Are motivational deficits part of the behavioural phenotype of Down syndrome?

Motivational deficits are generally accepted to be part of the behavioural phenotype associated with Down syndrome (DS). A motivational profile comprising low or inconsistent levels of task persistence, avoidance of challenging activities and over-dependence on adult direction has been described. However, comparisons are usually made between children with DS and those who are developing typically, without the inclusion of samples with intellectual disability (ID) from aetiologies other than DS. Such comparisons are needed to determine the extent to which motivational deficits are specific to DS, as opposed to being a feature of ID generally. Methods: The current study collected data about the personality-motivation profiles of children in three groups matched for mental age. They consisted of 80 typically developing (TD)3–7 year old children, 62 children with DS aged 7–15 years, and 54 children with moderate ID aged 7–15 years. Parents completed the 37-item EZ-Personality Questionnaire (EZPQ; Zigler et al., 2002), a measure of personality-motivational functioning. Results: There were significant differences between TD children and those with ID on all EZPQ scales. In most respects children with DS did not differ significantly from others with moderate ID, although they were rated as having greater expectancy of success and fewer negative reactions. Conclusion: The finding that children with DS are less motivated than TD children of the same mental age is consistent with previous studies in which parents have rated motivation. It seems, however, that motivation difficulties are associated with ID more generally, rather than being specific to those with DS. The study raises questions about phenotypic versus experiential effects on motivation for children with ID.

The role of the recycling endosome in regulating lamellipodia formation and macrophage migration

Cell migration is a highly complex process that requires the extension of cell membrane in the direction of travel. This membrane is continuously remodeled to expand the leading edge and alter its membrane properties. For a long time it has been known that there is a continual flow of polarized membrane traffic towards the leading edge during migration and that this trafficking is essential for cell migration. However, there is little information on how the cell coordinates exocytosis at the leading edge. It is also unclear whether these internal membranes are incorporated into the leading edge or are just delivering the necessary proteins for migration to occur. We have shown that recycling endosome membrane is incorporated into the plasma membrane at the leading edge to expand the membrane and at the same time delivers receptors to the leading edge to mediate migration. In order for this to happen the surface Q-SNARE complex Stx4/SNAP23 translocates to the leading edge where it binds to the R-SNARE VAMP3 on the recycling endosome allowing incorporation into the plasma membrane. Loss of any one of the components of this complex reduces efficient lamellipodia formation and restrains cell migration.

Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

The macrophage-inducible C-type lectin, Mincle, is an essential component of the innate immune response to Candida albicans

The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-_ by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.

The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds

Lithium (Li) has been widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders. Li+ ions are thought to enhance the remyelination of peripheral nerves and also stimulate the proliferation of neural progenitor cells and retinoblastoma cells via activation of the Wnt/β-catenin signalling pathway. Until now there have been no studies reporting the biological effects of released Li+ in bioactive scaffolds on cemetogenesis in periodontal tissue engineering applications. In this study, we incorporated parts of Li+ ions into the mesoporous bioactive glass (MBG) scaffolds and showed that this approach yielded scaffolds with a favourable composition, microstructure and mesopore properties for cell attachment, proliferation, and cementogenic differentiation of human periodontal ligament-derived cells (hPDLCs). We went on to investigate the biological effects of Li+ ions themselves on cell proliferation and cementogenic differentiation. The results showed that 5% Li+ ions incorporated into MBG scaffolds enhanced the proliferation and cementogenic differentiation of hPDLCs on scaffolds, most likely via activation of Wnt/β-catenin signalling pathway. Further study demonstrated that Li+ ions by themselves significantly enhanced the proliferation, differentiation and cementogenic gene expression of PDLCs. Our results indicate that incorporation of Li+ ions into bioactive scaffolds is a viable means of enhancing the Wnt canonical signalling pathway to stimulate cementogenic differentiation of PDLCs.

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

The surface-structure sensitivity of dioxygen activation in the anatase-photocatalyzed oxidation reaction

The reaction pathways by which oxygen is incorporated into the substrate in the photocatalytic oxidation of terephthalic acid (TPTA) are vastly different on {001} and {101} facets of an anatase single crystal. This was established by controlling the percentage of {101} and {001} facets, isotopically tracing the origins of the hydroxy group, and studying dioxygen consumption and variance in the concentration of hydroxylation intermediate.

Combined VEGF and PDGF treatment reduces secondary degeneration after spinal cord injury

Trauma to the spinal cord creates an initial physical injury damaging neurons, glia, and blood vessels, which then induces a prolonged inflammatory response, leading to secondary degeneration of spinal cord tissue, and further loss of neurons and glia surrounding the initial site of injury. Angiogenesis is a critical step in tissue repair, but in the injured spinal cord angiogenesis fails; blood vessels formed initially later regress. Stabilizing the angiogenic response is therefore a potential target to improve recovery after spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) can initiate angiogenesis, but cannot sustain blood vessel maturation. Platelet-derived growth factor (PDGF) can promote blood vessel stability and maturation. We therefore investigated a combined application of VEGF and PDGF as treatment for traumatic spinal cord injury, with the aim to reduce secondary degeneration by promotion of angiogenesis. Immediately after hemisection of the spinal cord in the rat we delivered VEGF and PDGF and to the injury site. One and 3 months later the size of the lesion was significantly smaller in the treated group compared to controls, and there was significantly reduced gliosis surrounding the lesion. There was no significant effect of the treatment on blood vessel density, although there was a significant reduction in the numbers of macrophages/microglia surrounding the lesion, and a shift in the distribution of morphological and immunological phenotypes of these inflammatory cells. VEGF and PDGF delivered singly exacerbated secondary degeneration, increasing the size of the lesion cavity. These results demonstrate a novel therapeutic intervention for SCI, and reveal an unanticipated synergy for these growth factors whereby they modulated inflammatory processes and created a microenvironment conducive to axon preservation/sprouting.

Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

Bicep femoris voluntary activation deficits contribute to eccentric knee flexor weakness following intermittent running

INTRODUCTION: Hamstring strain injuries (HSI) are the predominant non-contact injury in many sports. Eccentric hamstring muscle weakness following intermittent running has been implicated within the aetiology of HSI. This weakness following intermittent running is often greater eccentrically than concentrically, however the cause of this unique, contraction mode specific phenomenon is unknown. AIM: To determine if this preferential eccentric decline in strength is caused by declines in voluntary hamstring muscle activation. METHODS: Fifteen recreationally active males completed 18 × 20m overground sprints. Maximal strength (concentric and eccentric knee flexor and concentric knee extensor) was determined isokinetically at the velocities of ±1800.s-1 and ±600.s- while hamstring muscle activation was assessed using surface electromyography, before and 15 minutes after the running protocol. RESULTS: Overground intermittent running caused greater eccentric (27.2 Nm; 95% CI = 11.2 to 43.3; p=0.0001) than concentric knee flexor weakness (9.3 Nm; 95% CI = -6.7 to 25.3; P=0.6361). Following the overground running, voluntary activation levels of the lateral hamstrings showed a significant decline (0.08%; 95% CI = 0.045 to 0.120; P<0.0001). In comparison, medial hamstring activation showed no change following intermittent running. CONCLUSION: Eccentric hamstring strength is decreased significantly following intermittent overground running. Voluntary activation deficits in the biceps femoris muscle are responsible for some portion of this weakness. The implications of this finding are significant because the biceps femoris muscle is the most frequently strained of all the hamstring muscles and because fatigue appears to play an important part in injury occurrence.

Do voluntary activation deficits contribute to eccentric weakness following intermittent running?

Hamstring strain injuries (HSI) are the predominant non-contact injury in many sports. Eccentric hamstring muscle weakness following intermittent running has been implicated within the aetiology of HSI. This weakness following intermittent running is often greater eccentrically than concentrically, however the cause of this unique, contraction mode specific phenomenon is unknown. PURPOSE: To determine if this preferential eccentric decline in strength is caused by declines in voluntary hamstring muscle activation. METHODS: Fifteen recreationally active males completed 18 × 20m overground sprints. Maximal strength (concentric and eccentric knee flexor and concentric knee extensor) was determined isokinetically at the velocities of ±1800.s-1 and ±600.s- while hamstring muscle activation was assessed using surface electromyography, before and 15 minutes after the running protocol. RESULTS: Overground intermittent running caused greater eccentric (27.2 Nm; 95% CI = 11.2 to 43.3; p=0.0001) than concentric knee flexor weakness (9.3 Nm; 95% CI = -6.7 to 25.3; P=0.6361). Following the overground running, voluntary activation levels of the lateral hamstrings showed a significant decline (0.08%; 95% CI = 0.045 to 0.120; P<0.0001). In comparison, medial hamstring activation showed an increased level of activation following intermittent running (0.12%; 95% CI = 0.049 to 0.030; P = 0.0102). CONCLUSION: Eccentric hamstring strength is decreased significantly following intermittent overground running. Voluntary activation deficits in the biceps femoris muscle are responsible for some portion of this weakness. The implications of this finding are significant because the biceps femoris muscle is the most frequently strained of all the hamstring muscles and because fatigue appears to play an important part in injury occurrence.

Declines in eccentric knee flexor weakness following repeat sprint running are related to declines in biceps femoris voluntary activation

Introduction: Hamstring strain injuries (HSI) are the predominant non-contact injury in many sports. Eccentric hamstring muscle weakness following intermittent running has been implicated within the aetiology of HSI. This weakness following intermittent running is sometimes greater eccentrically than concentrically, however the cause of this unique, contraction mode specific phenomenon is unknown. The purpose of this research was to determine whether declines in knee flexor strength following overground repeat sprints are caused by declines in voluntary activation of the hamstring muscles. Methods: Seventeen recreationally active males completed 3 sets of 6 by 20m overground sprints. Maximal isokinetic concentric and eccentric knee flexor and concentric knee extensor strength was determined at ±1800.s-1 and ±600.s-1 while hamstring muscle activation was assessed using surface electromyography, before and 15 minutes after the running protocol. Results: Overground repeat sprint running resulted in a significant decline in eccentric knee flexor strength (31.1 Nm; 95% CI = 21.8 to 40.3 Nm; p < 0.001). However, concentric knee flexor strength was not significantly altered (11.1 Nm; 95% CI= -2.8 to 24.9; p=0.2294). Biceps femoris voluntary activation levels displayed a significant decline eccentrically (0.067; 95% CI=0.002 to 0.063; p=0.0325). However, there was no significant decline concentrically (0.025; 95% CI=-0.018 to 0.043; p=0.4243) following sprinting. Furthermore, declines in average peak torque at -1800.s-1 could be explained by changes in hamstring activation (R2 = 0.70). Moreover, it was change in the lateral hamstring muscle activity that was related to the decrease in knee flexor torque (p = 0.0144). In comparison, medial hamstring voluntary activation showed no change for either eccentric (0.06; 95% CI = -0.033 to 0.102; p=0.298) or concentric (0.09; 95% CI = -0.03 to 0.16; p=0.298) muscle actions following repeat sprinting. Discussion: Eccentric hamstring strength is decreased significantly following overground repeat sprinting. Voluntary activation deficits in the biceps femoris muscle explain a large portion of this weakness. The implications of these findings are significant as the biceps femoris muscle is the most frequently strained of the knee flexors and fatigue is implicated in the aetiology of this injury.