Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons

Tendon is composed of rope-like fascicles, bound together by interfascicular matrix (IFM). Our previous work shows that the IFM is critical for tendon function, facilitating sliding between fascicles to allow tendons to stretch. This function is particularly important in energy storing tendons, which experience extremely high strains during exercise, and therefore require the capacity for considerable inter-fascicular sliding and recoil. This capacity is not required in positional tendons. Whilst we have previously described the quasi-static properties of the IFM, the fatigue resistance of the IFM in functionally distinct tendons remains unknown. We therefore tested the hypothesis that fascicles and IFM in the energy storing equine superficial digital flexor tendon (SDFT) are more fatigue resistant than those in the positional common digital extensor tendon (CDET). Fascicles and IFM from both tendon types were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results demonstrated that both fascicles and IFM from the energy storing SDFT were able to resist a greater number of cycles before failure than those from the positional CDET. Further, SDFT fascicles and IFM exhibited less hysteresis over the course of testing than their counterparts in the CDET. This is the first study to assess the fatigue resistance of the IFM, demonstrating that IFM has a functional role within tendon and contributes significantly to tendon mechanical properties. These data provide important advances into fully characterising tendon structure-function relationships

Effect of fatigue loading on structure and functional behaviour of fascicles from energy-storing tendons

Tendons can broadly be categorized according to their function: those that act purely to position the limb and those that have an additional function as energy stores. Energy-storing tendons undergo many cycles of large deformations during locomotion, and so must be able to extend and recoil efficiently, rapidly and repeatedly. Our previous work has shown rotation in response to applied strain in fascicles from energy-storing tendons, indicating the presence of helical substructures which may provide greater elasticity and recovery. In the current study, we assessed how preconditioning and fatigue loading affect the ability of fascicles from the energy-storing equine superficial digital flexor tendon to extend and recoil. We hypothesized that preconditioned samples would exhibit changes in microstructural strain response, but would retain their ability to recover. We further hypothesized that fatigue loading would result in sample damage, causing further alterations in extension mechanisms and a significant reduction in sample recovery. The results broadly support these hypotheses: preconditioned samples showed some alterations in microstructural strain response, but were able to recover following the removal of load. However, fatigue loaded samples showed visual evidence of damage and exhibited further alterations in extension mechanisms, characterized by decreased rotation in response to applied strain. This was accompanied by increased hysteresis and decreased recovery. These results suggest that fatigue loading results in a compromised helix substructure, reducing the ability of energy-storing tendons to recoil. A decreased ability to recoil may lead to an impaired response to further loading, potentially increasing the likelihood of injury

Molecular and biochemical characterisation of novel glycosyltransferases in Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan. The anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB and also targets the biosynthesis of the important immunomodulatory molecule lipoarabinomannan (LAM), through inhibition of Mt-EmbC. A bioinformatics approach identified putative integral membrane proteins in Mycobacterium smegmatis, M. tuberculosis and the closely related species Corynebacterium glutamicum, with features common to the GT-C superfamily of glycosyltransferases. A novel arabinofuranosyltransferase, AftC, was deleted from both M. smegmatis and C. glutamicum and shown to be an internal branching α(1→3) arabinofuranosyltransferase involved in arabinogalactan biosynthesis. Further studies revealed a truncated LAM whereby the arabinan domain was severely reduced and consisted of a simple linear arabinan of approximately 12-15 α(1→5) linked Araf residues. This mutant LAM was also shown to be a potent stimulator of TNF-α production using a human macrophage cell line, thus illustrating that masking of the mannan core by arabinan in wild type LAM alters its ability in the production of this cytokine. We also describe a further arabinofuranosyltransferase, AftB. Deletion of its orthologue in C. glutamicum resulted in a viable mutant and biochemical analysis revealed the complete absence of terminal β(1→2)-linked arabinofuranosyl residues. Further analysis confirmed AftB as a terminal β(1→2) arabinofuranosyltransferase, which was also insensitive to EMB. The bioinformatic search for cell wall glycosyltransferases led to the identification of a rhamnosyltransferase in C. glutamicum, RptA. Deletion resulted in a reduction of terminal-rhamnopyranosyl linked residues and as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues. Furthermore, analysis of base-stable extractable lipids from C. glutamium revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and represent potential new drug targets.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

An investigation in to the cellular basis of tendon degeneration.

SIGLEAvailable from British Library Document Supply Centre- DSC:DX178303 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Chemical markers of human tendon health identified using Raman spectroscopy: potential for in vivo assessment

The purpose of this study is to determine whether age-related changes to tendon matrix molecules can be detected using Raman spectroscopy. Raman spectra were collected from human Achilles (n = 8) and tibialis anterior (n = 8) tendon tissue excised from young (17 ± 3 years) and old (72 ± 7 years) age groups. Normalised Raman spectra underwent principal component analysis (PCA), to objectively identify differences between age groups and tendon types. Certain Raman band intensities were correlated with levels of advanced glycation end-product (AGE) collagen crosslinks, quantified using conventional destructive biochemistry techniques. Achilles and tibialis anterior tendons in the old age group demonstrated significantly higher overall Raman intensities and fluorescence levels compared to young tendons. PCA was able to distinguish young and old age groups and different tendon types. Raman intensities differed significantly for several bands, including those previously associated with AGE crosslinks, where a significant positive correlation with biochemical measures was demonstrated. Differences in Raman spectra between old and young tendon tissue and correlation with AGE crosslinks provides the basis for quantifying age-related chemical modifications to tendon matrix molecules in intact tissue. Our results suggest that Raman spectroscopy may provide a powerful tool to assess tendon health and vitality in the future

Biosynthesis of mycobacterial arabinogalactan: identification of a novel (13)arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG (13)-linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor -D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) and a newly synthesized linear (15)-linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching (13) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials

Abstract: Background: Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits. Results: Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available. Conclusions: These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology. Graphical abstract

Gender differences in experiences of ART services in South Africa: a mixed methods study

Objectives: A mixed methods study exploring gender differences in patient profiles and experiences of ART services, along the access dimensions of availability, affordability and acceptability, in two rural and two urban areas of South Africa. methods Structured exit interviews (n = 1266) combined with in-depth interviews (n = 20) of women and men enrolled in ART care. results Men attending ART services were more likely to be employed (29% vs. 20%, P = 0.001) and were twice as likely to be married ⁄ co-habiting as women (42% vs. 22% P = 0.001). Men had known their HIV status for a shorter time (mean 32 vs. 36 months, P = 0.021) and were also less likely to disclose their status to non-family members (17% vs. 26%, P = 0.001). From both forms of data collection, a key finding was the role of female partners in providing social support and facilitating use of services by men. The converse was true for women who relied more on extended families and friends than on partners for support. Young, unmarried and unemployed men faced the greatest social isolation and difficulty. There were no major gender differences in the health system (supply side) dimensions of access. conclusions Gender differences in experiences of HIV services relate more to social than health system factors. However, the health system could be more responsive by designing services in ways that enable earlier and easier use by men.Web of Scienc

A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans