Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis.

Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes, and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently, we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis muscle-stage larvae (MSL) secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds to miRNA without exhibiting sequence specificity. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.

A case of asymptomatic uterine incarceration in a patient undergoing dilation and evacuation.

Uterine incarceration is a rare pregnancy complication that occurs when the uterine fundus becomes trapped beneath the sacral promontory. We present a case of incarceration in a patient seeking dilation and evacuation and a discussion of strategies for uterine reduction and cervical preparation.

"Healthy Kids"-A capacity building approach for the early childhood education and care sector.

ISSUE ADDRESSED: Queensland children have a higher level of developmental vulnerability compared to the Australian average. This paper reports on Healthy Kids-a capacity building strategy for the early childhood education and care (ECEC) sector targeting communities experiencing socio-economic and child development vulnerabilities. These communities may face additional barriers when engaging and participating in health promotion models. This paper reports on the development, key components and principles of a capacity building model referred to as Healthy Kids, that strategically responds to these barriers and supports these communities. METHODS: The development of the Healthy Kids model emerged through a quality improvement process that included an environmental scan, and review of existing capacity building, health promotion, and workforce development approaches. It also involved consultation and engagement with the ECEC sector. RESULTS: Evidence indicates Healthy Kids to be an innovative health promotion model focussed on building capacity through a workforce development strategy for the ECEC sector in a way that is accessible, low cost, and sustainable. SO WHAT?: This paper shares a model for building capacity through the establishment of localised cross-sector communities of practice across a large geographic region with a centralised coordinating hub. The hub and spoke model has facilitated community ownership to grow and be sustained over time. This model offers opportunities for partnerships, transferability, and contextualisation for those interested in contemporary health promotion, capacity building, and workforce development. The model offers an approach for those willing to step outside traditional boundaries to work across sectors and settings to achieve sustainable knowledge and skills, processes and resources that enables a collective commitment to improving health outcomes.

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction.

The calcium-sensing receptor is a homodimeric class C G protein-coupled receptor (GPCR) that senses extracellular Ca2+ (Ca2+ o ) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein-dependent signaling via twin Cysteine-rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR-GABAB1 and CaSR-GABAB2 chimeras subject to GABAB -dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK-293 cells were assessed for Ca2+ o -stimulated Ca2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop-2 or intraloop-3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild-type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type-1 (FHH-1), supported maximal signaling with reduced Ca2+ o potency. Thus two WT VFT domains were required for normal Ca2+ o potency and there was a pronounced gene-dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene-dosage effect. Finally, we observed that the Ca2+ o -stimulated CaSR operated exclusively via signaling in-trans and not via combined in-trans and in-cis signaling. We consider how receptor asymmetry may support the underlying mechanisms. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Update on Canterbury Charity Hospital Trust activities 2013 to end of 2020: adapting to changing unmet secondary elective healthcare need.

AIM: To update activities of the Canterbury Charity Hospital (CCH) and its Trust over the eight-year period 2013 to end of 2020, following previous reports in 2010 and 2013. METHOD: CCH continued to provide free secondary elective healthcare services to some patients in the Canterbury Distinct Health Board (CDHB) region who were unable to access healthcare they needed through public hospitals and were unable to pay for private care. CCH's services were supplied by a large volunteer workforce, supported by a skeleton staff, and were financed solely by charitable giving. Changes occurred periodically in the quantity and nature of regional unmet healthcare need, largely due to changes in services provided by the CDHB. In order to accommodate these changes, major structural and infrastructural developments were necessitated at CCH. RESULTS: Many healthcare services at CCH remained the same as before this period but new changes occurred there as a result of: (i) establishment of a flexible sigmoidoscopy day clinic for the management of fresh rectal bleeding in those under 50 years of age; (ii) requirement for a sudden increase in counselling services immediately after the terror attacks at Christchurch mosques; (iii) expansion of the Dental and Oral Surgery Service; and (iv) interruption of CCH service provision by the COVID-19 pandemic. CONCLUSIONS: CCH continued to fill some of the regional unmet elective healthcare need. This is, however, a national problem as attested by the presence of a charity hospital in Auckland and another being planned for Invercargill. Hopefully present and future governments will appreciate that free universal access to secondary elective healthcare is not only a humane imperative, but also a sound economic investment.

Is Piperacillin-Tazobactam an Appropriate Empirical Agent for Hospital-Acquired Sepsis and Community-Acquired Septic Shock of Unknown Origin in Australia?

Early appropriate empirical antibiotics are critical for reducing mortality in sepsis. For hospital-acquired sepsis of unknown origin in Australia, piperacillin-tazobactam (TZP) is recommended as an empirical therapy. Anecdotally, some institutions also use TZP for community-acquired septic shock. This narrative review aimed to scrutinise the appropriateness of TZP as an empirical agent for undifferentiated hospital-acquired sepsis and community-acquired septic shock. An online database (Medline) was searched for relevant studies in adults published in the last 10 years. Studies were included if they addressed separately reported clinical outcomes related to a relevant aspect of TZP therapy in sepsis. Of 290 search results, no studies directly addressed the study aim. This review therefore explores several themes that emerged from the contemporary literature, all of which must be considered to fully interrogate the appropriateness of TZP use in this context. This review reveals the paucity and low quality of evidence available for TZP use in sepsis of unclear origin, while demonstrating the urgent need and equipoise for an Australian audit of TZP use in patients with sepsis of unknown origin.

Complicated placenta accreta spectrum: identifying a high-risk cohort.

OBJECTIVE: To assess differences in the perioperative complication rate between patients with placenta accreta spectrum (PAS) with and without complicating factors. METHODS: This retrospective cohort study included subjects who underwent cesarean hysterectomy with histology-proven PAS between 23 0/7 and 42 0/7 weeks gestational age (GA) from 1 July 2008 to 11 April 2017. Perioperative outcomes were compared between those with uncomplicated PAS and "complicated PAS," defined as PAS subjects who experienced ≥2 bleeding episodes, preterm premature rupture of membranes (PPROM), or premature contractions requiring tocolysis. RESULTS: Overall, 26 complicated PAS and 27 uncomplicated PAS cases were compared; no difference in the rate of perioperative complications was identified. An increased proportion of complicated PAS cases required blood product transfusion before delivery: 2 (40%), 3 (27.3%), and 2 patients (20%) for those with PPROM, preterm contractions, and ≥2 bleeding episodes respectively, compared to patients with uncomplicated PAS, having no transfusions (p = .001). Time of delivery was earlier for patients with complicated compared to uncomplicated PAS (median GA 30.9 [Q1 = 27.9; Q3 = 31.9] and 34.9 [Q1 = 32.1; Q3 = 35.7], p < .001). Median birthweights were lower (p < .0144) and maternal length of stay longer (p < .0012) for complicated PAS. CONCLUSION: Patients with complicated PAS were not at higher risk for perioperative complications but were associated with earlier delivery, required more antenatal blood transfusions, and had a longer LOS.

Neonatal hypoxic-ischaemic encephalopathy: Motor impairment beyond cerebral palsy.

BACKGROUND: Research investigating neuromotor function in the absence of cerebral palsy (CP) for children who had neonatal HIE is limited. AIMS: To investigate school-age neurological and neuromotor function, and correlations with attention, neonatal Magnetic Resonance Imaging (MRI), and neuromotor assessments at toddler age. METHODS: Twenty-seven children with neonatal HIE without CP who underwent hypothermia treatment and a comparison group of 20 children were assessed at age 5-7 years for Minor Neurological Dysfunction (MND; simplified Touwen), motor skills (Movement Assessment Battery for Children-2; MABC-2), parental concern over motor function (MABC Checklist), general cognition (Wechsler Preschool and Primary Scale of Intelligence-IV, WPPSI), and attention (DuPaul ADHD Rating Scale). Neurological examination and motor development, using Bayley-3 scales, at age 24-months was extracted from the clinical database. Clinical neonatal MRI was assessed for hypoxic-ischaemic injury. RESULTS: In the HIE group, MND was more prevalent (p = 0.026) and M-ABC performance (total score p = 0.006; balance subtest p = 0.008) was worse; parents were more concerned about children's motor function (p = 0.011). HIE group inattention scores were higher (p = 0.032), which correlated with lower MABC-2 scores (rs = -0.590, p = 0.004). Neurological examination at 24-months correlated with MND (rs = 0.437, p = 0.033); Bayley-3 motor scores did not correlate with M-ABC-2 scores (rs = 368, p = 0.133). Neonatal MRI findings were not associated with school-age MND (rs = 0.140, p = 0.523) or MABC-2 (rs = 0.300, p = 0.165). CONCLUSIONS: Children with neonatal HIE, without CP, treated with hypothermia may be more likely to develop MND and motor difficulties than typically developing peers. Inattention may contribute to motor performance. In the absence of CP, neonatal MRI and toddler age assessment of motor development have limited predictive value for school-age outcome. Since this was an exploratory study with a small sample size, findings should be confirmed by a definite larger study.

Cardiovascular Disease Screening in Pregnancy.

Cardiovascular disease (CVD) has surpassed the traditional causes of pregnancy-related mortality, including hemorrhage and thromboembolism in the United States. CVD accounts for ~15.5% of all pregnancy-related deaths. Pregnancy is a "natural cardiovascular stress test" for a woman. The physiological changes in the maternal hemodynamics that are geared to accommodate the growing needs of the fetal-placental unit may also lead to symptoms that are indistinguishable from those of CVD, especially in the third trimester of pregnancy. It is imperative that an obstetric provider is able to differentiate symptoms of normal pregnancy from those of a pathologic process.

Fine-scale seascape genomics of an exploited marine species, the common cockle Cerastoderma edule, using a multimodelling approach.

Population dynamics of marine species that are sessile as adults are driven by oceanographic dispersal of larvae from spawning to nursery grounds. This is mediated by life-history traits such as the timing and frequency of spawning, larval behaviour and duration, and settlement success. Here, we use 1725 single nucleotide polymorphisms (SNPs) to study the fine-scale spatial genetic structure in the commercially important cockle species Cerastoderma edule and compare it to environmental variables and current-mediated larval dispersal within a modelling framework. Hydrodynamic modelling employing the NEMO Atlantic Margin Model (AMM15) was used to simulate larval transport and estimate connectivity between populations during spawning months (April-September), factoring in larval duration and interannual variability of ocean currents. Results at neutral loci reveal the existence of three separate genetic clusters (mean F ST = 0.021) within a relatively fine spatial scale in the north-west Atlantic. Environmental association analysis indicates that oceanographic currents and geographic proximity explain over 20% of the variance observed at neutral loci, while genetic variance (71%) at outlier loci was explained by sea surface temperature extremes. These results fill an important knowledge gap in the management of a commercially important and overexploited species, bringing us closer to understanding the role of larval dispersal in connecting populations at a fine geographic scale.

A multi-center evaluation of TECHNOSCREEN® ADAMTS-13 activity assay as a screening tool for detecting deficiency of ADAMTS-13.

BACKGROUND: Quantifying A disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS-13) activity enhances thrombotic thrombocytopenic purpura (TTP) diagnosis but most assays are time consuming, technically demanding, and mainly available in reference centers. OBJECTIVE: Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP. PATIENTS/METHODS: Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREEN® ADAMTS13 activity screening test in comparison with TECHNOZYM® ADAMTS-13 activity ELISA at two centers, and in-house fluorescence resonance energy transfer assay at the third center. The screening test indicates if ADAMTS-13 activity is at one of four level-indicator points: 0, 0.1, 0.4, or 0.8 IU/mL. RESULTS: Screen results were interpreted as binary data in that ADAMTS-13 activity was above or below the 0.1 IU/mL TTP clinical threshold. Combining all sites' data, the screen exhibited 88.7% sensitivity, 90.4% specificity, 74.6% positive predictive value, and 96.2% negative predictive value, comparable to published data for quantitative assays. Five samples with quantitative results below the threshold gave screen readings of 0.1 IU/mL and seven marginally above the threshold gave screen readings of zero. All would warrant plasma exchange while the level is quantified. Nine samples with normal/near normal results gave screens of zero and confirmatory quantifications would prompt early treatment withdrawal, as is current practice. One sample generated screen/quantitative results of 0.4/0.00 IU/mL respectively and was the only clear false-negative. CONCLUSIONS: The screening test provides more rapid ADAMTS-13 level evaluation than most currently available assays. Its simple operation renders it suitable for adoption in routine or specialist laboratory environments.

Bounce Forward: A School-Based Prevention Programme for Building Resilience in a Socioeconomically Disadvantaged Context.

Socioeconomic status is a strong predictor of normative development and well-being in young people. It is well-known that growing up in a socioeconomically disadvantaged context may lead to negative outcomes, both in childhood and in adulthood. Early intervention and prevention programmes are crucial for building resilience and improving health, well-being and equity. Bounce Forward is a school-based prevention programme implemented in Blackpool, a town in the United Kingdom facing multiple challenges. It was part of a whole town resilience approach and nascent global social movement known as the "Resilience Revolution." Between 2017 and 2019, the programme was delivered in all Year 5 classes at every primary school in Blackpool (n school = 36), reaching out to 3,134 students (ages 9-10; 50.4% male). The programme aimed to increase resilience in young people by building knowledge and skills about mental health and resilience through 10 sessions. In the current study, we longitudinally examined a range of protective factors, which are relevant to young people's resilience, as well as their mental health outcomes at three time points: before they participated in Bounce Forward, at the end of the programme, and 3-5 months later, when they started Year 6. The current sample included 441 Year 5 students (54.2% male) from 11 primary schools in Blackpool. Nineteen teaching staff also participated in the study and provided qualitative data regarding the impact of the programme on their students. Results showed improvement in some areas of young people's resilience after taking part in Bounce Forward. We also identified gender differences in several protective factors, indicating that boys may need further support. Teaching staff highlighted improvements in various areas; and also observed that their students have been using the strategies that they learnt from the programme. Altogether, findings suggested that young people benefitted from Bounce Forward. The programme is sustainable, offering a free to download teacher resource pack that allows schools to self-deliver it.

Expression of human CD46 and trans-complementation by murine adenovirus 1 fails to allow productive infection by a group B oncolytic adenovirus in murine cancer cells.

BACKGROUND: Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS: Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS: Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION: Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.

Emergency general surgery 'Hot Clinics' reduce admission rates and duration of inpatient stay.

OBJECTIVE: To determine the impact of a 'Hot Clinic' (HC) on emergency general surgery patient flow-through. DESIGN: Prospective service evaluation study. SETTING: HC is a four-bedded area coordinated by a specialist nurse. The HC consultant sees emergency patients referred from the emergency department, general practitioners or those in preceding 24 h considered suitable for interim discharge while awaiting investigations and HC reassessment. PATIENTS: All patients with acute abdominal pain were evaluated in three 4 week groups: before (group 1), 1 month (group 2) and 6 months after the HC was introduced (group 3). Interhospital transfers, intrahospital ward referrals and trauma patients were excluded. INTERVENTION: Introduction of consultant-led surgical HC every weekday afternoon. MAIN OUTCOME MEASURES: Proportion of patients admitted under general surgeons, length of inpatient stay and the proportion of patients referred again within 3 months were investigated. RESULTS: 1409 patients were referred, of which 1061 met the inclusion criteria: 307 in group 1, 326 in group 2 and 428 in group 3. There was no difference in gender distribution (p=0.759). Inpatient admissions were significantly reduced (85.0% vs 78.2% vs 54.4%; p<0.001) and the inpatient duration of stay was significantly shorter after HC introduction (median (IQR) (95% CI) 63.8 (29.0-111.6) (51.8 to 72.8) hours vs 48.8 (21.7-101.2) (42.0 to 55.6) hours vs 47.7 (20.9-92.7) (42.8 to 56.9) hours; p=0.011). CONCLUSIONS: Emergency general surgery HCs are associated with significant reductions in admission rates and inpatient bed occupancy. This service redesign has the potential to dramatically relieve pressure on acute surgical services.

Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

Internal validation of the DNAscan/ANDE™ Rapid DNA Analysis™ platform and its associated PowerPlex® 16 high content DNA biochip cassette for use as an expert system with reference buccal swabs.

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles.

Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators.

Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.

Structural mechanism of ligand activation in human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

Past Human Disturbance Effects upon Biodiversity are Greatest in the Canopy; A Case Study on Rainforest Butterflies.

A key part of tropical forest spatial complexity is the vertical stratification of biodiversity, with widely differing communities found in higher rainforest strata compared to terrestrial levels. Despite this, our understanding of how human disturbance may differentially affect biodiversity across vertical strata of tropical forests has been slow to develop. For the first time, how the patterns of current biodiversity vary between three vertical strata within a single forest, subject to three different types of historic anthropogenic disturbance, was directly assessed. In total, 229 species of butterfly were detected, with a total of 5219 individual records. Butterfly species richness, species diversity, abundance and community evenness differed markedly between vertical strata. We show for the first time, for any group of rainforest biodiversity, that different vertical strata within the same rainforest, responded differently in areas with different historic human disturbance. Differences were most notable within the canopy. Regenerating forest following complete clearance had 47% lower canopy species richness than regenerating forest that was once selectively logged, while the reduction in the mid-storey was 33% and at ground level, 30%. These results also show for the first time that even long term regeneration (over the course of 30 years) may be insufficient to erase differences in biodiversity linked to different types of human disturbance. We argue, along with other studies, that ignoring the potential for more pronounced effects of disturbance on canopy fauna, could lead to the underestimation of the effects of habitat disturbance on biodiversity, and thus the overestimation of the conservation value of regenerating forests more generally.