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

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

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

The quality of girls' diets declines and tracks across middle childhood

BACKGROUND: Food group intakes by US children are below recommendations and micronutrient inadequacies have been reported. There are few longitudinal data that focus on developmental changes in food and nutrient intake from early to middle childhood. We examined changes in nutrient and food group intakes over time and the tracking of intakes across middle childhood in a longitudinal sample of girls. METHODS: Three multiple-pass 24-hour diet recalls were conducted in a sample of 181 non-Hispanic White girls at ages 5, 7, and 9 years. Food and nutrient data were averaged across 3 days. Analyses of time effects were conducted using repeated measures analysis of variance and tracking of intakes was assessed via rank analysis. RESULTS: We found significant decreases in nutrient densities (intakes per 1000 kcal) of vitamins C and D, calcium, phosphorus, magnesium and zinc at age 9. Girls maintained their relative quartile positions for these micronutrients from ages 5–9. Analysis of food group data showed similar trends. At age 9, significantly fewer girls were meeting the recommendations for dairy, fruit and vegetable servings than at age 5 and girls also tended to remain in their respective quartiles over time, especially for fruit and dairy intakes. CONCLUSIONS: These results highlight the importance of developing healthy eating practices during early childhood when caretakers have considerable control over children's food intake

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

Girls’ Dairy Intake, Energy Intake, and Weight Status

We explored the relationships among girls’ weight status, dairy servings, and total energy intake. The hypothesis that consuming dairy could reduce risk for overweight was evaluated by comparing energy intake and weight status of girls who met or consumed less than the recommended three servings of dairy per day. Participants included 172 11-year-old non-Hispanic white girls, assessed cross-sectionally. Intakes of dairy, calcium, and energy were measured using three 24-hour recalls. Body mass index and body fat measures from dual-energy x-ray absorptiometry were obtained. Because preliminary analyses suggested systematic underreporting of energy intake, the relationships among dairy servings and measures of weight status were examined for the total sample and for subsamples of under-, plausible, and overreporters. Data for the total sample provided support for the hypothesized relationship among weight status, dairy servings, and energy intake. Thirty-nine percent of girls reported consuming the recommended ≥3 servings of dairy per day; these girls also reported higher energy intake but had lower body mass index z scores and body fat than the girls who consumed fewer than three dairy servings each day. Among plausible reporters, no relationship between dairy intake and weight status was noted. This discrepancy may be attributable to a high percentage (45%) of overweight underreporters in the total sample. Our findings reveal that reporting bias, resulting from the presence of a substantial proportion of underreporters of higher weight status, can contribute to obtaining spurious associations between dairy intake and weight status. These findings underscore the need for randomly controlled trials to assess the role of dairy in weight management. The prevalence of pediatric obesity has been rising for more than 20 years (1). There is evidence that increased intake of dairy foods and calcium may play a significant role in maintaining a healthful weight and moderating body fat (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15). However, results across studies have been inconsistent (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28), and this may be attributable to major challenges in using self-reported dietary intake data. Self-reported intakes tend to be subject to underreporting bias and the underreporting of energy intake tends to be positively related to weight status. Underreporters also tend to weigh more (29 and 30). Doubly labeled water techniques assessing energy expenditure suggest underreporting results in a 10% to 50% underestimation of actual energy intake and is a significant problem in older children (31). Doubly labeled water techniques are expensive and not feasible for large samples; thus, several methods have been developed that use estimated energy requirements to assess reporting bias (29 and 32). Therefore, in this study the method suggested by Huang and colleagues (29) was used to classify children as under-, plausible, or overreporters. The objective of this study was to assess the relationship among girls’ weight status, dairy servings, and total energy intake. The hypothesis that consuming dairy could reduce risk for overweight was evaluated by comparing energy intake and weight status of girls who met or consumed less than the recommended three servings of dairy per day. To explore the effect of reporting bias on this relationship, the hypothesis was evaluated using the total sample, and subgroups of girls identified as plausible, under-, or overreporters

Specialization of tendon mechanical properties results from interfascicular differences

Tendons transfer force from muscle to bone. Specific tendons, including the equine superficial digital flexor tendon (SDFT), also store and return energy. For efficient function, energy-storing tendons need to be more extensible than positional tendons such as the common digital extensor tendon (CDET), and when tested in vitro have a lower modulus and failure stress, but a higher failure strain. It is not known how differences in matrix organization contribute to distinct mechanical properties in functionally different tendons. We investigated the properties of whole tendons, tendon fascicles and the fascicular interface in the high-strain energy-storing SDFT and low-strain positional CDET. Fascicles failed at lower stresses and strains than tendons. The SDFT was more extensible than the CDET, but SDFT fascicles failed at lower strains than CDET fascicles, resulting in large differences between tendon and fascicle failure strain in the SDFT. At physiological loads, the stiffness at the fascicular interface was lower in the SDFT samples, enabling a greater fascicle sliding that could account for differences in tendon and fascicle failure strain. Sliding between fascicles prior to fascicle extension in the SDFT may allow the large extensions required in energy-storing tendons while protecting fascicles from damage

The Interfascicular Matrix of Energy Storing Tendons Houses Heterogenous Cell Populations Disproportionately Affected by Aging

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5th decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by aging. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells, and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by aging, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to establish heterogeneity in IFM cell populations, and to identify age-related alterations specific to IFM-localised cells

The interfascicular matrix enables fascicle sliding and recovery in tendon, and behaves more elastically in energy storing tendons

While the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the cost of locomotion. Energy storing tendons experience extremely high strains and need to be able to recoil efficiently for maximum energy storage and return. In the equine forelimb, the energy storing superficial digital flexor tendon (SDFT) has much higher failure strains than the positional common digital extensor tendon (CDET). However, we have previously shown that this is not due to differences in the properties of the SDFT and CDET fascicles (the largest tendon subunits). Instead, there is a greater capacity for interfascicular sliding in the SDFT which facilitates the greater extensions in this particular tendon (Thorpe et al., 2012). In the current study, we exposed fascicles and interfascicular matrix (IFM) from the SDFT and CDET to cyclic loading followed by a test to failure. The results show that IFM mechanical behaviour is not a result of irreversible deformation, but the IFM is able to withstand cyclic loading, and is more elastic in the SDFT than in the CDET. We also assessed the effect of ageing on IFM properties, demonstrating that the IFM is less able to resist repetitive loading as it ages, becoming stiffer with increasing age in the SDFT. These results provide further indications that the IFM is important for efficient function in energy storing tendons, and age-related alterations to the IFM may compromise function and predispose older tendons to injury