A Qualitative Enquiry of On-Farm Rules About Quad Bikes (ATVs): How Rules Are Determined and Implemented at a Farm Level in Rural Australia.

OBJECTIVE: Quad bikes are a leading cause of death and incident-related injury on farms, yet little is understood about rules used by farmers to ensure their safe operation. This study explored rules about quad bikes set by those who live or work on farms. Through the case of quad bikes, this study sought to understand how rules are determined and implemented at the farm level. SETTING: A mix of farm types and locations in rural Australia including Queensland, South Australia, and New South Wales. PARTICIPANTS: Eight farmers were interviewed and recruited from information sheets at farmers' markets, through a local health organisation, and a media release. DESIGN: Thematic analysis was used to transform data from eight semi-structured interviews with farmers in rural Australia. RESULTS: Data were distilled into two themes - "Rule content" described the explicit rules farmers had set on their properties, while the theme "Underlying rule principles" explored the values and norms which underpinned the creation and implementation of these rules. CONCLUSIONS: Through the case of quad bike rules, this study illustrated how rules are determined and implemented at the farm level. Perceptions of risk were tied to farmers being experts in their own environment and therefore able to mitigate risk. In contrast to injury data, reckless use of quad bikes was perceived to cause incidents, and this was the basis of rules for adults and children.

Robust diffraction-limited near-infrared-to-near-ultraviolet wide-field imaging from stratospheric balloon-borne platforms-Super-pressure Balloon-borne Imaging Telescope performance.

At a fraction of the total cost of an equivalent orbital mission, scientific balloon-borne platforms, operating above 99.7% of the Earth's atmosphere, offer attractive, competitive, and effective observational capabilities-namely, space-like seeing, transmission, and backgrounds-which are well suited for modern astronomy and cosmology. The Super-pressure Balloon-borne Imaging Telescope (SUPERBIT) is a diffraction-limited, wide-field, 0.5 m telescope capable of exploiting these observing conditions in order to provide exquisite imaging throughout the near-infrared to near-ultraviolet. It utilizes a robust active stabilization system that has consistently demonstrated a 48 mas 1σ sky-fixed pointing stability over multiple 1 h observations at float. This is achieved by actively tracking compound pendulations via a three-axis gimballed platform, which provides sky-fixed telescope stability at < 500 mas and corrects for field rotation, while employing high-bandwidth tip/tilt optics to remove residual disturbances across the science imaging focal plane. SUPERBIT's performance during the 2019 commissioning flight benefited from a customized high-fidelity science-capable telescope designed with an exceptional thermo- and opto-mechanical stability as well as a tightly constrained static and dynamic coupling between high-rate sensors and telescope optics. At the currently demonstrated level of flight performance, SUPERBIT capabilities now surpass the science requirements for a wide variety of experiments in cosmology, astrophysics, and stellar dynamics.

Hydroxytyrosol inhibits cancer stem cells and the metastatic capacity of triple-negative breast cancer cell lines by the simultaneous targeting of epithelial-to-mesenchymal transition, Wnt/ß-catenin and TGFß signaling pathways.

PURPOSE: This study was aimed to determine the impact of hydroxytyrosol (HT), a minor compound found in olive oil, on breast cancer stem cells (BCSCs) and the migration capacity of triple-negative breast cancer (TNBC) cell lines through the alteration of epithelial-to-mesenchymal transition (EMT) and embryonic signaling pathways. METHODS: BCSCs self-renewal was determined by the mammosphere-forming efficiency in SUM159PT, BT549, MDA-MB-231 and Hs578T TNBC cell lines. Flow cytometric analysis of CD44+/CD24-/low and aldehyde dehydrogenase positive (ALDH+) subpopulations, migration by the "wound healing assay", invasion and Western blot of EMT markers and TGFß signaling were investigated in SUM159PT, BT549 and MDA-MB-231 cell lines. Wnt/ß-catenin signaling was assessed by Western blot in BT549 cells expressing WNT1 and MDA-MB-231 cells. Changes in TGFß activity was determined by SMAD Binding Element (SBE) reporter assay. RESULTS: HT reduced BCSCs self-renewal, ALDH+ (aldehyde dehydrogenase) and CD44+/CD24-/low subpopulations, tumor cell migration and invasion. Consistently, HT suppressed Wnt/ß-catenin signaling by decreasing p-LRP6, LRP6, ß-catenin and cyclin D1 protein expression and the EMT markers SLUG, ZEB1, SNAIL and VIMENTIN. Finally, HT inhibited p-SMAD2/3 and SMAD2/3 in SUM159PT, BT549 and MDA-MB-231 cells, what was correlated with a less TGFß activity. CONCLUSION: In conclusion, we report for the first time the inhibitory role of HT on BCSCs and tumor cell migration by targeting EMT, Wnt/ß-catenin and TGFß signaling pathways. Our findings highlight the importance of the chemopreventive compound HT as a novel candidate to be investigated as an alternative targeted therapy for TNBC.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells.

Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a "mini-breast" organoid.

Microvesicle-mediated Wnt/ß-Catenin Signaling Promotes Interspecies Mammary Stem/Progenitor Cell Growth.

Signaling mechanisms that regulate mammary stem/progenitor cell (MaSC) self-renewal are essential for developmental changes that occur in the mammary gland during pregnancy, lactation, and involution. We observed that equine MaSCs (eMaSCs) maintain their growth potential in culture for an indefinite period, whereas canine MaSCs (cMaSCs) lose their growth potential in long term cultures. We then used this system to investigate the role of microvesicles (MVs) in promoting self-renewal properties. We found that Wnt3a and Wnt1 were expressed at higher levels in MVs isolated from eMaSCs compared with those from cMaSCs. Furthermore, eMaSC-MVs were able to induce Wnt/ß-catenin signaling in different target cells, including cMaSCs. Interestingly, the induction of Wnt/ß-catenin signaling in cMaSCs was prolonged when using eMaSC-MVs compared with recombinant Wnt proteins, indicating that MVs are not only important for transport of Wnt proteins, but they also enhance their signaling activity. Finally, we demonstrate that the eMaSC-MVs-mediated activation of the Wnt/ß-catenin signaling pathway in cMaSCs significantly improves the ability of cMaSCs to grow as mammospheres and, importantly, that this effect is abolished when eMaSC-MVs are treated with Wnt ligand inhibitors. This suggests that this novel form of intercellular communication plays an important role in self-renewal.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Antibiotic use for Vibrio infections: important insights from surveillance data.

BACKGROUND: There is a paucity of data on the in vivo efficacy of antibiotics for lethal Vibrio species. Analyses of long-term surveillance datasets may provide insights into use of antibiotics to decrease mortality. METHODS: The United States Centers for Disease Control and Prevention (CDC) Cholera and Other Vibrio Illness Surveillance (COVIS) dataset from 1990 to 2010, with 8056 records, was analysed to ascertain trends in antibiotics use and mortality. RESULTS: Two-thirds of patients (5243) were prescribed antibiotics - quinolones (56.1 %), cephalosporins (24.1 %), tetracyclines (23.5 %), and penicillins (15.4 %). Considering all Vibrio species, the only class of antibiotic associated with reduced odds of mortality was quinolone (odds ratio 0.56, 95 % CI 0.46-0.67). Patients with V. vulnificus treated according to CDC recommendations had lower mortality (quinolone alone: 16.7 %, 95 % CI 10.2-26.1; tetracycline plus cephalosporin: 21.7 %, 16.8-27.5; no antibiotic: 51.1 %, 45.6-56.7; each p < 0.001). Cephalosporin alone was associated with higher mortality (36.8 %, 28.2-46.3). For V. cholerae non-O1, non-O139, mortality rates were lower for quinolone (0 %, 0-2.0) or tetracycline (4.3 %, 1.2-14.5) compared to no antibiotic (9.3 %, 6.4-13.3). For all Vibrio species, mortality rates increased with number of antibiotics in the treatment regimen (p < 0.001). Treatment regimens that included quinolone were associated with lower mortality rates regardless of the number of antibiotics used. The main clinical syndromes of patients with V. vulnificus infection were septicaemia (53.1 %) and wound infections (30.6 %). Mortality among V. vulnificus patients with septicaemia was significantly higher than for other clinical syndromes (p < 0.001). In a multivariate regression model, mortality in cases with V. vulnificus was associated with presence of pre-existing conditions (ORs ranged from 4.52 to 10.30), septicaemia (OR 2.64, 95 % CI 1.92-3.63) and no antibiotic treatment (OR 7.89, 95 % CI 3.94-15.80). CONCLUSION: In view of the lack of randomized control trials, surveillance data may inform treatment decisions for potentially lethal Vibriosis. Considering all Vibrio species, use of quinolones is associated with lower mortality and penicillin alone is not particularly effective. For the most lethal species, V. vulnificus, treatment that includes either quinolone or tetracycline is associated with lower mortality than cephalosporin alone. We recommend treating patients who present with a clinical syndrome suggestive of V. vulnificus infection with a treatment regimen that includes a quinolone.

Both canonical and non-canonical Wnt signaling independently promote stem cell growth in mammospheres.

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/ß-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, ß-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/ß-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.

Medical graduates becoming rural doctors: rural background versus extended rural placement.

OBJECTIVES: To determine whether recruitment of rural students and uptake of extended rural placements are associated with students' expressed intentions to undertake rural internships and students' acceptance of rural internships after finishing medical school, and to compare any associations. DESIGN, SETTING AND PARTICIPANTS: Longitudinal study of three successive cohorts (commencing 2005, 2006, 2007) of medical students in the Sydney Medical Program (SMP), University of Sydney, New South Wales, using responses to self-administered questionnaires upon entry to and exit from the Sydney Medical School and data recorded in rolls. MAIN OUTCOME MEASURES: Students' expressed intentions to undertake rural internships, and their acceptance of rural internships after finishing medical school. RESULTS: Data from 448 students were included. The proportion of students preferring a rural career dropped from 20.7% (79/382) to 12.5% (54/433) between entry into and exit from the SMP. A total of 98 students took extended rural placements. Ultimately, 8.1% (35/434) accepted a rural internship, although 14.5% (60/415) had indicated a first preference for a rural post. Students who had undertaken an extended rural placement were more than three times as likely as those with rural backgrounds to express a first preference for a rural internship (23.9% v 7.7%; χ(2) = 7.04; P = 0.008) and more than twice as likely to accept a rural internship (21.3% v 9.9%; χ(2) = 3.85; P = 0.05). CONCLUSION: For the three cohorts studied, rural clinical training through extended placements in rural clinical schools had a stronger association than rural background with a preference for, and acceptance of, rural internship.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3ß activates TGF-ß signaling and induces epithelial-to-mesenchymal transition in breast cancer cells.

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3ß (GSK3ß), both in vitro and in vivo. Among other functions, GSK3ß is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3ß protein levels, increased TGF-ß signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3ß is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3ß is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.

LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.

Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) and loss (Lrp6(-)) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6(-/-) MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6(-/-) embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1.

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

Atypical protein kinase Cι is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated dishevelled 2.

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368-2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1δ, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993-1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of pPKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428-9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.

Eye health services for Aboriginal people in the western region of NSW, 2010.

AIM: To assess the availability, accessibility and uptake of eye health services for Aboriginal people in western NSW in 2010. METHODS: The use of document review, observational visits, key stakeholder consultation and service data reviews, including number of cataract operations performed, to determine regional service availability and use. RESULTS: Aboriginal people in western NSW have a lower uptake of tertiary eye health services, with cataract surgery rates of 1750 per million for Aboriginal people and 9702 per million for non-Aboriginal people. Public ophthalmology clinics increase access to tertiary services for Aboriginal people. CONCLUSION: Eye health services are not equally available and accessible for Aboriginal people in western NSW. Increasing the availability of culturally competent public ophthalmology clinics may increase access to tertiary ophthalmology services for Aboriginal people. The report of the review was published online, and outlines a list of recommendations.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir.

Wnt proteins that signal via the canonical Wnt/ß-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of ß-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, ß-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

AUSDRISK - application in general practice.

BACKGROUND: The Australian Type 2 Diabetes Risk Assessment Tool (AUSDRISK) has been promoted since July 2008. We studied its application rate and the profile of a sample of general practice patients within Central West New South Wales from June to December 2010. METHODS: Stage one assessed the awareness and application of AUSDRISK among general practitioners and general practice registrars. In stage two, the doctors used AUSDRISK and appropriate blood tests to screen patients aged 25-74 years who had not been previously diagnosed with diabetes. RESULTS: Seventy-eight doctors (response rate 45.1%) completed the survey. A total of 68.2% of general practice registrars and 23.2% of GPs were aware of AUSDRISK. Among the respondents 14.1% (95% CI: 6-22%) applied AUSDRISK in their usual practice, and 39.1% (95% CI: 31-47%) of the 151 patients had high AUSDRISK scores ≥15. DISCUSSION: Two years after the launch of AUSDRISK, the application rate of AUSDRISK is low. In this patient population, many patients had high AUSDRISK scores.