In vitro fermentation of oat and barley derived beta-glucans by human faecal microbiota

Fermentation of beta-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general beta-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a beta-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

What do we mean when we refer to Bacteroidetes populations in the human gastrointestinal microbiota?

Recent large-scale cloning studies have shown that the ratio of Bacteroidetes to Firmicutes may be important in the obesity-associated gut microbiota, but the species these phyla represent in this ecosystem has not been examined. The Bacteroidetes data from the recent Turnbaugh study were examined to determine those members of the phylum detected in human faecal samples. In addition, FISH analysis was performed on faecal samples from 17 healthy, nonobese donors using probe Bac303, routinely used by gut microbiologists to enumerate BacteroidesPrevotella populations in faecal samples, and another probe (CFB286) whose target range has some overlap with that of Bac303. Sequence analysis of the Turnbaugh data showed that 23/519 clones were chimeras or erroneous sequences; all good sequences were related to species of the order Bacteroidales, but no one species was present in all donors. FISH analysis demonstrated that approximately one-quarter of the healthy, nonobese donors harboured high numbers of Bacteroidales not detected by probe Bac303. It is clear that Bacteroidales populations in human faecal samples have been underestimated in FISH-based studies. New probes and complementary primer sets should be designed to examine numerical and compositional changes in the Bacteroidales during dietary interventions and in studies of the obesity-associated microbiota in humans and animal model systems.

Alternative strategies for activation of the natural lactoperoxidase system in cows' milk: trials in Tanzania

Thiocyanate content and lactoperoxidase activity of individual cow's milk of different breeds were determined, and the effects of different lactoperoxidase system (LP-s) activation strategies were compared. Lactoperoxidase activity varied significantly between Friesian and both Ayrshire and Tanzania Short Horn Zebu (TSHZ), but differences between Ayrshire and TSHZ were not significant. There was no significant variation in SCN- content between breeds. The LP-s was activated using three strategies based on SCN-: namely; equal concentrations of SCN- and H2O2 (7:7, 10:10, 15 :15 mg/l), excess SCN- concentrations (15:10, 20:10, 25:10 mg SCN-: H2O2/I), and excess H2O2 concentrations (10:15, 10:20, 10:25 mg SCN-: H2O2/I), plus a fourth strategy based on I- (15 : 15 mg I- : H2O2/I). The keeping quality (KQ) was assessed using pH, titratable acidity, clot on boiling and alcohol stability tests. All activation strategies enhanced the shelf life of milk (typically increasing KQ from around 10 to around 20 h), but it was clear that the effectiveness of the LP-s depends on the type and concentrations of the activators of the system. The LP-s activated using I- as an electron donor was more effective than the LP-s activated using SCN- as an electron donor, increasing the KQ by a further 6-8 h compared with SCN-.

Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 mu M) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

In vitro effects of synbiotic fermentation on the canine faecal microbiota.

Stirred, pH-controlled anaerobic batch cultures were used to investigate the in vitro effects of galacto-oligosaccharides (GOS) alone or combined with the probiotic Bifidobacterium bifidum 02 450B on the canine faecal microbiota of three different donors. GOS supported the growth of B. bifidum 02 450B throughout the fermentation. Quantitative analysis of bacterial populations by FISH revealed significant increases in Bifidobacterium spp. counts (Bif164) and a concomitant decrease in Clostridium histolyticum counts (Chis150) in the synbiotic-containing vessels compared with the controls and GOS vessels. Vessels containing probiotic alone displayed a transient increase in Bifidobacterium spp. and a transient decrease in Bacteroides spp. Denaturing gradient gel electrophoresis analysis showed that GOS elicited similar alterations in the microbial profiles of the three in vitro runs. However, the synbiotic did not alter the microbial diversity of the three runs to the same extent as GOS alone. Nested PCR using universal primers, followed by bifidobacterial-specific primers illustrated low bifidobacterial diversity in dogs, which did not change drastically during the in vitro fermentation. This study illustrates that the canine faecal microbiota can be modulated in vitro by GOS supplementation and that GOS can sustain the growth of B. bifidum 02 450B in a synbiotic combination.

In vitro examination of the effect of Orlistat on the ability of the faecal microbiota to utilize dietary lipids

Orlistat is an anti-obesity treatment with which several gastrointestinal (GI) side-effects are commonly associated in the initial stages of therapy. There is no physiological explanation as to why two-thirds of those who take the drug experience one or more side-effects. It has been hypothesized that the GI microbiota may protect from or contribute to these GI disturbances. Using in vitro batch culture and human gut model systems, studies were conducted to determine whether increased availability of dietary lipids and/or orlistat affect the composition and/or activity of the faecal microbiota. Results from 24-h batch culture fermentation experiments demonstrated no effect of orlistat in the presence or absence of a dietary lipid (olive oil) on the composition of bacterial communities [as determined by fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses], but did show there was great variability in the lipolytic activities of the microbiotas of individuals, as determined by gas chromatography analysis of long-chain fatty acids in samples. Subsequent studies focused on the effect of orlistat in the presence and absence of lipid in in vitro human gut model systems. Systems were run for 14 days with gut model medium (GMM) only (to steady state, SS), then fed at 12-h intervals with 50 mg orlistat, 2 g olive oil or a mixture of both for 14 days. FISH and DGGE were used to monitor changes in bacterial populations. Bacteria were cultivated from the GMM only (control) systems at SS. All strains isolated were screened for lipolytic activity using tributyrin agar. FISH and DGGE demonstrated that none of the compounds (singly or in combination) added to the systems had any notable effect on microbial population dynamics for any of the donors, although Subdoligranulum populations appeared to be inhibited by orlistat in the presence or absence of lipid. Orlistat had little or no effect on the metabolism of indigenous and added lipids in the fermentation systems, but there was great variability in the way the faecal microbiotas of the donors were able to degrade added lipids. Variability in lipid degradation could be correlated with the number and activity of isolated lipolytic bacteria. The mechanism by which orlistat and the GI microbiota cause side-effects in individuals is unknown, but several hypotheses have been proposed to account for their manifestation. The demonstration of great variability in the lipolytic activity of microbiotas to degrade lipids led to a large-scale cultivation-based study of lipolytic/lipase-positive bacteria present in the human faecal microbiota. Of 4,000 colonies isolated from 15 donors using five different agars, 378 strains were identified that had lipase activity. Molecular identification of strains isolated from five donors demonstrated that lipase activity is more prevalent in the human GI microbiota than previously thought, with members of the phyla Firmicutes, Bacteroidetes and Actinobacteria identified. Molecular identification and characterization of the substrate specificities of the strains will be carried out as part of ongoing work.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

‘Once a miner, always a miner’: Poverty and livelihood diversification in Akwatia, Ghana

This paper offers an alternative viewpoint on why people choose to engage in artisanal mining – the low tech mineral extraction and processing of mainly precious metals and stones – for extended periods in sub-Saharan Africa. Drawing upon experiences from Akwatia, Ghana’s epicentre of diamond production since the mid-1920s, the analysis challenges the commonly-held view that the region’s people are drawn to artisanal mining solely because of a desire ‘to get rich quick’. A combination of events, including the recent closure of Ghana Consolidated Diamonds Ltd’s industrial-scale operation and decreased foreign investment in the country’s diamond industry over concerns of it potentially harbouring ‘conflict’ stones from neighbouring Coˆte D’Ivoire, has had a debilitating economic impact on Akwatia. In an attempt to alleviate their hardships, many of the town’s so-called ‘lifetime’ diamond miners have managed to secure employment in neighbouring artisanal gold mining camps. But their decision has been condemned by many of the country’s policymakers and traditional leaders, who see it solely as a move to secure ‘fast money’. It is argued here, however, that these people pursue work in surrounding artisanal gold mining communities mainly because of poverty, and that their decision has more to do with a desire to immerse in activities with which they are familiar, that offer stable employment and consistent salaries, and provide immediate debt relief. Misdiagnosis of cases such as Akwatia underscores how unfamiliar policymakers and donors are with the dynamics of ASM in sub-Saharan Africa.

Recognition of greater diversity of Bacillus species and related bacteria in human faeces

In a study looking at the culturable, aerobic Actinobacteria associated with the human gastrointestinal tract, the vast majority of isolates obtained from dried human faeces belonged to the genus Bacillus and related bacteria. A total of 124 isolates were recovered from the faeces of 10 healthy adult donors. 16S rRNA gene sequence analyses showed the majority belonged to the families Bacillaceae (n = 81) and Paenibacillaceae (n = 3), with Bacillus species isolated from all donors. Isolates tentatively identified as Bacillus clausii (n = 32) and B. licheniformis (n = 28) were recovered most frequently, with the genera Lysinibacillus, Ureibacillus, Oceanobacillus, Ornithinibacillus and Virgibacillus represented in some donors. Phenotypic data confirmed the identities of isolates belonging to well-characterized species. Representatives of the phylum Actinobacteria were recovered in much lower numbers (n = 11). Many of the bacilli exhibited antimicrobial activity against one or more strains of Clostridium difficile, C. perfringens, Listeria monocytogenes and Staphylococcus aureus, with some (n = 12) found to have no detectable cytopathic effect on HEp-2 cells. This study has revealed greater diversity within gut-associated aerobic spore-formers than previous studies, and suggests that bacilli with potential as probiotics could be isolated from the human gut.

The synthesis and properties of [Fe3(CO)11(RCN)] derivatives, and a high-yield general route to mono- and di-substituted derivatives of [Fe3(CO)12]; the crystal and molecular structure of [Fe3(CO)11(NCC6H4-2-Me)]

The clusters [Fe3(CO)11(RCN)] (1: R = Me, C3H5, C6H5, or C6H4-2-Me) have been prepared at low temperature from [Fe3(CO)12] and RCN in the presence of Me3NO. Compounds 1 react essentially quantitatively with a wide range of two-electron donors, L, (viz.: CO, PPh3, P(OMe)3, PPh2H, PPh2Me, PF3, CyNC (Cy = cyclohexyl), P(OEt)3, SbPh3, PBu3, AsPh3, or SnR2 (R = CH(SiMe3)2)) to give [Fe3(CO)11L] (2). In some cases (2), on treatment with Me3NO and then L′ (L′ = a second two-electron donor) yields [Fe3(CO)10LL′] in high yield. The crystal and molecular structures of 1 (L = NCC6H4Me-2) have been determined by a full single crystal structure analysis, and shown to have an axial nitrile coordinated at the unique iron atom, with two CO groups bridging the other two metal atoms.

In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar and alginate seaweeds

Fermentation properties and prebiotic potential of novel low molecular weight polysaccharides (LMWPs) derived from agar and alginate bearing seaweeds was investigated. Ten LMWPs were supplemented to pH, temperature controlled anaerobic batch cultures inoculated with human feces from three donors, in triplicate. Microbiota changes were monitored using Fluorescent in-situ hybridization and short chain fatty acids, the fermentation end products were analysed using gas chromatography. Of the ten LMWPs tested, Gelidium seaweed CC2253 of molecular weight 64.64 KDa showed a significant increase in bifidobacterial populations from log(10) 8.06 at 0 h to log(10) 8.55 at 24 h (p = 0.018). For total bacterial populations, alginate powder CC2238 produced a significant increase from log(10) 9.01 at 0 h to log(10) 9.58 at 24 h (p = 0.032). No changes were observed in the other bacterial groups tested viz. Bacteroides, Lactobacilli/Enterococci, Eubacterium rectale/Clostridium coccoides and Clostridium histolyticum. The polysaccharides also showed significant increases in total SCFA production, particularly acetic and propionic acids, indicating that they were readily fermented. In conclusion, some LMWPs derived from agar and alginate bearing seaweeds were fermented by gut bacteria and exhibited potential to be used a novel source of prebiotics.

A 3D cloud-construction algorithm for the EarthCARE satellite mission

This article presents and assesses an algorithm that constructs 3D distributions of cloud from passive satellite imagery and collocated 2D nadir profiles of cloud properties inferred synergistically from lidar, cloud radar and imager data. It effectively widens the active–passive retrieved cross-section (RXS) of cloud properties, thereby enabling computation of radiative fluxes and radiances that can be compared with measured values in an attempt to perform radiative closure experiments that aim to assess the RXS. For this introductory study, A-train data were used to verify the scene-construction algorithm and only 1D radiative transfer calculations were performed. The construction algorithm fills off-RXS recipient pixels by computing sums of squared differences (a cost function F) between their spectral radiances and those of potential donor pixels/columns on the RXS. Of the RXS pixels with F lower than a certain value, the one with the smallest Euclidean distance to the recipient pixel is designated as the donor, and its retrieved cloud properties and other attributes such as 1D radiative heating rates are consigned to the recipient. It is shown that both the RXS itself and Moderate Resolution Imaging Spectroradiometer (MODIS) imagery can be reconstructed extremely well using just visible and thermal infrared channels. Suitable donors usually lie within 10 km of the recipient. RXSs and their associated radiative heating profiles are reconstructed best for extensive planar clouds and less reliably for broken convective clouds. Domain-average 1D broadband radiative fluxes at the top of theatmosphere(TOA)for (21 km)2 domains constructed from MODIS, CloudSat andCloud–Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) data agree well with coincidental values derived from Clouds and the Earth’s Radiant Energy System (CERES) radiances: differences betweenmodelled and measured reflected shortwave fluxes are within±10Wm−2 for∼35% of the several hundred domains constructed for eight orbits. Correspondingly, for outgoing longwave radiation∼65% are within ±10Wm−2.

Effect of elevated CO2 and high temperature on seed-set and grain quality of rice

Hybrid vigour may help overcome the negative effects of climate change in rice. A popular rice hybrid (IR75217H), a heat-tolerant check (N22), and a mega-variety (IR64) were tested for tolerance of seed-set and grain quality to high-temperature stress at anthesis at ambient and elevated [CO2]. Under an ambient air temperature of 29 °C (tissue temperature 28.3 °C), elevated [CO2] increased vegetative and reproductive growth, including seed yield in all three genotypes. Seed-set was reduced by high temperature in all three genotypes, with the hybrid and IR64 equally affected and twice as sensitive as the tolerant cultivar N22. No interaction occurred between temperature and [CO2] for seed-set. The hybrid had significantly more anthesed spikelets at all temperatures than IR64 and at 29 °C this resulted in a large yield advantage. At 35 °C (tissue temperature 32.9 °C) the hybrid had a higher seed yield than IR64 due to the higher spikelet number, but at 38 °C (tissue temperature 34–35 °C) there was no yield advantage. Grain gel consistency in the hybrid and IR64 was reduced by high temperatures only at elevated [CO2], while the percentage of broken grains increased from 10% at 29 °C to 35% at 38 °C in the hybrid. It is concluded that seed-set of hybrids is susceptible to short episodes of high temperature during anthesis, but that at intermediate tissue temperatures of 32.9 °C higher spikelet number (yield potential) of the hybrid can compensate to some extent. If the heat tolerance from N22 or other tolerant donors could be transferred into hybrids, yield could be maintained under the higher temperatures predicted with climate change.

A new parenthood paradigm for twenty-first century family law in England and Wales?

There are few other areas in family law where incongruence between the legal and social positions is as evident as that concerning parenthood. Recent cases involving lesbian couples and known sperm donors serve to highlight the increasing tension between the respective roles of biology, intention and functional parenting in the attribution of legal parental status. As both legislative and case-law developments have shown, intention is central in some circumstances, but not in others. The main claim of this paper is that this ad hoc approach leads to incoherent and unsatisfactory law: instead of striving to identify a status, what we are really looking to do is to identify the people who assume responsibility for a child. Drawing upon recent case-law, this paper explores how a conceptual reform of the law could result in a principled framework which would place formally recognised intention at the heart of parental status in order to reconnect legal duty with social reality for as many children and parents as possible. Moreover, it would ensure that parental status would not be dictated by the mode of conception of the child (natural or assisted). The analysis identifies the objectives of reform before proposing a new model which, while recognising the social importance of the biological parentage link, would reserve legal status for functional parenthood.