Studying the Human Gut Microbiota in the Trans-Omics Era - Focus on Metagenomics and Metabonomics

The human gut microbiota comprises a diverse microbial consortium closely co-evolved with the human genome and diet. The importance of the gut microbiota in regulating human health and disease has however been largely overlooked due to the inaccessibility of the intestinal habitat, the complexity of the gut microbiota itself and the fact that many of its members resist cultivation and are in fact new to science. However, with the emergence of 16S rRNA molecular tools and "post-genomics" high resolution technologies for examining microorganisms as they occur in nature without the need for prior laboratory culture, this limited view of the gut microbiota is rapidly changing. This review will discuss the application of molecular microbiological tools to study the human gut microbiota in a culture independent manner. Genomics or metagenomics approaches have a tremendous capability to generate compositional data and to measure the metabolic potential encoded by the combined genomes of the gut microbiota. Another post-genomics approach, metabonomics, has the capacity to measure the metabolic kinetic or flux of metabolites through an ecosystem at a particular point in time or over a time course. Metabonomics thus derives data on the function of the gut microbiota in situ and how it responds to different environmental stimuli e.g. substrates like prebiotics, antibiotics and other drugs and in response to disease. Recently these two culture independent, high resolution approaches have been combined into a single "transgenomic" approach which allows correlation of changes in metabolite profiles within human biofluids with microbiota compositional metagenomic data. Such approaches are providing novel insight into the composition, function and evolution of our gut microbiota.

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenyielenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis (2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox(R), a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox(R). Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Interactions between herbal and conventional medicines

Herb-drug interactions are subject to much interest at present, but for various reasons reports may be unreliable or unsubstantiated. Herbal medicines are variable in composition and quality, which may affect their interaction profile as well as the reliability of reports concerning them. In this review, clinical and experimental reports have been collated, evaluated and summarised, and the theoretical and clinical evidence presented. There is an explanation of the particular issues involved with herbal medicines as compared with conventional drugs, and reasons why comparisons may or may not be valid, which is intended for those without specialist experience in herbal products. It has become apparent that only a few herbal drugs have so far been cited in interaction reports, for example St John's Wort, Ginkgo biloba, Dan Shen, liquorice, Ma huang and garlic, and that the main drugs involved are those which are already susceptible to interactions with many other conventional drugs, such as warfarin, protease inhibitors and anti-cancer drugs. An attempt has been made to put the matter into perspective and recommendations have been given for health professionals to advise or develop strategies to safeguard patients, without resorting to speculation or scaremongering.

Which drugs cause preventable admissions to hospital? A systematic review

Aim: Previous systematic reviews have found that drug-related morbidity accounts for 4.3% of preventable hospital admissions. None, however, has identified the drugs most commonly responsible for preventable hospital admissions. The aims of this study were to estimate the percentage of preventable drug-related hospital admissions, the most common drug causes of preventable hospital admissions and the most common underlying causes of preventable drug-related admissions. Methods: Bibliographic databases and reference lists from eligible articles and study authors were the sources for data. Seventeen prospective observational studies reporting the proportion of preventable drug-related hospital admissions, causative drugs and/or the underlying causes of hospital admissions were selected. Included studies used multiple reviewers and/or explicit criteria to assess causality and preventability of hospital admissions. Two investigators abstracted data from all included studies using a purpose-made data extraction form. Results: From 13 papers the median percentage of preventable drug-related admissions to hospital was 3.7% (range 1.4-15.4). From nine papers the majority (51%) of preventable drug-related admissions involved either antiplatelets (16%), diuretics (16%), nonsteroidal anti-inflammatory drugs (11%) or anticoagulants (8%). From five studies the median proportion of preventable drug-related admissions associated with prescribing problems was 30.6% (range 11.1-41.8), with adherence problems 33.3% (range 20.9-41.7) and with monitoring problems 22.2% (range 0-31.3). Conclusions: Four groups of drugs account for more than 50% of the drug groups associated with preventable drug-related hospital admissions. Concentrating interventions on these drug groups could reduce appreciably the number of preventable drug-related admissions to hospital from primary care.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Multivariate analysis of the Fugl-Meyer outcome measures assessing the effectiveness of GENTLE/S robot-mediated stroke therapy

Background: Robot-mediated therapies offer entirely new approaches to neurorehabilitation. In this paper we present the results obtained from trialling the GENTLE/S neurorehabilitation system assessed using the upper limb section of the Fugl-Meyer ( FM) outcome measure. Methods: We demonstrate the design of our clinical trial and its results analysed using a novel statistical approach based on a multivariate analytical model. This paper provides the rational for using multivariate models in robot-mediated clinical trials and draws conclusions from the clinical data gathered during the GENTLE/S study. Results: The FM outcome measures recorded during the baseline ( 8 sessions), robot-mediated therapy ( 9 sessions) and sling-suspension ( 9 sessions) was analysed using a multiple regression model. The results indicate positive but modest recovery trends favouring both interventions used in GENTLE/S clinical trial. The modest recovery shown occurred at a time late after stroke when changes are not clinically anticipated. Conclusion: This study has applied a new method for analysing clinical data obtained from rehabilitation robotics studies. While the data obtained during the clinical trial is of multivariate nature, having multipoint and progressive nature, the multiple regression model used showed great potential for drawing conclusions from this study. An important conclusion to draw from this paper is that this study has shown that the intervention and control phase both caused changes over a period of 9 sessions in comparison to the baseline. This might indicate that use of new challenging and motivational therapies can influence the outcome of therapies at a point when clinical changes are not expected. Further work is required to investigate the effects arising from early intervention, longer exposure and intensity of the therapies. Finally, more function-oriented robot-mediated therapies or sling-suspension therapies are needed to clarify the effects resulting from each intervention for stroke recovery.

Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation

Porcine ear skin is widely used to study skin permeation and absorption of ester compounds, whose permeation and absorption profiles may be directly influenced by in situ skin esterase activity. Importantly, esterase distribution and activity in porcine ear skin following common protocols of skin handling and storage have not been characterised. Thus, we have compared the distribution and hydrolytic activity of esterases in freshly excised, frozen, heated and explanted porcine ear skin. Using an esterase staining kit, esterase activity was found to be localised in the stratum corneum and viable epidermis. Under frozen storage and a common heating protocol of epidermal sheet separation, esterase staining in the skin visibly diminished. This was confirmed by a quantitative assay using HPLC to monitor the hydrolysis of aspirin, in freshly excised, frozen or heated porcine ear skin. Compared to vehicle-only control, the rate of aspirin hydrolysis was approximately three-fold higher in the presence of freshly excised skin, but no different in the presence of frozen or heated skin. Therefore, frozen and heat-separated porcine ear skin should not be used to study the permeation of ester-containing permeants, in particular co-drugs and pro-drugs, whose hydrolysis or degradation can be modulated by skin esterases.

Colorectal cancer and the relationship between genes and the environment

Colorectal cancer (CRC) is a significant cause of morbidity and mortality in developed countries, with both genetic and environmental factors contributing to the etiology and progression of the disease. Several risk factors have been identified, including positive family history, red meat intake, smoking, and alcohol intake. Protective factors include vegetables, calcium, hormone replacement therapy, folate, nonsteroidal anti-inflammatory drugs, and physical activity. The interaction between these environmental factors, in particular diet and genes, is an area of growing interest. Currently, oncogenes, tumor suppressor genes, and mismatch repair genes are believed to play an essential role in colorectal carcinogenesis. When considering the genetics of CRC, only 10% of cases are inherited and only 2-6% can be ascribed to the highly penetrant genes, such as APC, hMLH and hMSH2. Lower penetrance genes combined with a Western-style diet contribute to the majority of sporadic CRCs. The purpose of this article is to give a brief overview of the epidemiologic studies that have been conducted and present the major findings. Here, we examine the molecular events in CRC, with particular focus on the interaction between genes and environment, and review the most current research in this area.

Synthesis, structural characterization and catalytic activity of a multifunctional enzyme mimetic oxoperoxovanadium(v) complex

The synthesis and structural characterization of a novel oxoperoxovanadium(v) complex [VO(O-2)(PAH)-(phen)] containing the ligands 2-phenylacetohydroxamic acid (PAHH) and 1,10-phenanthroline (phen) has been accomplished. The oxoperoxovanadium(v) complex was found to mimic both vanadate-dependent haloperoxidase (VHPO) activity as well as nuclease activity through effective interaction with DNA. The complex is the first example of a structurally characterized stable oxoperoxovanadium(v) complex with a coordinated bi-dentate hydroximate moiety (-CONHO-) from 2-phenylacetohydroximate (PAH). The oxoperoxovanadium(v) complex has been used as catalyst for the peroxidative bromination reaction of some unsaturated alcohols (e.g. 4-pentene-1-ol, 1-octene-3-ol and 9-decene-1-ol) in the presence of H2O2 and KBr. The catalytic products have been characterized by GC-MS analysis and spectrophotometric methods. The DNA binding of this complex has been established with CT DNA whereas the DNA cleavage was demonstrated with plasmid DNA. The interactions of the complex with DNA have been monitored by electronic absorption and fluorescence emission spectroscopy. Viscometric measurements suggest that the compound is a DNA intercalator. The nuclease activity of this complex was confirmed by gel electrophoresis studies.

Pollen-based differentiation of Amazonian rainforest communities and implications for lowland palaeoecology in tropical South America

An ongoing controversy in Amazonian palaeoecology is the manner in which Amazonian rainforest communities have responded to environmental change over the last glacial–interglacial cycle. Much of this controversy results from an inability to identify the floristic heterogeneity exhibited by rainforest communities within fossil pollen records. We apply multivariate (Principal Components Analysis) and classification (Unweighted Pair Group with Arithmetic Mean Agglomerative Classification) techniques to floral-biometric, modern pollen trap and lake sediment pollen data situated within different rainforest communities in the tropical lowlands of Amazonian Bolivia. Modern pollen rain analyses from artificial pollen traps show that evergreen terra firme (well-drained), evergreen terra firme liana, evergreen seasonally inundated, and evergreen riparian rainforests may be readily differentiated, floristically and palynologically. Analogue matching techniques, based on Euclidean distance measures, are employed to compare these pollen signatures with surface sediment pollen assemblages from five lakes: Laguna Bella Vista, Laguna Chaplin, and Laguna Huachi situated within the Madeira-Tapajós moist forest ecoregion, and Laguna Isirere and Laguna Loma Suarez, which are situated within forest patches in the Beni savanna ecoregion. The same numerical techniques are used to compare rainforest pollen trap signatures with the fossil pollen record of Laguna Chaplin.

Technical note: Organics-induced fluorescence in Raman studies of sulfuric acid aerosols

Fluorescence is a troublesome side effect in laboratory Raman studies on sulfuric acid solutions and aerosol particles. We performed experiments showing that organic matter induces fluorescence in H2SO4/H2O solutions. The intensity of the fluorescence signal appears to be almost independent of the concentration of the organic substances, but depends strongly on the sulfuric acid concentration. The ubiquity of organic substances in the atmosphere, their relatively high abundance, and the insensitivity of the fluorescence with respect to their concentrations will render most acidic natural aerosols subject to absorption and fluorescence, possibly influencing climate forcing. We show that, while fluorescence may in the future become a valuable tool of aerosol diagnostics, the concurrent absorption is too small to significantly affect the atmosphere's radiative balance.

Evaluation of Earth system models using modern and palaeo-observations: the state-of-the-art

Earth system models are increasing in complexity and incorporating more processes than their predecessors, making them important tools for studying the global carbon cycle. However, their coupled behaviour has only recently been examined in any detail, and has yielded a very wide range of outcomes, with coupled climate-carbon cycle models that represent land-use change simulating total land carbon stores by 2100 that vary by as much as 600 Pg C given the same emissions scenario. This large uncertainty is associated with differences in how key processes are simulated in different models, and illustrates the necessity of determining which models are most realistic using rigorous model evaluation methodologies. Here we assess the state-of-the-art with respect to evaluation of Earth system models, with a particular emphasis on the simulation of the carbon cycle and associated biospheric processes. We examine some of the new advances and remaining uncertainties relating to (i) modern and palaeo data and (ii) metrics for evaluation, and discuss a range of strategies, such as the inclusion of pre-calibration, combined process- and system-level evaluation, and the use of emergent constraints, that can contribute towards the development of more robust evaluation schemes. An increasingly data-rich environment offers more opportunities for model evaluation, but it is also a challenge, as more knowledge about data uncertainties is required in order to determine robust evaluation methodologies that move the field of ESM evaluation from "beauty contest" toward the development of useful constraints on model behaviour.

Evaluation of biospheric components in Earth system models using modern and palaeo-observations: the state-of-the-art

Earth system models (ESMs) are increasing in complexity by incorporating more processes than their predecessors, making them potentially important tools for studying the evolution of climate and associated biogeochemical cycles. However, their coupled behaviour has only recently been examined in any detail, and has yielded a very wide range of outcomes. For example, coupled climate–carbon cycle models that represent land-use change simulate total land carbon stores at 2100 that vary by as much as 600 Pg C, given the same emissions scenario. This large uncertainty is associated with differences in how key processes are simulated in different models, and illustrates the necessity of determining which models are most realistic using rigorous methods of model evaluation. Here we assess the state-of-the-art in evaluation of ESMs, with a particular emphasis on the simulation of the carbon cycle and associated biospheric processes. We examine some of the new advances and remaining uncertainties relating to (i) modern and palaeodata and (ii) metrics for evaluation. We note that the practice of averaging results from many models is unreliable and no substitute for proper evaluation of individual models. We discuss a range of strategies, such as the inclusion of pre-calibration, combined process- and system-level evaluation, and the use of emergent constraints, that can contribute to the development of more robust evaluation schemes. An increasingly data-rich environment offers more opportunities for model evaluation, but also presents a challenge. Improved knowledge of data uncertainties is still necessary to move the field of ESM evaluation away from a "beauty contest" towards the development of useful constraints on model outcomes.

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 104–106 M–1 and stoichiometries, n1, of 2–13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 103–105 M–1 and stoichiometries, n2, of 16–30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern–Volmer quenching constant, KSV.

Dietary iron depletion at weaning imprints low microbiome diversity and this is not recovered with oral nano Fe(III)

Alterations in the gut microbiota have been recently linked to oral iron. We conducted two feeding studies including an initial diet-induced iron-depletion period followed by supplementation with nanoparticulate tartrate-modified ferrihydrite (Nano Fe(III): considered bioavailable to host but not bacteria) or soluble ferrous sulfate (FeSO4: considered bioavailable to both host and bacteria). We applied denaturing gradient gel electrophoresis and fluorescence in situ hybridization for study-1 and 454-pyrosequencing of fecal 16S rRNA in study-2. In study-1, the within-community microbial diversity increased with FeSO4 (P = 0.0009) but not with Nano Fe(III) supplementation. This was confirmed in study-2, where we also showed that iron depletion at weaning imprinted significantly lower within- and between-community microbial diversity compared to mice weaned onto the iron-sufficient reference diet (P < 0.0001). Subsequent supplementation with FeSO4 partially restored the within-community diversity (P = 0.006 in relation to the continuously iron-depleted group) but not the between-community diversity, whereas Nano Fe(III) had no effect. We conclude that (1) dietary iron depletion at weaning imprints low diversity in the microbiota that is not, subsequently, easily recovered; (2) in the absence of gastrointestinal disease iron supplementation does not negatively impact the microbiota; and (3) Nano Fe(III) is less available to the gut microbiota.