Production of Pseudomonas aeruginosa Intercellular Small Signaling Molecules in Human Burn Wounds

Pseudomonas aeruginosa has developed a complex cell-to-cell communication system that relies on low-molecular weight excreted molecules to control the production of its virulence factors. We previously characterized the transcriptional regulator MvfR, that controls a major network of acute virulence functions in P. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (HAQs)-4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Though HHQ and PQS are produced in infected animals, their ratios differ from those in bacterial cultures. Because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human P. aeruginosa acute wound infection and whether their ratio is similar to that observed in P. aeruginosa-infected mice. We found that a clinically relevant P. aeruginosa isolate produced detectable levels of HAQs with ratios of HHQ and PQS that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. These molecules could be isolated from wound tissue as well as from drainage liquid. These results demonstrate for the first time that HAQs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection.

Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Integer filling facfor phases and isospin in vertical diatomic artificial molecules

Integer filling factor phases of many-electron vertically coupled diatomic artificial quantum dot molecules are investigated for different values of the interdot coupling. The experimental results are analyzed within local-spin density functional theory for which we have determined a simple lateral confining potential law that can be scaled for the different coupling regimes, and Hartree-Fock theory. Maximum density droplets composed of electrons in both bonding and antibonding or just bonding states are revealed, and interesting isospin-flip physics appears for weak interdot coupling when the systematic depopulation of antibonding states leads to changes in isospin.

Pinning of quantized vortices in helium drops by dopant atoms and molecules

Using a density functional method, we investigate the properties of liquid 4He droplets doped with atoms (Ne and Xe) and molecules ( SF6 and hydrogen cyanide). We consider the case of droplets having a quantized vortex pinned to the dopant. A liquid-drop formula is proposed that accurately describes the total energy of the complex and allows one to extrapolate the density functional results to large N. For a given impurity, we find that the formation of a dopant+vortex+4HeN complex is energetically favored below a critical size Ncr. Our results support the possibility to observe quantized vortices in helium droplets by means of spectroscopic techniques.

Dissociation of vertical semiconductor diatomic artificial molecules

We investigate the dissociation of few-electron circular vertical semiconductor double quantum dot artificial molecules at 0 T as a function of interdot distance. A slight mismatch introduced in the fabrication of the artificial molecules from nominally identical constituent quantum wells induces localization by offsetting the energy levels in the quantum dots by up to 2 meV, and this plays a crucial role in the appearance of the addition energy spectra as a function of coupling strength particularly in the weak coupling limit.

Lattice-gas model of orientable molecules: Application to liquid crystals

We have analyzed a two-dimensional lattice-gas model of cylindrical molecules which can exhibit four possible orientations. The Hamiltonian of the model contains positional and orientational energy interaction terms. The ground state of the model has been investigated on the basis of Karl¿s theorem. Monte Carlo simulation results have confirmed the predicted ground state. The model is able to reproduce, with appropriate values of the Hamiltonian parameters, both, a smectic-nematic-like transition and a nematic-isotropic-like transition. We have also analyzed the phase diagram of the system by mean-field techniques and Monte Carlo simulations. Mean-field calculations agree well qualitatively with Monte Carlo results but overestimate transition temperatures.

Effect of a transverse magnetic field on resonant magnetization tunneling in high-spin molecules

The recent observation of steps at regular intervals of magnetic field in the hysteresis loops of oriented crystals of the spin-10 molecular magnet Mn12O12(CH3COO)16(H2O)4 has been attributed to resonant tunneling between spin states. Here, we investigate the effect on the relaxation rate of applying the magnetic field at an angle with respect to the easy axis of magnetization. We find that the position of the resonances is independent of the transverse component of the field, and is determined solely by the longitudinal component. On the other hand, a transverse field significantly increases the relaxation rate, both on and off resonance. We discuss classical and quantum mechanical interpretations of this effect

The congenital soidum diarrhea and Spint2: from the molecules to the disease

La diarrhée congénitale de sodium est une maladie génétique très rare. Les enfants touchés par cette maladie présentent une diarrhée aqueuse sévère accompagnée d'une perte fécale de sodium et bicarbonates causant une déshydratation hyponatrémique et une acidose métabolique. Des analyses génétiques ont identifié des mutations du gène Spint2 comme cause de cette maladie. Le gène Spint2 code pour un inhibiteur de sérine protéase transmembranaire exprimé dans divers épithéliums tels que ceux du tube digestif ou des tubules rénaux. Le rôle physiologique de Spint2 n'est pas connu. De plus, aucun partenaire physiologique de Spint2 n'a été identifié et le mécanisme d'inhibition par Spint2 nous est peu connu. Le but de ce projet est donc d'obtenir de plus amples informations concernant la fonction et le rôle de Spint2 dans le contexte de la diarrhée congénitale de sodium, cela afin de mieux comprendre la physiopathologie des diarrhées et peut-être d'identifier de nouvelles cibles thérapeutiques. Un test fonctionnel dans les ovocytes de Xenopus a identifié les sérine protéases transmembranaires CAPI et Tmprssl3 comme potentielles cibles de Spint2 dans la mesure où ces deux protéases n'étaient plus bloquées par le mutant de Spint2 Y163C qui est associé avec la diarrhée congénitale de sodium. Des expériences fonctionnelles et biochimiques plus poussées suggèrent que l'inhibition de Tmprssl3 par Spint2 est le résultat d'une interaction complexe entre ces deux protéines. Les effets des sérine protéases transmembranaires sur l'échangeur Na+-H+ NHE3, qui pourrait être impliqué dans la pathogenèse de la diarrhée congénitale de sodium ont aussi été testés. Un clivage spécifique de NHE3 par la sérine protéase transmembranaire Tmprss3 a été observé lors d'expériences biochimiques. Malheureusement, la pertinence physiologique de ces résultats n'a pas pu être évaluée in vivo, étant donné que le modèle de souris knockout conditionnel de Spint2 que nous avons créé ne montrait une réduction de l'expression de Spint2 que de 50% et aucun phénotype. En résumé, ce travail met en évidence deux nouveaux partenaires possibles de Spint2, ainsi qu'une potentielle régulation de NHE3 par des sérine protéases transmembranaires. Des expériences supplémentaires faites dans des modèles animaux et lignées cellulaires sont requises pour évaluer la pertinence physiologique de ces données et pour obtenir de plus amples informations au sujet de Spint2 et de la diarrhée congénitale de sodium. - The congenital sodium diarrhea is a very rare genetic disease. Children affected by this condition suffer from a severe diarrhea characterized by watery stools with a high fecal loss of sodium and bicarbonates, resulting in hyponatremic dehydration and metabolic acidosis. Genetic analyses have identified mutations in the Spint2 gene as a cause of this disease. The spint2 gene encodes a transmembrane serine protease inhibitor expressed in various epithelial tissues including the gastro-intestinal tract and renal tubules. The physiological role of Spint2 is completely unknown. In addition, physiological partners of Spint2 are still to be identified and the mechanism of inhibition by Spint2 remains elusive. Therefore, the aim of this project was to get insights about the function and the role of Spint2 in the context of the congenital sodium diarrhea in order to better understand the pathophysiology of diarrheas and maybe identify new therapeutic targets. A functional assay in Xenopus oocytes identified the membrane-bound serine proteases CAPI and Tmprssl3 as potential targets of Spint2 because both proteases were no longer inhibited by the mutant Spint2 Y163C that has been associated with the congenital diarrhea. Further functional and biochemical experiments suggested that the inhibition of Tmprssl3 by Spint2 occurs though a complex interaction between both proteins. The effects of membrane-bound serine proteases on the Na+-H+ exchanger NHE3, which has been proposed to be involved in the pathogenesis of the congenital sodium diarrhea, were also tested. A specific cleavage of NHE3 by the membrane-bound serine protease Tmprss3 was observed in biochemical experiments. Unfortunately, the physiological relevance of these results could not be assessed in vivo since the conditional Spint2 knockout mouse model that we generated showed a reduction in Spint2 expression of only 50% and displayed no phenotype. Briefly, this work provides two new potential partners of Spint2 and emphasizes a putative regulation of NHE3 by membrane-bound serine proteases. Further work done in animal models and cell lines is required to assess the physiological relevance of these results and to obtain additional data about Spint2 and the congenital diarrhea.

Dpp/BMP signaling in flies: From molecules to biology.

Decapentaplegic (Dpp), the fly homolog of the secreted mammalian BMP2/4 signaling molecules, is involved in almost all aspects of fly development. Dpp has critical functions at all developmental stages, from patterning of the eggshell to the determination of adult intestinal stem cell identity. Here, we focus on recent findings regarding the transcriptional regulatory logic of the pathway, on a new feedback regulator, Pentagone, and on Dpp's roles in scaling and growth of the Drosophila wing.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.

Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.