The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

"Breath holding" as a thermoregulation strategy in the deep-diving scalloped hammerhead shark.

Fish moving between different thermal environments experience heat exchange via conduction through the body wall and convection from blood flow across the gills. We report a strategy of preventing convective heat loss at the gills during excursions into deep, cold water by the tropical scalloped hammerhead shark (Sphryna lewini). Adult scalloped hammerhead sharks dive rapidly and repeatedly from warm (~26°C) surface waters to depths exceeding 800 meters with temperatures as low as 5°C. Biologgers attached to adult sharks show that warm muscle temperatures were maintained throughout the deepest portion of each dive. Substantive cooling only occurred during the latter stages of the ascent phase and, once initiated, was rapid. Heat transfer coefficient modeling indicated that convective heat transfer was suspended, probably by suppressing gill function during deep dives. This previously unobserved strategy has broad similarities to marine mammal "breath hold" diving.

Operant conditioning as a tool to assess hearing abilities in sharks.

Sharks (elasmobranchs) are an ancient, diverse group of fishes, representing a basal stage in the evolution of vertebrate hearing. Yet, our understanding of behavioural measures of hearing abilities in sharks is limited. To address this, an operant conditioning paradigm was designed, and scalloped hammerhead Sphyrna lewini and rig (spotted estuary smooth hound) Mustelus lenticulatus were successfully trained to respond to pure-tone acoustic stimuli from an underwater speaker. After 2-3 weeks of training, both species showed distinctive responses to these acoustic stimuli and retained this behaviour when reinforced. S. lewini responded to a 400 Hz pulsed tone with an abrupt increase in tailbeat frequency (97 beats per 30 s vs. 69 beats for a 2 kHz control and 70 beats for no signal) and sustained vigorous swimming (arousal response) for at least 30 s. In response to a 200 Hz pulsed tone, M. lenticulatus visited a target area under the speaker significantly more frequently (13.4 ± 4.3 times per minute vs. 1.4 ± 1.5 times for a 1.2 kHz control and 0.9 ± 0.01 times for no signal) and swam circles under the speaker to search for food. The authors used S. lewini arousal responses to pure-tone stimuli of 40, 80, 200, 400, 600 and 800 Hz to generate a provisional hearing-threshold curve. The results show that S. lewini adapts to low-frequency hearing (greatest sensitivity at 200 Hz, upper limit 800 Hz), which is like other coastal pelagic sharks that have been investigated so far. Despite challenges operant acoustic conditioning studies are a viable method for revealing auditory capabilities of sharks.

Provisioning ecotourism does not increase tiger shark site fidelity.

A perennial criticism of provisioning ecotourism is that it alters the natural behavior and ecology of the target species by providing an artificial food source. Here we evaluate its impact on the long-term site fidelity patterns of tiger sharks in French Polynesia. We hypothesized that a significant impact of provisioning would lead to (1) increases in individual site fidelity over time, and (2) an increase in the number of resident individuals over time. Of 53 individuals photo-identified and monitored during > 500 dives over five years, 10 individuals accounted for > 75% of all sightings, whereas 35 sharks were sighted very infrequently. Even the most frequently observed tiger sharks exhibited overall low fidelity at the site and showed no increase in site fidelity over time. Furthermore, the number of tiger sharks sighted during each dive did not increase. The observed patterns of tiger shark sightings were best explained by natural movements, including general roaming within home ranges along the coastline and seasonal migrations. Despite the apparent lack of impact of provisioning ecotourism on tiger shark ecology in Tahitian waters, it would be prudent to implement a strict code of conduct during any future provisioning activities to maximize the safety of participants and animals involved.

First tiger shark Galeocerdo cuvier bite in 75 years in French Polynesia (Eastern Central Pacific).

We analyzed a tiger shark (estimated 2.8 m total length) bite on a snorkeler. The removal of the terminal part of the leg suggests a predatory motivation for the bite. This is the first documented bite by a tiger shark in French Polynesia waters for the past 75 years.

Suspected predatory bites on a snorkeler by an oceanic whitetip shark Carcharhinus longimanus off Moorea island (French Polynesia).

Understanding why sharks bite humans is essential for developing strategies to prevent these incidents. Here, we use bite wound characteristics and eye witness descriptions of shark behavior to determine the likely motivation for several bites perpetrated by an oceanic whitetip (OWT) shark Carcharhinus longimanus on an adult female snorkeler off Moorea island (French Polynesia) in October 2019. The victim was snorkeling with others in pelagic waters as part of an organized whale-watching tour when the shark-without any warning behavior-bit her at least three times resulting in severe injuries with substantial loss of soft tissue from the chest and both forearms. The victim survived these injuries thanks to rapid and effective first aid provided by her companions. The sudden, unprovoked and repeated bites with substantial tissue removal are consistent with predatory behavior although the dominance hypothesis cannot be fully ruled out. This would be the first case of a predatory shark bite ever documented in French Polynesia in over 70 years of data collection. Given the routine association of OWT sharks with cetaceans, in-water whale watching activities should adopt appropriate risk management strategies in regions hosting this species of shark.

Development and successful real-world use of a transfer DNA technique to identify species involved in shark bite incidents.

Identifying the species involved in shark bite incidents is an ongoing challenge but is important to mitigate risk. We developed a sampling protocol to identify shark species from DNA transferred to inanimate objects during bite incidents. To develop and refine the technique, we swabbed shark bite impressions on surfboards and wetsuit neoprene collected under semicontrolled conditions. Methods were tested experimentally and then successfully used to identify the species involved in a real-world shark bite incident. Thirty-two of 33 bite impressions yielded sufficient DNA sequences for species identification, producing barcodes from five test species, including dusky, Galapagos, bull, tiger, and white shark. The latter three species collectively account for a majority of shark bites worldwide. Our method successfully identified the species (Galeocerdo cuvier) responsible for a fatal shark bite on December 8th, 2020 on the island of Maui, from swab samples collected from the victim's surfboard 49 h after the bite incident. Our experimental results demonstrate that shark species can be accurately identified from transfer DNA recovered from bite impressions on surfboards and wetsuit neoprene. The successful use of our method in the real-world incident shows great potential for the practicality of this tool. We recommend DNA swabbing as a routine part of the forensic analysis of shark bites to help identify the species involved in human-shark interactions.

Similar circling movements observed across marine megafauna taxa.

Advances in biologging technology have enabled 3D dead-reckoning reconstruction of marine animal movements at spatiotemporal scales of meters and seconds. Examining high-resolution 3D movements of sharks (Galeocerdo cuvier, N = 4; Rhincodon typus, N = 1), sea turtles (Chelonia mydas, N = 3), penguins (Aptenodytes patagonicus, N = 6), and marine mammals (Arctocephalus gazella, N = 4; Ziphius cavirostris, N = 1), we report the discovery of circling events where animals consecutively circled more than twice at relatively constant angular speeds. Similar circling behaviors were observed across a wide variety of marine megafauna, suggesting these behaviors might serve several similar purposes across taxa including foraging, social interactions, and navigation.

Erratum to "World librarians: A socio-technical system providing library search services to offline schools and libraries in Malawi" [World Dev. Perspect. (1-9) (2020) 100234].

[This corrects the article DOI: 10.1016/j.wdp.2020.100234.].

World librarians: A socio-technical system providing library search services to offline schools and libraries in Malawi.

World Librarians is a socio-technical system that strives to solve the information access problem many remote offline schools and libraries have in lesser developed contexts. In this Case Report, we describe the system we have developed over the course of three years, where we first establish solar-powered computer labs in remote schools and libraries in Malawi, and then provide them digital information that they want, rather than what we in the Global North think they need. After providing background on these issues, we describe the socio-technical underpinnings and workflow in the World Librarians program. This involves the establishment of "Requester nodes" in the offline schools and libraries, the management of these deployments using a cloud-based WL app, and the operations of the WL "Searcher node" at the University of Massachusetts Amherst, as well as a novel micro-payment system that enables the transfer of large digital datasets through the use of teacher or librarian cell phones and data plans. We close the report with findings from a preliminary survey suggesting that the WL program is making a positive impact on the schools and libraries served. At its core, WL represents a global librarian support system working to remove the barriers to educational information for all global citizens, with central attention and awareness to Global North/South information power dynamics.

Diel patterns in swimming behavior of a vertically migrating deepwater shark, the bluntnose sixgill (Hexanchus griseus).

Diel vertical migration is a widespread behavioral phenomenon where organisms migrate through the water column and may modify behavior relative to changing environmental conditions based on physiological tolerances. Here, we combined a novel suite of biologging technologies to examine the thermal physiology (intramuscular temperature), fine-scale swimming behavior and activity (overall dynamic body acceleration as a proxy for energy expenditure) of bluntnose sixgill sharks (Hexanchus griseus) in response to environmental changes (depth, water temperature, dissolved oxygen) experienced during diel vertical migrations. In the subtropical waters off Hawai'i, sixgill sharks undertook pronounced diel vertical migrations and spent considerable amounts of time in cold (5-7°C), low oxygen conditions (10-25% saturation) during their deeper daytime distribution. Further, sixgill sharks spent the majority of their deeper daytime distribution with intramuscular temperatures warmer than ambient water temperatures, thereby providing them with a significant thermal advantage over non-vertically migrating and smaller-sized prey. Sixgill sharks exhibited relatively high rates of activity during both shallow (night) and deep (day) phases and contrary to our predictions, did not reduce activity levels during their deeper daytime distribution while experiencing low temperature and dissolved oxygen levels. This demonstrates an ability to tolerate the low oxygen conditions occurring within the local oxygen minimum zone. The novel combination of biologging technologies used here enabled innovative in situ deep-sea natural experiments and provided significant insight into the behavioral and physiological ecology of an ecologically important deepwater species.

Excised Femoral Heads in Hip Fracture Patients: Is Osteoporosis Worse Than Cancer?

Introduction Annually 80,000 hip fractures are treated at an estimated cost of two billion pounds. The 2011 guidance from the Royal College of Pathologists recommended all specimens where there is fracture through or below the articular surface should be examined to exclude/ identify an underlying cause (pathological fracture). The questions posed in this study are three-fold. Firstly, how does our practice for hip fracture patients comply with the above audit standards? Secondly, what is the prognostic significance of a past medical history of malignancy on survival? Thirdly, is there any other prognostic survival difference attributable to the diagnosis concluded from the histological analysis of the excised femoral head specimens? Methods A retrospective analysis of all hip fractures receiving joint arthroplasty was undertaken between January 2011 and March 2014. Mortality was recorded for a minimum follow-up of 30 months post-operatively. Each excised femoral head was histologically examined by a single consultant histopathologist, and all pre-operative X-rays were reviewed by a consultant radiologist. Histological diagnoses were recorded, and statistical analysis including Kaplan-Meier survival was performed. Results A total of 327 consecutive fractures were identified. Out of 187 specimens sent for analysis, only two revealed metastatic deposits in patients with known disseminated malignancy. A previous medical history of malignancy did not confer a significant increase in mortality over a five-year postoperative period (p = 0.42). A histological diagnosis of osteoporosis significantly increased mortality over a five-year postoperative period (p = 0.004). A comparative analysis found that patients with a histological diagnosis of osteoporosis had the poorest survival. Conclusion A histological femoral head analysis may diagnose previously undiagnosed osteoporosis, allowing the clinician to intervene in a disease process, which if left untreated, can lead to a significant increase in mortality.

Habitat geography around Hawaii's oceanic islands influences tiger shark (Galeocerdo cuvier) spatial behaviour and shark bite risk at ocean recreation sites.

We compared tiger shark (Galeocerdo cuvier) spatial behaviour among 4 Hawaiian Islands to evaluate whether local patterns of movement could explain higher numbers of shark bites seen around Maui than other islands. Our sample consisted of 96 electronically-tagged (satellite and acoustic transmitters) tiger sharks, individually tracked for up to 6 years. Most individuals showed fidelity to a specific 'home' island, but also swam between islands and sometimes ranged far (up to 1,400 km) offshore. Movements were primarily oriented to insular shelf habitat (0-200 m depth) in coastal waters, and individual sharks utilized core-structured home ranges within this habitat. Core utilization areas of large tiger sharks were closer to high-use ocean recreation sites around Maui, than around Oahu. Tiger sharks routinely visited shallow ocean recreation sites around Maui and were detected on more days overall at ocean recreation sites around Maui (62-80%) than Oahu (<6%). Overall, our results suggest the extensive insular shelf surrounding Maui supports a fairly resident population of tiger sharks and also attracts visiting tiger sharks from elsewhere in Hawaii. Collectively these natural, habitat-driven spatial patterns may in-part explain why Maui has historically had more shark bites than other Hawaiian Islands.

Combining abundance and performance data reveals how temperature regulates coastal occurrences and activity of a roaming apex predator.

The redistribution of species has emerged as one of the most pervasive impacts of anthropogenic climate warming, and presents many societal challenges. Understanding how temperature regulates species distributions is particularly important for mobile marine fauna such as sharks given their seemingly rapid responses to warming, and the socio-political implications of human encounters with some dangerous species. The predictability of species distributions can potentially be improved by accounting for temperature's influence on performance, an elusive relationship for most large animals. We combined multi-decadal catch data and bio-logging to show that coastal abundance and swimming performance of tiger sharks Galeocerdo cuvier are both highest at ~22°C, suggesting thermal constraints on performance may regulate this species' distribution. Tiger sharks are responsible for a large proportion of shark bites on humans, and a focus of controversial control measures in several countries. The combination of distribution and performance data moves towards a mechanistic understanding of tiger shark's thermal niche, and delivers a simple yet powerful indicator for predicting the location and timing of their occurrences throughout coastlines. For example, tiger sharks are mostly caught at Australia's popular New South Wales beaches (i.e. near Sydney) in the warmest months, but our data suggest similar abundances will occur in winter and summer if annual sea surface temperatures increase by a further 1-2°C.

Strong trans-Pacific break and local conservation units in the Galapagos shark (Carcharhinus galapagensis) revealed by genome-wide cytonuclear markers.

The application of genome-wide cytonuclear molecular data to identify management and adaptive units at various spatio-temporal levels is particularly important for overharvested large predatory organisms, often characterized by smaller, localized populations. Despite being "near threatened", current understanding of habitat use and population structure of Carcharhinus galapagensis is limited to specific areas within its distribution. We evaluated population structure and connectivity across the Pacific Ocean using genome-wide single-nucleotide polymorphisms (~7200 SNPs) and mitochondrial control region sequences (945 bp) for 229 individuals. Neutral SNPs defined at least two genetically discrete geographic groups: an East Tropical Pacific (Mexico, east and west Galapagos Islands), and another central-west Pacific (Lord Howe Island, Middleton Reef, Norfolk Island, Elizabeth Reef, Kermadec, Hawaii and Southern Africa). More fine-grade population structure was suggested using outlier SNPs: west Pacific, Hawaii, Mexico, and Galapagos. Consistently, mtDNA pairwise ΦST defined three regional stocks: east, central and west Pacific. Compared to neutral SNPs (FST = 0.023-0.035), mtDNA exhibited more divergence (ΦST = 0.258-0.539) and high overall genetic diversity (h = 0.794 ± 0.014; π = 0.004 ± 0.000), consistent with the longstanding eastern Pacific barrier between the east and central-west Pacific. Hawaiian and Southern African populations group within the west Pacific cluster. Effective population sizes were moderate/high for east/west populations (738 and 3421, respectively). Insights into the biology, connectivity, genetic diversity, and population demographics informs for improved conservation of this species, by delineating three to four conservation units across their Pacific distribution. Implementing such conservation management may be challenging, but is necessary to achieve long-term population resilience at basin and regional scales.

Emission Factors for Selected Semivolatile Organic Chemicals from Burning of Tropical Biomass Fuels and Estimation of Annual Australian Emissions.

This study reveals that open-field biomass burning can be an important source of various semivolatile organic chemicals (SVOCs) to the atmosphere including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and a range of pesticides. Emission factors (EFs) for 39 individual SVOCs are determined from burning of various fuel types that are common in tropical Australia. Emissions of PAHs are found to be sensitive to differences in combustion efficiencies rather than fuel types, reflecting a de novo formation mechanism. In contrast, revolatilization may be important for other SVOCs such as PCBs. On the basis of the EFs determined in this work, estimates of the annual emissions of these SVOCs from Australian bushfires/wildfires are achieved, including, for example, ∑PAHs (160 (min)-1100 (max) Mg), ∑PCBs (14-300 kg), ∑PBDEs (8.8-590 kg), α-endosulfan (6.5-200 kg), and chlorpyrifos (up to 1400 kg), as well as dioxin toxic equivalents (TEQs) of ∑dioxin-like-PCBs (0.018-1.4 g). Emissions of SVOCs that are predominantly revolatilized appear to be related to their use history, with higher emissions estimated for chemicals that had a greater historical usage and were banned only recently or are still in use.

Coral reef grazer-benthos dynamics complicated by invasive algae in a small marine reserve.

Blooms of alien invasive marine algae have become common, greatly altering the health and stability of nearshore marine ecosystems. Concurrently, herbivorous fishes have been severely overfished in many locations worldwide, contributing to increases in macroalgal cover. We used a multi-pronged, interdisciplinary approach to test if higher biomass of herbivorous fishes inside a no-take marine reserve makes this area more resistant to invasive algal overgrowth. Over a two year time period, we (1) compared fish biomass and algal cover between two fished and one unfished patch reef in Hawai'i, (2) used acoustic telemetry to determine fidelity of herbivorous fishes to the unfished reef, and (3) used metabarcoding and next-generation sequencing to determine diet composition of herbivorous fishes. Herbivore fish biomass was significantly higher in the marine reserve compared to adjacent fished reefs, whereas invasive algal cover differed by species. Herbivorous fish movements were largely confined to the unfished patch reef where they were captured. Diet analysis indicated that the consumption of invasive algae varied among fish species, with a high prevalence of comparatively rare native algal species. Together these findings demonstrate that the contribution of herbivores to coral reef resilience, via resistance to invasive algae invasion, is complex and species-specific.

Functional Outcome and Complications at 2.5 Years Following Volar Locking Plate Fixation of Distal Radius Fractures.

Distal radius fractures are increasingly treated by internal fixation, but there have been relatively few studies relating to functional outcome at 12 months or more. The aim of this study was to ascertain the patient reported function of the wrist at a minimum of 12 months following fixation of a distal radius fracture, the time taken to return to work, and the complication rate. We conducted a retrospective review of 187 consecutive patients treated by a specialist hand and wrist trauma team at a tertiary referral unit over a 5 year period. Mean age was 57.3 years (range 16-93). Median time to surgery was 4 days (interquartile range 2-9). Median follow up was 31 months (interquartile range 23-41 months). The median PRWE score was 3; (range 0-83). There was no difference in outcome in patients who had surgery delayed by greater than 2 weeks (p > 0.05). The median time to return to work was 5 weeks (interquartile range 1-8 weeks). There were 15 complications (8 %) including 3 tendon injuries. We have demonstrated an early return to work in patients who were employed, a low complication rate, and highly favourable functional outcomes at a mean of 30 months postoperatively. We recommend the use of the DVR plate and involvement of a dedicated hand and wrist trauma team for treatment of unstable fractures of the distal radius.

Unexpected Positive Buoyancy in Deep Sea Sharks, Hexanchus griseus, and a Echinorhinus cookei.

We do not expect non air-breathing aquatic animals to exhibit positive buoyancy. Sharks, for example, rely on oil-filled livers instead of gas-filled swim bladders to increase their buoyancy, but are nonetheless ubiquitously regarded as either negatively or neutrally buoyant. Deep-sea sharks have particularly large, oil-filled livers, and are believed to be neutrally buoyant in their natural habitat, but this has never been confirmed. To empirically determine the buoyancy status of two species of deep-sea sharks (bluntnose sixgill sharks, Hexanchus griseus, and a prickly shark, Echinorhinus cookei) in their natural habitat, we used accelerometer-magnetometer data loggers to measure their swimming performance. Both species of deep-sea sharks showed similar diel vertical migrations: they swam at depths of 200-300 m at night and deeper than 500 m during the day. Ambient water temperature was around 15°C at 200-300 m but below 7°C at depths greater than 500 m. During vertical movements, all deep-sea sharks showed higher swimming efforts during descent than ascent to maintain a given swimming speed, and were able to glide uphill for extended periods (several minutes), indicating that these deep-sea sharks are in fact positively buoyant in their natural habitats. This positive buoyancy may adaptive for stealthy hunting (i.e. upward gliding to surprise prey from underneath) or may facilitate evening upward migrations when muscle temperatures are coolest, and swimming most sluggish, after spending the day in deep, cold water. Positive buoyancy could potentially be widespread in fish conducting daily vertical migration in deep-sea habitats.

Impact of smoke from prescribed burning: Is it a public health concern?

UNLABELLED: Given the increase in wildfire intensity and frequency worldwide, prescribed burning is becoming a more common and widespread practice. Prescribed burning is a fire management tool used to reduce fuel loads for wildfire suppression purposes and occurs on an annual basis in many parts of the world. Smoke from prescribed burning can have a substantial impact on air quality and the environment. Prescribed burning is a significant source of fine particulate matter (PM2.5 aerodynamic diameter<2.5µm) and these particulates are found to be consistently elevated during smoke events. Due to their fine nature PM2.5 are particularly harmful to human health. Here we discuss the impact of prescribed burning on air quality particularly focussing on PM2.5. We have summarised available case studies from Australia including a recent study we conducted in regional Victoria, Australia during the prescribed burning season in 2013. The studies reported very high short-term (hourly) concentrations of PM2.5 during prescribed burning. Given the increase in PM2.5 concentrations during smoke events, there is a need to understand the influence of prescribed burning smoke exposure on human health. This is important especially since adverse health impacts have been observed during wildfire events when PM2.5 concentrations were similar to those observed during prescribed burning events. Robust research is required to quantify and determine health impacts from prescribed burning smoke exposure and derive evidence based interventions for managing the risk. IMPLICATIONS: Given the increase in PM2.5 concentrations during PB smoke events and its impact on the local air quality, the need to understand the influence of PB smoke exposure on human health is important. This knowledge will be important to inform policy and practice of the integrated, consistent, and adaptive approach to the appropriate planning and implementation of public health strategies during PB events. This will also have important implications for land management and public health organizations in developing evidence based objectives to minimize the risk of PB smoke exposure.