Insular Regions Research Articles

Mild Traumatic Brain Injury Results in Significant and Lasting Cortical Demyelination.

Despite contributing to neurocognitive deficits, intracortical demyelination after traumatic brain injury (TBI) is understudied. This study uses magnetic resonance imaging (MRI) to map intracortical myelin and its change in healthy controls and after mild TBI (mTBI). Acute mTBI involves reductions in relative myelin content primarily in lateral occipital regions. Demyelination mapped ~6 months post-injury is significantly more severe than that observed in typical aging (p < 0.05), with temporal, cingulate, and insular regions losing more myelin (30%, 20%, and 16%, respectively) than most other areas, although occipital regions experience 22% less demyelination. Thus, occipital regions may be more susceptible to primary injury, whereas temporal, cingulate and insular regions may be more susceptible to later manifestations of injury sequelae. The spatial profiles of aging- and mTBI-related chronic demyelination overlap substantially; exceptions include primary motor and somatosensory cortices, where myelin is relatively spared post-mTBI. These features resemble those of white matter demyelination and cortical thinning during Alzheimer's disease, whose risk increases after mTBI.

Neural basis for anxiety and anxiety-related physiological responses during a driving situation: an fMRI study.

Although the exteroceptive and interoceptive prediction of a negative event increases a person’s anxiety in daily life situations, the relationship between the brain mechanism of anxiety and the anxiety-related autonomic response has not been fully understood. In this functional magnetic resonance imaging (fMRI) study, we examined the neural basis of anxiety and anxiety-related autonomic responses in a daily driving situation. Participants viewed a driving video clip in the first-person perspective. During the video clip, participants were presented with a cue to indicate whether a subsequent crash could occur (attention condition) or not (safe condition). Enhanced activities in the anterior insula, bed nucleus of the stria terminalis, thalamus, and periaqueductal gray, and higher sympathetic nerve responses (pupil dilation and peripheral arterial stiffness) were triggered by the attention condition but not with the safe condition. Autonomic response-related functional connectivity was detected in the visual cortex, cerebellum, brainstem, and MCC/PCC with the right anterior insula and its adjacent regions as seed regions. Thus, the right anterior insula and adjacent regions, in collaboration with other regions play a role in eliciting anxiety based on the prediction of negative events, by mediating anxiety-related autonomic responses according to interoceptive information.

Alterations in resting-state functional activity and connectivity for major depressive disorder appetite and weight disturbance phenotypes.

Major depressive disorder (MDD) is often accompanied by changes in appetite and weight. Prior task-based functional magnetic resonance imaging (fMRI) findings suggest these MDD phenotypes are associated with altered reward and interoceptive processing. Using resting-state fMRI data, we compared the fractional amplitude of low-frequency fluctuations (fALFF) and seed-based connectivity (SBC) among hyperphagic (n = 77), hypophagic (n = 66), and euphagic (n = 42) MDD groups and a healthy comparison group (n = 38). We examined fALFF and SBC in a mask restricted to reward [nucleus accumbens (NAcc), putamen, caudate, ventral pallidum, and orbitofrontal cortex (OFC)] and interoceptive (anterior insula and hypothalamus) regions and also performed exploratory whole-brain analyses. SBC analyses included as seeds the NAcc and also regions demonstrating group differences in fALFF (i.e. right lateral OFC and right anterior insula). All analyses used threshold-free cluster enhancement. Mask-restricted analyses revealed stronger fALFF in the right lateral OFC, and weaker fALFF in the right anterior insula, for hyperphagic MDD v. healthy comparison. We also found weaker SBC between the right lateral OFC and left anterior insula for hyperphagic MDD v. healthy comparison. Whole-brain analyses revealed weaker fALFF in the right anterior insula, and stronger SBC between the right lateral OFC and left precentral gyrus, for hyperphagic MDD v. healthy comparison. Findings were no longer significant after controlling for body mass index, which was higher for hyperphagic MDD. Our results suggest hyperphagic MDD may be associated with altered activity in and connectivity between interoceptive and reward regions.

Convergent and divergent gray matter volume abnormalities in unaffected first-degree relatives and ultra-high risk individuals of schizophrenia

High-risk populations of schizophrenia can be mainly identified as genetic high-risk based on putative endophenotypes or ultra-high-risk (UHR) based on clinically manifested symptoms. Previous studies have consistently shown brain structural abnormalities in both genetic high-risk and UHR individuals. In this study, we aimed to disentangle the convergent and divergent pattern of gray matter alterations between UHR and unaffected first-degree relatives from genetic high-risk individuals. We used structural MRI scans and voxel-based morphometry method to examine gray matter volume (GMV) differences among 23 UHR subjects meeting the Structured Interview for Prodromal Syndromes (SIPS) criteria, 18 unaffected first-degree relatives (UFDR), 26 first-episode schizophrenia patients (FES) and 54 healthy controls (CN). We found that a number of brain regions exhibited a monotonically decreasing trend of GMV from CN to UFDR to UHR to FES. Compared with CN, the UHR subjects showed significant decreases of GMV similar to the patients in the inferior temporal gyrus, fusiform gyrus, middle occipital gyrus, insula, and limbic regions. Moreover, the UHR transformed subgroup had significantly lower GMV than UHR non-transformed subgroup in the right inferior temporal/fusiform gyrus. On the other hand, the UFDR subjects only showed significant GMV decreases in the inferior temporal gyrus and fusiform. Moreover, we found GMV in the occipital lobe was negatively correlated with the UHR subjects’ composite positive symptom of SIPS, and GMV in the cerebellum was positively correlated with FES subjects’ symptom severity. Our results suggest that GMV deficits and regional dysfunction are evident prior to the onset of psychosis and are more prominent in the UHR than the UFDR individuals.

Examining Grey Matter Structural Abnormalities in Young People Exposed to Childhood Maltreatment and Peer Victimisation

AimsEarly-life interpersonal stress, particularly childhood maltreatment (CM), is associated with neurobiological abnormalities. However, few studies have investigated the neural effects of peer victimisation (PV). This study examines the common and specific associations between CM, PV and brain structural alterations in healthy youths.MethodsGrey matter volume (GMV) and cortical thickness (CT) data were collected from 105 age-and gender-matched healthy youths (34 CM, 35 PV and 36 controls). Region-of-interest (ROI) and whole-brain analyses were conducted.ResultsFor the ROI, the CM group had smaller GMV than controls in left IFG, bilateral anterior insula, postcentral and lingual regions, which were associated with higher emotional abuse, along with smaller insular GMV than the PV group. The PV group had smaller left lingual GMV than controls, which was positively associated with age of bully onset. At the whole-brain level, both CM and PV groups had smaller GMV than controls in a cluster comprising left post/pre-central, inferior frontal, insula, superior parietal and supramarginal gyri. The PV group alone had increased CT in a cluster comprising left superior frontal, anterior cingulate and medial orbitofrontal gyri, which was related to greater cyberbullying.ConclusionEarly-life interpersonal stress from carers and peers is associated with common structural alterations of the inferior frontal-limbic, sensory and lingual regions involved in cognitive control, emotion and sensory processing. The findings of a CM-specific reduced anterior insular GMV and a PV-specific increased CT in the left medial prefrontal cluster is intriguing and underscores the unique negative effects of CM and PV, particularly cyberbullying.

Structural and Functional MRI Brain Changes in Patients with Schizophrenia Following Electroconvulsive Therapy: A Systematic Review.

Schizophrenia (SZ) is a severe psychiatric disorder typically characterized by multidimensional psychotic syndromes. Electroconvulsive therapy (ECT) is a treatment option for medication-resistant patients with SZ or treating acute symptoms. Although the efficacy of ECT has been demonstrated in clinical use, its therapeutic mechanisms in the brain remain elusive. This study aimed to summarize brain changes on structural magnetic resonance imaging (sMRI) and functional MRI (fMRI) after ECT. According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic review was carried out. The PubMed and Medline databases were systematically searched using the following medical subject headings (MeSH): (electroconvulsive therapy OR ECT) AND (schizophrenia) AND (MRI OR fMRI OR DTI OR DWI). This review yielded 12 MRI studies, including 4 with sMRI, 5 with fMRI and 3 with multimodal MRI. Increases in volumes of the hippocampus and its adjacent regions (parahippocampal gyrus and amygdala), as well as the insula and frontotemporal regions, were noted after ECT. fMRI studies found ECT-induced changes in different brain regions/networks, including the hippocampus, amygdala, default model network, salience network and other regions/networks that are thought to highly correlate with the pathophysiologic characteristics of SZ. The results of the correlation between brain changes and symptom remissions are inconsistent. Our review provides evidence supporting ECT-induced brain changes on sMRI and fMRI in SZ and explores the relationship between these changes and symptom remission.

Common and stimulus-type-specific brain representations of negative affect.

The brain contains both generalized and stimulus-type-specific representations of aversive events, but models of how these are integrated and related to subjective experience are lacking. We combined functional magnetic resonance imaging with predictive modeling to identify representations of generalized (common) and stimulus-type-specific negative affect across mechanical pain, thermal pain, aversive sounds and aversive images of four intensity levels each. This allowed us to examine how generalized and stimulus-specific representations jointly contribute to aversive experience. Stimulus-type-specific negative affect was largely encoded in early sensory pathways, whereas generalized negative affect was encoded in a distributed set of midline, forebrain, insular and somatosensory regions. All models specifically predicted negative affect rather than general salience or arousal and accurately predicted negative affect in independent samples, demonstrating robustness and generalizability. Common and stimulus-type-specific models were jointly important for predicting subjective experience. Together, these findings offer an integrated account of how negative affect is constructed in the brain and provide predictive neuromarkers for future studies.

Learning under social versus nonsocial uncertainty: A meta-analytic approach.

Much of the uncertainty that clouds our understanding of the world springs from the covert values and intentions held by other people. Thus, it is plausible that specialized mechanisms that compute learning signals under uncertainty of exclusively social origin operate in the brain. To test this hypothesis, we scoured academic databases for neuroimaging studies involving learning under uncertainty, and performed a meta‐analysis of brain activation maps that compared learning in the face of social versus nonsocial uncertainty. Although most of the brain activations associated with learning error signals were shared between social and nonsocial conditions, we found some evidence for functional segregation of error signals of exclusively social origin during learning in limited regions of ventrolateral prefrontal cortex and insula. This suggests that most behavioral adaptations to navigate social environments are reused from frontal and subcortical areas processing generic value representation and learning, but that a specialized circuitry might have evolved in prefrontal regions to deal with social context representation and strategic action.

Post-Covid tourism planning: based on the Azores residents’ perceptions about the development of regional tourism

ABSTRACT During 2020, in the context of COVID-19 pandemic, many touristic destinations noticed a drop above 70% of tourists compared to the previous year - 2019. Moreover, some destinations registered a drop in more than 80% in overnight stays compared to 2019. Therefore, in the current period of economic crisis, residents' perceptions of the impacts of tourism may differ substantially from those relating to periods of economic prosperity. Furthermore, the island destinations are more likely to suffer the adverse effects of tourism arising from their inadequate planning. The present study aims to know the current perceptions of residents in an insular region concerning the impacts of tourism development on their islands of residence. In this sense, several statistical methods were used in the scope of a quantitative approach, including some algorithms of Cluster Analysis, and Categorical Principal Component Analysis. Throughout this study, it is possible to identify the “Improve public infrastructure” as the most positive impact of this new tourism paradigm for this ultra-peripheral territory. It becomes clear that the Azores regional actors should follow the sustainable development strategy (including sustainable tourism) if this destination desires to prosper in the post-pandemic period as a potential rural tourism destination.

The cost of attentional reorienting on conscious visual perception: an MEG study.

How do attentional networks influence conscious perception? To answer this question, we used magnetoencephalography in human participants and assessed the effects of spatially nonpredictive or predictive supra-threshold peripheral cues on the conscious perception of near-threshold Gabors. Three main results emerged. (i) As compared with invalid cues, both nonpredictive and predictive valid cues increased conscious detection. Yet, only predictive cues shifted the response criterion toward a more liberal decision (i.e. willingness to report the presence of a target under conditions of greater perceptual uncertainty) and affected target contrast leading to 50% detections. (ii) Conscious perception following valid predictive cues was associated to enhanced activity in frontoparietal networks. These responses were lateralized to the left hemisphere during attentional orienting and to the right hemisphere during target processing. The involvement of frontoparietal networks occurred earlier in valid than in invalid trials, a possible neural marker of the cost of re-orienting attention. (iii) When detected targets were preceded by invalid predictive cues, and thus reorienting to the target was required, neural responses occurred in left hemisphere temporo-occipital regions during attentional orienting, and in right hemisphere anterior insular and temporo-occipital regions during target processing. These results confirm and specify the role of frontoparietal networks in modulating conscious processing and detail how invalid orienting of spatial attention disrupts conscious processing.

Mapping brain-wide excitatory projectome of primate prefrontal cortex at submicron resolution and comparison with diffusion tractography.

Resolving trajectories of axonal pathways in the primate prefrontal cortex remains crucial to gain insights into higher-order processes of cognition and emotion, which requires a comprehensive map of axonal projections linking demarcated subdivisions of prefrontal cortex and the rest of brain. Here, we report a mesoscale excitatory projectome issued from the ventrolateral prefrontal cortex (vlPFC) to the entire macaque brain by using viral-based genetic axonal tracing in tandem with high-throughput serial two-photon tomography, which demonstrated prominent monosynaptic projections to other prefrontal areas, temporal, limbic, and subcortical areas, relatively weak projections to parietal and insular regions but no projections directly to the occipital lobe. In a common 3D space, we quantitatively validated an atlas of diffusion tractography-derived vlPFC connections with correlative green fluorescent protein-labeled axonal tracing, and observed generally good agreement except a major difference in the posterior projections of inferior fronto-occipital fasciculus. These findings raise an intriguing question as to how neural information passes along long-range association fiber bundles in macaque brains, and call for the caution of using diffusion tractography to map the wiring diagram of brain circuits.

Lesion-symptom mapping corroborates lateralization of verbal and nonverbal memory processes and identifies distributed brain networks responsible for memory dysfunction

Memory disorders are a common consequence of cerebrovascular accident (CVA). However, uncertainties remain about the exact anatomical correlates of memory impairment and the material-specific lateralization of memory function in the brain.We used lesion-symptom mapping (LSM) in patients with first-time CVA to identify which brain structures are pivotal for verbal and nonverbal memory and to re-examine whether verbal and nonverbal memory functions are lateralized processes in the brain.The cognitive performance of a relatively large cohort of 114 patients in five classic episodic memory tests was analysed with factor analysis. Two factors were extracted that distinguished the verbal and nonverbal components of these memory tests, and their scores were subsequently tested for anatomical correlates by combining univariate and multivariate LSM.LSM analysis revealed for the verbal factor exclusively left-hemispheric insular, subcortical and adjacent white matter regions and for the nonverbal factor exclusively right-hemispheric temporal, occipital, insular, subcortical and adjacent white matter structures.These results corroborate the long-standing hypothesis of a material-specific lateralization of memory function in the brain and confirm a robust association between right temporal lobe lesions and nonverbal memory dysfunction. The right-hemispheric correlates for the nonverbal aspects of episodic memory include not only classic memory structures in the medial temporal lobe but also a more distributed network that includes cortical and subcortical structures also known for implicit memory processes.

Firm local embeddedness in an insular region: The Åland Islands compared to Finland

The present study analyzes the difference between the Åland Islands — an insular and peripheral part of Finland — and Finland as a whole in terms of firm local embeddedness. The analysis utilizes matched employee-employer longitudinal data for all businesses in Finland, including the Åland Islands, from 2006 to 2014. Local embeddedness is modelled both as tenure (the number of years a key stakeholder in a firm has lived in the same municipality as the firm) and by calculating the geographical distance the key stakeholder lives from the focal firm. Contrary to our expectations, we find that for our tenure measure of local embeddedness, firms are actually less locally embedded in the peripheral region than in the larger country. However, our distance measure of local embeddedness performs as expected with firms in the peripheral region. We hypothesize that that there may be an optimal level of local embeddedness, above which a local firm does not necessarily gain by further increasing its local embeddedness.

Evolution of Neuroimaging Findings in Severe COVID-19 Patients with Initial Neurological Impairment: An Observational Study.

Background and Objectives: Cerebral complications related to the COVID-19 were documented by brain MRIs during the acute phase. The purpose of the present study was to describe the evolution of these neuroimaging findings (MRI and FDG-PET/CT) and describe the neurocognitive outcomes of these patients. Methods: During the first wave of the COVID-19 outbreak between 1 March and 31 May 2020, 112 consecutive COVID-19 patients with neurologic manifestations underwent a brain MRI at Strasbourg University hospitals. After recovery, during follow-up, of these 112 patients, 31 (initially hospitalized in intensive care units) underwent additional imaging studies (at least one brain MRI). Results: Twenty-three men (74%) and eight women (26%) with a mean age of 61 years (range: 18–79) were included. Leptomeningeal enhancement, diffuse brain microhemorrhages, acute ischemic strokes, suspicion of cerebral vasculitis, and acute inflammatory demyelinating lesions were described on the initial brain MRIs. During follow-up, the evolution of the leptomeningeal enhancement was discordant, and the cerebral microhemorrhages were stable. We observed normalization of the vessel walls in all patients suspected of cerebral vasculitis. Four patients (13%) demonstrated new complications during follow-up (ischemic strokes, hypoglossal neuritis, marked increase in the white matter FLAIR hyperintensities with presumed vascular origin, and one suspected case of cerebral vasculitis). Concerning the grey matter volumetry, we observed a loss of volume of 3.2% during an average period of approximately five months. During follow-up, the more frequent FDG-PET/CT findings were hypometabolism in temporal and insular regions. Conclusion: A minority of initially severe COVID-19 patients demonstrated new complications on their brain MRIs during follow-up after recovery.

P540. Psychotic Spectrum Disorder Depressive Subtype and Increased Perfusion in Temporal and Insular Regions

Microvascular pathologies are common in both psychotic spectrum (PSD) and major depression disorders. In this study, we applied a novel three compartment intravoxel incoherent motion and free water imaging (IVIM-FWI) diffusion model to investigate perfusion pathologies in PSD depressive and non-depressive subtypes.

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic.

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

Survey on the presence of Leishmania sp. in peridomestic rodents from the Emilia-Romagna Region (North-Eastern Italy).

Leishmaniasis is a neglected vector-borne parasitic disease caused in Italy only by the species Leishmania infantum of the Leishmania donovani complex, which is the causative agent of the zoonotic visceral leishmaniasis (VL) and the sporadic cutaneous leishmaniasis (CL) in humans, and of the canine leishmaniasis (CanL). The disease is considered endemic in southern, central, and insular Italian regions and recognizes phlebotomine sand flies as vector and dogs as main reservoir. Among northern Italian region, Emilia-Romagna shows peculiar epidemiological situation and recent studies are questioning the role of dog as main reservoir of L. infantum. Due to their synanthropic relationship with humans, rodents have been tested for Leishmania spp. in several European countries. The aim of this study was to assess the presence of Leishmania spp. in peridomestic rodents in the Emilia-Romagna. The study was carried out on 136 peridomestic rodents collected by professional pest control services: 47 brown rats (Rattus norvegicus), 39 black rats (Rattus rattus) and 50 mice (Mus musculus). Specimens of earlobe skin, spleen, liver and prescapular lymph nodes were tested with a real-time PCR. Fifteen (11%) rodents, tested positive for Leishmania spp. in particular five brown rats (10.6%), five black rats (12.8%) and five mice (10%). Positivity was obtained from different target organs. These findings revealed the presence of Leishmania spp. in peridomestic rodents of Emilia-Romagna Region, also in two species never tested before in Italy, namely R. norvegicus and M. musculus.

The Determinants of Vaccine Literacy in the Italian Population: Results from the Health Literacy Survey 2019.

Vaccines are among the most important public health achievements of the last century; however, vaccine awareness and uptake still face significant challenges and the COVID-19 pandemic has only exacerbated this phenomenon. Vaccine Literacy (VL) is the ability to find, understand and judge immunisation-related information to make appropriate immunisation decisions. A cross-sectional study on a sample of 3500 participants, representative of the Italian adult population aged 18+ years, was conducted in Italy in 2021. A validated questionnaire, including sections on health literacy (HL), sociodemographic characteristics, risk factors, and lifestyles of respondents, was used. VL was measured by four items (item 19, 22, 26 and 29) of the HL section. While 67.6% of the respondents had a “good” (47.5%) or “sufficient” (20.1%) level of VL, 32.4% had “limited” VL levels. Although the overall VL level was quite high, many participants reported difficulties in dealing with vaccination information, particularly those with a lower educational level, those living in southern and insular regions of Italy, those with greater financial deprivation and those with a migration background. Improving VL in Italy should be a top priority in the political agenda, with special regard to socially and geographically disadvantaged communities.

The prefrontal cortex shows widespread decrease in H3 histamine receptor binding densities in rats with genetic generalized epilepsies

Distributions of brain H3 histamine receptors in regions of the prefrontal cortex were studied by assessing regional binding densities for [3 H](R)α-methylhistamine in coronal brain slices of normal rats and rats with genetically determined absence and/or audiogenic epilepsies.The three groups of epileptic rats displayed widespread significant decreases in H3 histamine receptor binding densities. A 20–25% decline was seen in the rostral aspects of the lateral prefrontal cortex, namely the granular, dysgranular, and dorsal agranular insular regions. The reduction was not specific for the epilepsy types. The same was observed in the rostral part of the primary cingulate cortex and the secondary midcingulate cortex. On borders of this core effect, several seizure-type specific declines were seen. Namely, the infralimbic, prelimbic and posterior agranular insular cortices demonstrated absence-epilepsy related reductions in the H3 histamine receptor binding densities. A decrease related to audiogenic seizures was noted in the rostral part of the piriform cortex.The pattern of widespread and seizure-type unspecific decline in H3 histamine receptor binding densities points to a common part of brain loops underlying generalized convulsive and non-convulsive types of epilepsy. It also might hint at putative seizure-related changes in the release of histamine from specific fibers innervating the prefrontal area.

Increasing of Blood Brain Barrier Permeability and the Association With Depression and Anxiety in Systemic Lupus Erythematosus Patients.

ObjectiveTo study changes in blood brain barrier (BBB) permeability in systemic lupus erythematosus (SLE) patients, and explore the association between the alterations in BBB permeability and depression/anxiety in SLE.MethodsBrain dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) images were collected from 42 SLE patients and 23 healthy controls (HCs). Based on the Patlak pharmacokinetic model, the Ktrans value of each voxel in the whole brain of each subject was calculated. BBB permeability indicator (the Ktrans value) between SLE patients and healthy control group was compared. Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA) were used to assess the mental health of SLE patients. The difference in BBB permeability was compared on SLE patients with depression/anxiety, SLE patients without depression/anxiety and HCs by ANOVA analysis.ResultsThe Ktrans value of the right insular region of the SLE group was significantly higher than that of the healthy control group. And the Ktrans value of the right insular region in SLE patients with depression/anxiety was significantly increased compared with SLE patients without depression/anxiety and HCs.ConclusionsSLE patients have increased BBB permeability, mainly in the right insular area. The increased BBB permeability in the right insular region is associated with the depression/anxiety in SLE patients.