Microencapsulation of a synbiotic into PLGA/alginate multiparticulate gels

Probiotic bacteria have gained popularity as a defence against disorders of the bowel. However, the acid sensitivity of these cells results in a loss of viability during gastric passage and, consequently, a loss of efficacy. Probiotic treatment can be supplemented using ‘prebiotics’, which are carbohydrates fermented specifically by probiotic cells in the body. This combination of probiotic and prebiotic is termed a ‘synbiotic’. Within this article a multiparticulate dosage form has been developed, consisting of poly(d,l-lactic-co-glycolic acid) (PLGA) microcapsules containing prebiotic Bimuno™ incorporated into an alginate–chitosan matrix containing probiotic Bifidobacterium breve. The aim of this multiparticulate was that, in vivo, the probiotic would be protected against gastric acid and the release of the prebiotic would occur in the distal colon. After microscopic investigation, this synbiotic multiparticulate was shown to control the release of the prebiotic during in vitro gastrointestinal transit, with the release of galacto-oligosaccharides (GOS) initially occurred over 6 h, but with a triphasic release pattern giving further release over 288 h. Encapsulation of B. breve in multiparticulates resulted in a survival of 8.0 ± 0.3 log CFU/mL cells in acid, an improvement over alginate–chitosan microencapsulation of 1.4 log CFU/mL. This was attributed to increased hydrophobicity by the incorporation of PLGA particles.

The bioactivity of composite Fmoc-RGDS-collagen gels

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to nonfunctionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

Lipopeptides: from self-assembly to bioactivity

This Feature Article discusses several classes of lipopeptide with important biomedical applications as antimicrobial and antifungal agents, in immune therapies and in personal care applications among others. Two main classes of lipopeptide are considered: (i) bacterially-expressed lipopeptides with a cyclic peptide headgroup and (ii) linear lipopeptides (with one or more lipid chains) based on bio-derived and bio-inspired amino acid sequences with current clinical applications. The applications are briefly summarized, and the biophysical characterization of the molecules is reviewed, with a particular focus on self-assembly. For several of these types of biomolecule, the formation of micelles above a critical micelle concentration has been observed while others form bilayer structures, depending on conditions of pH and temperature. As yet, there are few studies on the possible relationship between self-assembly into structures such as micelles and bioactivity of this class of molecule although this is likely to attract further attention.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for Cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Wearied plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls. (C) 2003 Annals of Botany Company.

Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 106 mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.

Suitability of cryopreservation for the long-term storage of rare and endangered plant species: a case history for cosmos atrosanguineus

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono-and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity. (c) 2006 Elsevier Inc. All rights reserved.

Metabolism of tea flavonoids in the gastrointestinal tract

There is considerable interest in the bioavailability of flavan-3-ols such as tea catechins and their bioactivity in vivo. Although flavanols such as catechin and epicatechin have long been characterized as powerful antioxidants in vitro, evidence suggests that these compounds undergo significant metabolism and conjugation during absorption in the small intestine and in the colon. In the small intestine these modifications lead primarily to the formation of glucuronide conjugates that are more polar than the parent flavanol and are marked for renal excretion. Other phase II processes lead to the production of O-methylated forms that have reduced antioxidant potential via the methylation of the B-ring catechol. Significant modification of flavanols also occurs in the colon where the resident microflora degrade them to smaller phenolic acids, some of which may be absorbed. Cell, animal and human studies have confirmed such metabolism by the detection of flavanol metabolites in the circulation and tissues. This review will highlight the major sites of flavanol metabolism in the gastrointestinal tract and the processes that give rise to potential bioactive forms of flavan-3-ols in vivo.

Self-assembly of amphiphilic peptides

The self-assembly of amphiphilic peptides is reviewed. The review covers surfactant-like peptides with amphiphilicity arising from the sequence of natural amino acids, and also peptide amphiphiles (PAs) in which lipid chains are attached to hydrophilic peptide sequences containing charged residues. The influence of the secondary structure on the self-assembled structure and vice versa is discussed. For surfactant-like peptides structures including fibrils, nanotubes, micelles and vesicles have been reported. A particularly common motif for PAs is beta-sheet based fibrils, although other structures have been observed. In these structures, the peptide epitope is presented at the surface of the nanostructure, providing remarkable bioactivity. Recent discoveries of potential, and actual, applications of these materials in biomedicine and bionanotechnology are discussed.

Cross-cultural comparison of three medicinal floras and implications for bioprospecting strategies

Ethnopharmacological relevance One of the major drawbacks of using ethnomedicinal data to direct testing of plants which may find pharmaceutical use is that certain plants without bioactivity might be traditionally used. An accepted way of highlighting bioactive plants is to compare usage in different cultures. This approach infers that presumed independent discovery by different cultures provides evidence for bioactivity. Although several studies have made cross-cultural comparisons, they focussed on closely related cultures, where common patterns might be the result of common cultural traditions. The aim of this study was to compare three independent ethnomedicinal floras for which similarities can be more robustly interpreted as independent discoveries, and therefore likely to be indication for efficacy. Materials and methods Data from the literature were compiled about the ethnomedicinal floras for three groups of cultures (Nepal, New Zealand and the Cape of South Africa), selected to minimise historical cultural exchange. Ethnomedicinal applications were divided in 13 categories of use. Regression and binomial analyses were performed at the family level to highlight ethnomedicinal “hot” families. General and condition-specific analyses were carried out. Results from the three regions were compared. Results Several “hot” families (Anacardiaceae, Asteraceae, Convolvulaceae, Clusiaceae, Cucurbitaceae, Euphorbiaceae, Geraniaceae, Lamiaceae, Malvaceae, Rubiaceae, Sapindaceae, Sapotaceae and Solanaceae) were recovered in common in the general analyses. Several families were also found in common under different categories of use. Conclusions Although profound differences are found in the three ethnomedicinal floras, common patterns in ethnomedicinal usage are observed in widely disparate areas of the world with substantially different cultural traditions. As these similarities are likely to stem from independent discoveries, they strongly suggest that underlying bioactivity might be the reason for this convergent usage. The global distribution of prominent usage of families used in common obtained by this study and the wider literature is strong evidence that these families display exceptional potential for discovery of previously overlooked or new medicinal plants and should be placed in high priority in bioscreening studies and conservation schemes.

Gut microbial catabolism of grape seed flavan-3-ols by human faecal microbiota. Targetted analysis of precursor compounds, intermediate metabolites and end-products.

In vitro batch culture fermentations were conducted with grape seed polyphenols and human faecal microbiota, in order to monitor both changes in precursor flavan-3-ols and the formation of microbial-derived metabolites. By the application of UPLC-DAD-ESI-TQ MS, monomers, and dimeric and trimeric procyanidins were shown to be degraded during the first 10 h of fermentation, with notable inter-individual differences being observed between fermentations. This period (10 h) also coincided with the maximum formation of intermediate metabolites, such as 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3′,4′-dihydroxyphenyl)-valeric acid, and of several phenolic acids, including 3-(3,4-dihydroxyphenyl)-propionic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxymandelic acid, and gallic acid (5–10 h maximum formation). Later phases of the incubations (10–48 h) were characterised by the appearance of mono- and non-hydroxylated forms of previous metabolites by dehydroxylation reactions. Of particular interest was the detection of γ-valerolactone, which was seen for the first time as a metabolite from the microbial catabolism of flavan-3-ols. Changes registered during fermentation were finally summarised by a principal component analysis (PCA). Results revealed that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone was a key metabolite in explaining inter-individual differences and delineating the rate and extent of the microbial catabolism of flavan-3-ols, which could finally affect absorption and bioactivity of these compounds.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78 % procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation, however no significant correlation coefficients (P>0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

CLSM method for the dynamic observation of pH change within polymer matrices for oral delivery

If acid-sensitive drugs or cells are administered orally, there is often a reduction in efficacy associated with gastric passage. Formulation into a polymer matrix is a potential method to improve their stability. The visualization of pH within these materials may help better understand the action of these polymer systems and allow comparison of different formulations. We herein describe the development of a novel confocal laser-scanning microscopy (CLSM) method for visualizing pH changes within polymer matrices and demonstrate its applicability to an enteric formulation based on chitosan-coated alginate gels. The system in question is first shown to protect an acid-sensitive bacterial strain to low pH, before being studied by our technique. Prior to this study, it has been claimed that protection by these materials is a result of buffering, but this has not been demonstrated. The visualization of pH within these matrices during exposure to a pH 2.0 simulated gastric solution showed an encroachment of acid from the periphery of the capsule, and a persistence of pHs above 2.0 within the matrix. This implies that the protective effect of the alginate-chitosan matrices is most likely due to a combination of buffering of acid as it enters the polymer matrix and the slowing of acid penetration.