Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Photo-cross-linked poly(D,L-lactide)-based networks. Structural characterization by HR-MAS NMR spectroscopy and hydrolytic degradation behavior

To date, biodegradable networks and particularly their kinetic chain lengths have been characterized by analysis of their degradation products in solution. We characterize the network itself by NMR analysis in the solvent-swollen state under magic angle spinning conditions. The networks were prepared by photoinitiated cross-linking of poly(dl-lactide)−dimethacrylate macromers (5 kg/mol) in the presence of an unreactive diluent. Using diffusion filtering and 2D correlation spectroscopy techniques, all network components are identified. By quantification of network-bound photoinitiator fragments, an average kinetic chain length of 9 ± 2 methacrylate units is determined. The PDLLA macromer solution was also used with a dye to prepare computer-designed structures by stereolithography. For these networks structures, the average kinetic chain length is 24 ± 4 methacrylate units. In all cases the calculated molecular weights of the polymethacrylate chains after degradation are maximally 8.8 kg/mol, which is far below the threshold for renal clearance. Upon incubation in phosphate buffered saline at 37 °C, the networks show a similar mass loss profile in time as linear high-molecular-weight PDLLA (HMW PDLLA). The mechanical properties are preserved longer for the PDLLA networks than for HMW PDLLA. The initial tensile strength of 47 ± 2 MPa does not decrease significantly for the first 15 weeks, while HMW PDLLA lost 85 ± 5% of its strength within 5 weeks. The physical properties, kinetic chain length, and degradation profile of these photo-cross-linked PDLLA networks make them most suited materials for orthopedic applications and use in (bone) tissue engineering.

Effects of opening in the cross girders of the flat bottom rail wagons to the load transferring mechanisms when used as road bridge deck

A Flat Bed Rail Wagon (FBRW) has been proposed as an alternative solution for replacing bridges on low traffic volume roads. The subject matter for this paper is to investigate the impediment to load transfer from cross girders to main girder, through visually identifiable structural flaws. Namely, the effect of having large openings at close proximity to the connection of the main girder to the cross girder of a FBRW was examined. It was clear that openings locally reduce the section modulus of the secondary members; however it was unclear how these reductions would affect the load transfer to the main girder. The results are presented through modeling grillage action for which the loads applied onto the FBRW were distributed through cross girders to the main girder.

Surface effects on the dual-mode vibration of <110> silver nanowires with different cross-sections

Dual-mode vibration of nanowires has been reported experimentally through actuation of the nanowire at its resonance frequency, which is expected to open up a variety of new modalities for the NEMS that could operate in the nonlinear regime. In the present work, we utilize large scale molecular dynamics simulations to investigate the dual-mode vibration of <110> Ag nanowires with triangular, rhombic and truncated rhombic cross-sections. By incorporating the generalized Young-Laplace equation into Euler-Bernoulli beam theory, the influence of surface effects on the dual-mode vibration is studied. Due to the different lattice spacing in principal axes of inertia of the {110} atomic layers, the NW is also modeled as a discrete system to reveal the influence from such specific atomic arrangement. It is found that the <110> Ag NW will under a dual-mode vibration if the actuation direction is deviated from the two principal axes of inertia. The predictions of the two first mode natural frequencies by the classical beam model appear underestimated comparing with the MD results, which are found to be enhanced by the discrete model. Particularly, the predictions by the beam theory with the contribution of surface effects are uniformly larger than the classical beam model, which exhibit better agreement with MD results for larger cross-sectional size. However, for ultrathin NWs, current consideration of surface effects is still experiencing certain inaccuracy. In all, for all different cross-sections, the inclusion of surface effects is found to reduce the difference between the two first mode natural frequencies. This trend is observed consistent with MD results. This study provides a first comprehensive investigation on the dual-mode vibration of <110> oriented Ag NWs, which is supposed to benefit the applications of NWs that acting as a resonating beam.

Cross-sectional study of a microsatellite marker in the low densitity lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

A linkage and cross-sectional study of hypertension and obesity using a poly(A) Alu-repeat polymorphism at the glucagon receptor gene locus (17q25)

1. Previous glucagon receptor gene (GCGR) studies have shown a Gly40Ser mutation to be more prevalent in essential hypertension and to affect glucagon binding affinity to its receptor. An Alu-repeat poly(A) polymorphism colocalized to GCGR was used in the present study to test for association and linkage in hypertension as well as association in obesity development. 2. Using a cross-sectional approach, 85 hypertensives and 95 normotensives were genotyped using polymerase chain reaction primers flanking the Alu-repeat. Both hypertensive and normotensive populations were subdivided into lean and obese categories based on body mass index (BMI) to determine involvement of this variant in obesity. For the linkage study, 89 Australian Caucasian hypertension affected sibships (174 sibpairs) were genotyped and the results were analysed using GENE-HUNTER, Mapmaker Sibs, ERPA and SPLINK (all freely available from http://linlkage.rockefeller. edu/soft/list.html). 3. Cross-sectional results for both hypertension and obesity were analysed using Chi-squared and Monte Carlo analyses. Results did not show an association of this variant with either hypertension (χ2 = 6.9, P = 0.14; Monte Carlo χ2 = 7.0, P = 0.11; n = 5000) or obesity (χ2 = 3.3, P = 0.35; Monte Carlo χ2 = 3.26, P = 0.34; n = 5000). In addition, results from the linkage study using hypertensive sib-pairs did not indicate linkage of the poly(A) repent with hypertension. Hence, results did not indicate a role far the Alu-repeat in either hypertension or obesity. However, as the heterozygosity of this poly(A) repeat is low (35%), a larger number of hypertensive sib-pairs may be required to draw definitive conclusions.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Diporphyrins linked by two-atom bridges synthestic, structural and theoretical studies

The syntheses, properties and electronic structures of a series of porphyrin dimers connected by two-atom bridges were compared. The study found that an azo linker results in the most efficient electronic communication between the two porphyrin rings, and is the superior connector for dimers, trimers and oligomers in the design of nonlinear optical materials. This has implications for the design of molecular probes and sensors, photodynamic therapy, microfabrication, and three-dimensional optical data storage. The research led to the synthesis of a number of new porphyrin monomers and dimers, which were characterised using structural, spectroscopic and spectrometric techniques.

Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Molecular sliding filament model for muscular contraction based on multiscale investigation

A multiscale approach that bridges the biophysics of the actin molecules at nanoscale and the biomechanics of actin filament at microscale level is developed and used to evaluate the mechanical performances of actin filament bundles. In order to investigate the contractile properties of skeletal muscle which is induced by the protein motor of myosin, a molecular model is proposed in the prediction of the dynamic behaviors of skeletal muscle based on classic sliding filament model. Randomly distributed myosin motors are applied on a 2.2 μm long sarcomere, whose principal components include actin and myosin filaments. It can be found that, the more myosin motors on the sarcomere, the faster the sarcomere contracts. The result demonstrates that the sarcomere shortening speed cannot increase infinitely by the modulation of myosin, thus providing insight into the self-protective properties of skeletal muscles. This molecular filament sliding model provides a theoretical way to evaluate the properties of skeletal muscles, and contributes to the understandings of the molecular mechanisms in the physiological phenomenon of muscular contraction.

Mechanics of microfilaments networks : a cross-scales study

The mechanical properties of microfilament networks are systematically summarized at different special scales in this paper. We have presented the mechanical models of single microfilaments and microfilament networks at microscale. By adopting a coarse-grained simulation strategy, the mechanical stability of microfilaments related cellular structures are analysed. Structural analysis is conducted to microfilament networks to understand the stress relaxation under compression. The nanoscale molecular mechanisms of the microfilaments deformation is also summarized from the viewpoint of molecular dynamics simulation. This paper provides the fundaments of multiscale modelling framework for the mechanical behaviours simulation of hierarchical microfilament networks.

Cross-scale biophysics modelling of F-actin cytoskeleton in cell

This thesis is a comparative study of the modelling of mechanical behaviours of F-actin cytoskeleton which is an important structural component in living cells. A new granular model was developed for F-actin cytoskeleton based on the concept of multiscale modelling. This framework overcomes difficulties encountered in physical modelling of cytoskeleton in conventional continuum mechanics modelling, and the computational challenges in all-atom molecular dynamics simulation. The thermostat algorithm was further modified to better predict the thermodynamic properties of F-actin cytoskeleton in modelling. This multiscale modelling framework was applied in explaining the physical mechanisms of cytoskeleton responses to external mechanical loads.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.