Prebiotics and resistance to gastrointestinal infections

Acute gut disorder is a cause for significant medicinal and economic concern. Certain individual pathogens of the gut, often transmitted in food or water, have the ability to cause severe discomfort. There is a need to manage such conditions more effectively. The route of reducing the risk of intestinal infections through diet remains largely unexplored. Antibiotics are effective at inhibiting pathogens; however, these should not be prescribed in the absence of disease and therefore cannot be used prophylactically. Moreover, their indiscriminate use has reduced effectiveness. Evidence has accumulated to suggest that some of the health-promoting bacteria in the gut (probiotics) can elicit a multiplicity of inhibitory effects against pathogens. Hence, an increase in their numbers should prove effective at repressing pathogen colonisation if/when infectious agents enter the gut. As such, fortification of indigenous bifidobacteria/lactobacilli by using prebiotics should improve protection. There are a number of potential mechanisms for lactic acid bacteria to reduce intestinal infections. Firstly, metabolic endproducts such as acids excreted by these micro-organisms may lower the gut pH to levels below those at which pathogens are able to effectively compete. Also, many lactobacilli and bifidobacteria species are able to excrete natural antibiotics, which can have a broad spectrum of activity. Other mechanisms include an improved immune stimulation, competition for nutrients and blocking of pathogen adhesion sites in the gut. Many intestinal pathogens like type 1 fimbriated Escherichia coli, salmonellae and campylobacters utilise oligosaccharide receptor sites in the gut. Once established, they can then cause gastroenteritis through invasive and/or toxin forming properties. One extrapolation of the prebiotic concept is to simulate such receptor sites in the gut lumen. Hence, the pathogen is 'decoyed' into not binding at the host mucosal interface. The combined effects of prebiotics upon the lactic acid flora and anti-adhesive strategies may lead towards new dietary interventions against food safety agents.

Does reactive surface area depend on grain size? Results from pH 3, 25 circle C far-from-equilibrium flow-through dissolution experiments on anorthite and biotite

Laboratory determined mineral weathering rates need to be normalised to allow their extrapolation to natural systems. The principle normalisation terms used in the literature are mass, and geometric- and BET specific surface area (SSA). The purpose of this study was to determine how dissolution rates normalised to these terms vary with grain size. Different size fractions of anorthite and biotite ranging from 180-150 to 20-10 mu m were dissolved in pH 3, HCl at 25 degrees C in flow through reactors under far from equilibrium conditions. Steady state dissolution rates after 5376 h (anorthite) and 4992 h (biotite) were calculated from Si concentrations and were normalised to initial- and final- mass and geometric-, geometric edge- (biotite), and BET SSA. For anorthite, rates normalised to initial- and final-BET SSA ranged from 0.33 to 2.77 X 10(-10) mol(feldspar) m(-2) s(-1), rates normalised to initial- and final-geometric SSA ranged from 5.74 to 8.88 X 10(-10) mol(feldspar) m(-2) s(-1) and rates normalised to initial- and final-mass ranged from 0.11 to 1.65 mol(feldspar) g(-1) s(-1). For biotite, rates normalised to initial- and final-BET SSA ranged from 1.02 to 2.03 X 10(-12) mol(biotite) m(-2) s(-1), rates normalised to initial- and final-geometric SSA ranged from 3.26 to 16.21 X 10(-12) mol(biotite) m(-2) s(-1), rates normalised to initial- and final-geometric edge SSA ranged from 59.46 to 111.32 x 10(-12) mol(biotite) m(-2) s(-1) and rates normalised to initial- and final-mass ranged from 0.81 to 6.93 X 10(-12) mol(biotite) g(-1) s(-1). For all normalising terms rates varied significantly (p <= 0.05) with grain size. The normalising terms which gave least variation in dissolution rate between grain sizes for anorthite were initial BET SSA and initial- and final-geometric SSA. This is consistent with: (1) dissolution being dominated by the slower dissolving but area dominant non-etched surfaces of the grains and, (2) the walls of etch pits and other dissolution features being relatively unreactive. These steady state normalised dissolution rates are likely to be constant with time. Normalisation to final BET SSA did not give constant ratios across grain size due to a non-uniform distribution of dissolution features. After dissolution coarser grains had a greater density of dissolution features with BET-measurable but unreactive wall surface area than the finer grains. The normalising term which gave the least variation in dissolution rates between grain sizes for biotite was initial BET SSA. Initial- and final-geometric edge SSA and final BET SSA gave the next least varied rates. The basal surfaces dissolved sufficiently rapidly to influence bulk dissolution rate and prevent geometric edge SSA normalised dissolution rates showing the least variation. Simple modelling indicated that biotite grain edges dissolved 71-132 times faster than basal surfaces. In this experiment, initial BET SSA best integrated the different areas and reactivities of the edge and basal surfaces of biotite. Steady state dissolution rates are likely to vary with time as dissolution alters the ratio of edge to basal surface area. Therefore they would be more properly termed pseudo-steady state rates, only appearing constant because the time period over which they were measured (1512 h) was less than the time period over wich they would change significantly. (c) 2006 Elsevier Inc. All rights reserved.

Characterization of mineral surfaces using FIB and TEM: a case study of naturally weathered alkali feldspars

Using a focused ion beam (FIB) instrument, electron-transparent samples (termed foils) have been cut from the naturally weathered surfaces of perthitic alkali feldspars recovered from soils overlying the Shap granite, northwest England. Characterization of these foils by transmission electron microscopy (TEM) has enabled determination of the crystallinity and chemical composition of near-surface regions of the feldspar and an assessment of the influence of intragranular microtextures on the microtopography of grain surfaces and development of etch pits. Damage accompanying implantation of the 30 kV Ga+ ions used for imaging and deposition of protective platinum prior to ion milling creates amorphous layers beneath outer grain surfaces, but can be overcome by coating grains with > 85 nm of gold before FIB work. The sidewalls of the foil and feldspar surrounding original voids are also partially amorphized during later stages of ion milling. No evidence was found for the presence of amorphous or crystalline weathering products or amorphous "leached layers" immediately beneath outer grain surfaces. The absence of a leached layer indicates that chemical weathering of feldspar in the Shap soils is stoichiometric, or if non-stoichiometric, either the layer is too thin to resolve by the TEM techniques used (i.e., <=similar to 2.5 nm) or an insufficient proportion of ions have been leached from near-surface regions so that feldspar crystallinity is maintained. No evidence was found for any difference in the mechanisms of weathering where a microbial filament rests on the feldspar surface. Sub-micrometer-sized steps on the grain surface have formed where subgrains and exsolution lamellae have influenced the propagation of fractures during physical weathering, whereas finer scale corrugations form due to compositional or strain-related differences in dissolution rates of albite platelets and enclosing tweed orthoclase. With progressive weathering, etch pits that initiated at the grain surface extend into grain interiors as etch tubes by exploiting preexisting networks of nanopores that formed during the igneous history of the grain. The combination of FIB and TEM techniques is an especially powerful way of exploring mechanisms of weathering within the "internal zone" beneath outer grain surfaces, but results must be interpreted with caution owing to the ease with which artifacts can be created by the high-energy ion and electron beams used in the preparation and characterization of the foils.

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: Preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

Molecular interactions between the penetration enhancer 1,8-cineole and human skin

Penetration enhancers are chemicals that temporarily and reversibly diminish the barrier function of the outermost layer of skin, the stratum corneum, to facilitate drug delivery to and through the tissue. In the current study, the complex mechanisms by which 1,8-cineole, a potent terpene penetration enhancer, disrupts the stratum corneum barrier is investigated using post-mortem skin samples. In order to validate the use of excised tissue for these and related studies, a fibre optical probe coupled to an FT-Raman spectrometer compared spectroscopic information for human skin recorded from in vivo and in vitro sampling arrangements. Spectra from full-thickness (epidermis and dermis) post-mortem skin samples presented to the spectrometer with minimal sample preparation (cold acetone rinse) were compared with the in vivo system (the forearms of human volunteers). No significant differences in the Raman spectra between the in vivo and in vitro samples were observed, endorsing the use of post-mortem or surgical samples for this investigational work. Treating post-mortem samples with the penetration enhancer revealed some unexpected findings: while evidence for enhancer-induced disruption of the barrier lipid packing in the stratum corneum was detected in some samples, spectra from other samples revealed an increase in lipid order on treatment with the permeation promoter. These findings are consistent with phase-separation of the enhancer within the barrier lipid domains as opposed to homogeneous disruption of the lipid lamellae. Copyright (C) 2006 John Wiley & Sons, Ltd.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG

Background: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. Results: The cells were grown at pH 5, 5.5, 6 (temperature 37 °C) and at pH 6.5 (temperature 25 °C, 30 °C and 37 °C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37 oC). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. Conclusions: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.

A study of the microstructure and condition of thin sheets of painting support ivory using SEM and ESEM

This paper outlines a study of the microstructure of thin sheets of ivory used as a painting support for portrait miniatures. Warping of the ivory support is one of the main problems commonly found in portrait miniatures from the late eighteenth century and early nineteenth century. Portrait miniatures from this period are painted on very thin sheets of ivory that are often only 0.2 mm in thickness. Warping can lead to cracking of the ivory and can also accentuate flaking of the paint layer. The problem of warping in ivory has thus been of long-term interest to conservators who deal with portrait miniatures, including those at the Victoria and Albert (V&A) Museum. The causes of warping are complex. However, it should be noted that artists normally stuck the thin ivory sheets onto paper or card before commencing the painting. The possible causes of warping therefore are thought to relate to the differential reactions of the ivory/adhesive/paper or card layers to changes in relative humidity (RH). It is well known that ivory is hygroscopic and anisotropic.1 However, only a few scientific studies have been carried out related to this subject and systematic analyses of the morphological and microstructural changes due to changes in RH or moisture in such thin sheets of ivory have yet to be investigated.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

Child anxiety and parenting in England and Italy: the moderating role of maternal warmth

BACKGROUND: Parenting factors have been implicated in the aetiology and maintenance of child anxiety. Most research has been correlational with little experimental or longitudinal work. Cross-cultural comparison could be illuminating. A comparison of Italian and British children and their mothers was conducted. METHODS: A sample of 8- to 10-year old children, 60 Italian and 49 English, completed the Spence Child Anxiety Scale. Mothers also completed two questionnaires of parenting: the Skills of Daily Living Checklist (assessing maternal autonomy granting) and the Parent-Child Interaction Questionnaire (assessing maternal intrusiveness). Parenting was assessed in two video-recorded blindly rated mother-child interaction tasks, the 'belt-buckling tasks and the 'etch-a-sketch', providing objective indices of overcontrol, warmth, lack of autonomy granting, and overprotection. RESULTS: There were no differences between the children in overall anxiety and specific forms of anxiety. Parenting, however, was markedly different for the two countries. Compared to English mothers, on the two questionnaires, Italian mothers were significantly less autonomy granting and more intrusive; and in terms of the observed indices, a significantly greater proportion of the Italian mothers displayed a high level of both overprotection and overcontrol, and a low level of autonomy granting. Notably, Italian mothers evidenced significantly more warmth than English mothers; and maternal warmth was found to moderate the impact of self-reported maternal intrusiveness on the level of both overall child anxiety and the level of child separation anxiety; and it also moderated the relationship between both observed maternal intrusiveness and overall child anxiety and observed maternal overprotectiveness and child separation anxiety. CONCLUSIONS: Although, compared to the British mothers, the Italian mothers were more likely to evidence high levels of parenting behaviours previously found to be anxiogenic, the high levels of warmth displayed by these mothers to their children appears to have neutralised the adverse impact of these behaviours.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

Serum protein layers on parylene-C and silicon oxide: effect on cell adhesion

Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.

Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling

Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.

Bio-fabrication and physiological self-release of tissue equivalents using smart peptide amphiphile templates

In this study we applied a smart biomaterial formed from a self-assembling, multi-functional synthetic peptide amphiphile (PA) to coat substrates with various surface chemistries. The combination of PA coating and alignment-inducing functionalised substrates provided a template to instruct human corneal stromal fibroblasts to adhere, become aligned and then bio-fabricate a highlyordered, multi-layered, three-dimensional tissue by depositing an aligned, native-like extracellular matrix. The newly-formed corneal tissue equivalent was subsequently able to eliminate the adhesive properties of the template and govern its own complete release via the action of endogenous proteases. Tissues recovered through this method were structurally stable, easily handled, and carrier-free. Furthermore, topographical and mechanical analysis by atomic force microscopy showed that tissue equivalents formed on the alignment-inducing PA template had highly-ordered, compact collagen deposition, with a two-fold higher elastic modulus compared to the less compact tissues produced on the non-alignment template, the PA-coated glass. We suggest that this technology represents a new paradigm in tissue engineering and regenerative medicine, whereby all processes for the biofabrication and subsequent self-release of natural, bioprosthetic human tissues depend solely on simple templatetissue feedback interactions.

An adhesive elastomeric supramolecular polyurethane healable at body temperature

In this paper, we report the synthesis and healing ability of a non-cytotoxic supramolecular polyurethane network whose mechanical properties can be recovered efficiently (> 99%) at the temperature of the human body (37 ºC). Rheological analysis revealed an acceleration in the drop of the storage modulus above 37 ºC, on account of the dissociation of the supramolecular polyurethane network, and this decrease in viscosity enables the efficient recovery of the mechanical properties. Microscopic and mechanical characterisation has shown that this material is able to recover mechanical properties across a damage site with minimal contact required between the interfaces and also demonstrated that the mechanical properties improved when compared to other low temperature healing elastomers or gel-like materials. The supramolecular polyurethane was found to be non-toxic in a cytotoxicity assay carried out in human skin fibroblasts (cell viability > 94% and non-significantly different compared to the untreated control). This supramolecular network material also exhibited excellent adhesion to pig skin and could be healed completely in situ post damage indicating that biomedical applications could be targeted, such as artificial skin or wound dressings with supramolecular materials of this type.