire un état des lieux sur le risque éthylphénol au Liban en s'intéressant à la présence d'un précurseur (acide p-coumarique), du microorganisme responsable (Brettanomyces) et du produit final (4-éthylphénol) dans les vins élaborés dans ce pays. Une forte hétérogénéité de concentrations en acide p-coumarique a été observée avec des valeurs variant de 0 à 31,4 mg.L-1. Des niveaux importants de 4-éthylphénols de l'ordre de 1,367 mg.L-1 ont été détectés sur certains vins. Un dépistage du contaminant microbien a permis de confirmer pour la première fois la présence de Brettanomyces au Liban, les proportions restant toutefois assez faibles (3 % des échantillons testés). Une étude génétique a caractérisé les souches retenues qui se sont montrées diverses au sein de l'espèce. Le travail a porté ensuite sur l'analyse cinétique des étapes réactionnelles constituant le processus enzymatique de la bioconversion des substrats acide p-coumarique et 4-vinlphénol en 4-éthylphénol pour 5 souches de Brettanomyces bruxellensis d'origines libanaises et françaises. La variabilité entre les souches s'est exprimée aux niveaux génétique et cinétique. Des profils hétérogènes de bioréaction ont été mis en évidence en fonction de la nature des souches. L'analyse du bilan-matière a révélé l'existence probable de phénomènes d'adsorption sur les parois des Brettanomyces qui sont souche-dépendants. La dernière partie a été consacrée à l'évaluation du lien entre quantité de biomasse et production de 4-éthylphénol ainsi qu'à l'influence de quelques paramètres environnementaux (pH, source d'ammonium et milieu de culture) sur la cinétique réactionnelle.Wine sensory alterations due to the presence of Brettanomyces yeasts are characterized by an increased content of volatile phenols such as 4-ethylphenol. The first aim of this work was to make an inventory of the "ethylphenol" risks in Lebanon by focusing on the presence of one precursor (p-coumaric acid), the microorganism provoking these risks (Brettanomyces) and the final product (4-ethylphenol) in wines produced in this country. High heterogeneity of p-coumaric acid concentration was observed with values ranging from 0 to 31,4 mg.L-1. Significant levels of 4-ethylphenols of about 1,367 mg L-1 have been detected in some wines. Screening of microbial contaminants confirmed the presence of Brettanomyces for the first time in Lebanon, with proportions remaining relatively low (3 % of samples tested). A genetic study has characterized the selected strains which are shown to be various within the species. The second objective of this study was the kinetic analysis of the reaction steps constituting the bioconversion enzymatic process of both substrates p-coumaric acid and 4-vinlphenol into 4-ethylphenol for 5 strains of Brettanomyces bruxellensis of different origins (Lebanon and France). Variability between strains was expressed at both levels, genetic and kinetic. Heterogeneous bioreaction profiles were identified according to strain's nature. The mass balance analysis revealed the possible existence of adsorption phenomena on the cell walls of Brettanomyces which are strain-dependent. The last part was devoted to the evaluation of the relationship between biomass concentration and production of 4-ethylphenol as well as the influence of some environmental parameters (pH, ammonium source and culture medium) on the reaction's kinetic.TOULOUSE-INP (315552154) / SudocSudocFranceF

A new method for the detection of early contamination of red wine by Brettanomyces bruxellensis using Pseudomonas putida 4-ethylphenol methylene hydroxylase (4-EPMH)

Brettanomyces/Dekkera bruxellensis is a cause of major concern for the winemaking industry worldwide. If a slight presence of this spoilage yeast in red wine adds a Brett character, a strong contamination has irreversible and detrimental effects on the organoleptic qualities due to the production of volatile phenols such as 4-ethylphenol. Time is a key factor in the treatment of B. bruxellensis contaminations. Nowadays, the diagnostic and quantification resources available are time consuming and too expensive, making them either inadequate or inaccessible to most of the winemakers. This study was focused on a new, easy to use, inexpensive method that could allow winemakers to directly detect B. bruxellensis contamination in red wine at an early stage, hence, reducing wine spoilage. In this work, the ability of Pseudomonas putida 4-ethylphenol methylene hydroxylase was tested in order to catabolize the 4-ethylphenol and to elaborate an enzymatic assay with the purpose of detecting early contaminations by B. bruxellensis in red wine. We have developed a colorimetric enzymatic assay, based on the redox state of the 4-ethylphenol methylene hydroxylase co-factor, cytochrome C, that can detect and quantify low concentrations of 4-ethylphenol. The range of concentrations detected is well below the level detectable by the human nose. Combined to an enrichment step, this method allows the detection of B. bruxellensis at an initial concentration of less than 10 cells per ml

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42\ub74% vs 44\ub72%; absolute difference \u20131\ub769 [\u20139\ub758 to 6\ub711] p=0\ub767; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5\u20138] vs 6 [5\u20138] cm H2O; p=0\ub70011). ICU mortality was higher in MICs than in HICs (30\ub75% vs 19\ub79%; p=0\ub70004; adjusted effect 16\ub741% [95% CI 9\ub752\u201323\ub752]; p<0\ub70001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0\ub780 [95% CI 0\ub775\u20130\ub786]; p<0\ub70001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status. Funding: No funding

Evaluation of Brettanomyces risk in Lebanese vineyards and kinetic study of the bioconversion of p-coumaric acid into 4-ethylphenol

Les altérations sensorielles des vins dues à la présence des levures du genre Brettanomyces se caractérisent par une augmentation de la teneur en phénols volatils tel que le 4-éthylphénol. Le premier objectif de ce travail était de faire un état des lieux sur le risque « éthylphénol » au Liban en s'intéressant à la présence d'un précurseur (acide p-coumarique), du microorganisme responsable (Brettanomyces) et du produit final (4-éthylphénol) dans les vins élaborés dans ce pays. Une forte hétérogénéité de concentrations en acide p-coumarique a été observée avec des valeurs variant de 0 à 31,4 mg.L-1. Des niveaux importants de 4-éthylphénols de l'ordre de 1,367 mg.L-1 ont été détectés sur certains vins. Un dépistage du contaminant microbien a permis de confirmer pour la première fois la présence de Brettanomyces au Liban, les proportions restant toutefois assez faibles (3 % des échantillons testés). Une étude génétique a caractérisé les souches retenues qui se sont montrées diverses au sein de l'espèce. Le travail a porté ensuite sur l'analyse cinétique des étapes réactionnelles constituant le processus enzymatique de la bioconversion des substrats acide p-coumarique et 4-vinlphénol en 4-éthylphénol pour 5 souches de Brettanomyces bruxellensis d'origines libanaises et françaises. La variabilité entre les souches s'est exprimée aux niveaux génétique et cinétique. Des profils hétérogènes de bioréaction ont été mis en évidence en fonction de la nature des souches. L'analyse du bilan-matière a révélé l'existence probable de phénomènes d'adsorption sur les parois des Brettanomyces qui sont souche-dépendants. La dernière partie a été consacrée à l'évaluation du lien entre quantité de biomasse et production de 4-éthylphénol ainsi qu'à l'influence de quelques paramètres environnementaux (pH, source d'ammonium et milieu de culture) sur la cinétique réactionnelle.Wine sensory alterations due to the presence of Brettanomyces yeasts are characterized by an increased content of volatile phenols such as 4-ethylphenol. The first aim of this work was to make an inventory of the "ethylphenol" risks in Lebanon by focusing on the presence of one precursor (p-coumaric acid), the microorganism provoking these risks (Brettanomyces) and the final product (4-ethylphenol) in wines produced in this country. High heterogeneity of p-coumaric acid concentration was observed with values ranging from 0 to 31,4 mg.L-1. Significant levels of 4-ethylphenols of about 1,367 mg L-1 have been detected in some wines. Screening of microbial contaminants confirmed the presence of Brettanomyces for the first time in Lebanon, with proportions remaining relatively low (3 % of samples tested). A genetic study has characterized the selected strains which are shown to be various within the species. The second objective of this study was the kinetic analysis of the reaction steps constituting the bioconversion enzymatic process of both substrates p-coumaric acid and 4-vinlphenol into 4-ethylphenol for 5 strains of Brettanomyces bruxellensis of different origins (Lebanon and France). Variability between strains was expressed at both levels, genetic and kinetic. Heterogeneous bioreaction profiles were identified according to strain's nature. The mass balance analysis revealed the possible existence of adsorption phenomena on the cell walls of Brettanomyces which are strain-dependent. The last part was devoted to the evaluation of the relationship between biomass concentration and production of 4-ethylphenol as well as the influence of some environmental parameters (pH, ammonium source and culture medium) on the reaction's kinetic

Prospective study to evaluate the number and the location of biopsies in rapid urease test for diagnosis of Helicobacter Pylori

Helicobacter pylori (H. pylori) can cause a wide variety of illnesses such as peptic ulcer disease, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The diagnosis and eradication of H. pylori are crucial. The diagnosis of H. pylori is usually based on the rapid urease test (RUT) and gastric antral biopsy for histology. The aim of this study is to evaluate the numbers of needed biopsies and their location (antrum/fundus) to obtain optimal result for the diagnosis of H. pylori. Three hundred fifty consecutive patients were recruited, 210 fulfill the inclusion criteria and had nine gastric biopsies for the detection of H. pylori infection: two antral for the first RUT (RUT1), one antral and one fundic for the second (RUT2), one antral for the third (RUT3) and two antral with two fundic for histology (HES, Giemsa, PAS). The reading of the 3 types of RUT was performed at 1 hour, 3 hours and 24 hours and biopsies were read by two experienced pathologists not informed about the result of RUT. Results of RUT were considered positive if H. pylori was found on histology of at least one biopsy. The RUT1 at 1h, 3h and 24h has a sensitivity of 72%, 82% and 89% and a specificity of 100%, 99% and 87% respectively. The positive predictive value (PPV) was 100%, 99% and 85% respectively and the negative predictive value (NPV) of 81%, 87% and 90%. The RUT2 at 1h, 3h and 24h, respectively, had a sensitivity of 86%, 87% and 91% and a specificity of 99%, 97% and 90%. The PPV was 99%, 96% and 88% and NPV of 89%, 90%, 94%. The RUT3 at 1h, 3h and 24h, respectively, had a sensitivity of 70%, 74% and 84% and a specificity of 99%, 99% and 94%. The PPV was 99%, 99% and 92% and NPV of 79%, 81% and 87%. The best sensitivity and specificity were obtained for RUT1 read at 3h, for RUT2 read 1h and 3h, and the RUT3 read at 24h.This study demonstrates that the best sensitivity and specificity of rapid test for urease is obtained when fundic plus antral biopsy specimens are used with a reading time at 3 hours

Radiation Treatment Timing and Dose Delivery: Effects on Bladder Cancer Cells in 3D in Vitro Culture

While radical cystectomy remains the primary treatment of choice for bladder cancer, increased evidence supports the use of bladder-preservation strategies based on adjuvant radiotherapy. This highlights the need for a better understanding of bladder cancer radiosensitivity to different types of treatment deliveries. The purpose of this study is to analyze the effect of treatment time, dose and fractionation on the number and sizes of grown three-dimensional (3D) bladder cancer spheres, and to assess the capacity of the linear-quadratic model in describing the response of cells cultured in 3D. 3D MatrigelTM-based cultures were employed to enrich for cancer stem cells (CSCs) from three human bladder cancer cell lines, RT4, T24 and UM-UC-3. Three single dose radiation treatments were performed at different time points after plating, and sphere number and sizes were assessed. Anti-CD44 immunofluorescence, clonogenic assay and anti-γH2AX staining were also performed to analyze the cell lines’ radiosensitivity. The radiosensitivity of spheres was dependent on the treatment timing after plating. Current linear quadratic dose fractionation models were shown to over-estimate radiosensitivity in 3D models. Our results showed the importance of treatment timing on the radio-response of bladder cancer spheres. We also demonstrated that bladder cancer spheres are more resistant to dose-fractionation than the estimation from the theoretical linear-quadratic model

Radiation Treatment Timing and Dose Delivery: Effects on Bladder Cancer Cells in 3D in Vitro Culture

While radical cystectomy remains the primary treatment of choice for bladder cancer, increased evidence supports the use of bladder-preservation strategies based on adjuvant radiotherapy. This highlights the need for a better understanding of bladder cancer radiosensitivity to different types of treatment deliveries. The purpose of this study is to analyze the effect of treatment time, dose and fractionation on the number and sizes of grown three-dimensional (3D) bladder cancer spheres, and to assess the capacity of the linear-quadratic model in describing the response of cells cultured in 3D. 3D MatrigelTM-based cultures were employed to enrich for cancer stem cells (CSCs) from three human bladder cancer cell lines, RT4, T24 and UM-UC-3. Three single dose radiation treatments were performed at different time points after plating, and sphere number and sizes were assessed. Anti-CD44 immunofluorescence, clonogenic assay and anti-γH2AX staining were also performed to analyze the cell lines’ radiosensitivity. The radiosensitivity of spheres was dependent on the treatment timing after plating. Current linear quadratic dose fractionation models were shown to over-estimate radiosensitivity in 3D models. Our results showed the importance of treatment timing on the radio-response of bladder cancer spheres. We also demonstrated that bladder cancer spheres are more resistant to dose-fractionation than the estimation from the theoretical linear-quadratic model