Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for ‘metabolic retroversion’

Dietary sources of methylamines such as choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It is recognized that the gut microbiota is able to convert choline to TMA in a fermentation-like process. Similarly, PC and carnitine are converted to TMA by the gut microbiota. It has been suggested that TMAO is subject to ‘metabolic retroversion’ in the gut (i.e. it is reduced to TMA by the gut microbiota, with this TMA being oxidized to produce TMAO in the liver). Sixty-six strains of human faecal and caecal bacteria were screened on solid and liquid media for their ability to utilize trimethylamine N-oxide (TMAO), with metabolites in spent media profiled by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. Enterobacteriaceae produced mostly TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria (enterococci, streptococci, bifidobacteria) produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA from TMAO. The presence of TMAO in media increased the growth rate of Enterobacteriaceae; while it did not affect the growth rate of lactic acid bacteria, TMAO increased the biomass of these bacteria. The positive influence of TMAO on Enterobacteriaceae was confirmed in anaerobic, stirred, pH-controlled batch culture fermentation systems inoculated with human faeces, where this was the only bacterial population whose growth was significantly stimulated by the presence of TMAO in the medium. We hypothesize that dietary TMAO is used as an alternative electron acceptor by the gut microbiota in the small intestine/proximal colon, and contributes to microbial population dynamics upon its utilization and retroversion to TMA, prior to absorption and secondary conversion to TMAO by hepatic flavin-containing monooxygenases. Our findings support the idea that oral TMAO supplementation is a physiologically-stable microbiota-mediated strategy to deliver TMA at the gut barrier.

Predictive Variables Affecting Transfusion Requirements in Orthotopic Liver Transplantation

INTRODUCTION AND AIMS: Adult orthotopic liver transplantation (OLT) is associated with considerable blood product requirements. The aim of this study was to assess the ability of preoperative information to predict intraoperative red blood cell (RBC) transfusion requirements among adult liver recipients. METHODS: Preoperative variables with previously demonstrated relationships to intraoperative RBC transfusion were identified from the literature: sex, age, pathology, prothrombin time (PT), factor V, hemoglobin (Hb), and platelet count (plt). These variables were then retrospectively collected from 758 consecutive adult patients undergoing OLT from 1997 to 2007. Relationships between these variables and intraoperative blood transfusion requirements were examined by both univariate analysis and multiple linear regression analysis. RESULTS: Univariate analysis confirmed significant associations between RBC transfusion and PT, factor V, Hb, Plt, pathology, and age (P values all < .001). However, stepwise backward multivariate analysis excluded variables Plt and factor V from the multiple regression linear model. The variables included in the final predictive model were PT, Hb, age, and pathology. Patients suffering from liver carcinoma required more blood products than those suffering from other pathologies. Yet, the overall predictive power of the final model was limited (R(2) = .308; adjusted R(2) = .30). CONCLUSION: Preoperative variables have limited predictive power for intraoperative RBC transfusion requirements even when significant statistical associations exist, identifying only a small portion of the observed total transfusion variability. Preoperative PT, Hb, age, and liver pathology seem to be the most significant predictive factors but other factors like severity of liver disease, surgical technique, medical experience in liver transplantation, and other noncontrollable human variables may play important roles to determine the final transfusion requirements.

Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

Effects of hypothermia on brain glucose metabolism in acute liver failure: a H/C-nuclear magnetic resonance study.

Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35°C) on brain metabolic pathways using combined 1H and 13C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. :1H and 13C NMR spectroscopy using [1-13C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37°C (normothermic) or 35°C (hypothermic), and appropriate sham-operated controls. At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% ± 7.7% and 42.2% ± 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% ± 15.6% and 267.3% ± 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% ± 48.7% and 707.9% ± 65.7% (P < 0.001), of control respectively, resulting in increased fractional 13C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. Mild hypothermia (35°C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Mild hypothermia prevents brain edema and attenuates up-regulation of the astrocytic benzodiazepine receptor in experimental acute liver failure.

BACKGROUND/AIMS: Mild hypothermia has proven useful in the clinical management of patients with acute liver failure. Acute liver failure in experimental animals results in alterations in the expression of genes coding for astrocytic proteins including the "peripheral-type" (astrocytic) benzodiazepine receptor (PTBR), a mitochondrial complex associated with neurosteroid synthesis. To gain further insight into the mechanisms whereby hypothermia attenuates the neurological complications of acute liver failure, we investigated PTBR expression in the brains of hepatic devascularized rats under normothermic (37 degrees C) and hypothermic (35 degrees C) conditions. METHODS: PTBR mRNA was measured using semi-quantitative RT-PCR in cerebral cortical extracts and densities of PTBR sites were measured by quantitative receptor autoradiagraphy. Brain pregnenolone content was measured by radioimmunoassay. RESULTS: At coma stages of encephalopathy, animals with acute liver failure manifested a significant increase of PTBR mRNA levels. Brain pregnenolone content and [(3)H]PK 11195 binding site densities were concomitantly increased. Mild hypothermia prevented brain edema and significantly attenuated the increased receptor expression and pregnenolone content. CONCLUSIONS: These findings suggest that an attenuation of PTBR up-regulation resulting in the prevention of increased brain neurosteroid content represents one of the mechanisms by which mild hypothermia exerts its protective effects in ALF.

The orphan nuclear receptor, liver receptor homolog-1 (LRH-1, NR5A2) regulates decidualization

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Conjugated linoleic acid and human health: a critical evaluation of the evidence

Purpose of review: This review critically evaluates studies investigating the effects of conjugated linoleic acid on human health, including effects on body composition, blood lipids, liver metabolism, insulin sensitivity and immune function. It focuses mainly on human intervention studies, but includes some reference to animal and cellular studies which provide insight into potential molecular mechanisms of action of conjugated linoleic acid. Recent findings: Human studies continue to report inconsistent effects of conjugated linoleic acid on human health. Some of these reports are based on overinterpretation of marginal effects of supplementation. Recent data suggest that the effects of the substance may be isomer dependent and that cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acids have opposing effects on blood lipids and on metabolism in adipocytes and hepatic cells. Summary: Claims that conjugated linoleic acid is beneficial for health remain as yet unconvincing. Human studies investigating the effects of conjugated linoleic acid supplements have tended to use mixtures of isomers and have been inconsistent. More recent studies have attempted to use relatively pure preparations of single isomers and these studies suggest that the effects of conjugated linoleic acid may be isomer-specific. These recent data suggest a relative detrimental effect of trans-10, cis-12 conjugated linoleic acid on blood lipids. There appears to be little effect of conjugated linoleic acid on immune function and the effects on insulin sensitivity remain unclear.

Probiotics and cancer: from in vitro to human studies

Studies in cell cultures and animal models provide evidence that probiotics can beneficially influence various stages in development of colon cancer including tumor initiation, promotion and metastasis. For example, oral administration of Lactobacillus and Bifidobacterium strains can prevent genotoxic damage to the colonic epithelium (considered to be an early stage of the carcinogenic process). Administration to rats of probiotics reduced the incidence of carcinogen-induced pre-cancerous lesions (aberrant crypt foci) in the colon. Furthermore a combination of Bifidobacterium longum and inulin (a prebiotic) was more effective than either treatment alone. In this latter study, the dietary treatments were given after exposure to the carcinogen, which suggests that the protective effects were being exerted at the promotional phase of carcinogenesis. L. acidophilus feeding has been shown to decrease the incidence of colon tumors in rats challenged with a carcinogen and B. longum reduced the incidence of carcinogeninduced colon, liver and mammary tumors. There is limited evidence from epidemiological studies for protective effects of products containing probiotics in humans, but a number of recent dietary intervention studies in healthy subjects and in polyp and cancer patients have yielded promising results on the basis of biomarkers of cancer risk and grade of colorectal tumors.

In vitro fermentation of oat bran obtained by debranning with a mixed culture of human fecal bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.

A metabolic system-wide characterisation of the pig: a model for human physiology

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major inter-species differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS

The first application of high field NMR spectroscopy (800 MHz for 1H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional 1H–1H TOCSY and 1H–13C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

A metabolic system-wide characterisation of the pig: a model for human physiology

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major interspecies differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.

Staphylococcus aureus MnhF mediates cholate efflux and facilitates survival under human colonic conditions

Resistance to the innate defences of the intestine is crucial for the survival and carriage of Staphylococcus aureus, a common coloniser of the human gut. Bile salts produced by the liver and secreted into the intestines are one such group of molecules with potent anti-microbial activity. The mechanisms by which S. aureus is able to resist such defences in order to colonize and survive in the human gut are unknown. Here we show that mnhF confers resistance to bile salts, which can be abrogated by efflux pump inhibitors. MnhF mediates efflux of radiolabelled cholic acid in both S. aureus and when heterologously expressed in Escherichia coli, rendering them resistant. Deletion of mnhF attenuated survival of S. aureus in an anaerobic three stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine.