Embryonic heart rate is higher in species that experience greater nest predation risk during incubation.

Avian eggs develop outside of the female body, and therefore embryonic development is subject to multiple internal (physiological) and external (ecological) factors. Embryonic developmental rate has important consequences for survival. Within species, embryos that develop too quickly often experience deformities, disorders, or mortality, while embryos that develop slowly risk inviability and increase the time they are exposed to various sources of mortality in the nest. These contrasting forces may lead to interspecific variation in developmental rates. We investigated potential factors affecting embryonic heart rate (EHR), a proxy of development, across 14 passerine species in the field. More specifically, we investigated if nest predation risk, clutch size, seasonality, and egg volume influenced EHR. From previous research, we expected, and found, that EHR was positively associated with embryonic age and egg temperature. Species with greater nest predation risk had higher EHR, shorter incubation periods, and lower nest temperature variance. EHR increased as the season progressed and with egg volume, while EHR declined with clutch size. Bird species exhibit varying strategies to increase nestling and fledgling survival in response to predation risk, and these results suggest that variation in embryonic development may be related to species-specific differences in nest predation risk.

Host avian species and environmental conditions influence the microbial ecology of brood parasitic brown-headed cowbird nestlings: What rules the roost?

The role of species interactions, as well as genetic and environmental factors, all likely contribute to the composition and structure of the gut microbiome; however, disentangling these independent factors under field conditions represents a challenge for a functional understanding of gut microbial ecology. Avian brood parasites provide unique opportunities to investigate these questions, as brood parasitism results in parasite and host nestlings being raised in the same nest, by the same parents. Here we utilized obligate brood parasite brown-headed cowbird nestlings (BHCO; Molothrus ater) raised by several different host passerine species to better understand, via 16S rRNA sequencing, the microbial ecology of brood parasitism. First, we compared faecal microbial communities of prothonotary warbler nestlings (PROW; Protonotaria citrea) that were either parasitized or non-parasitized by BHCO and communities among BHCO nestlings from PROW nests. We found that parasitism by BHCO significantly altered both the community membership and community structure of the PROW nestling microbiota, perhaps due to the stressful nest environment generated by brood parasitism. In a second dataset, we compared faecal microbiotas from BHCO nestlings raised by six different host passerine species. Here, we found that the microbiota of BHCO nestlings was significantly influenced by the parental host species and the presence of an inter-specific nestmate. Thus, early rearing environment is important in determining the microbiota of brood parasite nestlings and their companion nestlings. Future work may aim to understand the functional effects of this microbiota variability on nestling performance and fitness.

Is "memory-for-when" universal? Group and individual variability in temporal position memory for words, faces, and classrooms.

Many contemporary theories of memory assume that everyone automatically stores temporal contextual information about all types of encountered information, yet most studies on this topic have used words and ignored individual differences. Five experiments accumulated evidence that explicit storage of temporal context information does not appear to occur automatically for all people and types of memoranda. We collected judgments of temporal position (memory-for-when) for words (Experiments 1 & 3), faces (Experiments 2A, 3, 4, and 5), and classrooms (Experiments 2B & 3). At the group level, for each of these memoranda memory-for-when was sensitive to the original input position and showed a temporal primacy effect reflecting better memory for position for items near the beginning of the list, indicating some automatic storage of temporal context information. However, memory-for-when was significantly better for words than classrooms, with faces in the middle. Moreover, individuals varied dramatically in their ability to indicate memory-for-when, especially for classrooms where many people performed at or near chance. Taken together, the data suggest that explicit memory-for-when may be dissociable from the more implicit use of temporal contextual information that is theorised to occur during free recall.

stelfi: An R package for fitting Hawkes and log-Gaussian Cox point process models.

Modelling spatial and temporal patterns in ecology is imperative to understand the complex processes inherent in ecological phenomena. Log-Gaussian Cox processes are a popular choice among ecologists to describe the spatiotemporal distribution of point-referenced data. In addition, point pattern models where events instigate others nearby (i.e., self-exciting behaviour) are becoming increasingly popular to infer the contagious nature of events (e.g., animal sightings). While there are existing R packages that facilitate fitting spatiotemporal point processes and, separately, self-exciting models, none incorporate both. We present an R package, stelfi, that fits spatiotemporal self-exciting and log-Gaussian Cox process models using Template Model Builder through a range of custom-written C++ templates. We illustrate the use of stelfi's functions fitting models to Sasquatch (bigfoot) sightings data within the USA. The structure of these data is typical of many seen in ecology studies. We show, from a temporal Hawkes process to a spatiotemporal self-exciting model, how the models offered by the package enable additional insights into the temporal and spatial progression of point pattern data. We present extensions to these well-known models that include spatiotemporal self-excitation and joint likelihood models, which are better suited to capture the complex mechanisms inherent in many ecological data. The package stelfi offers user-friendly functionality, is open source, and is available from CRAN. It offers the implementation of complex spatiotemporal point process models in R for applications even beyond the field of ecology.

Surface Passivation by Quantum Exclusion: On the Quantum Efficiency and Stability of Delta-Doped CCDs and CMOS Image Sensors in Space.

Radiation-induced damage and instabilities in back-illuminated silicon detectors have proved to be challenging in multiple NASA and commercial applications. In this paper, we develop a model of detector quantum efficiency (QE) as a function of Si-SiO2 interface and oxide trap densities to analyze the performance of silicon detectors and explore the requirements for stable, radiation-hardened surface passivation. By analyzing QE data acquired before, during, and after, exposure to damaging UV radiation, we explore the physical and chemical mechanisms underlying UV-induced surface damage, variable surface charge, QE, and stability in ion-implanted and delta-doped detectors. Delta-doped CCD and CMOS image sensors are shown to be uniquely hardened against surface damage caused by ionizing radiation, enabling the stability and photometric accuracy required by NASA for exoplanet science and time domain astronomy.

Phenotypic signatures of urbanization? Resident, but not migratory, songbird eye size varies with urban-associated light pollution levels.

Urbanization now exposes large portions of the earth to sources of anthropogenic disturbance, driving rapid environmental change and producing novel environments. Changes in selective pressures as a result of urbanization are often associated with phenotypic divergence; however, the generality of phenotypic change remains unclear. In this study, we examined whether morphological phenotypes in two residential species (Carolina Wren [Thryothorus ludovicianus] and Northern Cardinal [Cardinalis cardinalis]) and two migratory species (Painted Bunting [Passerina ciris], and White-eyed Vireo [Vireo griseus]), differed between urban core and edge habitats in San Antonio, Texas, USA. More specifically, we examined whether urbanization, associated sensory pollution (light and noise) and brightness (open, bright areas cause by anthropogenic land use) influenced measures of avian body (mass and frame size) and lateral eye size. We found no differences in body size between urban core and edge habitats for all species except the Painted Bunting, in which core-urban individuals were smaller. Rather than a direct effect of urbanization, this was due to differences in age structure between habitats, with urban-core areas consisting of higher proportions of younger buntings which are, on average, smaller than older birds. Residential birds inhabiting urban-core areas had smaller eyes compared to their urban-edge counterparts, resulting from a negative association between eye size and light pollution and brightness across study sites; notably, we found no such association in the two migratory species. Our findings demonstrate how urbanization may indirectly influence phenotypes by altering population demographics and highlight the importance of accounting for age when assessing factors driving phenotypic change. We also provide some of the first evidence that birds may adapt to urban environments through changes in their eye morphology, demonstrating the need for future research into relationships among eye size, ambient light microenvironment use, and disassembly of avian communities as a result of urbanization.

Using individual-based bioenergetic models to predict the aggregate effects of disturbance on populations: A case study with beaked whales and Navy sonar.

Anthropogenic activities can lead to changes in animal behavior. Predicting population consequences of these behavioral changes requires integrating short-term individual responses into models that forecast population dynamics across multiple generations. This is especially challenging for long-lived animals, because of the different time scales involved. Beaked whales are a group of deep-diving odontocete whales that respond behaviorally when exposed to military mid-frequency active sonar (MFAS), but the effect of these nonlethal responses on beaked whale populations is unknown. Population consequences of aggregate exposure to MFAS was assessed for two beaked whale populations that are regularly present on U.S. Navy training ranges where MFAS is frequently used. Our approach integrates a wide range of data sources, including telemetry data, information on spatial variation in habitat quality, passive acoustic data on the temporal pattern of sonar use and its relationship to beaked whale foraging activity, into an individual-based model with a dynamic bioenergetic module that governs individual life history. The predicted effect of disturbance from MFAS on population abundance ranged between population extinction to a slight increase in population abundance. These effects were driven by the interaction between the temporal pattern of MFAS use, baseline movement patterns, the spatial distribution of prey, the nature of beaked whale behavioral response to MFAS and the top-down impact of whale foraging on prey abundance. Based on these findings, we provide recommendations for monitoring of marine mammal populations and highlight key uncertainties to help guide future directions for assessing population impacts of nonlethal disturbance for these and other long-lived animals.

Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified Sorghum bicolor.

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 µg g-1 DW) compared to null segregants (4.2 µg g-1 DW) and wild-type control (Tx430; 3.7 µg g-1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g-1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

Rapid and highly efficient morphogenic gene-mediated hexaploid wheat transformation.

The successful employment of morphogenic regulator genes, Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2), for Agrobacterium-mediated transformation of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) has been reported to improve transformation by inducing rapid somatic embryo formation. Here, we report two morphogenic gene-mediated wheat transformation methods, either with or without morphogenic and marker gene excision. These methods yield independent-transformation efficiency up to 58% and 75%, respectively. In both cases, the tissue culture duration for generating transgenic plants was significantly reduced from 80 to nearly 50 days. In addition, the transformation process was significantly simplified to make the procedure less labor-intensive, higher-throughput, and more cost-effective by eliminating the requirement for embryonic axis excision, bypassing the necessity for prolonged dual-selection steps for callus formation, and obviating the prerequisite of cytokinin for shoot regeneration. Furthermore, we have demonstrated the flexibility of the methods and generated high-quality transgenic events across multiple genotypes using herbicide (phosphinothricin, ethametsulfuron)- and antibiotic (G418)-based selections.

Thoracoscopic Left Atrial Appendage Exclusion for Atrial Fibrillation Patients Intolerant to Anticoagulation.

OBJECTIVE: Outcomes associated with isolated epicardial left atrial appendage (LAA) exclusion in atrial fibrillation (AF) patients with a contraindication or intolerance to anticoagulation are not clearly defined, especially in patients with prior stroke. This study evaluated the perioperative safety, medication use, and stroke outcomes for isolated thoracoscopic LAA exclusion for stroke prevention. METHODS: A single-center retrospective study was performed of adults undergoing isolated thoracoscopic LAA exclusion with an epicardial exclusion device without a concomitant surgical procedure. Descriptive statistics were performed. RESULTS: Twenty-five patients met the inclusion criteria. The cohort was 68% male (n = 17) with a mean age of 76.4 ± 6.5 years, a mean preoperative CHA2DS2-VASc score of 4.2 ± 1.4, and a mean preoperative HAS-BLED score of 2.68 ± 1.03. Seventeen patients (68%) had nonparoxysmal AF. There were 11 patients with intolerance of anticoagulation due to intracranial hemorrhage (44%), 6 due to gastrointestinal bleeding (24%), and 4 due to genitourinary bleeding (16%). All procedures were performed thoracoscopically with 100% technical success-the mean LAA stump length was 5.5 ± 2.3 mm on intraoperative transesophageal echocardiography. The median hospital length of stay was 2 (interquartile range [IQR] 1, 6.5) days. The median follow-up time was 430 (IQR 125, 972) days. During follow-up, 1 patient with cerebral angiopathy had temporary neurologic deficits at an outside institution, with brain imaging showing no evidence of ischemic injury. There were no other thromboembolic events over the 38.8 postoperative patient-years examined. All patients were off anticoagulation at last follow-up. CONCLUSIONS: This study shows the perioperative safety, technical success, freedom from anticoagulation, and stroke outcomes of isolated thoracoscopic LAA exclusion in patients with AF at high risk for thromboembolic disease.

Short-to Mid-Term Survivorship of a Patient-specific Unicompartmental Knee Arthroplasty Implant Cast from a Three-Dimensional Printed Mold.

The purpose of this study was to determine early survivorship and complication rates associated with the implantation of a new patient-specific unicompartmental knee implant cast from a three-dimensional (3D) printed mold, introduced in 2012. We retrospectively reviewed 92 consecutive patients who underwent unicompartmental knee arthroplasty (UKA) with a patient-specific implant cast from a 3D printed mold between September 2012 and October 2015. The early results of a patient-specific UKA implant were favorable in our cohort, with survivorship free from reoperation of 97% at an average 4.5 years follow-up. Future studies are necessary to investigate the long-term performance of this implant. Survivorship of a patient-specific unicompartmental knee arthroplasty implant cast from a 3D printed mold.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum.

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

Ototoxicity of Long-Term α-Difluoromethylornithine for Skin Cancer Prevention.

OBJECTIVE: Evaluate the effects of α-difluoromethylornithine (DFMO) on hearing thresholds as part of a randomized, double-blind, placebo-controlled trial. METHODS: Subjects were randomized and assigned to the control (placebo) or experimental (DFMO) group. DFMO or placebo were administered orally (500 mg/m2 /day) for up to 5 years. RESULTS: Subjects taking DFMO had, on average, increased hearing thresholds from baseline across the frequency range compared to subjects in the control group. Statistical analysis revealed this was significant in the lower frequency range. CONCLUSIONS: This randomized controlled trial revealed the presence of increased hearing thresholds associated with long-term DFMO use. As a whole, DFMO may help prevent and treat certain types of cancers; however, it can result in some degree of hearing loss even when administered at low doses. This study further highlights the importance of closely monitoring hearing thresholds in subjects taking DFMO. Laryngoscope, 133:676-682, 2023.

Ontogenetic effects of brood parasitism by the Brown-headed Cowbird on host offspring.

Nest-sharer avian brood parasites do not evict or otherwise kill host chicks, but instead inflict a range of negative effects on their nestmates that are mediated by interactions between the parasite and host life history traits. Although many of the negative fitness effects of avian brood parasitism are well documented across diverse host species, there remains a paucity of studies that have examined the impacts of parasitism across the entirety of host ontogeny (i.e., from when an egg is laid until independence). More specifically, few studies have examined the impact of brood parasitism on the pre- and post-fledging development, physiology, behavior, and survival of host offspring. To help fill this knowledge gap, we assessed the effects of brood parasitism by Brown-headed Cowbirds (Molothrus ater) across the ontogeny (incubation, nestling, and post-fledging period) of nine sympatrically breeding host species in central Illinois, USA; due to sample sizes, impacts on the post-fledging period were only examined in two of the nine species. Specifically, we examined the impact of brood parasitism on ontogenetic markers including the embryonic heart rate, hatching rate, nestling period length, nest survival, and offspring growth and development. Additionally, in species in which we found negative impacts of cowbird parasitism on host nestmate ontogeny, we examined whether the difference in adult size between parasites and their hosts and their hatching asynchrony positively predicted variation in host costs across these focal taxa. We found that costs of cowbird parasitism were most severe during early nesting stages (reduction in the host clutch or brood size) and were predicted negatively by host size and positively by incubation length. In contrast, we only found limited costs of cowbird parasitism on other stages of host ontogeny; critically, post-fledging survival did not differ between host offspring that fledged alongside cowbirds and those that did not. Our findings (i) highlight the direct costs of cowbird parasitism on host fitness, (ii) provide evidence for when (the stage) those costs are manifested, and (iii) may help to explain why many anti-cowbird defenses of hosts have evolved for protection from parasitism during the laying and incubation stages.

Host community-wide patterns of post-fledging behavior and survival of obligate brood parasitic brown-headed cowbirds.

The antagonistic arms races between obligate brood parasites and their hosts provide critical insights into coevolutionary processes and constraints on the evolution of life history strategies. In avian brood parasites-a model system for examining host-parasite dynamics-research has primarily focused on the egg and nestling stage, while far less is known about the behavior and ecology of fledgling and juvenile brood parasites. To provide greater insights into the post-fledging period of generalist brood parasites, we used handheld and automated telemetry systems to examine the behavior and survival of fledgling brown-headed cowbirds (Molothrus ater). Our host community-wide analysis (data on cowbirds fledged from different host species were pooled) shows that fledgling cowbirds' follow patterns of movement and survival found across the post-fledging literature on parental passerine species. Cowbird fledgling survival was lowest during the first 3 days post-fledging, whereas daily rates of survival neared 100% after about 16 days post-fledging. Cowbird daytime post-fledging activity rates, perch heights, and distance from the natal area all increased with fledging age and young generally gained independence from host parents at 3-4 weeks post-fledging, with approximately the same latency as has been observed in studies on fledglings of cowbird host species. Our research demonstrates how automated telemetry systems can overcome past methodological limitations in post-fledging research and provides an important foundation for future studies examining adaptations that cowbirds and other brood parasites use to exploit hosts during the post-fledging period.

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum.

For many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

Discrete-space continuous-time models of marine mammal exposure to Navy sonar.

Assessing the patterns of wildlife attendance to specific areas is relevant across many fundamental and applied ecological studies, particularly when animals are at risk of being exposed to stressors within or outside the boundaries of those areas. Marine mammals are increasingly being exposed to human activities that may cause behavioral and physiological changes, including military exercises using active sonars. Assessment of the population-level consequences of anthropogenic disturbance requires robust and efficient tools to quantify the levels of aggregate exposure for individuals in a population over biologically relevant time frames. We propose a discrete-space, continuous-time approach to estimate individual transition rates across the boundaries of an area of interest, informed by telemetry data collected with uncertainty. The approach allows inferring the effect of stressors on transition rates, the progressive return to baseline movement patterns, and any difference among individuals. We apply the modeling framework to telemetry data from Blainville's beaked whale (Mesoplodon densirostris) tagged in the Bahamas at the Atlantic Undersea Test and Evaluation Center (AUTEC), an area used by the U.S. Navy for fleet readiness training. We show that transition rates changed as a result of exposure to sonar exercises in the area, reflecting an avoidance response. Our approach supports the assessment of the aggregate exposure of individuals to sonar and the resulting population-level consequences. The approach has potential applications across many applied and fundamental problems where telemetry data are used to characterize animal occurrence within specific areas.

Early-life patterns of growth are linked to levels of phenotypic trait covariance and postfledging mortality across avian species.

Life history studies have established that trade-offs between growth and survival are common both within and among species. Identifying the factor(s) that mediate this trade-off has proven difficult, however, especially at the among-species level. In this study, we examined a series of potentially interrelated traits in a community of temperate-zone passerine birds to help understand the putative causes and consequences of variation in early-life growth among species. First, we examined whether nest predation risk (a proven driver of interspecific variation in growth and development rates) was correlated with species-level patterns of incubation duration and nestling period length. We then assessed whether proxies for growth rate covaried with mean trait covariance strength (i.e., phenotypic correlations ( rp), which can be a marker of early-life stress) among body mass, tarsus length, and wing length at fledging. Finally, we examined whether trait covariance strength at fledging was related to postfledging survival. We found that higher nest predation risk was correlated with faster skeletal growth and that our proxies for growth corresponded with increased trait covariance strength ( rp), which subsequently, correlated with higher mortality in the next life stage (postfledging period). These results provide an indication that extrinsic pressures (nest predation) impact rates of growth, and that there are costs of rapid growth across species, expressed as higher mean rp and elevated postfledging mortality. The link between higher levels of trait covariance at fledging and increased mortality is unclear, but increased trait covariance strength may reflect reduced phenotypic flexibility (i.e., phenotypic canalization), which may limit an organism's capacity for coping with environmental or ecological variability.