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.

1,1,1-Trichloro-2,2-bis(4-ethoxyphenyl)ethane

In the title compound, C18H19Cl3O2, which is the 4-ethoxyphenyl analogue of the insecticidally active 4-methoxyphenyl compound methoxychlor, the dihedral angle between the two benzene rings is 60.38(13)deg. An intramolecular aromatic C-H...Cl interaction is present.

Bariatric surgery or medicine for type 2 diabetes?

Diet and medical treatment are the standard treatment for type 2 diabetes. In obese subjects with type 2 diabetes, bariatric surgery is effective in resolving diabetes. Two clinical trials comparing bariatric surgery to medical treatment were evaluated. Both the Surgical Treatment And Medications Potentially Eradicate Diabetes Efficiently (STAMPEDE) trial (laparoscopic Roux-En Y gastric bypass and sleeve gastrectomy) and the DIet and medical therapy versus BAriatric SurgerY in type 2 diabetes (DIBASY) trial (laparoscopic gastric bypass and biliopancreatic-diversion) showed that surgery was more effective than medical care in resolving or managing type 2 diabetes. Larger studies, or a compilation of studies, are needed to determine whether one of these procedures is better, or if they are all similarly effective, and this should also be weighed against the risk of the operations.

Augmenting public participation : enhancing planning outcomes through the use of social media and web 2.0

Social media and web 2.0 tools offer opportunities to devise novel participation strategies that can engage previously difficult to reach as well as new segments of society in urban planning. This paper examines participatory planning in the four local government areas of Brisbane City Council, Gold Coast City Council, Redland City Council, and Toowoomba Regional Council, all situated in South East Queensland, Australia. The paper discusses how social media and web 2.0 tools can deliver a more engaging planning experience to citizens, and investigates local government’s current use and receptiveness to social media tools for plan making and community engagement. The study’s research informed the development of criteria to assess the level of participation reached through the current use of social media and web 2.0 in the four local government areas. This resulted in an adaptation of the International Association for Public Participation (IAP2) Toolbox to integrate these new tools which is being presented to encourage further discussion and evaluation by planning professionals.

Isopropyl 2,2-bis(4-bromophenyl)-2-hydroxyacetate

In the structure of the title compound C17H16Br2O3, which is a restricted commercial acaricide (common name bromopropylate), has two independent and conformationally similar molecules in the asymmetric unit [dihedral angle between the planes of the two phenyl rings in each, 68.7(4) and 77.4(5)deg]. The C-atoms of the isopropyl group of one of the molecules are disordered over two sites with occupancies of 0.638 and 0.362. Minor non-merohedral twinning was also present in the crystal. Intermolecular hydrogen-bonding interactions involving the hydroxy groups and carboxyl O-atom acceptors give separate centrosymmetric homodimers through cyclic hydrogen-bonding motifs [graph set R2/2(10)].

Poly(2-oxazoline) hydrogel monoliths via thiol-ene coupling

Copoly(2-oxazoline)s, prepared by cationic ring-opening polymerization of 2-(dec-9-enyl)-2-oxazoline with either 2-methyl-2-oxazoline or 2-ethyl-2-oxazoline, have been crosslinked with small dithiol molecules under UV-irradiation to form homogeneous networks. In-situ monitoring of the crosslinking reaction by photo-rheology revealed network formation within minutes. The degree of swelling in water was found to be tunable by the hydrophilicity of the starting macromers and the proportion of alkene side arms. Furthermore, degradable hydrogels have been prepared based on a hydrolytically cleavable dithiol crosslinker.

Randomised controlled trial of an automated, interactive telephone intervention (TLC Diabetes) to improve type 2 diabetes management : Baseline findings and six-month outcomes

Background: Effective self-management of diabetes is essential for the reduction of diabetes-related complications, as global rates of diabetes escalate. Methods: Randomised controlled trial. Adults with type 2 diabetes (n = 120), with HbA1c greater than or equal to 7.5 %, were randomly allocated (4 × 4 block randomised block design) to receive an automated, interactive telephone-delivered management intervention or usual routine care. Baseline sociodemographic, behavioural and medical history data were collected by self-administered questionnaires and biological data were obtained during hospital appointments. Health-related quality of life (HRQL) was measured using the SF-36. Results: The mean age of participants was 57.4 (SD 8.3), 63 % of whom were male. There were no differences in demographic, socioeconomic and behavioural variables between the study arms at baseline. Over the six-month period from baseline, participants receiving the Australian TLC (Telephone-Linked Care) Diabetes program showed a 0.8 % decrease in geometric mean HbA1c from 8.7 % to 7.9 %, compared with a 0.2 % HbA1c reduction (8.9 % to 8.7 %) in the usual care arm (p = 0.002). There was also a significant improvement in mental HRQL, with a mean increase of 1.9 in the intervention arm, while the usual care arm decreased by 0.8 (p = 0.007). No significant improvements in physical HRQL were observed. Conclusions: These analyses indicate the efficacy of the Australian TLC Diabetes program with clinically significant post-intervention improvements in both glycaemic control and mental HRQL. These observed improvements, if supported and maintained by an ongoing program such as this, could significantly reduce diabetes-related complications in the longer term. Given the accessibility and feasibility of this kind of program, it has strong potential for providing effective, ongoing support to many individuals with diabetes in the future.

The Spartan-3E Tutorial 2 : Introduction to using the PicoBlaze Microcontroller Version 1.0

This tutorial is designed to help new users become familiar with using the PicoBlaze microcontroller with the Spartan-3E board. The tutorial gives a brief introduction to the PicoBlaze microcontroller, and then steps through the following: - Writing a small PicoBlaze assembly language (.psm) file, and stepping through the process of assembling the .psm file using KCPSM3; - Writing a top level VHDL module to connect the PicoBlaze microcontroller (KCPSM3 component) and the program ROM, and to connect the required input and output ports; - Connecting the top level module inputs and outputs to the switches, buttons and LEDs on the Spartan-3E board; - Downloading the program to the Spartan-3E board using the Project Navigator software.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

Emergency Health Services (EHS) : demand and service delivery models. [Monograph 2 : Queensland EHS users’ profile]

Emergency health is a critical component of Australia’s health system and one which is increasingly congested from growing demand and blocked access to inpatient beds. The Emergency Health Services Queensland (EHSQ) study aims to identify the factors driving increased demand for emergency health and to evaluate strategies which may safely reduce the future demand growth. This monograph addresses the characteristics of users of emergency health services with an aim to identify those that appear to contribute to demand growth. This study utilises data on patients treated by Emergency Departments (ED) and Queensland Ambulance Service (QAS) across Queensland. ED data was derived from the Emergency Department Information System (EDIS) for the period 2001-02 through to 2010-11. Ambulance data was extracted from the QAS’ Ambulance Information Management System (AIMS) and electronic Ambulance Report Form (eARF) for the period 2001-02 through to 2009-10. Due to discrepancies and comparability issues for ED data, this monograph compares data from the 2003-04 time period with 2010-11 data for 21 of the reporting EDs. Also a snapshot of users for the 2010-11 financial year for 31 reporting EDs is used to describe the characteristics of users and to compare those characteristics with population demographics. For QAS data, the 2002-03 and 2009-10 time periods were selected for detailed analyses to identify trends. • Demand for emergency health care services is increasing, representing both increased population and increased relative utilisation. Per capita demand for ED attention has increased by 2% per annum over the last decade and for ambulance attention by 3.7% per annum. • The growth in ED demand is prominent in more urgent triage categories with actual decline in less urgent patients. An estimated 55% of patients attend hospital EDs outside of normal working hours. There is no evidence that patients presenting out of hours are significantly different to those presenting within working hours; they have similar triage assessments and outcomes. • Patients suffering from injuries and poisoning comprise 28% of the ED workload (an increase of 65% in the study period), whilst declines of 32% in cardiovascular and circulatory conditions, and musculoskeletal problems have been observed. • 25.6% of patients attending EDs are admitted to hospital. 19% of admitted patients and 7% of patients who die in the ED are triage category 4 or 5 on arrival. • The average age of ED patients is 35.6 years. Demand has grown in all age groups and amongst both men and women. Men have higher utilisation rates for ED in all age groups. The only group where the growth rate in women has exceeded men is in the 20-29 age group; this growth is particularly in the injury and poisoning categories. • Considerable attention has been paid publicly to ED performance criteria. It is worth noting that 50% of all patients were treated within 33 minutes of arrival. • Patients from lower socioeconomic areas appear to have higher utilisation rates and the utilisation rate for indigenous people appears to exceed those of European and other backgrounds. The utilisation rates for immigrant people is generally less than that of Australian born however it has not been possible to eliminate the confounding impact of different age and socioeconomic profiles. • Demand for ambulance service is also increasing at a rate that exceeds population growth. Utilisation rates have increased by an average of 5% per annum in Queensland compared to 3.6% nationally, and the utilisation rate in Queensland is 27% higher than the national average. • The growth in ambulance utilisation has also been amongst the more urgent categories of dispatch and utilisation rates are higher in rural and regional areas than in the metropolitan area. The demand for ambulance increases with age but the growth in demand for ambulance service has been more prominent in younger age groups. These findings contribute significantly to an understanding of the growth in demand for emergency health. It shows that the growth is amongst patients in genuine need of emergency healthcare and public rhetoric that the congestion of emergency health services is due to inappropriate attendees is unable to be substantiated. The consistency of the growth in demand over the last decade reflects not only the changing demographics of the Australian population but also the changes in health status, standards of acute health care and other social factors. The growth is also amongst patients with acute injury and poisoning which is inconsistent with rates of chronic disease as a fundamental driver. We have also interviewed patients in regard to their decision making choices for acute health care and the factors that influence these decisions and this will be the subject of a third Monograph and publications.

Kaolinite particle sizes in the <2 µM range using laser scattering

The Clay Minerals Society Source Clay kaolinites, Georgia KGa-1 and KGa-2, have been subjected to particle size determinations by 1) conventional sedimentation methods, 2) electron microscopy and image analysis, and 3) laser scattering using improved algorithms for the interaction of light with small particles. Particle shape, size distribution, and crystallinity vary considerably for each kaolinite. Replicate analyses of separated size fractions showed that in the <2 µm range, the sedimentation/centrifugation method of Tanner and Jackson (1947) is reproducible for different kaolinite types and that the calculated size ranges are in reasonable agreement with the size bins estimated from laser scattering. Particle sizes determined by laser scattering must be calculated using Mie theory when the dominant particle size is less than ∼5 µm. Based on this study of two well-known and structurally different kaolinites, laser scattering, with improved data reduction algorithms that include Mie theory, should be considered an internally consistent and rapid technique for clay particle sizing.

An extended theory of planned behavior intervention for older adults With Type 2 diabetes and cardiovascular disease

A randomized controlled trial evaluated the effectiveness of a 4-wk extended theory of planned behavior (TPB) intervention to promote regular physical activity and healthy eating among older adults diagnosed with Type 2 diabetes or cardiovascular disease (N = 183). Participants completed TPB measures of attitude, subjective norm, perceived behavioral control, and intention, as well as planning and behavior, at preintervention and 1 wk and 6 wk postintervention for each behavior. No significant time-by-condition effects emerged for healthy eating. For physical activity, significant time-by-condition effects were found for behavior, intention, planning, perceived behavioral control, and subjective norm. In particular, compared with control participants, the intervention group showed short-term improvements in physical activity and planning, with further analyses indicating that the effect of the intervention on behavior was mediated by planning. The results indicate that TPB-based interventions including planning strategies may encourage physical activity among older people with diabetes and cardiovascular disease.

Dichotomy in the design studio 2.0 : adapting to new blended learning environments

In a study aimed at better understanding how students adapt to new blended studio learning environments, all undergraduate and masters of architecture students at a large school of architecture in Australia, learned a semester of architectural design in newly renovated, technology embedded, design studio environments. The renovations addressed the lessons learned from a 2011 pilot study of a second year architectural design studio learned in a high technology embedded prototype digital laboratory. The new design studios were purpose designed for the architecture students and adapted Student-Centred Active Learning Environment for Undergraduate Programs design principles. At the end of the semester, the students completed a questionnaire about their experiences of learning in the new design studio environments. Using a dual method qualitative approach, the questionnaire data were coded and extrapolated using both thematic analysis and grounded theory methodology. The results from these two approaches were compared, contrasted and finally merged, to reveal five distinct emerging themes, which were instrumental in offering resistance or influencing adaptation to, the new blended studio learning environments. This paper reports on the study, discusses the major contributors to resistance and adaptation, and proposes points for consideration when renovating or designing new blended studio learning environments. This research extends the 2011 pilot study by the same authors: ‘Dichotomy in the design studio: Adapting to new blended learning environments’.