Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization.

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.

Whale shark rhodopsin adapted to deep-sea lifestyle by a substitution associated with human disease.

Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with secondarily expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter feeding, was previously shown to possess the 'blue-shifted' rhodopsin (RHO), which is a signature of deep-sea adaptation. In this study, our spectroscopy of recombinant whale shark RHO mutants revealed that this blue shift is caused dominantly by an unprecedented spectral tuning site 94. In humans, the mutation at the site causes congenital stationary night blindness (CSNB) by reducing the thermal stability of RHO. Similarly, the RHO of deep-diving whale shark has reduced thermal stability, which was experimentally shown to be achieved by site 178 and 94. RHOs having the natural substitution at site 94 are also found in some Antarctic fishes, suggesting that the blue shift by the substitution at the CSNB site associated with the reduction in thermal stability might be allowed in cold-water deep-sea habitats.

Effects of mitral valve repair on valvular geometry and hemodynamics in dogs with myxomatous mitral valve disease.

OBJECTIVE: To investigate the effects of mitral valve repair on the geometry of the mitral valve complex, hemodynamics, and cardiac function of dogs with myxomatous mitral valve disease (MMVD). STUDY DESIGN: Retrospective cohort study. SAMPLE POPULATION: Dogs (n = 77) with stage C MMVD undergoing mitral valve repair under cardiopulmonary bypass. METHODS: Mitral valve geometry and cardiac function were assessed using echocardiography preoperatively, 1 week postoperatively and 3 months postoperatively. RESULTS: The coaptation length (0 [0-0] vs. 7 [6-8.5] mm, p < .001) and forward stroke volume index (1.5 ± 0.4 vs. 2.3 ± 0.6 mL/kg, p < .001) were higher at 3-months postoperatively than preoperatively, whereas the vertebral heart score (12.3 ± 1.2 vs. 10.8 ± 0.8 V, p < .001), left atrial-to-aortic ratio (2.2 ± 0.7 vs. 1.2 ± 0.3, p < .001), peak velocity of early diastolic transmitral flow (144 ± 34 vs. 91 ± 18 cm/s, p < .001), and regurgitant volume index (11.3 [8.2-14.0] vs. 1.6 [0.95-2.35] mL/kg, p < .001) were lower. Postoperatively, mitral valve geometry was completely changed within 1 week, whereas changes in vertebral heart score lasted for 3 months. CONCLUSION: Mitral valve repair changed valvular geometry and improved hemodynamics as assessed by follow-up echocardiography. CLINICAL SIGNIFICANCE: This study acts as reference for surgeons and cardiologists considering or evaluating the effects of mitral valve repair in dogs and provides useful data for the enhancement of relevant surgical techniques and the selection of relevant pre- and postoperative observations.

Ion Pairing of Cationic and Anionic Ir(III) Photosensitizers for Photocatalytic CO2 Reduction at Lipid-Membrane Surfaces.

We synthesized an ion pair comprising cationic and anionic Ir(III) photosensitizers ([Ir1+][Ir2-]) for photocatalytic CO2 reduction and showed that the cationic component imparts stability, while the cyclometalating ligands in the anionic component ensure effective visible-light absorption. The triplet excited state of [Ir1+] is the key photoredox species in this system and is mainly generated through the transfer of triplet excitation energy from the anionic moiety due to Coulombic interactions and appropriate triplet energy alignment between the two ionic components. The positive photosensitization effect of ion pairing was demonstrated by photocatalytic CO2 reduction in cooperation with a Re(I) molecular catalyst incorporated into a vesicle membrane.

Heterodonty and ontogenetic shift dynamics in the dentition of the tiger shark Galeocerdo cuvier (Chondrichthyes, Galeocerdidae).

The lifelong tooth replacement in elasmobranch fishes (sharks, rays and skates) has led to the assemblage of a great number of teeth from fossil and extant species, rendering tooth morphology an important character for taxonomic descriptions, analysing phylogenetic interrelationships and deciphering their evolutionary history (e.g. origination, divergence, extinction). Heterodonty (exhibition of different tooth morphologies) occurs in most elasmobranch species and has proven to be one of the main challenges for these analyses. Although numerous shark species are discovered and described every year, detailed descriptions of tooth morphologies and heterodonty patterns are lacking or are only insufficiently known for most species. Here, we use landmark-based 2D geometric morphometrics on teeth of the tiger shark Galeocerdo cuvier to analyse and describe dental heterodonties among four different ontogenetic stages ranging from embryo to adult. Our results reveal rather gradual and subtle ontogenetic shape changes, mostly characterized by increasing size and complexity of the teeth. We furthermore provide the first comprehensive description of embryonic dental morphologies in tiger sharks. Also, tooth shapes of tiger sharks in different ontogenetic stages are re-assessed and depicted in detail. Finally, multiple cases of tooth file reversal are described. This study, therefore, contributes to our knowledge of dental traits across ontogeny in the extant tiger shark G. cuvier and provides a baseline for further morphological and genetic studies on the dental variation in sharks. Therefore, it has the potential to assist elucidating the underlying developmental and evolutionary processes behind the vast dental diversity observed in elasmobranch fishes today and in deep time.

Regio- and stereoselective synthesis of tetrasubstituted (E)-(2-chloro-2-fluoro-1-iodovinyl)dimethylphenylsilane and its desilylative sonogashira coupling.

Trihalo-substituted vinylsilane was readily synthesized in 77% yield and in an excellent regio- and stereoselective manner from in situ-generated fluorosilylacetylene, followed by the addition of I2 and NCS. The silane was committed to sequential Sonogashira coupling with various terminal acetylenes under mild conditions, affording desilylated chlorofluoro-enyne adducts in moderate to good yields.

Long-term monitoring of egg-laying cycle using ultrasonography reveals the reproductive dynamics of circulating sex steroids in an oviparous catshark, Scyliorhinus torazame.

The many diverse reproductive strategies of elasmobranchs (sharks, skates and rays) from lecithotrophic oviparity to matrotrophic viviparity have attracted significant research attention. However, the endocrine control of elasmobranch reproduction is less well-documented largely due to their reproductive characteristics, such as a long reproductive cycle, and/or repeated internal fertilization using stored sperm in oviparous species. In the present study, for the first time, we succeeded in non-invasive monitoring of the continuing egg-laying cycle of the cloudy catshark Scyliorhinus torazame using portable ultrasound devices. Furthermore, long-term simultaneous monitoring of the egg-laying cycle and measurement of plasma sex steroids revealed cycling patterns of estradiol-17ß (E2), testosterone (T) and progesterone (P4). In particular, a decline in T followed by a reciprocal surge in plasma P4 were consistently observed prior to the appearance of the capsulated eggs, implying that P4 is likely associated with the ovulation and/or egg-case formation. While the cycling pattern of E2 was not as apparent as those of T and P4, threshold levels of E2 (>5 ng/mL) and T (>1 ng/mL) appeared to be crucial in the continuation of egg-laying cycle. The possibility to trace the dynamics of plasma sex steroids in a single individual throughout the reproductive cycles makes the catshark a useful model for regulatory and mechanistic studies of elasmobranch reproduction.

Determination of the triple oxygen isotopic composition of tropospheric ozone in terminal positions using a multistep nitrite-coated filter-pack system.

RATIONALE: The triple oxygen isotopic composition (Δ17 O) of tropospheric ozone (O3 ) is a useful tracer for identifying the source and is essential for clarifying the atmospheric chemistry of oxidants. However, the single nitrite-coated filter method is inaccurate owing to the nitrate blank produced through the reaction of nitrite and oxygen compounds other than O3 . METHODS: A multistep nitrite-coated filter-pack system is newly adopted to transfer the O-atoms in terminal positions of O3 to nitrite on each filter to determine the Δ17 O of O3 in terminal positions (denoted as Δ17 O(O3 )term ). The NO3 - produced by this reaction is chemically converted into N2 O, and continuous-flow isotope ratio mass spectrometry (CF-IRMS) is used to determine the oxygen isotopic compositions. RESULTS: The reciprocal of the NO3 - quantities on the nitrite-coated filters in each sample showed a strong linear relationship with Δ17 O of NO3 - . Using the linear relation, we corrected the changes in Δ17 O of NO3 - on the filters. We verified the accuracy of the new method through the measurement of artificial O3 with known Δ17 O(O3 )term value that had been determined from the changes in Δ17 O of O2 . The Δ17 O(O3 )term of tropospheric O3 was in agreement with previous studies. CONCLUSIONS: We accurately determined the δ18 O and Δ17 O values of tropospheric O3 by blank correction using our new method. Measurements of Δ17 O(O3 )term of the ambient troposphere showed 1.1 ± 0.7‰ diurnal variations between daytime (higher) and nighttime (lower) due likely to the formation of the temperature inversion layer at night.

Changes in sex steroid hormone levels reflect the reproductive status of captive female zebra sharks (Stegostoma fasciatum).

Captive breeding in aquaria is a useful means for ex situ preservation of threatened elasmobranch species. To promote captive breeding, it is important to determine the female reproductive status. However, information regarding reproductive status in female elasmobranchs is limited. Here, we used zebra sharks, Stegostoma fasciatum, as a model for elasmobranch reproduction in captivity. We investigated the relationships among changes in the sex steroid hormone levels, follicle size, and egg-laying period to develop indicators for the female reproductive status. We confirmed that mature female zebra sharks undergo an annual reproductive cycle. Additionally, we showed that the variations in sex steroid hormone levels correlated with reproductive status in mature female zebra sharks. Plasma estradiol-17ß (E2) concentrations increased two months before ovarian follicle development and decreased along with follicle regression. Interestingly, E2 levels were inversely correlated with water temperature (R = -0.901). Moreover, high levels of testosterone (T) correlated well with the laying period. These results strongly suggest that E2 is an indicator for ovarian follicle development, and that T is a useful indicator for both the onset and end of the egg-laying period in captive zebra sharks.

Source-Receptor Relationship Analysis of the Atmospheric Deposition of PAHs Subject to Long-Range Transport in Northeast Asia.

The source-receptor relationship analysis of PAH deposition in Northeast Asia was investigated using an Eulerian regional-scale aerosol chemical transport model. Dry deposition (DD) of PAH was controlled by wind flow patterns, whereas wet deposition (WD) depended on precipitation in addition to wind flow patterns. The contribution of WD was approximately 50-90% of the total deposition, except during winter in Northern China (NCHN) and Eastern Russia (ERUS) because of the low amount of precipitation. The amount of PAH deposition showed clear seasonal variation and was high in winter and low in summer in downwind (South Korea, Japan) and oceanic-receptor regions. In the downwind region, the contributions from NCHN (WD 28-52%; DD 54-55%) and Central China (CCHN) (WD 43-65%; DD 33-38%) were large in winter, whereas self-contributions (WD 20-51%; DD 79-81%) were relatively high in summer. In the oceanic-receptor region, the deposition amount decreased with distance from the Asian continent. The amount of DD was strongly influenced by emissions from neighboring domains. The contributions of WD from NCHN (16-20%) and CCHN (28-35%) were large. The large contributions from China in summer to the downwind region were linked to vertical transport of PAHs over the Asian continent associated with convection.

Combining multivariate statistics and the think-aloud protocol to assess Human-Computer Interaction barriers in symptom checkers.

Symptom checkers are software tools that allow users to submit a set of symptoms and receive advice related to them in the form of a diagnosis list, health information or triage. The heterogeneity of their potential users and the number of different components in their user interfaces can make testing with end-users unaffordable. We designed and executed a two-phase method to test the respiratory diseases module of the symptom checker Erdusyk. Phase I consisted of an online test with a large sample of users (n=53). In Phase I, users evaluated the system remotely and completed a questionnaire based on the Technology Acceptance Model. Principal Component Analysis was used to correlate each section of the interface with the questionnaire responses, thus identifying which areas of the user interface presented significant contributions to the technology acceptance. In the second phase, the think-aloud procedure was executed with a small number of samples (n=15), focusing on the areas with significant contributions to analyze the reasons for such contributions. Our method was used effectively to optimize the testing of symptom checker user interfaces. The method allowed kept the cost of testing at reasonable levels by restricting the use of the think-aloud procedure while still assuring a high amount of coverage. The main barriers detected in Erdusyk were related to problems understanding time repetition patterns, the selection of levels in scales to record intensities, navigation, the quantification of some symptom attributes, and the characteristics of the symptoms.

Gonadal Morphology, Histology, and Endocrinological Characteristics of Immature Female Whale Sharks, Rhincodon typus.

Captive breeding of whale sharks is one of the great challenges for aquariums. However, there is limited information available related to reproductive physiology due to the difficulty of sampling and long-term observation. In the present report, we provide information on the reproductive physiology of female whale sharks, which were incidentally captured as bycatch in a set-net off the coast of Okinawa, Japan. Total lengths of three captured female whale sharks were 403, 665, and 761 cm, respectively, at the time of their death. Collected paired ovaries differed in size between right and left. However, it seems not to determine which side of ovary becomes developed. Histological observations revealed that oocytes surrounded by follicle cell layers localized in the developed ovary, and most developed oocytes exhibited yolk vesicle stage. Additionally, in the largest specimen, there were low levels of three steroid hormones (Testosterone, Dihydrotestosterone, and Estradiol-17ß) that did not show seasonal variation. The present results indicate that even the whale shark over 7 m in TL are still histologically and endocrinologically immature. We expect that the present data will provide fundamental information related to reproductive physiology of female whale sharks, and will contribute to protection activities and increased success in captive breeding of whale sharks.

Fecal DNA analysis coupled with the sighting records re-expanded a known distribution of dugongs in Ryukyu Islands after half a century.

DNA analysis of large herbivore feces samples collected from seagrass beds at two distant sites (Irabu Island in Miyako Islands and Kushi in Okinawa Island) in the Ryukyu Islands proved that some of these feces were from dugongs, which had been treated in recent studies as extinct in this region since the last stranding of a deceased individual in 2019. In addition, local knowledge of sightings of animals thought to be dugongs and confirmed cases of dugong feeding trails since 2010 were compiled to estimate its recent distribution. This is the first scientific report on the presence of this mammal in the Ryukyu Islands within the last four years, and particularly in the Miyako Islands within the last half-century. As the Ryukyu Islands are known to be the northern limit of the dugong's fragmented distribution in East Asia, conservation efforts are therefore needed.

Propagation path of a flowering cherry (Cerasus × yedoensis) cultivar 'Somei-Yoshino' traced by somatic mutations.

In the long history of human relations with flowering cherry trees in Japan, 'Somei-Yoshino' occupies an exceptional position among a variety of flowering trees: it is a self-incompatible interspecific hybrid but has been enthusiastically planted by grafting throughout Japan, due most likely to its flamboyant appearance upon full bloom. Thus, 'Somei-Yoshino' gives us a rare opportunity to trace and investigate the occurrence and distribution of somatic mutations within a single plant species through analysis of the genomes of the clonally propagated trees grown under a variety of geographical and artificial environments. In the studies presented here, a total of 46 samples of 'Somei-Yoshino' trees were collected and their genomes were analyzed. We identified 684 single nucleotide mutations, of which 71 were present in more than two samples. Clustering analysis of the mutations indicated that the 46 samples were classified into eight groups, four of which included 36 of the 46 samples analyzed. Interestingly, all the four tree samples collected in Ueno Park of Tokyo were members of the four groups mentioned above. Based on comparative analysis of their mutations, one of the four trees growing in Ueno Park was concluded to be the closest to the original ancestor. We propose that somatic mutations may be used as tracers to establish the ancestral relationship amongst clonally propagated individuals.

Validity and reproducibility of food photographic estimation for evaluating meals in evacuation shelters.

Objective: The primary goal was to evaluate the validity of food photographic estimation for nutritional assessment compared with weighed food record (WFR). Methods: We evaluated the validity and reproducibility of photographic estimation of foods provided in evacuation shelters. We analyzed 35 meals served at 12 shelters in Kumamoto Prefecture in Japan, affected by a heavy rain disaster in 2020. In this context, we compared 21 senior students' portion size estimation by food photographs to WFR. In addition, we assigned five meals for each of the 21 senior students, and the same meal photograph was estimated by three students to test reproducibility. Results: No statistically significant difference was detected between the two methods regarding energy, the total grams of meal, the protein, and vitamins B1, B2, and C, except for salt. In addition, the students who never self-cooked underestimated the total grams. Conclusion: Food photographic estimation could simplify the nutritional assessment in evacuation shelters. However, unclear photographs and food items served by weight could weaken the estimation accuracy. According to previous studies and the applied postestimation questionnaire, photographs taken from specified angles and reference food photobooks for portion size estimation may improve accuracy.

Successful delivery of viviparous lantern shark from an artificial uterus and the self-production of lantern shark luciferin.

Our recent success in the long-term maintenance of lantern shark embryos in artificial uterine systems has provided a novel option for the medical treatment of premature embryos for captive viviparous elasmobranchs. The remaining issue with this system is that the embryos cannot survive the abrupt change in the chemical environment from artificial uterine fluid (AUF) to seawater during delivery. To overcome this issue, the present study developed a new protocol for seawater adaptation, which is characterized by a long-term and stepwise shift from AUF to seawater prior to delivery. This protocol was employed successfully, and the specimen survived for more than seven months after delivery, the longest captive record of the species. During the experiment, we unexpectedly detected bioluminescence of the embryonic lantern shark in the artificial uterus. This observation indicates that lantern sharks can produce luciferin, a substance for bioluminescence. This contradicts the recent hypothesis that lantern sharks lack the ability to produce luciferin and use luciferin obtained from food sources.

Usefulness of Simple Meal Screening Using Dietary Assessment Sheets for Evacuation Shelters.

To maintain the health of evacuees, meals provided at emergency shelters should be monitored and improvement measures should be taken where necessary. Time and human resources are limited during devastating disasters; thus, the authors have developed the Dietary Assessment Sheets for Evacuation Shelters. This study examines whether the quality of meals can be assessed using simple check items on the Dietary Assessment Sheets.A total of 28 Dietary Assessment Sheets for 98 meals provided 16-19 days after the heavy rains in Kumamoto in July 2020 at 12 shelters were used in the analysis. Dietitians working for shelters were requested to fill the Dietary Assessment Sheets and food record sheets provided by the Prefectural Government. If any subitem was checkmarked, it was considered "checked," and if left blank, it was considered "not checked" and divided into categorical groups. Based on the food record sheets and meal photos, contents of energy, protein, and vitamins B1, B2, and C were calculated for each meal. Energy and nutrient contents were compared between the categorical groups.The study revealed that nutritionally poor meals were characterized as those comprising only "Grain dishes," those that were not "Boxed meals," and those involving "Self-Defense Forces," "Dietitians," or "Others." Their energy, protein, and vitamin B1, B2, and C content was significantly lower than that of meals with marks on other checkboxes in the same category.Shelters that provide these meals should be given priority for nutrition assistance due to their poor diets. The results imply that the Dietary Assessment Sheets could serve as a simple tool to determine the shelters that require nutritional assistance.

Meal plans for meeting the reference values using food items available in shelters.

BACKGROUND: We have suggested "Revised Nutritional Reference Values for Feeding at Evacuation Shelters" (Revised RV) as a daily nutritional recommendation for meals served at evacuation shelters where poor diets had been reported. Since there are no meal examples to satisfy the Revised RV, our objectives were, for the future meal provision, to develop nutritionally adequate meal plans using the foods served at shelters in the past and to examine if the Revised RV could be met by changing combination of foods available. METHODS: In this case study using secondary data, we analyzed food weights of 86 meals served and recorded at 12 shelters after the heavy rains in July 2020. We obtained these data from Kumamoto Prefecture that was damaged and asked us dietary assessment for nutrition assistance. Foods were classified into 3 types according to the check mark in the record sheets: food aid (commercial packaged food), boxed meal, and hot meal service. We counted serving frequency of each food and analyzed nutritional differences by their combinations. Menus were devised by choosing foods that were served more frequently or were more nutritious among those served at shelters. The target values for one meal were set at 1/3 of the Revised RV for energy, protein, vitamins B1, B2, and C, and salt. RESULTS: None of the meals served in the shelters satisfied the target. We created 2 menus using food aid only: (#1 curry doughnut, milk with long shelf-life, and orange jelly) and (#2 salmon rice ball, ham and cheese sandwich, and vegetable juice); 1 menu by combination of boxed meal and food aid: (#3 boxed meal and vegetable juice); and 2 menus by combination of hot meal service and food aid: (#4 chicken meatball soup, packaged tofu, soy sauce, preprocessed white rice, and bottled green tea) and (#5 bamboo shoots rice, chicken and vegetable miso soup, and bottled green tea). Planned menus generally contained more energy, protein, and vitamins and less salt than the meals served. Their vitamin C contents were especially higher. CONCLUSION: Nutritionally adequate meals could be planned by changing the combination of foods available in shelters.

Dataset of motivational factors for using mobile health applications and systems.

We created and carried out a cross-sectional anonymous structured questionnaire on what motivates users of mobile health applications and wearables to share their collected health related data. The questionnaire was distributed online in English, French, and Norwegian. In addition, a flyer with information of where to locate the online questionnaire was distributed during a Swiss health conference. We used snowball sampling and encouraged participants to forward the questionnaires to friends, family, and others. Data were collected between October 2018 and March 2020. 58.1 % (n = 473) responded to the English survey, 34.3 % (n = 279) responded to the French survey, and 7.6 % (n = 62) responded to the Norwegian survey. The questionnaire contained 38 questions divided into seven themes: Background and health goals, Wearables and sensors, Mobile applications, Logging of health data, Data sharing- and integration, Social media and entertainment, and Demographics (age, gender, country of origin, chronic disease status, and chronic disease caretaker status). Answer options were single answer, multiple-choice, open-ended, or on a 4-point Likert scale. Questions were defined based on 16 in-person interviews with people without any chronic disorder, people with diabetes, and people with sickle cell disease. All questions were optional. Data were collected from 814 participants. All answers to the open-ended questions have been translated into English. This dataset is especially interesting for researchers interesting in what motivates people with and without chronic disease across countries to use mHealth tools and share their collected health data. Only a subset of variables has been analyzed so far and new research questions on motivation can potentially be answered using this dataset.

Emission and atmospheric transport of particulate PAHs in Northeast Asia.

The emission, concentration levels, and transboundary transport of particulate polycyclic aromatic hydrocarbons (PAHs) in Northeast Asia were investigated using particulate PAH measurements, the newly developed emission inventory (Regional Emission inventory in ASia for Persistent Organic Pollutants version, REAS-POP), and the chemical transport model (Regional Air Quality Model ver2 for POPs version, RAQM2-POP). The simulated concentrations of the nine particulate PAHs agreed well with the measured concentrations, and the results firmly established the efficacy of REAS/RAQM2-POP. It was found that the PAH concentrations in Beijing (China, source region), which were emitted predominantly from domestic coal, domestic biofuel, and other transformations of coal (including coke production), were approximately 2 orders of magnitude greater than those monitored at Noto (Japan, leeward region). In Noto, the PAH concentrations showed seasonal variations; the PAH concentrations were high from winter to spring due to contributions from domestic coal, domestic biofuel, and other transformations of coal, and low in summer. In summer, these contribution were decrease, instead, other sources, such as the on-road mobile source, were relatively increased compared with those in winter. These seasonal variations were due to seasonal variations in emissions from China, as well as transboundary transport across the Asian continent associated with meteorological conditions.