Amperometric tyrosinase based biosensor using an electrogenerated polythiophene film as an entrapment support

International audienceAn amperometric enzyme sensor using tyrosinase, also called polyphenol oxidase (PPO), was constructed for determination of phenolic compounds and herbicides. The enzyme was entrapped in a conducting polymer, poly 3,4-ethylenedioxythiophene (PEDT), electrochemically generated on a glassy carbon electrode. Several experimental parameters in the electropolymerisation process and working conditions were determined to optimise biosensor performances. Mono-phenol and di-phenol were tested in oxygenated solutions, by amperometric measurements at −200 mV (vs. SCE) in a batch system. The limit of detection of these molecules ranges from 5 to 500 nM. Detection of herbicides was obtained from the inhibition of tyrosinase electrode responses. The limit of detection for atrazine and diuron was 1 and 0.5 mg l−1 respectively. These data suggest that PEDT film is a promising PPO immobilisation method

Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides

International audienceAn optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level

Pierwotny śródczaszkowy pozaszkieletowy chirzęstniakomięsak śluzowaty

Extraskeletal myxoid chondrosarcomas (EMC) are extremely rare and are usually located in the deep soft tissues of the lower extremities. Less than 10 cases of intracranial EMC have been reported in the literature, making their management and early diagnosis difficult. We present a new case of intracranial EMC occurring in a 70-year-old woman presenting with a right frontal mass initially assumed to be a brain metastasis from breast adenocarcinoma. The optimal management of these tumours is also discussed. Analysis from the literature suggests that complete resection should be recommended, whenever feasible. Although the high risk for relapse after surgery encourages postoperative treatments, relative resistance to both radiotherapy and chemotherapy characterizes EMC. Future perspectives might include multimodal treatments with highly conformal radiotherapy modalities for dose escalation strategies or use of new molecules. Knowledge of these unusual malignant tumours will be the first step for improving patients’ outcome.Pozaszkieletowy chrzęstniakomięsak śluzowaty to wyjątkowo rzadki guz, który występuje zwykle głęboko w tkankach miękkich kończyn dolnych. Opisano mniej niż 10 przypadków tego guza umiejscowionych śródczaszkowo, co utrudnia wczesne rozpoznanie i leczenie. W pracy przedstawiono nowy przypadek śródczaszkowego chrzęstniakomięsaka śluzowatego u 70-letniej kobiety z guzem okolicy czołowej prawej, traktowanym początkowo jako przerzut gruczolakoraka sutka do mózgu. Omówiono optymalne leczenie tych guzów. Analiza piśmiennictwa wskazuje, że w miarę możliwości powinno się zalecać całkowite wycięcie. Duże ryzyko wznowy po leczeniu chirurgicznym skłania do podejmowania dodatkowego leczenia, ale guz charakteryzuje się względną opornością na radioterapię i chemioterapię. Przyszłe wielorakie metody leczenia mogłyby wykorzystywać radioterapię konformalną w celu zwiększenia dawki promieniowania lub zastosowanie nowych cząsteczek. Wiedza o tych rzadkich nowotworach złośliwych będzie pierwszym krokiem do poprawy wyników leczenia

Algal biosensors for aquatic ecosystems monitoring

International audienceThe harmful effect of toxic chemicals on natural ecosystems has led to an increasing demand for early-warning systems to detect those toxicants at very low concentrations levels. Whole-cell biosensors based either on chlorophyll fluorescence or enzyme (phosphatase and esterase) inhibition are constructed for real-time detection and on-line monitoring. Results show that these devices are sensitive to heavy metals and pesticides. The system allows the cells to operate in their natural environment which favours long term stability and reflects the toxic action mechanism providing therefore an ecological interest

Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the mecA+ Staphylococcus (i.e., resistant) strains treated with oxacillin

Exploitation de signaux biologiques pour la réalisation de capteurs environnementaux. Application à la construction d'un biocapteur à micro-algues immobilisées et d'une bioélectrode à enzyme immobilisée

Two biosensors, respectively based on micro algae and polyphenol oxidase (PPO), are presented in this study.Chlorophyll fluorescence is used to monitor the photosynthetic activity of micro-algae in the algal biosensor. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimization. Detection was obtained with immobilized micro algae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. This design enables to increase the statistical signal confidence and ecological representativeness when several micro algae strains are tested simultaneously. Chlorella vulgaris was used to optimize and validate the biosensor. The biosensor response using five algal strains simultaneously to pollutants was evaluated.The amperometric enzyme sensor is based on the entrapment of polyphenol oxidase in an electro generated film of poly(3,4-ethylenedioxythiophene) (PEDT). Phenolic compounds and PPO inhibitors can be detected. The electro polymerization process and working conditions were determined to optimize biosensor performance.The biosensors detection spectrum and sensitivity complement each other to detect toxic compounds. The algal biosensor can achieve detection of photosystem II herbicides at sub µg.l-1 concentration level, while the PPO bioelectrode can detect phenolic compounds at µg.l-1 level.Deux biocapteurs, respectivement fondés sur des cellules de micro-algues et sur la polyphénol oxydase (PPO), sont décrits dans cette étude.Le principe du biocapteur algal repose sur le suivi de l'activité photosynthétique des micro-algues par la mesure de la fluorescence chlorophyllienne. Le mode de culture en continu a été choisi afin d'obtenir des suspensions algales dont l'état physiologique des cellules est stable au cours du temps. Les cellules algales sont immobilisées sur- une membrane en fibre de quartz. Le biocapteur comporte cinq membranes algales. L'utilisation simultanée de cinq souches algales différentes permet d'augmenter la représentativité du signal mesuré d'un point de vue statistique ou écotoxicologique. Chlorella vulgaris a été utilisée lors des étapes d'optimisation et de validation du biocapteur. Le comportement de cinq souches algales a été étudié simultanément en l'absence et en la présence de polluants. Le biocapteur enzymatique est fondé sur l'immobilisation de la polyphénol oxydase dans un film électrogénéré de polymère : le poly(3,4-éthylènedioxythiophène) (PEDT). Il permet la détection directe de composés phénoliques et la détection indirecte d'inhibiteurs de cette enzyme. Les conditions d'élaboration et d'utilisation des bioélectrodes ont été optimisées.Par leur spectre de détection et leur sensibilité, ces deux biocapteurs sont des outils complémentaires. Le biocapteur algal est capable de détecter des concentrations en herbicides anti-PSII inférieures au µg.l-1, tandis que la bioélectrode à PPO détecte les composés phénoliques à des concentrations de l'ordre du µg.l-1

Biocapteur optique pour la détection de pesticides

Parmi les divers produits qui dégradent notre environnement, les herbicides font partie des | composés difficiles à détecter grâce à des méthodes simples. Les techniques chromatographiques liquides ou gazeuses (HPLC, GC) nécessitent une pré-concentration préalable du produit dans l'échantillon pour arriver à détecter ces produits présents souvent à très faibles concentrations. En outre, ces méthodes ne se prêtent pas à des mesures en continu. En raison de la toxicité de ces produits, des systèmes d'alerte précoce deviennent nécessaires pour avertir de l'apparition d'une pollution accidentelle. Les biocapteurs qui sont des dispositifs sensibles et peu coûteux permettent aussi de surveiller en continu la concentration des polluants présents dans les milieux aquatiques. Le biocapteur est conçu pour transformer un phénomène biochimique en un signal électrique : il est donc issu de la combinaison d'un composant biologique appelé biorécepteur et d'un transducteur qui représente en fait le mode de détection. Le biorécepteur sert à identifier l'espèce à détecter par modification de son système biologique. L'utilisation des micro-algues comme biorécepteurs apporte surtout un intérêt écotoxicologique étant donné que ces micro-organismes constituent le premier maillon de la chaîne alimentaire et qu'ils sont les cibles de composés toxiques tels que des herbicides

Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14.

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14

Early herpes and TTV DNAemia in septic shock patients: a pilot study

International audienceBackground: Septic shock patients exhibit an increased incidence of viral reactivation. Precise timing of such reactivation-as an early marker of immune suppression, or as a consequence of the later-is not known precisely. Here, using a fully designed nucleic acid extraction automated procedure together with tailored commercial PCR kits, we focused on the description of early reactivation within the first week of ICU admission of several herpes viruses and Torque Teno virus (TTV) in 98 septic shock patients. Results: Most of septic shock patients had at least one viremia event during the first week (88%). TTV and herpesviruses were detected in 56% and 53% of septic shock patient, respectively. The two most frequent herpesviruses detected within the first week were EBV (35%) and HSV1 (26%). Different kinetic were observed among herpesviruses, faster for EBV and HSV1 than for CMV and HHV6. Although no association was found between herpes viremia and secondary infections, patients with herpesviridae-related viremia were more severe, e.g., higher SOFA scores and plasma lactate levels. While reactivating only 1 virus was not associated with mortality, patients with multiple viremia events had higher ICU mortality. Surprisingly, EBV + TTV early reactivation seemed associated with a lower D28 mortality. No clear association was observed between viremia and immune biomarkers. Conclusion: Applying a semi-automated process of viral DNAemia determination to this cohort of 98 patients with septic shock, we observed that the number of patients with positive viremia increased during the first week in the ICU. Of note, there was no improvement in predicting the outcome when using viremia status. Nevertheless, this pilot study, introducing standardized procedures from extraction to detection, provides the basis for future standardized diagnostic criteria. A prospective longitudinal clinical study using these procedures will enable determination of whether such viremia is due to a lack of a latent virus control by the immune system or a true clinical viral infection