Prophylaxis of experimental endocarditis with antiplatelet and antithrombin agents : a role for long-term prevention of infective endocarditis in humans?

BACKGROUND: Infective endocarditis (IE) mostly occurs after spontaneous low-grade bacteremia. Thus, IE cannot be prevented by circumstantial antibiotic prophylaxis. Platelet activation following bacterial-fibrinogen interaction or thrombin-mediated fibrinogen-fibrin polymerization is a critical step in vegetation formation. We tested the efficacy of antiplatelet and antithrombin to prevent experimental IE. METHODS: A rat model of experimental IE following prolonged low-grade bacteremia mimicking smoldering bacteremia in humans was used. Prophylaxis with antiplatelets (aspirin, ticlopidine [alone or in combination], eptifibatide, or abciximab) or anticoagulants (antithrombin dabigatran etexilate or anti-vitamin K acenocoumarol) was started 2 days before inoculation with Streptococcus gordonii or Staphylococcus aureus. Valve infection was assessed 24 hours later. RESULTS: Aspirin plus ticlopidine, as well as abciximab, protected 45%-88% of animals against S. gordonii and S. aureus IE (P < .05). Dabigatran etexilate protected 75% of rats against IE due to S. aureus (P < .005) but failed to protect against S. gordonii (<30% protection). Acenocoumarol was ineffective. CONCLUSIONS: Antiplatelet and direct antithrombin agents may be useful in the prophylaxis of IE in humans. In particular, the potential dual benefit of dabigatran etexilate might be reconsidered for patients with prosthetic valves, who require life-long anticoagulation and in whom S. aureus IE is associated with high mortality.

Bitumens in the late Variscan hydrothermal vein-type uranium deposit of Pribram, Czech Republic: Sources, radiation-induced alteration, and relation to mineralization

The late Variscan (275-278 Ma) Pribram uranium deposit is one of the largest known accumulations of uraniferous bitumens in hydrothermal veins. The deposit extends along the northwestern boundary of the Central Bohemian pluton (345-335 Ma) with low-grade metamorphosed Late Proterozoic and unmetamorphosed Cambrian rocks. From a net uranium production of 41,742 metric tons (t), more than 6,000 t were extracted from bitumen-uraninite ores during 43 years of exploration and mining. Three morphological varieties of solid bitumen are recognized: globular, asphaltlike, and cokelike. While the globular bitumen is uranium free, the other two types are uraniferous. The amount of bitumen in ore veins gradually decreases toward the contact with the plutonic body and increases with depth. Two types of bitumen microtextures are recognized using high-resolution transmission electron microscopy: amorphous and microporous, the former being less common in uraniferous samples. A lower Raman peak area ratio (1,360/1,575 cm(-1)) in mineralized bitumens (0.9) compared with uranium-free samples (2.0) indicates a lower degree of microtextural organization in the latter The H/C and O/C atomic ratios in uranium-free bitumens (0.9-1.1 and 0.09, respectively) are higher than those in mineralized samples (H/C = 0.3-0.8, O/C = 0.03-0.09). The chloroform extractable matter yield is Very low in uranium-free bitumens (0.30-0.35% of the total organic carbon,TOC) and decreases with uranium content increase. The extracted solid uraniferous bitumen infrared spectra show depletion in aliphatic CH2 and CH3 groups compared to uranium-free samples. The concentration of oxygen-bearing functional groups relative to aromatic bonds in the IR spectra of uranium-free and mineralized bitumen, however, do not differ significantly. C-13 NMR confirmed than the aromaticity of a uraniferous sample is higher (F-ar = 0.61) than in the uranium-free bitumen (F-ar = 0.51). Pyrolysates from uraniferous and nonuraniferous bitumens do not differ significantly, being predominantly cresol, alkylphenols, alkylbenzenes, and alkylnaphthalenes. The liquid pyrolysate yield decreases significantly with increasing uranium content. The delta(13)C Values of bulk uranium-free bitumens and low-grade uraniferous, asphaltlike bitumens range from -43.6 to 52.3 per mil. High-grade, cokelike, uraniferous bitumens are more C-13 depleted (54.5 to -58.4 parts per thousand). In contrast to the very light isotopic ratios of the high-grade uraniferous cokelike bitumen bulk carbon, the individual n-alkanes and isoprenoids (pristane and phytane) extracted from the same sample are significantly C-13 enriched. The isotopic composition of the C13-24 n-alkanes extracted from the high-grade uraniferous sample (delta(13)C = -28.0 to 32.6 parts per thousand) are heavier compared with the same compounds in a uranium-free sample (delta(13)C = 31.9 to 33.8 parts per thousand). It is proposed that the bitumen source was the isotopically light (delta(13)C = 35.8 to 30.2 parts per thousand) organic matter of the Upper Proterozoic host rocks that were pyrolyzed during intrusion of the Central Bohemian pluton. The C-13- depleted pyrolysates were mobilized from the innermost part of the contact-metamorphic aureole, accumulated in structural traps in less thermally influenced parts of the sedimentary complex and were later extracted by hydrothermal fluids. Bitumens at the Pribram deposit are younger than the main part of the uranium mineralization and were formed through water-washing and radiation-induced polymerization of both the gaseous and liquid pyrolysates. Direct evidence for pyrolysate reduction of uranium in the hydrothermal system is difficult to obtain as the chemical composition of the original organic fluid phase was modified during water-washing and radiolytic alteration. However, indirect evidence-e.g., higher O/C atomic ratios in uranium-free bitumens (0.1) relative to the Upper Proterozoic source rocks (0.02-0.05), isotopically very light carbon in associated whewellite (delta(13)C = 31.7 to -28.4 parts per thousand), and the striking absence of bitumens in the pre-uranium, hematite stage of the mineralization-indicates that oxidation of organic fluids may have contributed to lowering of aO(2) and uraninite precipitation.

Characterization of fecal indicator bacteria in sediments cores from the largest freshwater lake of Western Europe (Lake Geneva, Switzerland)

This study characterized the fecal indicator bacteria (FIB), including Escherichia coli (E. coli) and Enteroccocus (ENT), disseminated over time in the Bay of Vidy, which is the most contaminated area of Lake Geneva. Sediments were collected from a site located at similar to 500 m from the present waste water treatment plant (WWTP) outlet pipe, in front of the former WWTP outlet pipe, which was located at only 300 m from the coastal recreational area (before 2001). E. coil and ENT were enumerated in sediment suspension using the membrane filter method. The FIB characterization was performed for human Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and human specific bacteroides by PCR using specific primers and a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bacterial cultures revealed that maximum values of 35.2 x 10(8) and 6.6 x 10(6) CFU g(-1) dry sediment for E. coil and ENT, respectively, were found in the sediments deposited following eutrophication of Lake Geneva in the 1970s. whereas the WWTP started operating in 1964. The same tendency was observed for the presence of human fecal pollution: the percentage of PCR amplification with primers ESP-1/ESP-2 for E. faecalis and E. faecium indicated that more than 90% of these bacteria were from human origin. Interestingly, the PCR assays for specific-human bacteroides HF183/HF134 were positive for DNA extracted from all isolated strains of sediment surrounding WWPT outlet pipe discharge. The MALDI-TOF MS confirmed the presence of general E. coli and predominance E. faecium in isolated strains. Our results demonstrated that human fecal bacteria highly increased in the sediments contaminated with WWTP effluent following the eutrophication of Lake Geneva. Additionally, other FIB cultivable strains from animals or adapted environmental strains were detected in the sediment of the bay. The approaches used in this research are valuable to assess the temporal distribution and the source of the human fecal pollution in aquatic environments. (C) 2011 Elsevier Inc. All rights reserved.

Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest ymax

Installations de compostage industriel : exposition aux bioaérosols et effets sur la santé des travailleurs

La quantité de déchets ménagers et industriels ne cesse d'augmenter. Le tri sélectif des ordures est devenu systématique dans beaucoup de municipalités européennes et le compostage industriel des matières organiques par maturation accélérée en système clos a acquis une place prépondérante. Cependant, ce type de compostage nécessite une forte concentration de divers micro-organismes. Il en résulte un risque microbiologique aéroporté encore mal évalué pour les travailleurs exposés à ces atmosphères chargées en bioaérosols (bactéries, champignons et endotoxines en suspension dans l'air). [Auteur]

Functional analysis of a conserved DNA methyltransferase in caulobacter crescentus

CcrM is a DNA methyltransferase that methylates the adenine in GANTC motifs in the chromo-some of the bacterial model Caulobacter crescentus. The loss of the CcrM homolog is lethal in C. crescentus and in several other species of Alphaproteobacteria. In this research, we used different experimental and bioinformatic approaches to determine why CcrM is so critical to the physiology of C. crescentus. We first showed that CcrM is a resident orphan DNA methyltransferase in non-Rickettsiales Alphaproteobacteria and that its gene is strictly conserved in this clade (with only one ex¬ception among the genomes sequenced so far). In C. crescentus, cells depleted in CcrM in rich medium quickly lose viability and present an elongated phenotype characteristic of an im¬pairment in cell division. Using minimal medium instead of rich medium as selective and main¬tenance substrate, we could generate a AccrM mutant that presents a viability comparable to the wild type strain and only mild morphological defects. On the basis of a transcriptomic ap¬proach, we determined that several genes essential for cell division were downregulated in the AccrM strain in minimal medium. We offered decisive arguments to support that the efficient transcription of two of these genes, ftsZ and mipZ, coding respectively for the Z-ring forming GTPase FtsZ and an inhibitor of FtsZ polymerization needed for the correct positioning of the Z- ring at mid-cell, requires the methylation of an adenine in a conserved GANTC motif located in their core promoter region. We propose a model, according to which the genome of C. crescentus encodes a transcriptional activator that requires a methylated adenine in a GANTC context to bind to DNA and suggest that this transcriptional regulator might be the global cell-cycle regulator GcrA. In addition, combining a classic genetic approach and in vitro evolution experiments, we showed that the mortality and cell division defects of the AccrM strain in rich medium are mainly due to limiting intracellular levels of the FtsZ protein. We also studied the dynamics of GANTC methylation in C. crescentus using the SMRT technol¬ogy developed by Pacific Biosciences. Our findings support the commonly accepted model, accord¬ing to which the methylation state of GANTC motifs varies during the cell cycle of C. crescentus: before the initiation of DNA replication, the GANTC motifs are fully-methylated (methylated on both strands); when the DNA gets replicated, the GANTC motifs become hemi-methylated (methyl¬ated on one strand only) and this occurs at different times during replication for different loci along the chromosome depending on their position relative to the origin of replication; the GANTC mo¬tifs are only remethylated after DNA replication has finished as a consequence of the massive and short-lived expression of CcrM in predivisional cells. About 30 GANTC motifs in the C. crescentus chromosome were found to be undermethylated in most of the bacterial population; these might be protected from CcrM activity by DNA binding proteins and some of them could be involved in methylation-based bistable transcriptional switches. - CcrM est une ADN méthyltransférase qui méthyle les adénines dans le contexte GANTC dans le génome de la bactérie modèle Caulobacter crescentus. La perte de l'homologue de CcrM chez C. crescentus et chez plusieurs autres espèces d'Alphaproteobactéries est létale. Dans le courant de cette recherche, nous tentons de déterminer pourquoi la protéine CcrM est cruciale pour la survie de C. crescentus. Nous démontrons d'abord que CcrM est une adénine méthyltransférase orpheline résidente, dont le gène fait partie du génome minimal partagé par les Alphaprotéobactéries non-Rickettsiales (à une exception près). Lorsqu'une souche de C. crescentus est privée de CcrM, sa viabilité décroît rapi¬dement et ses cellules présentent une morphologie allongée qui suggère que la division cellulaire est inhibée. Nous sommes parvenus à créer une souche AccrM en utilisant un milieu minimum, au lieu du milieu riche classiquement employé, comme milieu de sélection et de maintenance pour la souche. Lorsque nous avons étudié le transcriptome de cette souche de C. crescentus privée de CcrM, nous avons pu constater que plusieurs gènes essentiels pour le bon déroulement de la division cellulaire bactérienne étaient réprimés. En particulier, l'expression adéquate des gènes ftsZ et mipZ - qui codent, respectivement, pour FtsZ, la protéine qui constitue, au milieu de la cellule, un anneau protéique qui initie le processus de division et pour MipZ, un inhibiteur de la polymérisation de FtsZ qui est indispensable pour le bon positionnement de l'anneau FtsZ - est dépendante de la présence d'une adénine méthylée dans un motif GANTC conservé situé dans leur région promotrice. Nous présentons un modèle selon lequel le génome de C. crescentus code pour un facteur de transcription qui exige la présence d'une adénine méthylée dans un contexte GANTC pour s'attacher à l'ADN et nous suggérons qu'il pourrait s'agir du régulateur global du cycle cellulaire GcrA. En outre, nous montrons, en combinant la génétique classique et une approche basée sur l'évolution expérimentale, que la mortalité et l'inhibition de la division cellulaire caractéristiques de la souche àccrMeη milieu riche sont dues à des niveaux excessivement bas de protéine FtsZ. Nous avons aussi étudié la dynamique de la méthylation du chromosome de C. crescentus sur la base de la technologie SMRT développée par Pacific Biosciences. Nous confirmons le modèle communément accepté, qui affirme que l'état de méthylation des motifs GANTC change durant le cycle cellulaire de C. crescentus: les motifs GANTC sont complètement méthylés (méthylés sur les deux brins) avant de début de la réplication de l'ADN; ils deviennent hémi-méthylés (méthylés sur un brin seulement) une fois répliqués, ce qui arrive à différents moments durant la réplication pour différents sites le long du chromosome en fonction de leur position par rapport à l'origine de répli-cation; finalement, les motifs GANTC sont reméthylés après la fin de la réplication du chromosome lorsque la protéine CcrM est massivement, mais très transitoirement, produite. Par ailleurs, nous identifions dans le chromosome de C. crescentus environ 30 motifs GANTC qui restent en perma-nence non-méthylés dans une grande partie de la population bactérienne; ces motifs sont probable-ment protégés de l'action de CcrM par des protéines qui s'attachent à l'ADN et certains d'entre eux pourraient être impliqués dans des mécanismes de régulation générant une transcription bistable.

Identification and modelling of human breast cancer subtypes

Résumé Le cancer du sein est le cancer le plus commun chez les femmes et est responsable de presque 30% de tous les nouveaux cas de cancer en Europe. On estime le nombre de décès liés au cancer du sein en Europe est à plus de 130.000 par an. Ces chiffres expliquent l'impact social considérable de cette maladie. Les objectifs de cette thèse étaient: (1) d'identifier les prédispositions et les mécanismes biologiques responsables de l'établissement des sous-types spécifiques de cancer du sein; (2) les valider dans un modèle ín vivo "humain-dans-souris"; et (3) de développer des traitements spécifiques à chaque sous-type de cancer du sein identifiés. Le premier objectif a été atteint par l'intermédiaire de l'analyse des données d'expression de gènes des tumeurs, produite dans notre laboratoire. Les données obtenues par puces à ADN ont été produites à partir de 49 biopsies des tumeurs du sein provenant des patientes participant dans l'essai clinique EORTC 10994/BIG00-01. Les données étaient très riches en information et m'ont permis de valider des données précédentes des autres études d'expression des gènes dans des tumeurs du sein. De plus, cette analyse m'a permis d'identifier un nouveau sous-type biologique de cancer du sein. Dans la première partie de la thèse, je décris I identification des tumeurs apocrines du sein par l'analyse des puces à ADN et les implications potentielles de cette découverte pour les applications cliniques. Le deuxième objectif a été atteint par l'établissement d'un modèle de cancer du sein humain, basé sur des cellules épithéliales mammaires humaines primaires (HMECs) dérivées de réductions mammaires. J'ai choisi d'adapter un système de culture des cellules en suspension basé sur des mammosphères précédemment décrit et pat décidé d'exprimer des gènes en utilisant des lentivirus. Dans la deuxième partie de ma thèse je décris l'établissement d'un système de culture cellulaire qui permet la transformation quantitative des HMECs. Par la suite, j'ai établi un modèle de xénogreffe dans les souris immunodéficientes NOD/SCID, qui permet de modéliser la maladie humaine chez la souris. Dans la troisième partie de ma thèse je décris et je discute les résultats que j'ai obtenus en établissant un modèle estrogène-dépendant de cancer du sein par transformation quantitative des HMECs avec des gènes définis, identifiés par analyse de données d'expression des gènes dans le cancer du sein. Les cellules transformées dans notre modèle étaient estrogène-dépendantes pour la croissance, diploïdes et génétiquement normales même après la culture cellulaire in vitro prolongée. Les cellules formaient des tumeurs dans notre modèle de xénogreffe et constituaient des métastases péritonéales disséminées et du foie. Afin d'atteindre le troisième objectif de ma thèse, j'ai défini et examiné des stratégies de traitement qui permettent réduire les tumeurs et les métastases. J'ai produit un modèle de cancer du sein génétiquement défini et positif pour le récepteur de l'estrogène qui permet de modéliser le cancer du sein estrogène-dépendant humain chez la souris. Ce modèle permet l'étude des mécanismes impliqués dans la formation des tumeurs et des métastases. Abstract Breast cancer is the most common cancer in women and accounts for nearly 30% of all new cancer cases in Europe. The number of deaths from breast cancer in Europe is estimated to be over 130,000 each year, implying the social impact of the disease. The goals of this thesis were first, to identify biological features and mechanisms --responsible for the establishment of specific breast cancer subtypes, second to validate them in a human-in-mouse in vivo model and third to develop specific treatments for identified breast cancer subtypes. The first objective was achieved via the analysis of tumour gene expression data produced in our lab. The microarray data were generated from 49 breast tumour biopsies that were collected from patients enrolled in the clinical trial EORTC 10994/BIG00-01. The data set was very rich in information and allowed me to validate data of previous breast cancer gene expression studies and to identify biological features of a novel breast cancer subtype. In the first part of the thesis I focus on the identification of molecular apacrine breast tumours by microarray analysis and the potential imptìcation of this finding for the clinics. The second objective was attained by the production of a human breast cancer model system based on primary human mammary epithelial cells {HMECs) derived from reduction mammoplasties. I have chosen to adopt a previously described suspension culture system based on mammospheres and expressed selected target genes using lentiviral expression constructs. In the second part of my thesis I mainly focus on the establishment of a cell culture system allowing for quantitative transformation of HMECs. I then established a xenograft model in immunodeficient NOD/SCID mice, allowing to model human disease in a mouse. In the third part of my thesis I describe and discuss the results that I obtained while establishing an oestrogen-dependent model of breast cancer by quantitative transformation of HMECs with defined genes identified after breast cancer gene expression data analysis. The transformed cells in our model are oestrogen-dependent for growth; remain diploid and genetically normal even after prolonged cell culture in vitro. The cells farm tumours and form disseminated peritoneal and liver metastases in our xenograft model. Along the lines of the third objective of my thesis I defined and tested treatment schemes allowing reducing tumours and metastases. I have generated a genetically defined model of oestrogen receptor alpha positive human breast cancer that allows to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.

CB1 knockout mice display impaired functionality of 5-HT1A and 5-HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5-HT) systems was investigated by examining 5-HT-dependent behavioural and biochemical responses in CB1 receptor knockout mice. CB1 knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5-HT2A receptor selective agonist ()DOI, as well as a reduced hypothermic response following administration of the 5-HT1A receptor agonist (±)-8-OH-DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB1 knockout mice. However, the tricyclic antidepressant imipramine and the 5-HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB1 receptor knockout and wild-type mice. No differences were found between both genotypes with regard to 5-HT2A receptor and 5-HT1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of the 5-HT1A receptor agonist (±)-8-OH-DPAT to stimulate 35SGTPS binding was detected in the hippocampal CA1 area of CB1 receptor knockout mice. This study provides evidence that CB1 receptors are involved in the regulation of serotonergic responses mediated by 5-HT2A and 5-HT1A receptors, and suggests that a reduced coupling of 5-HT1A receptors to Gi/o proteins in the hippocampus might be involved in these effects.

BDNF impairment in the hippocampus is related to enhanced despair behavior in CB1 knockout mice

Stress can cause damage and atrophy of neurons in the hippocampus by deregulating the expression of neurotrophic factors that promote neuronal plasticity. The endocannabinoid system represents a physiological substrate involved in neuroprotection at both cellular and emotional levels. The lack of CB1 receptor alters neuronal plasticity and originates an anxiety-like phenotype in mice. In the present study, CB1 knockout mice exhibited an augmented response to stress revealed by the increased despair behavior and corticosterone levels showed in the tail suspension test and decreased brain derived neurotrophic factor (BDNF) levels in the hippocampus. Interestingly, local administration of BDNF in the hippocampus reversed the increased despair behavior of CB1 knockout mice, confirming the crucial role played by BDNF on the emotional impairment of these mutants. The neurotrophic deficiency seems to be specific for BDNF since no differences were found in the levels of NGF and NT-3, two additional neurotrophic factors. Moreover, BDNF impairment is not related to the activity of its specific receptor TrkB or the activity of the transcription factor CREB. These results suggest that the lack of CB1 receptor originates an enhanced response to stress and neuronal plasticity by decreasing BDNF levels in the hippocampus that lead to impairment in the responses to emotional disturbances.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Hidrossedimentologia e disponibilidade hídrica da bacia hidrográfica da Barragem de Poilão

TEIXEIRA, José João Lopes. Departamento de Engenharia Agrícola, Centro de Ciências Agrárias da Universidade Federal do Ceará, Agosto de 2011. Hidrossedimentologia e disponibilidade hídrica da bacia hidrográfica da Barragem de Poilão, Cabo Verde. Orientador: José Carlos de Araújo. Examinadores: George Leite Mamede, Pedro Henrique Augusto Medeiros. O Arquipélago de Cabo Verde, situado na costa ocidental africana, sofre influência do deserto de Saara tornando o clima caraterizado por pluviometria muito baixa e distribuída irregularmente no espaço e no tempo. As chuvas são muito concentradas, gerando grandes escoamentos para o mar. O aumento da disponibilidade hídrica requer além da construção e manutenção de infraestrutura de captação e conservação de águas pluviais, uma gestão eficiente destes recursos. Atualmente, constitui um dos eixos estratégicos da política do estado de Cabo Verde, a captação, armazenamento e mobilização de águas superficiais através de construção de barragens. Estudos do comportamento hidrológico e sedimentológico do reservatório e da sua bacia de contribuição constituem premissas básicas para um ótimo dimensionamento, gestão e monitoramento da referida infraestrutura. É neste sentido que o presente estudo objetivou sistematizar informações hidrológicas e sedimentológicas da bacia hidrográfica da Barragem de Poilão (BP) e apresentar proposta operacional de longo prazo. A área de estudo ocupa 28 km² a montante da Bacia Hidrográfica da Ribeira Seca (BHRS) na Ilha de Santiago. A altitude da bacia varia de 99 m, situada na cota da barragem, até 1394 m. Para o estudo, foram utilizados e sistematizados, série pluviométrica de 1973 a 2010, registos de vazão instantânea do período 1984 a 2000 e registos agroclimáticos da área de estudo (1981 a 2004). Para o preenchimento das falhas tanto dos escoamentos como da descarga sólida em suspensão, foi utilizado o método de curva chave. Para estimativa de produção de sedimentos na bacia, aplicou-se a Equação Universal de Perda de Solo (USLE) e a razão de aporte de sedimentos (SDR). O índice de retenção de sedimentos no reservatório foi estimado pelo método de Brune e a distribuição de sedimento pelo método empírico de redução de área descrito por Borland e Miller e, revisado por Lara. Para gerar e simular curvas de vazão versus garantia foi utilizado código computacional VYELAS, desenvolvido por Araújo e baseado na abordagem de Campos. Também foi avaliada a redução da vazão de retirada do período 2006 a 2026, provocado pelo assoreamento do reservatório. Concluiu-se que em média a precipitação anual é de 323 mm, concentrando-se 73% nos meses de agosto e setembro; a bacia de contribuição apresenta como valor um número de curva (CN) de 76, com abstração inicial (Ia) de 26 mm, coeficiente de escoamento de 19% e uma vazão anual afluente de 1,7 hm³(cv= 0,73); a disponibilidade hídrica para uma garantia de 85% é avaliada em 0,548 hm³/ano e não 0,671 hm³/ano como indica o projeto original. Com uma descarga sólida estimada em 22.185 m³/ano conclui-se que até o ano de 2026, a capacidade do reservatório reduz a uma taxa de 1,8 % ao ano, devido ao assoreamento, provocando uma redução de 41% da disponibilidade hídrica inicial. Nessa altura, as perdas por evaporação e sangria serão da ordem de 81% da vazão afluente de entrada no reservatório. Na base desses resultados se apresentou proposta de operação da BP.

Engineered aprotinin for improved stability of fibrin biomaterials.

Fibrin has been long used clinically for hemostasis and sealing, yet extension of use in other applications has been limited due to its relatively rapid resorption in vivo, even with addition of aprotinin or other protease inhibitors. We report an engineered aprotinin variant that can be immobilized within fibrin and thus provide extended longevity. When recombinantly fused to a transglutaminase substrate domain from α(2)-plasmin inhibitor (α(2)PI(1-8)), the resulting variant, aprotinin-α(2)PI(1-8), was covalently crosslinked into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Challenge with physiological plasmin concentrations revealed that aprotinin-α(2)PI(1-8)-containing matrices retained 78% of their mass after 3 wk, whereas matrices containing wild type (WT) aprotinin degraded completely within 1 wk. Plasmin challenge of commercial sealants Omrixil and Tisseel, supplemented with aprotinin-α(2)PI(1-8) or WT aprotinin, showed extended longevity as well. When seeded with human dermal fibroblasts, aprotinin-α(2)PI(1-8)-supplemented matrices supported cell growth for at least 33% longer than those containing WT aprotinin. Subcutaneously implanted matrices containing aprotinin-α(2)PI(1-8) were detectable in mice for more than twice as long as those containing WT aprotinin. We conclude that our engineered recombinant aprotinin variant can confer extended longevity to fibrin matrices more effectively than WT aprotinin in vitro and in vivo.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Contrôle biologique de la pourriture cendrée (Macrophomina phaseolina) du niébé par l’enrobage des semences avec Clonostachys rosea: Impact des conditions de conservation des semences traitées sur la performance du bioagent.

Dans les sols infestés avec Macrophomina phaseolina les semences de niébé ne germent pas ou bien la plantule est détruite plus ou moins rapidement après la germination. Clonostachys rosea est un champignon commun du sol reconnu comme un saprophyte avec une haute capacité de compétition sur les racines et dans le sol. Notre étude a pour objectif d’évaluer l’effet de C. rosea en traitement de semences du niébé conservées à 40°C sur le développement de la pourriture charbonneuse. L’effet de la durée de conservation sur la viabilité des spores et le pouvoir antagonique du bioagent a également été étudié dans les conditions semi contrôlé et au champ. Les isolats de C. rosea proviennent de la collection du laboratoire de phytopathologie de AGRHYMET. La production des spores a été effectuée sur le milieu PDA pendant 4 semaines. Les semences du niébé ont été traitées avec une suspension de spores à la concentration de 108 spores / ml à raison de 5 ml de suspension par kg de semences. Les semences traitées ont été séchées sous la hotte à flux laminaire pendant 2 h avant d’être semées ou conservées à 40°C. Des tests de viabilité des spores de C. rosea sur le milieu de culture PDA ont été effectués au laboratoire tous les 15 jours de conservation. Le taux de germination des semences sur papier buvard, évalué au cinquième jour après semis au laboratoire a été de 99 %. La forte moyenne de spores viables au jour de l’enrobage (J0) (>4,5 105), a chuté à 1700 spores par graines après 75 jours de conservation. En serre, le niveau de colonisation des tissus des plants de niébé se développant sur le sol infesté a été significativement plus faible chez les plants issus de graines enrobées (1009 microsclérotes/g tissus) que chez ceux issus de graines non enrobées (2356 microsclérotes/g tissus). Par rapport a la sévérité de la maladie, exprimée sur une échelle de 1-5, ont été notées une forte infection des plants du témoin Macrophomina (graines non traitées semées dans un sol infesté) et des attaques moins sévères sur les traitements G3M et G4M (graines traitées semées dans un sol infesté). Au champ dans l’essai semé avec des graines enrobées mais non conservées, l’association des isolats G3 et G4 a été nettement supérieure aux autres traitements par rapport aux rendements en fane et en graines. Par contre, par rapport au rendement, aucun effet des traitements n’a été observé dans l’essai semé avec les graines conservées pendant 30 jours.