Distribution and identification of molecular interactions between tomato roots and bacterial biofilms

Conference (2012, September)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

Intersubjectivity and intersubjectification: typology and operationalisation

Conference (2012, July 19)

Creation of model proteins to investigate the mechanism of aggregation of expanded-polyglutamine proteins. Insertion of polyglutamine tracts into the ß-lactamase BlaP

Doctoral thesis (2012)

Polyglutamine (polyQ) diseases are characterized by the formation of intranuclear amyloid-like aggregates by proteins containing an expansion of a polyQ tract above a threshold length. These insoluble ... [more ▼]

Polyglutamine (polyQ) diseases are characterized by the formation of intranuclear amyloid-like aggregates by proteins containing an expansion of a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis of the diseases. The only known common point between the causative proteins is the expanded polyQ tract, suggesting that their aggregation critically depends on the expansion of the polyQ tract above a threshold length. Several studies have however shown that the non-polyQ regions can also influence the aggregation behavior of polyQ proteins. In this work, polyQ chimeras were created by inserting different polyQ lengths in two positions (197-198 and 216-217) of the β-lactamase BlaP from Bacillus licheniformis 749/C. The structural and thermodynamic properties of the polyQ chimeras as well as their aggregating properties under native and denaturing conditions were investigated using a range of biophysical techniques including fluorescence, circular dichroism, absorbance, x-ray fiber diffraction and transmission electron microscopy. We have first created and characterized chimeras with 23, 30, 55 and 79Q inserted in position 197-198 (chimeras 197-198). None of these insertions modifies the structure of BlaP; they do, however, significantly destabilize the enzyme by 7.6-8.8 kJ/mol independently of the polyQ length. Similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the BlaP chimeras aggregate into amyloid-like fibrils. It is comprised between 55 and 79Q and between 30 and 55Q under native and denaturing conditions, respectively. This threshold value therefore depends on the structural integrity of BlaP and thus on the steric and/or conformational constraints applied by BlaP to the polyQ tract. We have then compared the properties of these chimeras with those of the chimeras containing polyQ of the same lengths in position 216-217 (chimeras 216-217). The tertiary structure of BlaP is slightly perturbed in the chimeras 216-217 and these chimeras are less stable than the chimeras 197-198. However, the urea-induced equilibrium unfolding experiments suggest that chimeras 216-217 populate a series of discrete intermediate states between the native and unfolded states or that their unfolded state significantly differs from that of chimeras 197-198. Finally, the aggregating properties of the polyQ chimeras 216-217 differ from those of chimeras 197-198. First, the threshold number of glutamines above which chimeras 216-217 readily form fibrils under native conditions (between 30 and 55Q) is lower than that observed for chimeras 197-198 (between 55 and 79Q) suggesting that the steric and/or conformational constraints imposed on the polyQ tract are lower when it is inserted in position 216-217. Secondly, the results obtained under both native and denaturing conditions indicate that the BlaP moiety could assist fibril formation by chimeras 216-217 while it has an inhibiting effect and no effect on fibril formation by the chimeras 197-198 with 55 and 79 glutamines, respectively. Taken together, these results show that the aggregating properties of BlaP chimeras result from a very complex interplay between the propensity of the polyQ tract to mediate fibril formation and the modulating effect of the BlaP moiety. BlaP chimeras present therefore valuable models to investigate in details how the properties of the host protein influence the aggregation behavior of expanded polyQ proteins. [less ▲]

Ajustement des parents d'enfants souffrant de mucoviscidose. Influence des facteurs psychosociaux et des stratégies de coping sur la qualité de vie

Book published by Editions universitaires europeennes (2012)

La prise en charge des enfants souffrant de maladie chronique s'est progressivement déplacée de l'hôpital à la maison où les responsabilités, concernant la gestion de la maladie, reposent sur les épaules ... [more ▼]

La prise en charge des enfants souffrant de maladie chronique s'est progressivement déplacée de l'hôpital à la maison où les responsabilités, concernant la gestion de la maladie, reposent sur les épaules de la famille. Les chercheurs, en reconnaissant ces changements, ont commencé à étudier le contexte familial de l'enfant avec une maladie chronique (Walker, Ford & Donald, 1987). Toutefois, la particularité de la prise en charge d’un enfant atteint de mucoviscidose a été peu étudiée. Par ailleurs, les connaissances dont nous disposons sur la manière dont les parents gèrent la prise en charge de la maladie de leur enfant sont principalement basées sur des recherches ne tenant compte que de la perspective des mères, en ignorant celle des pères (Hayes & Savage, 2008). Par conséquent, les objectifs de cette étude globale, de nature exploratoire, sont: (1) mettre en évidence les différentes variables qui peuvent jouer un rôle dans l’ajustement des parents à la maladie de leur enfant; (2) identifier l’impact de ces variables sur la qualité de vie de ces derniers; (3) tester l’existence de différences dans les résultats selon le sexe du parent. [less ▲]

Light induced photosynthetic electron transfer upon anaerobiosis in Chlamydomonas: Kinetics, electron sinks and setup of a fluorescence screen to identify new players

Conference (2012, June 15)

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from ... [more ▼]

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from reduced ferredoxin resulting in the production of molecular hydrogen. In this work, light-induced photosynthetic electron transfer after a prolonged dark-anaerobiosis period was studied by following the kinetics of chlorophyll fluorescence emission, P700 oxidation and proton-motive force formation and consumption during the first 3 seconds of illumination. We show that during the induction of photosynthesis, an hyd-dependent photosynthetic electron transfer operates at a maximal rate of 110 electrons per photosystem per second, that is about half the one measured in aerobiosis. The implication in this process of components of the linear, cyclic and chlororespiratory electron transfer pathways, as well as various electron sinks, are investigated thanks to the availability of mutants. In a next step, we screen an insertional mutant library (~3000 clones) on the basis of the fluorescence induction kinetics upon a shift from dark-anaerobiosis to light. Five mutants display the signature of mutants deficient for NADPH:PQ oxidoreductase or hyd activities. In particular, one is defective for hydrogenase HydG assembly factor. This mutant behaves exactly has the hydEF mutant, thus confirming that in vivo both the assembly factors are required for an efficient hydrogenase activity. [less ▲]

Mechanochemical Study of Conformational Transitions in a Single Synthetic Peptide Chain

Poster (2012, June 05)

The aim of this study is to investigate the mechanochemical behavior of homopolypeptides able to change their conformation is a stimuli-responsive way. The peptidic secondary structures were studied in ... [more ▼]

The aim of this study is to investigate the mechanochemical behavior of homopolypeptides able to change their conformation is a stimuli-responsive way. The peptidic secondary structures were studied in detail by atomic force microscopy (AFM) at the single-molecule level. Synthetic copolymers containing a polypeptide block were prepared by N-carboxyanhydride amino acid ring-opening polymerization. The polymer chains were grafted as a dilute brush onto gold surfaces via disulfide end-groups. Their mechanochemical behavior was then studied by AFM single-molecule force spectroscopy (SMFS). The investigated polypeptide blocks were based on poly(L-glutamic acid), which undergoes a transition from alpha-helix to random coil. This can be induced by external stimuli (pH, ionic strength, temperature) or simply by applying a force. We were able to study the mechanically driven unfolding of the peptide by stretching-release cycles of the biomacromolecule. Stretching the helical peptide resulted in original features in the force-distance traces. Plateaus that are specific for the helical conformation were detected, quantified and discussed. Pulling-relaxing SMFS experiments eventually led to a better understanding of the force induced unfolding of an alpha-helix and the reversibility of the phenomenon. [less ▲]

Pseudo-articulations à col parabolique

Report (2012)

Ce rapport donne la théorie des pseudo-articulations à col parabolique. Faisant suite à des travaux antérieurs du Laboratoire de Méthodes de Fabrication, il adopte la même démarche, à savoir:1)Solution ... [more ▼]

Ce rapport donne la théorie des pseudo-articulations à col parabolique. Faisant suite à des travaux antérieurs du Laboratoire de Méthodes de Fabrication, il adopte la même démarche, à savoir:1)Solution exacte; 2)Solution asymptotique; 3)Facteur correctif pour passer de la solution exacte à la solution asymptotique 4) Expression approchée de ce facteur. Cette démarche fournit des solutions très bien approchées et beaucoup plus simples à utiliser que celles de la littérature, un fait appréciable au niveau de la conception. [less ▲]

Low temperature assembly method of microfluidic bio-molecules detection device

in Suga, Tadatomo; Takagi, Hideki; Higurashi, Eiji (Eds.) 2012 3rd IEEE International Workshop on Low Temperature Bonding for 3D Integration (LTB-3D 2012) (2012, May 22)

We developed a microfluidic device for the detection of bio-molecules. The active part of the device is a biofunctionalized interdigitated capacitive sensor. The microsystem consists of a sensor on ... [more ▼]

We developed a microfluidic device for the detection of bio-molecules. The active part of the device is a biofunctionalized interdigitated capacitive sensor. The microsystem consists of a sensor on silicon chip, a microfluidic channel formed by photo-patternable resist and a plastic cover. We implemented a low temperature packaging process to assemble the sensor and prevent the biological material from degradation. [less ▲]

EFFECT OF PARECOXIB, A SELECTIVE COX-2 INHIBITOR, IN THE PREVENTION OF POSTOPERATIVE ADHESIONS IN A RAT MODEL

in Acta Chirurgica Belgica (2012, May), 112(3), 8444

Objectives: Postoperative peritoneal adhesions are frequent after abdominal surgery. Many preventive agents have been tried in animal models and in clinical trials, but up to now, there has been no ... [more ▼]

Objectives: Postoperative peritoneal adhesions are frequent after abdominal surgery. Many preventive agents have been tried in animal models and in clinical trials, but up to now, there has been no definitive strategy to prevent their formation. In this study, the effectiveness of parecoxib (Dynastat®), a selective cyclooxygenase-2 inhibitor, in preventing experimental intra-abdominal adhesions in rats was studied. Methods: Thirty male rats who underwent a primary surgical procedure aiming at inducing peritoneal injury to produce intraabdominal adhesion, were randomized in three groups: (A) control group, no therapy; (B) intraperitoneal (IP) parecoxib group; (C) intramuscular (IM) parecoxib group. Ten days later, a xyphopubic midline incision was performed and the whole abdominal cavity was explored to score the peritoneal adhesions. Results: Twenty-three rats developed adhesions, 9 (100%) in group A, 7 (70%) in group B and 4 (40%) in group C (P=0.01). The extent and severity scores of adhesion were significantly lower in groups B and C than those in control group (p<0.001). Type of adhesions was measured at 2.25 ± 0.67 in group A, 1.20 ± 0.86 in group B and 0.82 ± 0.80 in group C. This was significantly different between group A and B (p<0.001), between A and C (p<0.001). But no significant difference was found between group B and C (p=0.17). Conclusions: In this study, we found a significant effect on parecoxib in the prevention of postoperative adhesions. But, without avoiding completely the formation of adhesion, parecoxib reduces significantly extent and severity of postoperative adhesions in rats treated with IP or IM parecoxib administration. [less ▲]

INTRAPERITONEAL ADHESIONS AFTER OPEN OR LAPAROSCOPIC ABDOMINAL PROCEDURE: AN EXPERIMENTAL STUDY IN THE RAT

in Acta Chirurgica Belgica (2012, May), 112(3), 8546

Objectives: Adhesion formation is common after abdominal surgery. The incidence and severity of adhesion formation following open or laparoscopic surgery remain controversial. The role of CO2 ... [more ▼]

Objectives: Adhesion formation is common after abdominal surgery. The incidence and severity of adhesion formation following open or laparoscopic surgery remain controversial. The role of CO2 pneumoperitoneum is also largely discussed. This study aimed to compare adhesion formation following peritoneal injury by electrocoagulation performed through open or laparoscopic procedures in a rat model. Methods: Sixty male rats were randomized to undergo a 1.5cm peritoneal injury with unipolar cautery under general anesthesia: open surgery (group A, n=20), laparoscopic surgery with CO2 pneumoperitoneum (group B, n=20) and laparoscopic surgery with air pneumoperitoneum (group C, n=20). Duration of the procedures was fixed at 90 minutes in all groups, and pneumoperitoneum pressure at 10mmHg. Ten days later, the animals underwent a secondary laparotomy to score peritoneal adhesions using qualitative and quantitative parameters. Results: Forty-five rats developed at least one adhesion, respectively 95% in group A, 83% in group B and 55% in group C (P<0.01; Group C vs Group A, P<0.01). According to number, thickness, tenacity, vascularization, extent, type, and grading according to Zühkle classification, no significant difference was observed between groups A and B. The distribution of adhesions after open surgery was significantly different than after laparoscopic surgery (P<0.001). Interestingly, group C rats developed significantly less adhesions at the traumatized site, and their adhesions had less severe qualitative scores compared to open surgery (P<0.01). Conclusions: In this animal model, CO2 laparoscopic surgery did not decrease the formation of postoperative adhesion, compared to open surgery. The difference with the animals operated with air pneumoperitoneum emphasizes the role of CO2 in peritoneal injury leading to adhesion formation. [less ▲]