A core component of psychological therapy causes adaptive changes in computational learning mechanisms.

BACKGROUND: Cognitive distancing is an emotion regulation strategy commonly used in psychological treatment of various mental health disorders, but its therapeutic mechanisms are unknown. METHODS: 935 participants completed an online reinforcement learning task involving choices between pairs of symbols with differing reward contingencies. Half (49.1%) of the sample was randomised to a cognitive self-distancing intervention and were trained to regulate or 'take a step back' from their emotional response to feedback throughout. Established computational (Q-learning) models were then fit to individuals' choices to derive reinforcement learning parameters capturing clarity of choice values (inverse temperature) and their sensitivity to positive and negative feedback (learning rates). RESULTS: Cognitive distancing improved task performance, including when participants were later tested on novel combinations of symbols without feedback. Group differences in computational model-derived parameters revealed that cognitive distancing resulted in clearer representations of option values (estimated 0.17 higher inverse temperatures). Simultaneously, distancing caused increased sensitivity to negative feedback (estimated 19% higher loss learning rates). Exploratory analyses suggested this resulted from an evolving shift in strategy by distanced participants: initially, choices were more determined by expected value differences between symbols, but as the task progressed, they became more sensitive to negative feedback, with evidence for a difference strongest by the end of training. CONCLUSIONS: Adaptive effects on the computations that underlie learning from reward and loss may explain the therapeutic benefits of cognitive distancing. Over time and with practice, cognitive distancing may improve symptoms of mental health disorders by promoting more effective engagement with negative information.

A meta-analysis of cognitive flexibility in autism spectrum disorder.

Cognitive flexibility is a fundamental process that underlies adaptive behaviour in response to environmental change. Studies examining the profile of cognitive flexibility in autism spectrum disorder (ASD) have reported inconsistent findings. To address whether difficulties with cognitive flexibility are characteristic of autism, we conducted a random-effects meta-analysis and employed subgroup analyses and meta-regression to assess the impact of relevant moderator variables such as task, outcomes, and age. Fifty-nine studies were included and comprised of 2122 autistic individuals without intellectual disabilities and 2036 neurotypical controls, with an age range of 4 to 85 years. The results showed that autistic individuals have greater difficulties with cognitive flexibility, with an overall statistically significant small to moderate effect size. Subgroup analyses revealed a significant difference between task outcomes, with perseverative errors obtaining the largest effect size. In summary, the present meta-analysis highlights the existence of cognitive flexibility difficulties in autistic people, in the absence of learning disabilities, but also that this profile is characterised by substantial heterogeneity. Potential contributing factors are discussed.

A systematic review of the measures that have been used to assess surface characteristics in relation to their impact on walking and falling.

We investigated whether an impediment to progress in understanding the environmental factors that cause falls may be the difficulty in comparing results across studies because walking surfaces are poorly defined and underspecified. We conducted a systematic review of 384 studies from 370 articles that tested how different surfaces influenced human walking and falling. For each study, we report which categories of surfaces were used (indoor, outdoor, treadmill, virtual reality and qualitative), the nature of each surface (stairs, slopes, slippery, compliant, rough or default) and how information about each surface was measured. We found that minimal information was provided for many surfaces, making it impossible to meaningfully compare results for different types of surfaces across studies. We conclude that most published studies of walking and falling provide insufficient data to describe the surfaces that they used and we provide recommendations about how to improve the reporting of walking surfaces.

Dietary protein, lipid and insect meal on growth, plasma biochemistry and hepatic immune expression of lake whitefish (Coregonus clupeaformis).

Studies are lacking that investigate the dietary nutrient requirements of lake whitefish (Coregonus clupeaformis), a newly farmed fish species in Ontario, Canada. Dietary levels of protein and lipid must be optimized to ensure high growth performance for the commercial success of this species. Additionally, the inclusion of insect meal in the diet may improve growth and immune response. The objective of this study was to evaluate the effects of dietary protein:lipid ratios and insect meal as a feed additive on the growth performance and hepatic immune function of juvenile lake whitefish (301 ± 10 g). A 16-week (112 day) trial was performed with five diets including a commercial control diet (BCC), and four experimental diets with high or low levels of protein (54 and 48%, respectively) and lipid (18 and 12%, respectively). The high protein dietary groups contained 5% of full-fat black soldier fly larvae (Hermetia illucens). Fish weights, viscera, liver, and blood were collected for further analysis. Specific growth rate, thermal growth coefficient and weight gain were significantly higher in fish fed with the BCC and high protein high lipid (HPHL) diets. However, viscerosomatic index was found to be significantly higher in fish fed the BCC diet, thus HPHL is more optimal for non-visceral weight gain. Higher levels of plasma phosphorus, aspartate aminotransferase and potassium indicated poor growth and stress in fish fed low lipid diets. Relative expression of HSP70, involved in cellular repair, was significantly downregulated in fish fed high lipid diets, and no effects were found on the expression of innate immune and oxidative stress genes. Also, IL8 (CXCL8) and catalase were upregulated (non-significant) in fish fed the HPHL diet with the largest weight gain. No effects of insects were found on growth, plasma biochemistry or gene expression, which suggests 5% dietary inclusion was too low. Overall, we recommend a HPHL diet for the cultivation of lake whitefish based on improved growth performance, low viscera weight, improved plasma biochemistry and downregulation of cellular repair genes.

Uncertainty as a driver of the youth mental health crisis.

Mental health problems in young people have been on the rise for over a decade, with that trend accelerating during the pandemic. This review proposes that the catalyst effect of the pandemic offers insights into a key driver of increases in youth depression and anxiety: greater uncertainty. Uncertainty about many aspects of everyday life, including social connections, education, job security and health, increased during the pandemic, and this coincided with increasing rates of depression and anxiety. Lab-based developmental cognitive and clinical neuroscience research on tolerance of uncertainty and adolescent mental health shows that when adolescents fail to show age-typical tolerance of uncertainty, they are at greater risk of mental health problems. Avenues for future research to understand and promote tolerance of uncertainty in adolescents are proposed.

Transdiagnostic computations of uncertainty: towards a new lens on intolerance of uncertainty.

People radically differ in how they cope with uncertainty. Clinical researchers describe a dispositional characteristic known as "intolerance of uncertainty", a tendency to find uncertainty aversive, reported to be elevated across psychiatric and neurodevelopmental conditions. Concurrently, recent research in computational psychiatry has leveraged theoretical work to characterise individual differences in uncertainty processing. Under this framework, differences in how people estimate different forms of uncertainty can contribute to mental health difficulties. In this review, we briefly outline the concept of intolerance of uncertainty within its clinical context, and we argue that the mechanisms underlying this construct may be further elucidated through modelling how individuals make inferences about uncertainty. We will review the evidence linking psychopathology to different computationally specified forms of uncertainty and consider how these findings might suggest distinct mechanistic routes towards intolerance of uncertainty. We also discuss the implications of this computational approach for behavioural and pharmacological interventions, as well as the importance of different cognitive domains and subjective experiences in studying uncertainty processing.

Perceptual decision-making in autism as assessed by "spot the difference" visual cognition tasks.

Discriminating between similar figures proves to be a remarkably demanding task due to the limited capacity of our visual cognitive processes. Here we examine how perceptual inference and decision-making are modulated by differences arising from neurodiversity. A large sample of autistic (n = 140) and typical (n = 147) participants completed two forced choice similarity judgement tasks online. Each task consisted of "match" (identical figures) and "mismatch" (subtle differences between figures) conditions. Signal detection theory analyses indicated a response bias by the autism group during conditions of uncertainty. More specifically, autistic participants were more likely to choose the "mismatch" option, thus leading to more hits on the "mismatch" condition, but also more false alarms on the "match" condition. These results suggest differences in response strategies during perceptual decision-making in autism.

Transgenerational effects of zinc in zebrafish following early life stage exposure.

Although toxic effects of zinc (Zn) have been well established in the different developmental stages in fish, long-lasting effects of Zn exposure during embryonic development have not been explored. Exposure to an environmentally relevant Zn concentration of 10 µM (650 µg/L) during the first five days after fertilization did not affect survival, body weight, malformations or overall hatching success of F0 and F1 larvae. Zn exposure did, however, result in delayed hatching in both the F0 and F1 generations and caused significant changes in homeostasis of Zn and selenium (Se) in F0 and F1 fish. This was especially pronounced when F1 embryos from Zn-exposed parents were treated with 30 µM (2000 µg/L) Zn. In the F0 generation, skewed sex ratio towards males and changes in homeostasis of Zn, Se and manganese (Mn) in the brain, gill, liver and gonad of adult fish were also observed. These changes were associated with altered expression of Zn- and Mn-regulatory genes and sex differentiation genes in F0 and F1 fish. The present study suggests that fish may carry memory from embryo-larval Zn exposure into adulthood and further to the next generation. The present study shows that ecotoxicological risk of an exposure to Zn during embryo-larval development may persist long after recovery and may also manifest in the F1 generation.

Disrupted Dorsal Mid-Insula Activation During Interoception Across Psychiatric Disorders.

OBJECTIVE: Maintenance of bodily homeostasis relies on interoceptive mechanisms in the brain to predict and regulate bodily state. While altered neural activation during interoception in specific psychiatric disorders has been reported in many studies, it is unclear whether a common neural locus underpins transdiagnostic interoceptive differences. METHODS: The authors conducted a meta-analysis of neuroimaging studies comparing patients with psychiatric disorders with healthy control subjects to identify brain regions exhibiting convergent disrupted activation during interoception. Bibliographic, neuroimaging, and preprint databases through May 2020 were searched. A total of 306 foci from 33 studies were extracted, which included 610 control subjects and 626 patients with schizophrenia, bipolar or unipolar depression, posttraumatic stress disorder, anxiety, eating disorders, or substance use disorders. Data were pooled using a random-effects model implemented by the activation likelihood estimation algorithm. The preregistered primary outcome was the neuroanatomical location of the convergence of peak voxel coordinates. RESULTS: Convergent disrupted activation specific to the left dorsal mid-insula was found (Z=4.47, peak coordinates: -36, -2, 14; volume: 928 mm3). Studies directly contributing to the cluster included patients with bipolar disorder, anxiety, major depression, anorexia, and schizophrenia, assessed with task probes including pain, hunger, and interoceptive attention. A series of conjunction analyses against extant meta-analytic data sets revealed that this mid-insula cluster was anatomically distinct from brain regions involved in affective processing and from regions altered by psychological or pharmacological interventions for affective disorders. CONCLUSIONS: These results reveal transdiagnostic, domain-general differences in interoceptive processing in the left dorsal mid-insula. Disrupted mid-insular activation may represent a neural marker of psychopathology and a putative target for novel interventions.

Biomechanical adaptation to post-stroke visual field loss: a systematic review.

BACKGROUND: Homonymous visual field defects represent the most frequent type of visual field loss after stroke, affecting nearly 30% of individuals with unilateral post-chiasmal brain damage. This review aimed to gather the available evidence on the biomechanical changes to visual field loss following stroke. METHODS: A systematic review was conducted inclusive of randomised controlled trials, cohort studies, before-after studies and case-controlled studies. Studies including adult and paediatric participants that investigated eye, head, or body movements in post-stroke visual field loss during visual exploration tasks were included. Search terms included a range of MESH terms as well as alternative terms relating to stroke, visual field loss, hemianopia, visual functions and scanning behaviour. Articles were selected by two authors independently. Data were extracted by one author and verified by a second. All included articles were assessed for risk of bias using checklists appropriate to the study design. RESULTS: Thirty-six articles (1123 participants) were included in the overall review (Kappa 0.863) and categorised into simulated or true visual field loss (typically hemianopia). Seven studies identified the biomechanical alterations to simulated hemianopia compared to normal performance. Twenty-nine studies detailed eye, head and body movement parameters in true hemianopia. Hemianopic participants and healthy adults with simulated hemianopia differed significantly from controls in various fixation and saccade parameters as indicated by increased number and duration of fixations, number and duration of saccades and scan path length with shorter mean saccadic amplitude. Under simulated hemianopia, participants were consistently biased towards the sighted visual field while gaze behaviour in true hemianopia was biased in the direction of the blind hemifield. CONCLUSIONS: There is considerable evidence on the altered eye movements that occur in true hemianopia and in healthy adults with simulated hemianopia. Successful performance in naturalistic tasks of visual exploration appears to be related to compensatory mechanisms of visual exploratory behaviour, namely, an increase in the amplitude and peak velocity of saccades, widening horizontally the distribution of eye movements, and a shift of the overall distribution of saccades into the blind field. This review highlights the lack of studies reporting head and other body movement parameters in hemianopia. Further studies with robust methodology and large sample sizes involving participants with post-stroke visual field loss are needed. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020194403.

The effect of uncertainty on prediction error in the action perception loop.

Among all their sensations, agents need to distinguish between those caused by themselves and those caused by external causes. The ability to infer agency is particularly challenging under conditions of uncertainty. Within the predictive processing framework, this should happen through active control of prediction error that closes the action-perception loop. Here we use a novel, temporally-sensitive, behavioural proxy for prediction error to show that it is minimised most quickly when volatility is high and when participants report agency, regardless of the accuracy of the judgement. We demonstrate broad effects of uncertainty on accuracy of agency judgements, movement, policy selection, and hypothesis switching. Measuring autism traits, we find differences in policy selection, sensitivity to uncertainty and hypothesis switching despite no difference in overall accuracy.

The Computational, Pharmacological, and Physiological Determinants of Sensory Learning under Uncertainty.

The ability to represent and respond to uncertainty is fundamental to human cognition and decision-making. Noradrenaline (NA) is hypothesized to play a key role in coordinating the sensory, learning, and physiological states necessary to adapt to a changing world, but direct evidence for this is lacking in humans. Here, we tested the effects of attenuating noradrenergic neurotransmission on learning under uncertainty. We probed the effects of the ß-adrenergic receptor antagonist propranolol (40 mg) using a between-subjects, double-blind, placebo-controlled design. Participants performed a probabilistic associative learning task, and we employed a hierarchical learning model to formally quantify prediction errors about cue-outcome contingencies and changes in these associations over time (volatility). Both unexpectedness and noise slowed down reaction times, but propranolol augmented the interaction between these main effects such that behavior was influenced more by prior expectations when uncertainty was high. Computationally, this was driven by a reduction in learning rates, with people slower to update their beliefs in the face of new information. Attenuating the global effects of NA also eliminated the phasic effects of prediction error and volatility on pupil size, consistent with slower belief updating. Finally, estimates of environmental volatility were predicted by baseline cardiac measures in all participants. Our results demonstrate that NA underpins behavioral and computational responses to uncertainty. These findings have important implications for understanding the impact of uncertainty on human biology and cognition.

Keep your head down: Maintaining gait stability in challenging conditions.

BACKGROUND: Peripheral vision often deteriorates with age, disrupting our ability to maintain normal locomotion. Laboratory based studies have shown that lower visual field loss, in particular, is associated with changes in gaze and gait behaviour whilst walking and this, in turn, increases the risk of falling in the elderly. Separately, gaze and gait behaviours change and fall risk increases when walking over complex surfaces. It seems probable, but has not yet been established, that these challenges to stability interact. RESEARCH QUESTION: How does loss of the lower visual field affect gaze and gait behaviour whilst walking on a variety of complex surfaces outside of the laboratory? Specifically, is there a synergistic interaction between the effects on behaviour of blocking the lower visual field and increased surface complexity? METHODS: We compared how full vision versus simulated lower visual field loss affected a diverse range of behavioural measures (head pitch angle, eye angle, muscle coactivation, gait speed and walking smoothness as measured by harmonic ratios) in young participants. Participants walked over a range of surfaces of different complexity, including pavements, grass, steps and pebbles. RESULTS: In both full vision and blocked lower visual field conditions, surface complexity influenced gaze and gait behaviour. For example, more complex surfaces were shown to be associated with lowered head pitch angles, increased leg muscle coactivation, reduced gait speed and decreased walking smoothness. Relative to full vision, blocking the lower visual field caused a lowering of head pitch, especially for more complex surfaces. However, crucially, muscle coactivation, gait speed and walking smoothness did not show a significant change between full vision and blocked lower visual field conditions. Finally, head pitch angle, muscle coactivation, gait speed and walking smoothness were all correlated highly with each other. SIGNIFICANCE: Our study showed that blocking the lower visual field did not significantly change muscle coactivation, gait speed or walking smoothness. This suggests that young people cope well when walking with a blocked lower visual field, making minimal behavioural changes. Surface complexity had a greater effect on gaze and gait behaviour than blocking the lower visual field. Finally, head pitch angle was the only measure that showed a significant synergistic interaction between surface complexity and blocking the lower visual field. Together our results indicate that, first, a range of changes occur across the body when people walk over more complex surfaces and, second, that a relatively simple behavioural change (to gaze) suffices to maintain normal gait when the lower visual field is blocked, even in more challenging environments. Future research should assess whether young people cope as effectively when several impairments are simulated, representative of the comorbidities found with age.

Physical and perceptual measures of walking surface complexity strongly predict gait and gaze behaviour.

BACKGROUND: Walking surfaces vary in complexity and are known to affect stability and fall risk whilst walking. However, existing studies define surfaces through descriptions only. OBJECTIVE: This study used a multimethod approach to measure surface complexity in order to try to characterise surfaces with respect to locomotor stability. METHODS: We assessed how physical measurements of walking surface complexity compared to participant's perceptual ratings of the effect of complexity on stability. Physical measurements included local slope measures from the surfaces themselves and shape complexity measured using generated surface models. Perceptual measurements assessed participants' perceived stability and surface roughness using Likert scales. We then determined whether these measurements were indicative of changes to stability as assessed by behavioural changes including eye angle, head pitch angle, muscle coactivation, walking speed and walking smoothness. RESULTS: Physical and perceptual measures were highly correlated, with more complex surfaces being perceived as more challenging to stability. Furthermore, complex surfaces, as defined from both these measurements, were associated with lowered head pitch, increased muscle coactivation and reduced walking smoothness. SIGNIFICANCE: Our findings show that walking surfaces defined as complex, based on physical measurements, are perceived as more challenging to our stability. Furthermore, certain behavioural measures relate better to these perceptual and physical measures than others. Crucially, for the first time this study defined walking surfaces objectively rather than just based on subjective descriptions. This approach could enable future researchers to compare results across walking surface studies. Moreover, perceptual measurements, which can be collected easily and efficiently, could be used as a proxy for estimating behavioural responses to different surfaces. This could be particularly valuable when determining risk of instability when walking for individuals with compromised stability.

Look out: an exploratory study assessing how gaze (eye angle and head angle) and gait speed are influenced by surface complexity.

BACKGROUND: Most research investigating the connection between walking and visual behaviour has assessed only eye movements (not head orientation) in respect to locomotion over smooth surfaces in a laboratory. This is unlikely to reflect gaze changes found over the complex surfaces experienced in the real world, especially given that eye and head movements have rarely been assessed simultaneously. RESEARCH QUESTION: How does gaze (eye and head) angle and gait speed change when walking over surfaces of different complexity? METHODS: In this exploratory study, we used a mobile eye tracker to monitor eye movements and inertia measurement unit sensors (IMUs) to measure head angle whilst subjects (n = 11) walked over surfaces with different complexities both indoors and outdoors. Gait speed was recorded from ankle IMUs. RESULTS: Overall, mean gaze angle was lowest over the most complex surface and this surface also elicited the slowest mean gait speed. The head contributed increasingly to the lowering of gaze with increased surface complexity. Less complex surfaces showed no significant difference between gaze and gait behaviour. SIGNIFICANCE: This study supports previous research showing that increased surface complexity is an important factor in determining gaze and gait behaviour. Moreover, it provides the novel finding that head movements provide important contributions to gaze location. Our future research aims are to further assess the role of the head in determining gaze location during locomotion across a greater range of complex surfaces to determine the key surface characteristics that influence gaze during gait.

ZnT8 Haploinsufficiency Impacts MIN6 Cell Zinc Content and ß-Cell Phenotype via ZIP-ZnT8 Coregulation.

The zinc transporter ZnT8 (SLC30A8) localises to insulin secretory granules of ß-cells where it facilitates zinc uptake for insulin crystallisation. ZnT8 abundance has been linked to ß-cell survival and functional phenotype. However, the consequences of ZnT8 haploinsufficiency for ß-cell zinc trafficking and function remain unclear. Since investigations in human populations have shown SLC30A8 truncating polymorphisms to decrease the risk of developing Type 2 Diabetes, we hypothesised that ZnT8 haploinsufficiency would improve ß-cell function and maintain the endocrine phenotype. We used CRISPR/Cas9 technology to generate ZnT8 haploinsufficient mouse MIN6 ß-cells and showed that ZnT8 haploinsufficiency is associated with downregulation of mRNAs for Slc39a8 and Slc39a14, which encode for the zinc importers, Znt- and Irt-related proteins 8 (ZIP8) and 14 (ZIP14), and with lowered total cellular zinc content. ZnT8 haploinsufficiency disrupts expression of a distinct array of important ß-cell markers, decreases cellular proliferation via mitogen-activated protein (MAP) kinase cascades and downregulates insulin gene expression. Thus, ZnT8 cooperates with zinc importers of the ZIP family to maintain ß-cell zinc homeostasis. In contrast to the hypothesis, lowered ZnT8 expression reduces MIN6 cell survival by affecting zinc-dependent transcription factors that control the ß-cell phenotype.

A Neuroanatomical Substrate Linking Perceptual Stability to Cognitive Rigidity in Autism.

Overly stable visual perception seen in individuals with autism spectrum disorder (ASD) is related to higher-order core symptoms of the condition. However, the neural basis by which these seemingly different symptoms are simultaneously observed in individuals with ASD remains unclear. Here, we aimed to identify such a neuroanatomical substrate linking perceptual stability to autistic cognitive rigidity, a part of core restricted, repetitive behaviors (RRBs). First, using a bistable visual perception test, we measured the perceptual stability of 22 high-functioning adults with ASD and 22 age-, IQ-, and sex-matched typically developing human individuals and confirmed overstable visual perception in autism. Next, using a spontaneous task-switching (TS) test, we showed that the individuals with ASD were more likely to repeat the same task voluntarily and spontaneously, and such rigid TS behavior was associated with the severity of their RRB symptoms. We then compared these perceptual and cognitive behaviors and found a significant correlation between them for individuals with ASD. Finally, we found that this behavioral link was supported by a smaller gray matter volume (GMV) of the posterior superior parietal lobule (pSPL) in individuals with ASD. Moreover, this smaller GMV in the pSPL was also associated with the RRB symptoms and replicated in two independent datasets. Our findings suggest that the pSPL could be one of the neuroanatomical mediators of cognitive and perceptual inflexibility in autism, which could help a unified biological understanding of the mechanisms underpinning diverse symptoms of this developmental disorder.SIGNIFICANCE STATEMENT Behavioral studies show perceptual overstability in autism spectrum disorder (ASD). However, the neural mechanisms by which such sensory symptoms can coexist and often correlate with seemingly separate core symptoms remain unknown. Here, we have identified such a key neuroanatomical substrate. We have revealed that overstable sensory perception of individuals with ASD is linked with their cognitive rigidity, a part of core restricted, repetitive behavior symptoms, and such a behavioral link is underpinned by a smaller gray matter volume in the posterior superior parietal lobule in autism. These findings uncover a key neuroanatomical mediator of autistic perceptual and cognitive inflexibility and would ignite future studies on how the core symptoms of ASD interact with its unique sensory perception.

Getting a grasp on action-specific scaling: A response to Witt (2017).

Can higher level cognition directly influence visual spatial perception? Many recent studies have claimed so, on the basis that manipulating cognitive factors (e.g., morality, emotion, action capacity) seems to directly affect perception. However, Firestone and Scholl (Behavioral and Brain Sciences, 39, 1-77, 2016) argued that such studies often fall prey to at least one of six pitfalls. They further argued that if an effect could be accounted for by any of these pitfalls, it is not a true demonstration of a top-down influence of cognition on perception. In response to Firestone and Scholl (Behavioral and Brain Sciences, 39, 1-77, 2016), Witt (Psychonomic Bulletin & Review, 24(4), 999-1021, 2017) discussed four action-specific scaling effects which, she argued, withstand all six pitfalls and thus demonstrate true perceptual changes caused by differences in action capacity. Her third case study was the influence of apparent grasping capacity on perceived object size. In this article, we provide new interpretations of previous findings and assess recent data which suggest that this effect is not, in fact, perceptual. Instead, we believe that many earlier studies showing this effect are subject to one or more of the pitfalls outlined by Firestone and Scholl (Behavioral and Brain Sciences, 39, 1-77, 2016). We substantiate our claims with recent empirical evidence from our laboratory which suggests that neither actual nor perceived grasping capacity directly influence perceived object size. We conclude that studies manipulating grasping capacity do not provide evidence for the action-specific account because variation in this factor does not directly influence size perception.