Bacterial, SCFA and gas profiles of a range of food ingredients following in vitro fermentation by human colonic microbiota.

It is now apparent that there is a strong link between health and nutrition and this can be seen clearly when we talk of obesity. The food industry is trying to capitalise on this by adapting high sugar/fat foods to become healthier alternatives. In confectionery food ingredients can be used for a range of purposes including sucrose replacement. Many of these ingredients may also evade digestion in the upper gut and be fermented by the gut microbiota upon entering the colon. This study was designed to screen a range of ingredients and their activities on the gut microbiota. In this study we screened a range of these ingredients in triplicate batch culture fermentations with known prebiotics as controls. Changes in bacteriology were monitored using FISH. SCFA were measured by GC and gas production was assessed during anaerobic batch fermentations. Bacterial enumeration showed significant increases (P ≤ 0.05) in bifidobacteria and lactobacilli with polydextrose and most polyols with no significant increases in Clostridium histolyticum/perfringens. SCFA and gas formation indicated that the substrates added to the fermenters were being utilised by the gut microbiota. It therefore appears these ingredients exert some prebiotic activity in vitro. Further studies, particularly in human volunteers, are necessary.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Konjac glucomannan hydrolysate beneficially modulates bacterial composition and activity within the faecal microbiota

The prebiotic potential of a konjac glucomannan hydrolysate (GMH) was investigated in vitro using batch cultures inoculated with human faeces. Bacterial enumeration was carried out using the culture independent technique, fluorescent in situ hybridisation (FISH), and short chain fatty acid (SCFA) production was monitored by gas chromatography. The populations of Bifidobacterium genus, Lactobacillus–Enterococcus group and the Atopobium group all significantly increased after GMH and inulin fermentation. The Bacteroides–Prevotella group had a lower end population after GMH fermentation while inulin gave an increase, although these differences were not significant. No significant differences in SCFA concentrations were observed between inulin and GMH. As with inulin, GMH produced selective stimulation of beneficial gut microbiota and a favourable SCFA profile. In order to confirm a beneficial effect of GMH further in vivo studies involving healthy human volunteers should be considered.

Pseudomonas syringae pv. coryli, the causal agent of bacterial twig dieback of Corylus avellana

Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution.

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

Investigation of the faecal microbiota of kittens: monitoring bacterial succession and effect of diet

Weaning is a stressful process for kittens, and is often associated with diarrhoea and the onset of infectious diseases. The gastrointestinal microbiota plays an essential role in host well-being, including improving homeostasis. Composition of the gastrointestinal microbiota of young cats is poorly understood, and the impact of diet on the kitten microbiota unknown. The aims of this study were to monitor the faecal microbiota of kittens and determine the effect(s) of diet on its composition. Bacterial succession was monitored in two groups of kittens (at 4 and 6 weeks, and 4 and 9 months of age) fed different foods. Age-related microbial changes revealed significantly different counts of total bacteria, lactic acid bacteria, Desulfovibrionales, Clostridium cluster IX and Bacteroidetes between 4-week- and 9-month-old kittens. Diet-associated differences in the faecal microbiota of the two feeding groups were evident. In general, fluorescence in situ hybridization analysis demonstrated bifidobacteria, Atopobium group, Clostridium cluster XIV and lactic acid bacteria were dominant in kittens. Denaturing gradient gel electrophoresis profiling showed highly complex and diverse faecal microbiotas for kittens, with age- and/or food-related changes seen in relation to species richness and similarity indices. Four-week-old kittens harboured more diverse and variable profiles than those of weaned kittens.

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Self-assembly of Fmoc-tetrapeptides based on the RGDS cell adhesion motif

Self-assembly in aqueous solution has been investigated for two Fmoc [Fmoc ¼ N-(fluorenyl)-9-methoxycarbonyl] tetrapeptides comprising the RGDS cell adhesion motif from fibronectin or the scrambled sequence GRDS. The hydrophobic Fmoc unit confers amphiphilicity on the molecules, and introduces aromatic stacking interactions. Circular dichroism and FTIR spectroscopy show that the self-assembly of both peptides at low concentration is dominated by interactions among Fmoc units, although Fmoc-GRDS shows b-sheet features, at lower concentration than Fmoc-RGDS. Fibre X-ray diffraction indicates b-sheet formation by both peptides at sufficiently high concentration. Strong alignment effects are revealed by linear dichroism experiments for Fmoc-GRDS. Cryo-TEM and smallangle X-ray scattering (SAXS) reveal that both samples form fibrils with a diameter of approximately 10 nm. Both Fmoc-tetrapeptides form self-supporting hydrogels at sufficiently high concentration. Dynamic shear rheometry enabled measurements of the moduli for the Fmoc-GRDS hydrogel, however syneresis was observed for the Fmoc-RGDS hydrogel which was significantly less stable to shear. Molecular dynamics computer simulations were carried out considering parallel and antiparallel b-sheet configurations of systems containing 7 and 21 molecules of Fmoc-RGDS or Fmoc-GRDS, the results being analyzed in terms of both intermolecular structural parameters and energy contributions.

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.

Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth

Consumption of anthocyanins has been related with beneficial health effects. However, bioavailability studies have shown low concentration of anthocyanins in plasma and urine. In this study, we have investigated the bacterial-dependent metabolism of malvidin-3-glucoside, gallic acid and a mixture of anthocyanins using a pH-controlled, stirred, batch-culture fermentation system reflective of the distal human large intestine conditions. Most anthocyanins have disappeared after 5 h incubation while gallic acid remained constant through the first 5 h and was almost completely degraded following 24 h of fermentation. Incubation of malvidin-3-glucoside with fecal bacteria mainly resulted in the formation of syringic acid, while the mixture of anthocyanins resulted in formation of gallic, syringic and p-coumaric acids. All the anthocyanins tested enhanced significantly the growth of Bif idobacterium spp. and Lactobacillus−Enterococcus spp. These results suggest that anthocyanins and their metabolites may exert a positive modulation of the intestinal bacterial population.