Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Comparative analysis of the physical properties of murine and human S100A7: Insight into why zinc piracy is mediated by human but not murine S100A7.

S100 proteins are a subfamily of EF-hand calcium-binding proteins found primarily in vertebrate animals. They are distinguished by binding of transition metals and functioning in both the intracellular and extracellular milieu. S100A7 functions in the protection of the skin and mucous membranes and is a biomarker in inflammatory skin disease. A recent study of Neisseria gonorrhoeae infection revealed that human but not murine S100A7 could be used to evade host nutritional immunity. To understand the molecular basis for this difference, we carried out a comparative analysis of the physical and structural properties of human and murine S100A7. The X-ray crystal structure of Ca2+-loaded mouse S100A7 (mS100A7) was determined to 1.69 Å resolution, and Ca2+-induced conformational changes were assessed by NMR. Unlike human S100A7 (hS100A7), which exhibits conformational changes in response to binding of Ca2+, no significant changes in mS100A7 were detected. Dynamic light scattering, circular dichroism, and a competition chelator assay were used to compare the Zn2+ affinity and the effects of ion binding on mS100A7 versus hS100A7. Alignment of their sequences revealed a substantial difference in the C-terminal region, which is an important mediator of protein-protein interactions, suggesting a rationale for the specificity of N. gonorrhoeae for hS100A7. These data, along with more detailed analysis of S100A7 sequence conservation across different species, support the proposal that, although hS100A7 is highly conserved in many mammals, the murine protein is a distinct ortholog. Our results highlight the potential limitations of using mouse models for studying bacterial infections in humans.

Adherence Enables Neisseria gonorrhoeae to Overcome Zinc Limitation Imposed by Nutritional Immunity Proteins.

Neisseria gonorrhoeae (Gc) must overcome the limitation of metals such as zinc to colonize mucosal surfaces in its obligate human host. While the zinc-binding nutritional immunity proteins calprotectin (S100A8/A9) and psoriasin (S100A7) are abundant in human cervicovaginal lavage fluid, Gc possesses TonB-dependent transporters TdfH and TdfJ that bind and extract zinc from the human version of these proteins, respectively. Here we investigated the contribution of zinc acquisition to Gc infection of epithelial cells of the female genital tract. We found that TdfH and TdfJ were dispensable for survival of strain FA1090 Gc that was associated with Ect1 human immortalized epithelial cells, when zinc was limited by calprotectin and psoriasin. In contrast, suspension-grown bacteria declined in viability under the same conditions. Exposure to murine calprotectin, which Gc cannot use as a zinc source, similarly reduced survival of suspension-grown Gc, but not Ect1-associated Gc. We ruled out epithelial cells as a contributor to the enhanced growth of cell-associated Gc under zinc limitation. Instead, we found that attachment to glass was sufficient to enhance bacterial growth when zinc was sequestered. We compared the transcriptional profiles of WT Gc adherent to glass coverslips or in suspension, when zinc was sequestered with murine calprotectin or provided in excess, from which we identified open reading frames that were increased by zinc sequestration in adherent Gc. One of these, ZnuA, was necessary but not sufficient for survival of Gc under zinc-limiting conditions. These results show that adherence protects Gc from zinc-dependent growth restriction by host nutritional immunity proteins.

Neutrophil-Associated Responses to Vibrio cholerae Infection in a Natural Host Model.

Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.

The Zinc Transporter ZnuABC Is Critical for the Virulence of Chromobacterium violaceum and Contributes to Diverse Zinc-Dependent Physiological Processes.

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked reductions in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed low capacities to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subversion of zinc-based host nutritional immunity.

Parametric human modelling to determine body surface area covered by sun-protective clothing.

Solar ultraviolet radiation (UVR) is the main environmental risk-factor for cancer of the skin. Sun-protective clothing provides a physical barrier that reduces the UVR dose reaching the skin and European and Australian standards for sun-protective clothing set minimum clothing coverage requirements. Body Surface Area Coverage by clothing (BSAC) is calculated by means of indirect or direct methods, which are laborious and do not support computer-based apparel design. To support the sun-safe specification and design of garments, parametric digital human models and protective clothing mesh covering the minimum Body Surface Area specified in AS/NZS 4399:2017, were created making use of MakeHuman v1.1.1 and Blender software. The Whole Body Surface Area (WBSA) and the BSAC were calculated employing code developed in Blender. Thus, different groups of subjects were analysed to explore BSAC. The method assists in the evaluation of exposed body areas in a wider spectrum of different occupations. Practitioner summary: Sun-protective clothing provides a physical barrier that reduces the UVR dose reaching the skin's surface. Body Surface Area Coverage (BSAC) by clothing is an important determinant of the sun protective capabilities of a garment. In this study, BSAC is calculated using parametric digital human modelling. Abbreviation: UVR: (Solar) ultraviolet radiation; DHM: digital human modeling; BSA: body surface area; BSAC: body surface area coverage (by clothing); BSANC: body surface area not covered (by clothing); WBSA: whole body surface area; BCC: basal cell carcinoma; SCC: squamous cell carcinoma; UPF: ultraviolet protection factor; GPF: garment protection factor.

Modifiable Risk-factors for Keratinocyte Cancers in Australia: A Case-control Study.

Keratinocyte cancer is the most common malignancy in Caucasians. The aim of this study was to investigate risk-factors responsible for development of keratinocyte cancer in Australia. A case-control study was conducted, including 112 cases of squamous cell carcinoma (SCC), 95 cases of basal cell carcinoma (BCC) and 122 controls. Freckling during adolescence (SCC: odds ratio (OR) 1.04, p < 0.01; BCC: OR 1.05, p < 0.01), propensity to sunburn (SCC: OR 2.75, p = 0.01, BCC: OR 2.68 p = 0.01) and high cumulative sun-exposure (SCC: OR 2.43, p = 0.04; BCC: OR 2.36 p = 0.04) were independent risk-factors for both SCC and BCC. This study provides further evidence that a sun-sensitive phenotype and excessive sun-exposure during adulthood contribute to the risk of developing keratinocyte cancer. Wearing a hat, long-sleeved shirts, and sunscreen did not significantly reduce the risk of keratinocyte cancer in this study.

The geospatial relationship of pterygium and senile cataract with ambient solar ultraviolet in tropical Ecuador.

Tropical Ecuador presents a unique climate in which we study the relationship between the ambient levels of solar ultraviolet radiation and eye disease in the absence of a latitudinal gradient. The national distribution of surface ultraviolet, taking into account MODIS and OMI satellite observation of aerosol, ozone, surface albedo, local elevation and cloud fractions measured during 2011, was compared with the national pterygium (WHO ICD H11) and senile cataract (WHO ICD H25) incidence projected from the 2010 National Institute of Statistics and Census (Ecuador). Public Health Ministry projections for age categories 0 to 39, 40 to 59 and 60+ years were compared to surface ultraviolet irradiance data in 1040 parishes. Correlations drawn between modelled surface ultraviolet and eye disease incidence show a significant increase in both pterygium and senile cataract in the highest ambient exposure regions of the Pacific coast and western lowlands with incidence rates of 34.39 and 16.17 per 100 000 residents respectively. The lowest rates of incidence for pterygium (6.89 per 100 000) and senile cataract (2.90 per 100 000) were determined in high altitude sites and are attributed here to increased daily cloud fraction for parishes located in the Andean mountain range. The South American Andes experience the highest solar UV exposures on Earth and report frequent high incidence of keratinocyte cancer. Our results show the high Andes to be the location of the lowest eye disease incidence suggesting that both pterygium and senile cataract are the result of cumulative exposure to solar ultraviolet. These findings have clear implications for the agricultural workers and fishermen of the lowland districts of Ecuador, contrary to conventional understanding that greater risks are faced in locations of high altitude.

Sun protective behaviors at an outdoor entertainment event in Australia.

An observational study was conducted to examine the use of sun protective hats, clothing, and sunglasses of people attending an outdoor entertainment event in an area of high-to-extreme ultraviolet radiation in New South Wales, Australia. Armidale is unique, as it is a highly-elevated area, almost 1000 m above sea level, and temperatures are often mild with very high-to-extreme levels of ultraviolet radiation. Four trained data collectors observed attendees as they entered the event, and recorded their use of sun protective hats, clothing, and sunglasses. While more than half of the attendees wore sun protective hats, only 14% wore sun protective clothing. Broad-brimmed hats were considered sun protective, while sun protective clothing was defined by shirts with at least three-quarter-length sleeves. Females were more likely to wear both a sun protective hat and clothing than males, and children were less protected than adults. Legislative changes are required to ensure that organizers of outdoor events have a legal responsibility to provide a safe environment for attendees, including strategies to help reduce ultraviolet radiation exposure.

Investigating the patterns and determinants of seasonal variation in vitamin D status in Australian adults: the Seasonal D Cohort Study.

BACKGROUND: Vitamin D status generally varies seasonally with changing solar UVB radiation, time in the sun, amount of skin exposed, and, possibly, diet. The Seasonal D Study was designed to quantify the amplitude and phase of seasonal variation in the serum concentration of 25-hydroxyvitamin D, (25OH)D)) and identify the determinants of the amplitude and phase and those of inter-individual variability in seasonal pattern. METHODS: The Seasonal D Study collected data 2-monthly for 12 months, including demographics, personal sun exposure using a diary and polysulphone dosimeters over 7 days, and blood for serum 25(OH)D concentration. The study recruited 333 adults aged 18-79 years living in Canberra (35°S, n = 168) and Brisbane (27°South, n = 165), Australia. DISCUSSION: We report the study design and cohort description for the Seasonal D Study. The study has collected a wealth of data to examine inter- and intra-individual seasonal variation in vitamin D status and serum 25(OH)D levels in Australian adults.

The contributions of solar ultraviolet radiation exposure and other determinants to serum 25-hydroxyvitamin D concentrations in Australian adults: the AusD Study.

The Quantitative Assessment of Solar UV [ultraviolet] Exposure for Vitamin D Synthesis in Australian Adults (AusD) Study aimed to better define the relationship between sun exposure and serum 25-hydroxyvitamin D (25(OH)D) concentration. Cross-sectional data were collected between May 2009 and December 2010 from 1,002 participants aged 18-75 years in 4 Australian sites spanning 24° of latitude. Participants completed the following: 1) questionnaires on sun exposure, dietary vitamin D intake, and vitamin D supplementation; 2) 10 days of personal ultraviolet radiation dosimetry; 3) a sun exposure and physical activity diary; and 4) clinical measurements and blood collection for 25(OH)D determination. Our multiple regression model described 40% of the variance in 25(OH)D concentration; modifiable behavioral factors contributed 52% of the explained variance, and environmental and demographic or constitutional variables contributed 38% and 10%, respectively. The amount of skin exposed was the single strongest contributor to the explained variance (27%), followed by location (20%), season (17%), personal ultraviolet radiation exposure (8%), vitamin D supplementation (7%), body mass index (weight (kg)/height (m)(2)) (4%), and physical activity (4%). Modifiable behavioral factors strongly influence serum 25(OH)D concentrations in Australian adults. In addition, latitude was a strong determinant of the relative contribution of different behavioral factors.

Does being a "SunSmart School" influence hat-wearing compliance? An ecological study of hat-wearing rates at Australian primary schools in a region of high sun exposure.

BACKGROUND: Childhood sun exposure is an important risk factor for skin cancer. Anecdotal evidence suggests that hats are under-utilized by Australian primary school students. METHODS: The proportion of students and adult role-models wearing hats was observed at 36 primary schools (63.9% SunSmart schools [SSS]) in Townsville (latitude 19.3°S; high to extreme maximum daily UV-index year round), Queensland, Australia, from 2009 to 2011. RESULTS: Overall, 52.2% of 28,775 students and 47.9% of 2954 adults were observed wearing a hat. Hat use (all styles) among SSS and non-SunSmart school (NSSS) students was similar before (24.2% vs 20.5%; p=0.701), after (25.4% vs 21.7%; p=0.775) and during school-hours (93.0% vs 89.2%; p=0.649) except SSS students wore gold-standard (broad-brim/bucket/legionnaire) hats during school play-breaks more often in the warmer months (October-March) than NSSS students (54.7% vs 37.4%; p=0.02). Although the proportion of adults who wore hats (all styles) was similar at SSS and NSSS (48.2% vs 46.8%; p=0.974), fewer adults at SSS wore them before school (3.7% vs 10.2%; p=0.035). CONCLUSIONS: SunSmart status is not consistently associated with better hat-wearing behavior. The protective nature of hats and the proportion of school students and adult role-models wearing them could be improved, possibly by offering incentives to schools that promote sun-safety.

School sun-protection policies-does being SunSmart make a difference?

Evaluate the comprehensiveness of primary school sun-protection policies in tropical North Queensland, Australia. Pre-determined criteria were used to assess publicly available sun-protection policies from primary schools in Townsville (latitude 19.3°S; n = 43), Cairns (16.9°S; n = 46) and the Atherton Tablelands (17.3°S; n = 23) during 2009-2012. Total scores determined policy comprehensiveness. The relationship between policy score, SunSmart status and demographic characteristics was explored. At least 96.6% of primary schools sampled had a sun-protection policy. Although policies of Cancer Council accredited 'SunSmart' schools addressed more environmental, curriculum and review-related criteria than those of 'non-SunSmart' schools, the overall median score for both groups was low at 2 from a possible 12 (48.5% of SunSmart schools [SSSs]: inter-quartile range [IQR = 2.0-9.0] versus 65.9% of non-SSSs: [IQR = 2.0-3.0], P = 0.008). Most policies addressed hat wearing, while criteria related to shade provision at outdoor events, regular policy review and using the policy to plan outdoor events were poorly addressed. Although most primary schools in skin cancer-prone North Queensland have written sun-protection policies, the comprehensiveness of these policies could be vastly improved. These schools may require further support and advice to improve the comprehensive of their policies and incentives to continually implement them to achieve and maintain exemplary sun-protection compliance.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Changes in the pattern of sun exposure and sun protection in young children from tropical Australia.

BACKGROUND: Australia has one of the highest rates of skin cancer globally. Lifetime risk is associated with childhood sun exposure. OBJECTIVE: We sought to investigate whether skin cancer prevention programs have resulted in improvements in sun-exposure and sun-protection behavior among young children in tropical Australia. METHODS: Two cohorts of 12-to 35-month-old children from Townsville, Australia, were compared: cohort 1 was recruited from hospital birth records (1991) and cohort 2 was recruited via local child-care centers (1999-2002). Children's phenotypic characteristics were assessed. Parents completed questionnaires detailing children's demographic characteristics, and sun-exposure and sun-protective practices. RESULTS: Although 1-year-old children from cohort 2 spent more time in the sun than those from cohort 1 (median 2.2 vs 2.8 h/d; P = .002), a higher proportion almost always wore sunscreen and a swim-shirt year round. Although more 1-year-old children in cohort 2 had experienced a sunburn (35.5% vs 51.2%; P = .007), both cohort 2 age groups experienced fewer hours of sun exposure to the back of the trunk (P < .001), were less likely to have been sunburned on the back/shoulders (age 1 year 34.8% vs 10.1% and age 2 years 52% vs 10.1%; P < .001), and acquired fewer melanocytic nevi at these sites (P < .001). LIMITATIONS: There was potential for socially desirable responses (information bias). CONCLUSION: Although duration of sun exposure in early childhood did not decrease during an 8-year period, reported use of personal sun protection did. The observed increase in popularity of swim-shirts and sunscreen between cohorts coincided with the development of significantly fewer melanocytic nevi in these children.

An intervention to discourage Australian mothers from unnecessarily exposing their babies to the sun for therapeutic reasons.

Parents play a key role in children's sun-protective behaviour, with good sun-protective habits established early tending to be sustained. We designed a maternity hospital-based educational intervention to reduce myths that could result in mothers intentionally sunning their babies. Interviews were conducted with two cross-sections of healthy post-partum inpatients in the maternity ward of a large regional public hospital. The first group (n = 106) was recruited before the commencement of educational in-services for maternity nursing staff; the second group (n = 203) was interviewed after the last staff in-service session. More pre-intervention than post-intervention women reported they would expose their baby to sunlight to: treat suspected jaundice (28.8% vs. 13.3%; p < 0.001) or help their baby's skin adapt to sunlight (10.5% vs. 2.5%; p = 0.003). Fewer post-intervention women indicated they would sun themselves to treat breastfeeding-associated sore/cracked nipples (7.6% vs. 2%; p = 0.026). This educational intervention should be used to educate parents, health professionals and students.

Sun-Protective Clothing Worn Regularly during Early Childhood Reduces the Number of New Melanocytic Nevi: The North Queensland Sun-Safe Clothing Cluster Randomized Controlled Trial.

Numerous pigmented moles are associated with sun exposure and melanomarisk. This cluster randomized controlled trial aimed to determine if sun-protective clothing could prevent a significant proportion of the moles developing in young children (ACTRN12617000621314; Australian New Zealand Clinical Trials Registry. Twenty-five childcare centers in Townsville (19.25° S), Australia, were matched on shade provision and socioeconomic status. One center from each pair was randomized to the intervention arm and the other to the control arm. Children at 13 intervention centers wore study garments and legionnaire hats at childcare and received sun-protective swimwear and hats for home use, while children at the 12 control centers did not. The 1-35-month-old children (334 intervention; 210 control) were examined for moles at baseline (1999-2002) and were re-examined annually for up to 4 years. Both groups were similar at baseline. Children at intervention centers acquired fewer new moles overall (median 12.5 versus 16, p = 0.02; 0.46 versus 0.68 moles/month, p = 0.001) and fewer new moles on clothing-protected skin (6 vs. 8; p = 0.021 adjusted for confounding and cluster sampling) than controls. Intervention children had 24.3% fewer new moles overall (26.5 versus 35) and 31.6% (13 versus 19) fewer moles on clothing-protected skin than controls after 3.5 years. Sunlight's influence on nevogenesis is mitigated when children regularly wear UPF 30-50+ clothing covering half their body, implying that increased clothing cover reduces melanoma risk. Sun-protective clothing standards should mandate reporting of the percentage of garment coverage for childrenswear.

Serial Cross-Sectional Observations of Sun-Protective Behaviors at an Annual Outdoor Motorsport Event in Tropical Queensland, Australia.

Skin cancer, the most prevalent cancer in Caucasians residing at low latitudes, can primarily be prevented by avoiding overexposure to sunlight. Serial cross-sectional observations were conducted at an outdoor motorsport event held in Townsville, Queensland each July (Southern winter) to determine whether sun-protection habits changed over time. Most (71.1%) of the 1337 attendees observed (97.6% lightly pigmented skin, 64.0% male) wore a hat (any style shading the face), while few (18.5%) wore three-quarter or full-length sleeves. While hat-wearing rates (any style) were similar in 2009 (326, 72.6%) and 2013 (625, 70.4%), the use of sun-protective styles (wide-brimmed/bucket/legionnaires) decreased from 29.2% to 18.6% over the same period, primarily because the use of sun-protective hats halved (from 28.7% to 14.0%) among females, while decreasing from 29.4% to 21.1% in males. Although relatively few individuals wore sun-protective (three-quarter-length or full-length) sleeves regardless of year (OR = 0.117, P < 0.0001), encouragingly, the use of sun-protective sleeves more than doubled between 2009 (10.5%) and 2013 (22.5%). Interestingly females, albeit the minority, at this sporting event were less likely to wear a hat (OR = 0.473, P < 0.0001) than males. These findings highlight the need for continued momentum toward skin cancer primary prevention through sun protection with a dedicated focus on outdoor sporting settings.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

Slip, Slop, Slap, Slide, Seek and Sport: A Systematic Scoping Review of Sun Protection in Sport in Australasia.

Australia and New Zealand have the highest incidence of skin cancer. Sport is a fundamental part of Australasian culture, beginning in childhood, often with life-long participation. Participating in outdoor sports can contribute significantly to the lifetime ultraviolet radiation (UVR) dose individuals receive and their risk of developing skin cancer. This systematic scoping review explores the use of sun-protection by outdoor sporting participants in Australasia and considers how sun-protection practices may be improved and better evaluated in the community. A search of electronic databases using the search strategy "sun protection" AND "sport" AND "Australia" yielded 17 studies published in English from January 1992 to August 2021. Study methods included using UV-dosimeters to measure individual UVR-exposure; remote estimates of clothing-adjusted UVR-exposure; direct observation of sun-protection practices; and self-reported sun-exposure and sun-protection. Despite 40 years of 'Slip, Slop, Slap' campaigns in Australia, the use of sun-protection in most outdoor sports is inadequate. The paucity of comparable data limited our analyses, demonstrating a need for standardized, objective evaluation tools. Such tools, if used across a range of sports, should inform the development of workable recommendations that sporting clubs could implement and adopt into policy, thus empowering them to better protect the health of their participants.