Pharmacogenomic Considerations for Anticoagulant Prescription in Patients with Hereditary Haemorrhagic Telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is a vascular dysplasia that commonly results in bleeding but with frequent indications for therapeutic anticoagulation. Our aims were to advance the understanding of drug-specific intolerance and evaluate if there was an indication for pharmacogenomic testing. Genes encoding proteins involved in the absorption, distribution, metabolism, and excretion of warfarin, heparin, and direct oral anticoagulants (DOACs) apixaban, rivaroxaban, edoxaban, and dabigatran were identified and examined. Linkage disequilibrium with HHT genes was excluded, before variants within these genes were examined following whole genome sequencing of general and HHT populations. The 44 genes identified included 5/17 actionable pharmacogenes with guidelines. The 76,156 participants in the Genome Aggregation Database v3.1.2 had 28,446 variants, including 9668 missense substitutions and 1076 predicted loss-of-function (frameshift, nonsense, and consensus splice site) variants, i.e., approximately 1 in 7.9 individuals had a missense substitution, and 1 in 71 had a loss-of-function variant. Focusing on the 17 genes relevant to usually preferred DOACs, similar variant profiles were identified in HHT patients. With HHT patients at particular risk of haemorrhage when undergoing anticoagulant treatment, we explore how pre-emptive pharmacogenomic testing, alongside HHT gene testing, may prove beneficial in reducing the risk of bleeding and conclude that HHT patients are well placed to be at the vanguard of personalised prescribing.

Rural Identity and LGBT Public Opinion in the United States.

Opposition to LGBT rights remains a contemporary fixture within the United States in spite of increasingly liberalizing attitudes toward LGBT individuals. In this paper, I argue that a potentially overlooked factor driving this opposition is rural identity-or an individual's psychological attachment to a rural area. Using data from the 2020 ANES, I find that rural identity predicts less favorable estimations of LGBT individuals. Rural identifiers are also less likely to support pro-LGBT policy measures than nonrural identifiers. Nevertheless, I find the magnitude of the effects of rural identity on anti-LGBT views to be surprisingly small. It is also the case that, on average, rural identifiers exhibit net-positive estimations of LGBT individuals and are broadly supportive of LGBT rights, suggesting that elected officials enacting anti-LGBT legislation in rural areas of the United States are potentially out of step with the preferences of their electorate. These findings also have implications for what it means to hold a rural identity beyond a generalized animosity toward urban areas, and for understanding urban-rural divergences in US public opinion on issues such as LGBT rights.

Monitoring medication and illicit drug consumption in a prison by wastewater-based epidemiology: Impact of COVID-19 restrictions.

Drug consumption in prisons is a concern for the safety of incarcerated people and staff. Typically, drug use prevalence in prisons is estimated through urinalysis and intelligence operations, which can be intrusive and stressful. An alternative approach, wastewater-based epidemiology (WBE), was used in this study to estimate the consumption of licit and illicit drugs for the entire population of a prison in Australia. Wastewater samples were collected from March to December 2020, covering periods of no restrictions and periods when prison access was restricted to prevent the transmission of COVID-19. Target biomarkers were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The average consumption of common illicit drugs (MDMA, methamphetamine and cocaine) over the sampling period in the prison (0.5 - 4.5 mg/1000 people/day) was two to three orders of magnitude lower than in the community population (254 - 1000 mg/1000 people/day). Comparison of WBE estimates against pharmacy dispensing data suggested potential illicit buprenorphine consumption at the prison. Methamphetamine and buprenorphine use decreased when no visitors were allowed (18% - 72% decrease for methamphetamine; about half decrease for buprenorphine) and increased once these restrictions were eased (22% - 39% increase for methamphetamine; 44% - 67% increase for buprenorphine). The changes in drug use may be attributed in part to a reduction of drug trafficking into the prison from visitors or non-essential staffs and in part to the reduced contribution of urine from staff who used toilets within the prison. This study provided useful information on the scale of illicit drug use and extra-medical use of licit drugs in prison, and its changes under different security conditions.

Correlates of support for international vaccine solidarity during the COVID-19 pandemic: Cross-sectional survey evidence from Germany.

During the COVID-19 pandemic, many residents of high-income countries (HICs) were eligible for COVID-19 vaccine boosters, while many residents of lower-income countries (LICs) had not yet received a first dose. HICs made some efforts to contribute to COVID-19 vaccination efforts in LICs, but these efforts were limited in scale. A new literature discusses the normative importance of an international redistribution of vaccines. Our analysis contributes an empirical perspective on the willingness of citizens in a HIC to contribute to such efforts (which we term international vaccine solidarity). We analyse the levels and predictors of international vaccine solidarity. We surveyed a representative sample of German adults (n = 2019) who participated in a two-wave YouGov online survey (w1: Sep 13-21, 2021 and w2: Oct 4-13, 2021). International vaccine solidarity is measured by asking respondents preferences for sharing vaccine supplies internationally versus using that supply as boosters for the domestic population. We examine a set of pre-registered hypotheses. Almost half of the respondents in our sample (48%) prioritize giving doses to citizens in less developed countries. A third of respondents (33%) prefer to use available doses as boosters domestically, and a fifth of respondents (19%) did not report a preference. In line with our hypotheses, respondents higher in cosmopolitanism and empathy, and those who support domestic redistribution exhibit more support for international dose-sharing. Older respondents (who might be more at risk) do not consistently show less support for vaccine solidarity. These results help us to get a better understanding of the way citizens' form preferences about a mechanism that redistributes medical supplies internationally during a global crisis.

Vaccine attributes and vaccine uptake in Hungary: evidence from a conjoint experiment.

BACKGROUND: In an ongoing public health crisis, the question of why some people are unwilling to take vaccines with particular attributes is an especially pertinent one, since low rates of vaccination mean that it will take longer for many nations to exit the coronavirus disease 2019 (COVID-19) pandemic. METHODS: In this article, we conduct a pre-registered conjoint experiment in Hungary (N = 2512), where respondents were asked about their attitudes towards hypothetical COVID-19 vaccines whose characteristics varied across a number of attributes. RESULTS: Results indicate that vaccine attributes matter for the likelihood of uptake when it comes to the prevalence of severe side effects, efficacy and country of origin. Moreover, we find that our pre-treatment measure of institutional trust moderates the effect of our treatment, as differences in vaccine attributes are larger for those with robust levels of institutional trust compared to those with weaker levels. CONCLUSION: Our findings suggest that institutional trust matters when it comes to understanding the relationship between vaccine attributes and likelihood of uptake.

Apparent Half-Lives of Chlorinated-Perfluorooctane Sulfonate and Perfluorooctane Sulfonate Isomers in Aviation Firefighters.

Elevated levels of perfluorooctane sulfonate (PFOS) and elevated detection frequency of chloro-substituted PFOS have been reported in Australian firefighters with historical exposure to aqueous-film forming foam (AFFF). The aim of this study is to estimate the apparent half-lives of Cl-PFOS and PFOS isomers in firefighters following the end of exposure to 3M-AFFF. Paired serum samples from 120 firefighters, collected approximately five years apart, were analyzed for 8-Cl-PFOS (8-chloroperfluoro-1-octanesulfonic acid) and PFOS isomers via targeted LC-MS/MS. Apparent half-life was estimated by assuming a first order-elimination model. Cl-PFOS was detected in 93% of all initial serum samples (

The Effects of Exercise Training on Glucose Homeostasis and Muscle Metabolism in Type 1 Diabetic Female Mice.

Although exercise training is an important recommendation for the management of type 1 diabetes (T1D), most of the available research studies predominantly focus on male subjects. Given the importance of sex as a biological variable, additional studies are required to improve the knowledge gap regarding sex differences in T1D research. Therefore, the purpose of this study was to examine the role of exercise training in mediating changes in glucose homeostasis and skeletal muscle metabolism in T1D female mice. Female mice were injected with streptozotocin (STZ) to induce T1D. Two weeks after STZ injection, control (CON) and STZ mice were exercise trained on a treadmill for 4 weeks. Aerobic exercise training failed to improve glucose tolerance, prevent the decrease in body weight and adipose tissue mass, or attenuate muscle atrophy in T1D female mice. However, insulin sensitivity was improved in T1D female mice after exercise training. Aerobic exercise training maintained skeletal muscle triglyceride content but did not prevent depletion of skeletal muscle or liver glycogen in T1D mice. Gene expression analysis suggested that T1D resulted in decreased glucose transport, decreased ketone body oxidation, and increased fatty acid metabolism in the skeletal muscle, which was not altered by exercise training. These data demonstrate that 4 weeks of aerobic exercise training of a moderate intensity is insufficient to counteract the negative effects of T1D in female mice, but does lead to an improvement in insulin sensitivity.

Stamping the vaccine passport? Public support for lifting COVID-19 related restrictions for vaccinated citizens in France, Germany, and Sweden.

During theCOVID-19pandemic,manycountries implementedrestrictionsto limit the spread of the SARS-CoV-2 virus (e.g. travel restrictions and lockdowns).One path to loosening restrictions is to do so selectively only for vaccinated individuals (e.g. by implementing vaccine passports domestically or as a prerequisite for international travel).Setting different rules based on people's vaccination statusis howevera contentious issue among health policy experts, government officials, and the public. Our analysis focuses on the levels and correlates of public support for the lifting of restrictions for the vaccinatedin April 2021, i.e. at a time when restrictions were in place and aselective lifting of these restrictions just for the vaccinatedwas debated in Europe.We use representative quota samples of the populations of France (N = 1,752), Germany (N = 1,759), and Sweden (N = 1,754). We find that a slight plurality support lifting restrictions for the vaccinated in France and Germany but not in Sweden. Vaccine hesitancy emerges as strong predictor of opposition to such a policy. Additionally, individuals who are already vaccinated (in France and Germany) and who are higher in risk-seeking express more support for the lifting of restrictions for the vaccinated. We discuss implications for the debate on vaccine passports.

Which vaccine attributes foster vaccine uptake? A cross-country conjoint experiment.

Why do people prefer one particular COVID-19 vaccine over another? We conducted a pre-registered conjoint experiment (n = 5,432) in France, Germany, and Sweden in which respondents rated the favorability of and chose between pairs of hypothetical COVID-19 vaccines. Differences in effectiveness and the prevalence of side-effects had the largest effects on vaccine preferences. Factors with smaller effects include country of origin (respondents are less favorable to vaccines of Chinese and Russian origin), and vaccine technology (respondents exhibited a small preference for hypothetical mRNA vaccines). The general public also exhibits sensitivity to additional factors (e.g. how expensive the vaccines are). Our data show that vaccine attributes are more important for vaccine preferences among those with higher vaccine favorability and higher risk tolerance. In our conjoint design, vaccine attributes-including effectiveness and side-effect prevalence-appear to have more muted effects among the most vaccine hesitant respondents. The prevalence of side-effects, effectiveness, country of origin and vaccine technology (e.g., mRNA vaccines) determine vaccine acceptance, but they matter little among the vaccine hesitant. Vaccine hesitant people do not find a vaccine more attractive even if it has the most favorable attributes. While the communication of vaccine attributes is important, it is unlikely to convince those who are most vaccine hesitant to get vaccinated.

Transformation and fate of pharmaceuticals, personal care products, and per- and polyfluoroalkyl substances during aerobic digestion of anaerobically digested sludge.

Post-anaerobic aerobic digestion (PAAD) is a promising strategy to further reduce the volume and improve the quality of anaerobically digested sludge (ADS). However, the effect of PAAD process on the fate of pharmaceuticals and personal care products (PPCPs) and per- and polyfluoroalkyl substances (PFAS) remains largely unknown. In this study, fourteen PPCPs and fifteen PFAS were detected in ADS and evaluated regarding their fate and transformation in a laboratory aerobic digester operated with a hydraulic retention time of 13 days under 22 ℃. Twelve PPCPs demonstrated significant (p < 0.05) decrease in their total concentrations (dissolved and adsorbed fractions combined) with six compounds presenting substantial transformation (> 80%) after aerobic digestion. On the contrary, PFAS were not removed and their concentrations were either increased (increasing ratio: 91 - 571%) or consistent in the sludge during PAAD process, suggesting their recalcitrance to post aerobic digestion. More than half of PPCPs and PFAS demonstrated medium to strong sorption onto solids with their solid fraction higher than 50% in the ADS. After PAAD process, sorption of four PPCPs and three PFAAs to solids was enhanced in sludge.

Direct injection analysis of oxypurinol and metformin in wastewater by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

The increasing global prevalence of gout and diabetes has led to a rise in the use of their respective medications, allopurinol and metformin. These are excreted via urine as oxypurinol and metformin and are discharged into wastewater and the environment. Current environmental monitoring of those two polar chemicals requires labour intensive and potentially inefficient sample pre-treatments, such as using solid-phase extraction or freeze-drying. This study validated a sensitive and simple method using direct-injection LC-MS/MS for the simultaneous measurement of oxypurinol and metformin in wastewater. The final method utilised a hydrophilic interaction liquid chromatography together with simple filtration through 0.2 µm regenerated cellulose filter followed by dilution in acetonitrile with a dilution factor of 10. The developed method was validated with the limit of quantifications (LOQ) of 0.11 and 0.34 µg/L for metformin and oxypurinol, respectively. The new method was applied to 42 influent wastewater samples and 6 effluent samples collected from 6 Australian wastewater treatment plants. Both compounds were detected well above the LOQ at concentrations 29-214 µg/L in influent and 2-53 µg/L in effluent for metformin, and 24-248 µg/L in influent and 4-81 µg/L in effluent for oxypurinol, demonstrating its high applicability.

Quantification of selected analgesics and their metabolites in influent wastewater by liquid chromatography tandem mass spectrometry.

Wastewater-based epidemiology (WBE) is an established tool to analyse patterns of community drug use. In recent times, monitoring the use of analgesics has become a public health priority. An analytical method was developed, optimised and validated for a broad range of common analgesics (non-steroidal anti-inflammatories and opioids) and their metabolites at trace concentrations in wastewater influent. Samples were analysed via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with an electrospray ionization source (ESI) in both positive and negative ion multiple reaction monitoring modes. Fifteen of the thirty-five biomarkers were validated for LC-MS/MS analysis by direct injection, while the other 20 biomarkers were present in wastewater at lower concentrations, requiring extraction by solid phase extraction (SPE) using Oasis HLB cartridges. The limit of quantification (LOQ) ranged between 17 ng/L - 191 ng/L and 13 ng/L -110 ng/L for direct injection and SPE, respectively. Recoveries for SPE ranged between 80% and 120%. Following validation, the method was applied to 28 wastewater samples from four municipal wastewater treatment plants. Twenty-eight of the thirty-five analgesic biomarkers were detected in the samples at concentrations of between 0.03 and 289 µg/L. Population normalised mass loads ranged from 10 mg/day/1000 (meperidine) to 72,000 mg/day/1000 (paracetamol) among all investigated compounds. The described method is a suitable analytical tool for wastewater-based epidemiology applications to estimate analgesics consumption.

Systematic Evaluation of the In-Sample Stability of Selected Pharmaceuticals, Illicit Drugs, and Their Metabolites in Wastewater.

The in-sample stability of selected pharmaceuticals, illicit drugs, and their metabolites in wastewater was assessed under six different conditions-untreated, addition of hydrochloric acid or sodium metabisulfite solution, combined with or without sterile filtration, and at four representative temperatures, at 35 °C for up to 28 days, 22 °C for 56 days, and 4 °C and -20 °C for 196 days, or freeze/thaw cycles for 24 weeks. Paracetamol, 6-monoacetylmorphine, morphine, and cocaine were poorly stable in untreated wastewater-e.g., with 50% transformation within 1.2-8.1 days at 22 °C, and acidification reduced their in-sample transformations. Acesulfame, carbamazepine, cotinine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), ketamine, norfentanyl, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and norbuprenorphine were highly or moderately stable over the observed period, even in untreated wastewater. Fitting of pseudo-first-order kinetics and the Arrhenius equation was used to develop a multistage transformation estimation model combined with an interactive tool to evaluate possible transformation scenarios of selected biomarkers for the processes from sampling to preanalysis. However, as the wastewater composition can vary between sites and over time, the variability of in-sample stability requires further exploration.

Expanding the indications for the bone anchored hearing system (BAHS) in patients with single sided deafness.

PURPOSE: Provide data to support expansion of FDA indications for the Bone anchored hearing system (BAHS). MATERIALS AND METHODS: This retrospective study in a tertiary otologic referral center included106 consecutive subjects who were implanted with a Bone Anchored Hearing System (BAHS) between January 2009 and January 2015 for single sided deafness. Subjects were divided into three groups by bone conduction pure tone average (PTA) of the better hearing ear: 0-20 dB (group 1), 21-40 dB (group 2) and 41-55 dB (group 3). All patients underwent BAHS implantation. Speech perception data (Hearing In Noise Test and Consonant-Nucleus-Consonant testing) was collected before and after surgical intervention. Patient-reported quality of life measures were obtained at least 6 months after activation. These included the Abbreviated Profile of Hearing Aid Benefit and Glasgow Benefit Inventory. RESULTS: All three groups of subjects demonstrated statistically significant improvement in outcome measures following BAHS. Subject reported quality of life outcome measures demonstrated significant improvement in disability from hearing loss and in quality of life. CONCLUSIONS: Patients with single sided deafness who have bone conduction thresholds worse than 20 dB in their contralateral ear are still able to benefit significantly from BAHS.

Wastewater-based prevalence trends of gout in an Australian community over a period of 8 years.

Gout is a rheumatic arthritis disease which poses a health burden. Monitoring the prevalence of gout is key to reduce the community burden of gout disease and associated health costs. Allopurinol has been used as a first line gout preventive medication in Australia which is metabolised into oxypurinol and excreted in urine. Wastewater-based epidemiology (WBE) was applied to estimate temporal trends of gout prevalence in an Australian community over eight-years via the quantification of oxypurinol in wastewater. A total of 180 wastewater samples collected between 2012 and 2019 were analysed for oxypurinol to estimate allopurinol consumption in a community in South East Queensland, Australia. Annual gout prevalence was estimated by daily defined doses (DDD) consumed and ranged from 24 to 32 DDD/day/1000, an equivalent gout prevalence of 2.3 to 3.2% over the eight-year period. A statistically significant increase in allopurinol consumption was observed over the period (Slope = 0.094, p = 0.0001), equating to year-on-year increases in gout prevalence of 3.6% per year. To the best of our knowledge, this is the first long-term gout prevalence study using wastewater, adding epidemiological and public health insights in the gout research field.

Trends in artificial sweetener consumption: A 7-year wastewater-based epidemiology study in Queensland, Australia.

A 7-year temporal trend study of artificial sweetener consumption was performed by determining per capital mass loads in 293 influent wastewater samples collected from a wastewater treatment plant in Australia between 2012 and 2018. Population-weighted per capita mass loads of the four detected artificial sweeteners ranged from 2.4 ± 0.8 mg d-1 p-1 for saccharin to 7.8 ± 2.0 mg d-1 p-1 for acesulfame over the study period. Negligible intra-week fluctuations were observed, however the consumption of acesulfame was seen to be significantly influenced by season with the highest consumption in summer. The consumption of sucralose and saccharin significantly increased with an annual increase rate of 10% and 6.0%. Cyclamate consumption declined over the same period with average annual decrease rate of 11%, which agrees with data from market surveys. Sucrose equivalence of total artificial sweeteners consumption showed an increase between 2012 and 2016, then decreased in 2018. This is the first long-term trend study of artificial sweetener consumption by wastewater analysis and highlights the feasibility to quantitatively measure artificial sweeter consumption over time.

Influences of Chemical Properties, Soil Properties, and Solution pH on Soil-Water Partitioning Coefficients of Per- and Polyfluoroalkyl Substances (PFASs).

The aim of this study was to assess the soil-water partitioning behavior of a wider range of per- and polyfluoroalkyl substances (PFASs) onto soils covering diverse soil properties. The PFASs studied include perfluoroalkyl carboxylates (PFCAs), perfluoroalkane sulfonates (PFSAs), fluorotelomer sulfonates (FTSs), nonionic perfluoroalkane sulfonamides (FASAs), cyclic PFAS (PFEtCHxS), per- and polyfluoroalkyl ether acids (GenX, ADONA, 9Cl-PF3ONS), and three aqueous film-forming foam (AFFF)-related zwitterionic PFASs (AmPr-FHxSA, TAmPr-FHxSA, 6:2 FTSA-PrB). Soil-water partitioning coefficients (log Kd values) of the PFASs ranged from less than zero to approximately three, were chain-length-dependent, and were significantly linearly related to molecular weight (MW) for PFASs with MW > 350 g/mol (R2 = 0.94, p < 0.0001). Across all soils, the Kd values of all short-chain PFASs (≤5 -CF2- moieties) were similar and varied less (<0.5 log units) compared to long-chain PFASs (>0.5 to 1.5  log units) and zwitterions AmPr- and TAmPr-FHxSA (∼1.5 to 2 log units). Multiple soil properties described sorption of PFASs better than any single property. The effects of soil properties on sorption were different for anionic, nonionic, and zwitterionic PFASs. Solution pH could change both PFAS speciation and soil chemistry affecting surface complexation and electrostatic processes. The Kd values of all PFASs increased when solution pH decreased from approximately eight to three. Short-chain PFASs were less sensitive to solution pH than long-chain PFASs. The results indicate the complex interactions of PFASs with soil surfaces and the need to consider both PFAS type and soil properties to describe mobility in the environment.

Determination of anabasine, anatabine, and nicotine biomarkers in wastewater by enhanced direct injection LC-MS/MS and evaluation of their in-sewer stability.

Wastewater-based epidemiology (WBE) has been used to estimate tobacco use in the population. However, the increased use of nicotine replacement therapies and e-cigarettes contributes to the load of nicotine metabolites in wastewater, causing over-estimation of tobacco use if nicotine metabolites were used in WBE back-estimation. This study aims to develop a rapid method for determining the tobacco-specific biomarkers, anabasine and anatabine, in wastewater and to evaluate their in-sewer stability for better estimation of tobacco use by WBE. An enhanced direct injection LC-MS/MS was developed to quantify anabasine and anatabine as well as nicotine biomarkers (nicotine, cotinine and hydroxycotinine). The method was optimal when wastewater was filtered through 0.2 µm RC syringe filters and a pre-conditioned SPE cartridge (Oasis HLB 1 cc, 30 mg) before 50 µL was injected into the LC-MS/MS system. Limits of quantification varied between 2.7 and 54.9 ng/L with recoveries from 76% to 103% for all five compounds. In sewer reactors, anabasine and anatabine were less stable than cotinine and hydroxycotinine. They were more stable in the gravity sewer reactor with <20% loss in 12 h than in the rising main sewer reactor with ~30% loss in the same period. We then applied the new method to 42 daily wastewater influent samples collected from an Australian wastewater treatment plant. The five biomarkers were detected in all samples with concentrations ranging from 9.2 to 7430 ng/L. All five compounds were positively correlated with one another. Our results suggested a high throughput analytical method for feasible application in anabasine and anatabine as biomarkers of tobacco use in routine wastewater monitoring.

National wastewater reconnaissance of artificial sweetener consumption and emission in Australia.

Artificial sweeteners are used as sugar substitutes in our daily lives yet consumption and release patterns are currently unknown in Australia. The spatial distribution of artificial sweetener consumption and WWTP effluent emission in Australia was estimated by wastewater analysis. Wastewater influent and effluent samples were collected from 69 WWTPs across Australia during the week of the 2016 Australian census. Mean population-weighted per capita loads for individual artificial sweeteners (cyclamate, aspartame, acesulfame, sucralose, saccharin) ranged from 0.12 ± 0.14 mg d-1p-1 for aspartame to 6.9 ± 2.8 mg d-1p-1 for acesulfame with 1004 kg of these artificial sweeteners being consumed daily in Australia. Significant removal of aspartame (100%), cyclamate (92 ± 18%) and saccharin (88 ± 21%) was observed during wastewater treatment. The average per capita release to the environment for individual artificial sweeteners (cyclamate, acesulfame, sucralose, saccharin) ranged from 230 ± 780 mg d-1 1000p-1 (cyclamate) to 3800 ± 1400 mg d-1 1000p-1 (sucralose). The daily release of artificial sweeteners from Australian WWTPs was estimated to be 142 kg suggesting that 14% of the artificial sweeteners consumed in Australia are released into the environment. To the best of our knowledge, this is the first wastewater study to estimate the occurrence and population-normalized artificial sweetener consumption and emission in Australia.