Multivalency in healable supramolecular polymers: the effect of supramolecular cross-link density on the mechanical properties and healing of noncovalent polymer networks

Polymers with the ability to heal themselves could provide access to materials with extended lifetimes in a wide range of applications such as surface coatings, automotive components and aerospace composites. Here we describe the synthesis and characterisation of two novel, stimuli-responsive, supramolecular polymer blends based on π-electron-rich pyrenyl residues and π-electron-deficient, chain-folding aromatic diimides that interact through complementary π–π stacking interactions. Different degrees of supramolecular “cross-linking” were achieved by use of divalent or trivalent poly(ethylene glycol)-based polymers featuring pyrenyl end-groups, blended with a known diimide–ether copolymer. The mechanical properties of the resulting polymer blends revealed that higher degrees of supramolecular “cross-link density” yield materials with enhanced mechanical properties, such as increased tensile modulus, modulus of toughness, elasticity and yield point. After a number of break/heal cycles, these materials were found to retain the characteristics of the pristine polymer blend, and this new approach thus offers a simple route to mechanically robust yet healable materials.

Stabilization of the i-motif structure by the intra-strand cross-link formation

The i-motif structures are formed by oligonucleotides containing cytosine tracts under acidic conditions. The folding of the i-motif under physiological conditions is of great interest because of its biological role. In this study, we investigated the effect of the intra-strand cross-link on the stability of the i-motif structure. The 4-vinyl-substituted analog of thymidine (T-vinyl) was incorporated into the 5′-end of the human telomere complementary strand, which formed the intra-strand cross-link with the internal adenine. The intra-strand cross-linked i-motif displayed CD spectra similar to that of the natural i-motif at acidic pH, which was transformed into a random coil with the increasing pH. The pH midpoint for the transition from the i-motif to random coil increased from pH 6.1 for the natural one to pH 6.8 for the cross-linked one. The thermodynamic parameters were obtained by measuring the thermal melting behaviors by CD and UV, and it was determined that the intra-strand cross-linked i-motif is stabilized due to a favorable entropy effect. Thus, this study has clearly indicated the validity of the intra-strand cross-linking for stabilization of the i-motif structure.

Mechanical effects of plant cell wall enzymes on xyloglucan/cellulose composites

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Design of redundant drive joint with adjustable stiffness and damping mechanism to improve joint admittance

Improving admittance of robotic joints is the key issue for making rehabilitation robots safe. This paper describes a design of Redundant Drive Joint (RD-Joint) which allows greater flexibility in the design of robotic mechanisms. The design strategy of the RD-Joint employs a systematic approach which consists of 1) adopting a redundant joint mechanism with internal kinematical redundancy to reduce effective joint inertia, and 2) adopting an adjustable admittance mechanism with a novel Cross link Reduction Mechanism and mechanical springs and dampers as a passive second actuator. First, the basic concepts used to construct the redundant drive joint mechanism are explained, in particular the method that allows a reduction in effective inertia at the output joint. The basic structure of the RD-Joint is introduced based on the idea of reduced inertia along with a method to include effective stiffness and damping. Then, the basic design of the adjustable admittance mechanism is described. Finally, a prototype of RD-joint is described and its expected characteristics are discussed.

Formation of dityrosine cross-links during breadmaking

To establish its significance during commercial breadmaking, dityrosine formation was quantified in flours and doughs of six commercial wheat types at various stages of the Chorleywood Bread Process. Dityrosine was formed mainly during mixing and baking, at the levels of nmol/g dry weight. Good breadmaking flours tended to exhibit higher dityrosine content in the final bread than low quality ones, but no relationship was found for dityrosine as a proportion of flour protein content, indicating that the latter was still a dominant factor in the analysis. There was no correlation between gluten yield of the six wheat types and their typical dityrosine concentrations, suggesting that dityrosine crosslinks were not a determinant factor for gluten formation. Ascorbic acid was found to inhibit dityrosine formation during mixing and proving, and have no significant effect on dityrosine in the final bread. Hydrogen peroxide promoted dityrosine formation, which suggests a radical mechanism involving endogenous peroxidases might be the responsible for dityrosine formation during breadmaking.

Amniotic fluid and umbilical cord plasma corticotropin-releasing factor (CRF), CRF-binding protein, adrenocorticotropin, and cortisol concentrations in intraamniotic infection and inflammation at term

Context: Pregnant tissues express corticotropin-releasing factor (CRF), a peptide modulating fetal and placental ACTH and cortisol secretion. These actions are modulated by the locally expressed CRF-binding protein (CRF-BP). Objective: The objective of the study was to determine whether CRF, CRF-BP, ACTH, and cortisol concentrations change in amniotic fluid and umbilical cord plasma in the presence of intraamniotic infection/inflammation (IAI) in women with spontaneous labor at term. Design: This was a cross-sectional study. Setting: The study was conducted at a tertiary referral center for obstetric care. Patients: Patients included women in active labor at term with (n = 39) and without (controls; n = 78) IAI. Main Outcome Measures: Amniotic fluid and umbilical cord plasma concentrations of CRF, CRF-BP, ACTH, and cortisol measured by RIA and immunoradiometric assays were measured. Results: In patients with IAI, amniotic fluid CRF (0.97 +/- 0.18 ng/ml) and CRF-BP (33.06 +/- 5.54 nmol/liter) concentrations were significantly (P < 0.001) higher than in controls (CRF: 0.32 +/- 0.04 ng/ml; CRF-BP: 14.69 +/- 2.79 ml). The umbilical cord plasma CRF and CRF-BP concentrations were significantly (P < 0.001 for all) higher in women with IAI than in controls (CRF: 2.96 +/- 0.35 ng/ml vs. 0.38 +/- 0.18 ng/ml; CRF-BP: 152.12 +/- 5.94 nmol/liter vs. 106.9 +/- 5.97 nmol/liter). In contrast, amniotic fluid and umbilical cord plasma ACTH and cortisol concentrations did not differ between groups. Conclusions: Amniotic fluid and umbilical cord plasma CRF and CRF-BP concentrations are increased in women with spontaneous labor at term and IAI. CRF-BP may modulate CRF actions on ACTH and cortisol secretion, playing a pivotal role in limiting the inflammatory process and thus avoiding an overactivation of the fetal/placental hypothalamus-pituitary-adrenal axis at birth.

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner

Amyloid fibrils are typically rigid, unbrariched structures with diameters of similar to 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low PH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding, material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underling protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression. (c) 2006 Elsevier Ltd. All rights reserved.

Stress relaxation behavior of wheat dough, gluten, and gluten protein fractions

Relaxation behavior was measured for dough, gluten and gluten protein fractions obtained from the U.K. biscuitmaking flour, Riband, and the U.K. breadmaking flour, Hereward. The relaxation spectrum, in which relaxation times (tau) are related to polymer molecular size, for dough showed a broad molecular size distribution, with two relaxation processes: a major peak at short times and a second peak at times longer than 10 sec, which is thought to correspond to network structure, and which may be attributed to entanglements and physical cross-links of polymers. Relaxation spectra of glutens were similar to those for the corresponding doughs from both flours. Hereward gluten clearly showed a much more pronounced second peak in relaxation spectrum and higher relaxation modulus than Riband gluten at the same water content. In the gluten protein fractions, gliadin and acetic acid soluble glutenin only showed the first relaxation process, but gel protein clearly showed both the first and second relaxation processes. The results show that the relaxation properties of dough depend on its gluten protein and that gel protein is responsible for the network structure for dough and gluten.

Glucokinase Regulatory Protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome

Glucokinase Regulatory Protein (GCKR) plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been of considerable interest, due to their potential to reduce metabolic syndrome (MetS) risk. Objective To examine whether genetic variability at the GCKR gene locus was associated with the degree of insulin resistance, plasma concentrations of C-reactive protein (CRP) and n-3 PUFA in MetS subjects. Design Homeostasis model assessment of insulin resistance (HOMA-IR), HOMA-B, plasma concentrations of C-peptide, CRP, fatty acid composition and the GCKR rs1260326-P446L polymorphism, were determined in a cross-sectional analysis of 379 subjects with MetS participating in the LIPGENE dietary cohort. Results Among subjects with n-3 PUFA levels below the population median, carriers of the common C/C genotype had higher plasma concentrations of fasting insulin (P = 0.019), C-peptide (P = 0.004), HOMA-IR (P = 0.008) and CRP (P = 0.032) as compared with subjects carrying the minor T-allele (Leu446). In contrast, homozygous C/C carriers with n-3 PUFA levels above the median showed lower plasma concentrations of fasting insulin, peptide C, HOMA-IR and CRP, as compared with individuals with the T-allele. Conclusions We have demonstrated a significant interaction between the GCKR rs1260326-P446L polymorphism and plasma n-3 PUFA levels modulating insulin resistance and inflammatory markers in MetS subjects. Further studies are needed to confirm this gene-diet interaction in the general population and whether targeted dietary recommendations can prevent MetS in genetically susceptible individuals.

A randomised, double-blind, placebo controlled cross-over study to determine the gastrointestinal effects of consumption of arabinoxylanoligosaccharides enriched bread in healthy volunteers

BACKGROUND: Prebiotics are food ingredients, usually non-digestible oligosaccharides, that are selectively fermented by populations of beneficial gut bacteria. Endoxylanases, altering the naturally present cereal arabinoxylans, are commonly used in the bread industry to improve dough and bread characteristics. Recently, an in situ method has been developed to produce arabinoxylan-oligosaccharides (AXOS) at high levels in breads through the use of a thermophilic endoxylanase. AXOS have demonstrated potentially prebiotic properties in that they have been observed to lead to beneficial shifts in the microbiota in vitro and in murine, poultry and human studies. METHODS: A double-blind, placebo controlled human intervention study was undertaken with 40 healthy adult volunteers to assess the impact of consumption of breads with in situ produced AXOS (containing 2.2 g AXOS) compared to non-endoxylanase treated breads. Volatile fatty acid concentrations in faeces were assessed and fluorescence in situ hybridisation was used to assess changes in gut microbial groups. Secretory immunoglobulin A (sIgA) levels in saliva were also measured. RESULTS: Consumption of AXOS-enriched breads led to increased faecal butyrate and a trend for reduced iso-valerate and fatty acids associated with protein fermentation. Faecal levels of bifidobacteria increased following initial control breads and remained elevated throughout the study. Lactobacilli levels were elevated following both placebo and AXOS-breads. No changes in salivary secretory IgA levels were observed during the study. Furthermore, no adverse effects on gastrointestinal symptoms were reported during AXOS-bread intake. CONCLUSIONS: AXOS-breads led to a potentially beneficial shift in fermentation end products and are well tolerated.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Use of miniaturized protein arrays for Escherichia coli O serotyping

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Grid computing solutions for distributed repositories of protein folding and unfolding simulations

The P-found protein folding and unfolding simulation repository is designed to allow scientists to perform analyses across large, distributed simulation data sets. There are two storage components in P-found: a primary repository of simulation data and a data warehouse. Here we demonstrate how grid technologies can support multiple, distributed P-found installations. In particular we look at two aspects, first how grid data management technologies can be used to access the distributed data warehouses; and secondly, how the grid can be used to transfer analysis programs to the primary repositories --- this is an important and challenging aspect of P-found because the data volumes involved are too large to be centralised. The grid technologies we are developing with the P-found system will allow new large data sets of protein folding simulations to be accessed and analysed in novel ways, with significant potential for enabling new scientific discoveries.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.