Measurement of the Electrogenicity of Bile Salt/H+ Antiport in Escherichia coli

The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H+ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that functions physiologically in protection of bacterial cells against bile salts - imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an electrogenic antiport reaction (Paul et al., 2014). This protocol describes, using fluorometry, how to detect electrogenic antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆Ψ. The method exploits changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the probe Oxonol V in response to changes in membrane potential due to the MdtM-catalysed sodium cholate/H+ exchange reaction. The protocol can be adapted to detect activity of any secondary active antiporter that couples the electrogenic translocation of H+ across a biological membrane to that of its counter-substrate, and may be used to unmask otherwise camouflaged transport activities and physiological roles.

Synthesis and characterization of main-chain bile acid-based degradable polymers

Les acides biliaires sont des composés naturels existants dans le corps humain. Leur biocompatibilité, leur caractère amphiphile et la rigidité de leur noyau stéroïdien, ainsi que l’excellent contrôle de leurs modifications chimiques, en font de remarquables candidats pour la préparation de matériaux biodégradables pour le relargage de médicaments et l'ingénierie tissulaire. Nous avons préparé une variété de polymères à base d’acides biliaires ayant de hautes masses molaires. Des monomères macrocycliques ont été synthétisés à partir de diènes composés de chaînes alkyles flexibles attachées à un noyau d'acide biliaire via des liens esters ou amides. Ces synthèses ont été réalisées par la fermeture de cycle par métathèse, utilisant le catalyseur de Grubbs de première génération. Les macrocycles obtenus ont ensuite été polymérisés par ouverture de cycle, entropiquement induite le catalyseur de Grubbs de seconde génération. Des copolymères ont également été préparés à partir de monolactones d'acide ricinoléique et de monomères cycliques de triester d’acide cholique via la même méthode. Les propriétés thermiques et mécaniques et la dégradabilité de ces polymères ont été étudiées. Elles peuvent être modulées en modifiant les différents groupes fonctionnels décorant l’acide biliaire et en ayant recours à la copolymérisation. La variation des caractéristiques physiques de ces polymères biocompatibles permet de moduler d’autres propriétés utiles, tel que l’effet de mémoire de forme qui est important pour des applications biomédicales.

Polyamides and polyesters made of bile acids in the main chain

La préparation de polymères à base d’acides biliaires, molécules biologiques, a attiré l'attention des chercheurs en raison des applications potentielles dans les domaines biomédicaux et pharmaceutiques. L’objectif de ce travail est de synthétiser de nouveaux biopolymères dont la chaîne principale est constituée d’unités d’acides biliaires. La polymérisation par étapes a été adoptée dans ce projet afin de préparer les deux principales classes de polymères utilisés en fibres textiles: les polyamides et les polyesters. Des monomères hétéro-fonctionnels à base d’acides biliaires ont été synthétisés et utilisés afin de surmonter le déséquilibre stoechiométrique lors de la polymérisation par étapes. Le dérivé de l’acide lithocholique modifié par une fonction amine et un groupement carboxylique protégé a été polymérisé en masse à températures élevées. Les polyamides obtenus sont très peu solubles dans les solvants organiques. Des polyamides et des polyesters solubles en milieu organique ont pu être obtenus dans des conditions modérées en utilisant l’acide cholique modifié par des groupements azide et alcyne. La polymérisation a été réalisée par cycloaddition azoture-alcyne catalysée par l'intermédiaire du cuivre(Ι) avec deux systèmes catalytiques différents, le bromure de cuivre(I) et le sulfate de cuivre(II). Seul le bromure de cuivre(Ι) s’est avéré être un catalyseur efficace pour le système, permettant la préparation des polymères avec un degré de polymérisation égale à 50 et une distribution monomodale de masse moléculaire (PDI ˂ 1.7). Les polymères synthétisés à base d'acide cholique sont thermiquement stables (307 °C ≤ Td ≤ 372 °C) avec des températures de transition vitreuse élevées (137 °C ≤ Tg ≤ 167 °C) et modules de Young au-dessus de 280 MPa, dépendamment de la nature chimique du lien.

Traitement de l'arthrose canine : étude d'efficacité d'une diète riche en moule verte (Perna canaliculus) et validité des méthodes cliniques d'évaluation de l'effet traitement

Introduction: L’arthrose est une maladie fréquente chez le chien. Même si des traitements palliatifs ont prouvé leur efficacité pour soulager les signes cliniques associés, aucun n’a permis de restaurer la structure des cartilages ou la fonction des patients. De nouvelles modalités de traitement sont proposées mais très peu d’évidences existent quant à leur efficacité. Un problème récurrent pour apporter plus d’évidence est le manque d’outils d’évaluation dûment validés. Objectif: Evaluer l’efficacité d’une diète contenant de la moule verte, ainsi que la validité de différentes méthodes pour évaluer les effets de ce traitement sur la condition arthrosique de chiens de propriétaire. Méthodes: Une étude prospective croisée contrôlée en aveugle a été conduite sur 30 chiens de propriétaires. Leur condition était stable et non traitée. Ils ont reçu une diète contrôle pendant 30 jours puis la diète thérapeutique pendant 60 jours. A J0 (inclusion), J30 (fin de la période contrôle) et J90 (fin du traitement), les concentrations plasmatiques en acides gras ω–3 (EPA et DHA), le pic de force verticale (PVF – critère de référence), un questionnaire multifactoriel standardisé (MFQ), et la conductance électrodermique (EDA) ont été mesurés. Deux fois par semaine, les propriétaires remplissaient un questionnaire sur la fonction spécifique à leur animal (CODI). L’activité locomotrice (actimétrie) a été enregistrée en continu sur 7 chiens. Résultats: Aucun effet secondaire n’a été documenté excepté une augmentation du poids des chiens durant la période traitement. La localisation -thoracique ou pelvienne- du membre étudié et le poids ont interagi avec PVF. Les taux d’EPA et DHA ont augmenté durant la période traitement (p=0.026 et p<0.001, respectivement), tout comme PVF corrigé pour l’effet du poids et de la localisation du membre (p=0.003). La validité technique de PVF a été vérifiée. La validité technique et selon-critère du CODI ont été vérifiées. MFQ n’a pas montré d’homogénéité suffisante entre ses différents composants, bien que les résultats du composite convergent partiellement avec PVF. L’EDA n’a pas montré de validité technique suffisante. L’actimétrie a montré une validité technique satisfaisante ainsi que des résultats convergeant avec ceux de PVF. Conclusions: La méthode choisie comme référence a montré un effet bénéfique de la diète testée et sa validité technique a été vérifiée. À la fois, CODI et l’actimétrie ont été validés. La validité d’EDA et de MFQ a été remise en question. L’actimétrie a donné des résultats prometteurs, probablement complémentaires de PVF.

AST-120 (spherical carbon adsorbent) lowers ammonia levels and attenuates brain edema in bile duct-ligated rats

The pathogenesis of hepatic encephalopathy is multifactorial, involving gut-derived toxins such as ammonia, which has been demonstrated to induce oxidative stress. Therefore, a primary hepatic encephalopathy treatment target is reducing ammonia production in the gastrointestinal tract. AST-120, an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600 m(2) /g, acts as a sink for neurotoxins and hepatotoxins present in the gut. We evaluated the capacity of AST-120 to adsorb ammonia in vitro and to lower blood ammonia, oxidative stress and brain edema in cirrhotic rats. Cirrhosis was induced in rats by bile duct ligation for 6 weeks. AST-120 was administered by gavage preventively for 6 weeks (0.1, 1, and 4 g/kg/day). In addition, AST-120 was evaluated as a short-term treatment for 2 weeks and 3 days (1 g/kg/day) and as a sink to adsorb intravenously infused ammonium acetate. In vitro, AST-120 efficiently adsorbed ammonia. Ammonia levels significantly decreased in a dose-dependent manner for all AST-120-treated bile duct-ligated rats (nontreated: 177.3 ± 30.8 μM; AST-120, 0.1 g/kg/day: 121.9 ± 13.8 μM; AST-120, 1 g/kg/day: 80.9 ± 30.0 μM; AST-120, 4 g/kg/day: 48.8 ± 19.6 μM) and significantly correlated with doses of AST-120 (r = -0.6603). Brain water content and locomotor activity normalized after AST-120 treatments, whereas arterial reactive oxygen species levels remained unchanged. Furthermore, AST-120 significantly attenuated a rise in arterial ammonia after ammonium acetate administration (intravenously). Conclusion:AST-120 treatment decreased arterial ammonia levels, normalized brain water content and locomotor activity but did not demonstrate an effect on systemic oxidative stress. Also, AST-120 acts as an ammonia sink, efficiently removing blood-derived ammonia. Additional studies are warranted to evaluate the effects of AST-120 on hepatic encephalopathy in patients with advanced liver disease. (HEPATOLOGY 2011;).

La educación de los sordomudos y ciegos en el BILE.

Se analizan las referencias que aparecen en el BILE (Boletín de la Institución Libre de Enseñanza) sobre la educación de los deficientes auditivos y oculares. Se puede afirmar la preocupación de los institucionistas por dar a conocer la situación real de los afectados por este tipo de deficiencias físicas, así como dar a conocer las tendencias pedagógicas y médicas que se estaban poniendo en práctica en otros lugares del mundo. El BILE encarnó el espíritu reformador e innovador de la enseñanza en nuestro país, el esfuerzo para que España avanzará en la formación de sus jóvenes, el de crear centros vanguardistas en la concepción de pedagogía en todos sus ámbitos, así como en la aplicación de las nuevas corrientes pedagógicas en su organización.

El boletín de la institución libre de enseñanza BILE.

En este artículo presento un análisis descriptivo del Boletín de la Institución Libre de Enseñanza en sus dos épocas de existencia. Periodos delimitados por los acontecimientos de nuestra guerra civil. La PRIMERA es la más importante por su duración, abarca sesenta años de publicación ininterrumpida, desde el año 1877 hasta la mitad de 1936. La SEGUNDA época del B.I.L.E, es relativamente reciente, reaparece en 1987 y lo edita la Fundación Francisco Giner de los Ríos. El revisar que misión se propuso esta revista, sus comienzos, evolución y estructuras contribuirá al convencimiento de que el Boletín constituye una fuente documental valiosísima e imprescindible para el estudioso de la Institución Libre de Enseñanza y también para quien quiera profundizar en la evolución de la Historia de la Educación Española de finales del siglo XIX y comienzos del XX. Especialmente es fuente necesaria para conocer nuestra incorporación al Movimiento Pedagógico de la Escuela Nueva, directrices educativas que fueron seguidas por la I.L.E y de cuyos principios y realizaciones ha quedado constancia en las páginas de su Boletín.

Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries

A total of 94 European eels (Anguilla anguilla) were collected from five estuaries in the UK. The deconjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) in the bile of the eels were separated using HPLC. Six PAH metabolites were identified: 1-hydroxy (1-OH) metabolites of phenanthrene, pyrene and chrysene; and the 1-OH, 3-OH and 7,8 dihydrodiol metabolites of benzo[a]pyrene (BaP). The mean concentration of the six metabolites was greatest in eels from the Tyne (49 muM) followed by the Wear (33 muM), Tees (19 muM), Thames (4 muM) and Severn (2 muM) estuaries. Although 1-OH pyrene was always the dominant compound, there were significant differences (P<0.05) between sites and between estuaries for some metabolites. Normalising the molar concentration of the bile metabolites to the bile biliverdin absorbance reduced sample variation. When the metabolites identified were-each expressed as a percentage of the total detected, the metabolite profile was characteristic for each estuary. (C) 2002 Elsevier Science B.V. All rights reserved.

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutics

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultraperformance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats.Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0±10.4%) andheart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-β-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influencedbymicrobial activitiesormodulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

Aims: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. Methods and Results: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. Conclusions: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. Significance and Impact of Study: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.