Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R2 = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Straightforward biodegradable nanoparticle generation through megahertz-order ultrasonic atomization

Simple and reliable formation of biodegradable nanoparticles formed from poly-ε-caprolactone was achieved using 1.645 MHz piston atomization of a source fluid of 0.5% w/v of the polymer dissolved in acetone; the particles were allowed to descend under gravity in air 8 cm into a 1 mM solution of sodium dodecyl sulfate. After centrifugation to remove surface agglomerations, a symmetric monodisperse distribution of particles φ 186 nm (SD=5.7, n=6) was obtained with a yield of 65.2%. © 2006 American Institute of Physics.

Spectroscopic characterization and solubility investigation on the effects of As(V) on mineral structure tooeleite (Fe6(AsO3)4SO4(OH)4·H2O)

Tooeleite is an unique ferric arsenite sulfate mineral, which has the potential significance of directly fixing As(III) as mineral trap. The tooeleite and various precipitates were hydrothermally synthesized under the different of initial As(III)/As(V) molar ratios and characterized by XRD, FTIR, XPS and SEM. The crystallinity of tooeleite decreases with the amount of As(V). The precipitate is free of any crystalline tooeleite at the level of that XRD could detect when the ratio of As(III)/As(V) of 7:3 and more. The characteristic bands of tooeleite are observed in 772, 340, 696 and 304 cm−1, which are assigned to the ν1, ν2, ν3 and ν4 vibrations of AsO33−. These intensities of bands gradually decreases with the presence of As(V) and its increasing. An obviously wide band is observed in 830 cm−1, which is the ν1 vibration of AsO4. The result of XPS reveals that the binding energies of As3d increase from 44.0 eV to 45.5 eV, which indicates that the amount of As(V) in the precipitates increases. The concentrations of arsenic released of these precipitates are 350–650 mg/L. The stability of tooeleite decreases by comparison when the presence of coexisting As(V) ions.

Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies. © 2011 Duncan et al.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction: Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods: PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results: Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions: This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

The dominant 55kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55. kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55. kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55. kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Physical mechanisms underlying the strain-rate-dependent mechanical behavior of kangaroo shoulder cartilage

Due to anatomical and biomechanical similarities to human shoulder, kangaroo was chosen as a model to study shoulder cartilage. Comprehensive enzymatic degradation and indentation tests were applied on kangaroo shoulder cartilage to study mechanisms underlying its strain-rate-dependent mechanical behavior. We report that superficial collagen plays a more significant role than proteoglycans in facilitating strain-rate-dependent behavior of kangaroo shoulder cartilage. By comparing the mechanical properties of degraded and normal cartilages it was noted that proteoglycan and collagen degradation significantly compromised strain-rate-dependent mechanical behavior of the cartilage. Superficial collagen contributed equally to the tissue behavior at all strain-rates. This is different to studies reported on knee cartilage and confirms the importance of superficial collagen on shoulder cartilage mechanical behavior. A porohyperelastic numerical model also indicated that collagen disruption would lead to faster damage of the shoulder cartilage than when proteoglycans are depleted.

Preliminary investigations into the use of a functionalised polymer to reduce diffusion in Fricke gel dosimeters

Purpose A modification of the existing PVA-​FX hydrogel has been made to investigate the use of a functionalised polymer in a Fricke gel dosimetry system to decrease Fe3+ diffusion. Methods The chelating agent, xylenol orange, was chem. bonded to the gelling agent, polyvinyl alc. (PVA) to create xylenol orange functionalised PVA (XO-​PVA)​. A gel was created from the XO-​PVA (20​% w​/v) with ferrous sulfate (0.4 mM) and sulfuric acid (50 mM)​. Results This resulted in an optical d. dose sensitivity of 0.014 Gy-​1, an auto-​oxidn. rate of 0.0005 h-​1, and a diffusion rate of 0.129 mm2 h-​1; an 8​% redn. compared to the original PVA-​FX gel, which in practical terms adds approx. 1 h to the time span between irradn. and accurate read-​out. Conclusions Because this initial method of chem. bonding xylenol orange to polyvinyl alc. has inherently low conversion, the improvement on existing gel systems is minimal when compared to the drawbacks. More efficient methods of functionalising polyvinyl alc. with xylenol orange must be developed for this system to gain clin. relevance.

Inter-lamellar shear resistance confers compressive stiffness in the intervertebral disc: An image-based modelling study on the bovine caudal disc

The intervertebral disc withstands large compressive loads (up to nine times bodyweight in humans) while providing flexibility to the spinal column. At a microstructural level, the outer sheath of the disc (the annulus fibrosus) comprises 12–20 annular layers of alternately crisscrossed collagen fibres embedded in a soft ground matrix. The centre of the disc (the nucleus pulposus) consists of a hydrated gel rich in proteoglycans. The disc is the largest avascular structure in the body and is of much interest biomechanically due to the high societal burden of disc degeneration and back pain. Although the disc has been well characterized at the whole joint scale, it is not clear how the disc tissue microstructure confers its overall mechanical properties. In particular, there have been conflicting reports regarding the level of attachment between adjacent lamellae in the annulus, and the importance of these interfaces to the overall integrity of the disc is unknown. We used a polarized light micrograph of the bovine tail disc in transverse cross-section to develop an image-based finite element model incorporating sliding and separation between layers of the annulus, and subjected the model to axial compressive loading. Validation experiments were also performed on four bovine caudal discs. Interlamellar shear resistance had a strong effect on disc compressive stiffness, with a 40% drop in stiffness when the interface shear resistance was changed from fully bonded to freely sliding. By contrast, interlamellar cohesion had no appreciable effect on overall disc mechanics. We conclude that shear resistance between lamellae confers disc mechanical resistance to compression, and degradation of the interlamellar interface structure may be a precursor to macroscopic disc degeneration.

Defining the role of common variation in the genomic and biological architecture of adult human height

Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated approximately 2,000, approximately 3,700 and approximately 9,500 SNPs explained approximately 21%, approximately 24% and approximately 29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/beta-catenin and chondroitin sulfate-related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.

Experimental and numerical investigation of strain-rate-dependent behavior of kangaroo shoulder cartilage

This thesis introduces a new animal model, kangaroo, to biomechanical investigations of shoulder cartilage research. It examines the effect of cartilage structure and constituents on tissue behavior and its adaptation to mechanical loading. In doing so, the study explains the relationship of tissue's functional behaviors to its structure and constituents which has important implications for tissue engineering strategies catering joint specific cartilage tissue generation.

The structure of glycosaminoglycans and their interactions with proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three-dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.