An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation.

RATIONALE: Lymphatic vasculature plays important roles in tissue fluid homeostasis maintenance and in the pathology of human diseases. Yet, the molecular mechanisms that control lymphatic vessel maturation remain largely unknown. OBJECTIVE: We analyzed the gene expression profiles of ex vivo isolated lymphatic endothelial cells to identify novel lymphatic vessel expressed genes and we investigated the role of semaphorin 3A (Sema3A) and neuropilin-1 (Nrp-1) in lymphatic vessel maturation and function. METHODS AND RESULTS: Lymphatic and blood vascular endothelial cells from mouse intestine were isolated using fluorescence-activated cell sorting, and transcriptional profiling was performed. We found that the axonal guidance molecules Sema3A and Sema3D were highly expressed by lymphatic vessels. Importantly, we found that the semaphorin receptor Nrp-1 is expressed on the perivascular cells of the collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1 but not with an antibody that blocks VEGF-A binding to Nrp-1 resulted in a complex phenotype of impaired lymphatic vessel function, enhanced perivascular cell coverage, and abnormal lymphatic vessel and valve morphology. CONCLUSIONS: Together, these results reveal an unanticipated role of Sema3A-Nrp-1 signaling in the maturation of the lymphatic vascular network likely via regulating the perivascular cell coverage of the vessels thus affecting lymphatic vessel function and lymphatic valve development.

Devolatilization-induced pressure build-up: Implications for reaction front movement and breccia pipe formation

Generation of fluids during metamorphism can significantly influence the fluid overpressure, and thus the fluid flow in metamorphic terrains. There is currently a large focus on developing numerical reactive transport models, and with it follows the need for analytical solutions to ensure correct numerical implementation. In this study, we derive both analytical and numerical solutions to reaction-induced fluid overpressure, coupled to temperature and fluid flow out of the reacting front. All equations are derived from basic principles of conservation of mass, energy and momentum. We focus on contact metamorphism, where devolatilization reactions are particularly important owing to high thermal fluxes allowing large volumes of fluids to be rapidly generated. The analytical solutions reveal three key factors involved in the pressure build-up: (i) The efficiency of the devolatilizing reaction front (pressure build-up) relative to fluid flow (pressure relaxation), (ii) the reaction temperature relative to the available heat in the system and (iii) the feedback of overpressure on the reaction temperature as a function of the Clapeyron slope. Finally, we apply the model to two geological case scenarios. In the first case, we investigate the influence of fluid overpressure on the movement of the reaction front and show that it can slow down significantly and may even be terminated owing to increased effective reaction temperature. In the second case, the model is applied to constrain the conditions for fracturing and inferred breccia pipe formation in organic-rich shales owing to methane generation in the contact aureole.

Prevention of atelectasis formation during the induction of general anesthesia in morbidly obese patients

Résumé: La formation des atélectasies durant l'induction de l'anesthésie générale est plus importante chez le patient obèse morbide. Nous avons démontré dans des travaux de recherche antérieurs que l'utilisation de la PEEP (Pression Positive en Fin d'Expiration) durant l'induction de l'anesthésie prévient la formation d'atélectasies chez des patients non obèses. Par conséquent, nous voulions étudier l'efficacité de la pression positive en fin d'expiration chez le patient obèse morbide dans la prévention de la formation d'atélectasies. Nous avons fait une étude de 23 patients obèses morbides (BMI > 35 kg / m2) dans 2 groupes. Dans le groupe utilisant la pression positive en fin d'expiration, les patients respiraient 100% d'oxygène pendant 5 minutes par l'intermédiaire d'un masque facial type CPAP avec une pression de 10 cm H20. Après l'induction de l'anesthésie, nous avons ventilé les patients au masque facial avec une PEEP de 10 cm H20. Dans le groupe de contrôle, nous avons procédé au même type d'induction sans utiliser la pression positive en fin d'expiration. La surface de poumon atélectatique a été évaluée par tomographie (CT scann). L'étude des échanges gazeux se faisait à 2 reprises, à partir de gazométries réalisées juste avant l'induction de l'anesthésie puis juste après l'intubation. Après l'induction de l'anesthésie et l'intubation, les patients du groupe de contrôle présentaient une quantité d'atélectasies plus importante que les patients du groupe où la PEEP avait été utilisée (10.4% + 4.8% dans le groupe de contrôle versus 1.3% dans le groupe utilisant la pression positive en fin d'expiration p < 0.001). Après l'intubation, en présence d'une fraction inspirée en oxygène à 100%, la Pa02 était significativement supérieure dans le groupe ayant utilisé la pression positive en fin d'expiration en comparaison avec le groupe de contrôle (respectivement 457 ± 130 mmHg versus 315 ± 100 mmHg). Nous avons conclu que chez le patient obèse morbide, le recours à la pression positive en fin d'expiration lors de l'induction de l'anesthésie permet de prévenir largement la formation d'atélectasies et s'accompagne d'une meilleure oxygénation. Abstract: Atelectasis caused by general anesthesia is increased in morbidly obese patients. We have shown that application of positive end-expiratory pressure (PEEP) during the induction of anesthesia prevents atelectasis formation in nonobese patients. We therefore studied the efficacy of PEEP in morbidly obese patients to prevent atelectasis. Twenty-three adult morbidly obese patients (b ody mass index >35 kg/m2) were randomly assigned to one of two groups. In the PEEP group, patients breathed 100% oxygen (5 min) with a continuous positive airway pressure of 10 cm H20 and, after the induction, mechanical ventilation via a face mask with a PEEP of 10 cm H2O. In the control group, the same induction was applied but without continuous positive airway pressure or PEEP. Atelectasis, determined by computed tomography, and blood gas analysis were measured twice: before the induction and directly after intubation. After endotracheal intubation, patients of the control group showed an increase in the amount of atelectasis, which was much larger than in the PEEP group (10.4% -± 4.8% in control group versus 1.7% ± 1.3% in PEEP group; P <0.001). After in.tubation with a fraction of inspired oxygen of 1.0, Pao, was significantly higher in the PEEP group compared with the control group (457 ±- 130 mm Hg versus 315 ± 100 mm Hg, respectively; P = 0.035) We conclude that in morbidly obese patients, atelectasis formation is largely prevented by PEEP applied during the anesthetic induction and is associated with a better oxygenation.

Biofilm formation at the solid-liquid and air-liquid interfaces by Acinetobacter species.

BACKGROUND The members of the genus Acinetobacter are Gram-negative cocobacilli that are frequently found in the environment but also in the hospital setting where they have been associated with outbreaks of nosocomial infections. Among them, Acinetobacter baumannii has emerged as the most common pathogenic species involved in hospital-acquired infections. One reason for this emergence may be its persistence in the hospital wards, in particular in the intensive care unit; this persistence could be partially explained by the capacity of these microorganisms to form biofilm. Therefore, our main objective was to study the prevalence of the two main types of biofilm formed by the most relevant Acinetobacter species, comparing biofilm formation between the different species. FINDINGS Biofilm formation at the air-liquid and solid-liquid interfaces was investigated in different Acinetobacter spp. and it appeared to be generally more important at 25°C than at 37°C. The biofilm formation at the solid-liquid interface by the members of the ACB-complex was at least 3 times higher than the other species (80-91% versus 5-24%). In addition, only the isolates belonging to this complex were able to form biofilm at the air-liquid interface; between 9% and 36% of the tested isolates formed this type of pellicle. Finally, within the ACB-complex, the biofilm formed at the air-liquid interface was almost 4 times higher for A. baumannii and Acinetobacter G13TU than for Acinetobacter G3 (36%, 27% & 9% respectively). CONCLUSIONS Overall, this study has shown the capacity of the Acinetobacter spp to form two different types of biofilm: solid-liquid and air-liquid interfaces. This ability was generally higher at 25°C which might contribute to their persistence in the inanimate hospital environment. Our work has also demonstrated for the first time the ability of the members of the ACB-complex to form biofilm at the air-liquid interface, a feature that was not observed in other Acinetobacter species.

Use of fluorescent proteins to monitor the regulation of antifungal activity in root-associated Pseudomonas fluorescens CHA0

SUMMARY Roots of crop plants are the target of soil-borne pathogens, mainly fungi that cause considerable damage to plant health. By antagonizing these pathogens, some root-colonizing pseudomonads provide plants with efficient biological protection from disease. Pseudomonas fluorescens CHAO is a soil bacterium with the ability to suppress a considerable range of root diseases. A major characteristic conferring biocontrol capacity to this strain is the production of antifungal compounds, in particular 2,4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the biosyntheses of these metabolites is complex and involves several regulatory systems responding to multiple environmental signals. In the present work, we have developed reporter systems based on green (GFP) and red fluorescent (DsRed) proteins to monitor regulation of antifungal gene expression in vitro and on plant roots. Stable and unstable GFP-based reporter fusions to the DAPG and PLT biosynthetic genes allowed us to demonstrate that P. fluorescens CHAO keeps the two antifungal compounds at a fine-tuned balance that can be affected by environmental signals. A GFP-based screening technique helped us to identify two novel regulators of balanced antibiotic production, i.e. MvaT and MvaV that are functionally and structurally related to the nucleoid-binding protein H-NS. They act in concert as global regulators of DAPG and PLT production and other biocontrol-related traits in P. fluorescens CHAO, and are essential for the bacterium's capacity to control a root disease caused by Pythium. The combined use of autofluorescent reporters, flow cytometry, and epifluorescence microscopy permitted us to visualize and quantify the expression of DAPG and PLT biosynthetic genes on roots. A GFP- and DsRed-based two-color approach was then developed to further improve the sensitivity of the flow cytometric quantitation method. The findings of this study shed more light on the complex regulatory mechanisms controlling antifungal activity of P. filuorescens in the rhizosphere. RESUME 4 e Les racines de plantes de culture sont la cible de divers pathogènes, principalement des champignons, qui nuisent gravement à la santé des plantes. Certains pseudomonades colonisant les racines peuvent avoir un effet antagoniste sur les pathogènes et protéger ainsi les plantes de manière efficace. Pseudomonas fluorescens CHAO est une bactérie du sol ayant la capacité de supprimer une gamme considérable de maladies racinaires. Une des caractéristiques principales conférant la capacité de biocontrôle à cette souche, est la production de composés antifongiques, en particulier le 2,4-diacétyphloroglucinol (DAPG) et la pyolutéorine (PLT). La régulation de la biosynthèse de ces métabolites est complexe et implique plusieurs systèmes régulateurs répondant à de multiples signaux environnementaux. Dans ce travail, nous avons développé des systèmes rapporteurs basés sur des protéines fluorescentes verte (GFP) et rouge (DsRed), afin d'étudier la régulation de l'expression des gènes d'antifongiques in vitro et sur les racines des plantes. Des fusions GFP stables et instables rapportrices de l'expression des gènes de biosynthèse du DAPG et de la PLT nous ont permis de démontrer que P. fluorescens CHAO gère les deux antifongiques dans une balance finement régulée pouvant être affectée par des signaux environnementaux. Une technique de criblage basée sur la GFP nous a permis d'identifier deux nouveaux régulateurs de la production d'antibiotiques, MvaT et MvaV, apparentés à la protéine H-NS liant l'ADN, Elles agissent de concert en tant que régulateurs globaux sur la production de DAPG et de PLT, ainsi que sur d'autres éléments relatifs au biocontrôle chez P. fluorescens CHAO. De plus, elles sont essentielles à la bactérie pour contrôler une maladie racinaire causée par Pythium. L'utilisation combinée de rapporteurs autofluorescents, de cytométrie de flux et de microscopie à épifluorescence nous a permis de visualiser et de quantifier l'expression des gènes de biosynthèse du DAPG et de la PLT sur les racines. Une approche utilisant simultanément la GFP et la DsRed a ensuite été développée afin d'améliorer la sensibilité de la méthode de quantification par cytométrie de flux. Les résultats de cette étude ont apporté plus de lumière sur les mécanismes régulateurs complexes contrôlant l'activité antifongique de P. fluorescens dans la rizosphère.

The antimicrobial susceptibility, biofilm formation and genotypic profiles of Staphylococcus haemolyticus from bloodstream infections

We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.

Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.

Quantification of abscisic Acid in a single maize root.

Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate.

Blockade of both alpha1A- and alpha1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery.

Attenuation of early restenosis after percutaneous coronary intervention (PCI) is important for the successful treatment of coronary artery disease. Some clinical studies have shown that hypertension is a risk factor for early restenosis after PCI. These findings suggest that alpha(1)-adrenergic receptors (alpha(1)-ARs) may facilitate restenosis after PCI because of alpha(1)-AR's remarkable contribution to the onset of hypertension. In this study, we examined the neointimal formation after vascular injury in the femoral artery of alpha(1A)-knockout (alpha(1A)-KO), alpha(1B)-KO, alpha(1D)-KO, alpha(1A)-/alpha(1B)-AR double-KO (alpha(1AB)-KO), and wild-type mice to investigate the functional role of each alpha(1)-AR subtype in neointimal formation, which is known to promote restenosis. Neointimal formation 4 wk after wire injury was significantly (P < 0.05) smaller in alpha(1AB)-KO mice than in any other group of mice, while blood pressures were not altered in any of the groups of mice after wire injury compared with those before it. These results suggest that lack of both alpha(1A)- and alpha(1B)-ARs could be necessary to inhibit neointimal formation in the mouse femoral artery.