Changes In Brain Organization Research Articles

Dynamic nonreversibility view of intrinsic brain organization and brain dynamic analysis of repetitive transcranial magnitude stimulation.

Intrinsic neural activities are characterized as endless spontaneous fluctuation over multiple time scales. However, how the intrinsic brain organization changes over time under local perturbation remains an open question. By means of statistical physics, we proposed an approach to capture whole-brain dynamics based on estimating time-varying nonreversibility and k-means clustering of dynamic varying nonreversibility patterns. We first used synthetic fMRI to investigate the effects of window parameters on the temporal variability of varying nonreversibility. Second, using real test-retest fMRI data, we examined the reproducibility, reliability, biological, and physiological correlation of the varying nonreversibility substates. Finally, using repetitive transcranial magnetic stimulation-fMRI data, we investigated the modulation effects of repetitive transcranial magnetic stimulation on varying nonreversibility substate dynamics. The results show that: (i) as window length increased, the varying nonreversibility variance decreased, while the sliding step almost did not alter it; (ii) the global high varying nonreversibility states and low varying nonreversibility states were reproducible across multiple datasets and different window lengths; and (iii) there were increased low varying nonreversibility states and decreased high varying nonreversibility states when the left frontal lobe was stimulated, but not the occipital lobe. Taken together, these results provide a thermodynamic equilibrium perspective of intrinsic brain organization and reorganization under local perturbation.

Differential contribution of between and within-brain coupling to movement synchronization.

A fundamental characteristic of the human brain that supports behavior is its capacity to create connections between brain regions. A promising approach holds that during social behavior, brain regions not only create connections with other brain regions within a brain, but also coordinate their activity with other brain regions of an interaction partner. Here we ask whether between-brain and within-brain coupling contribute differentially to movement synchronization. We focused on coupling between the inferior frontal gyrus (IFG), a brain region associated with the observation-execution system, and the dorsomedial prefrontal cortex (dmPFC), a region associated with error-monitoring and prediction. Participants, randomly divided into dyads, were simultaneously scanned with functional near infra-red spectroscopy (fNIRS) while performing an open-ended 3D hand movement task consisting of three conditions: back-to-back movement, free movement, or intentional synchronization. Results show that behavioral synchrony was higher in the intentional synchrony compared with the back-to-back and free movement conditions. Between-brain coupling in the IFG and dmPFC was evident in the free movement and intentional synchrony conditions but not in the back-to-back condition. Importantly, between-brain coupling was found to positively predict intentional synchrony, while within-brain coupling was found to predict synchronization during free movement. These results indicate that during intentional synchronization, brain organization changes such that between-brain networks, but not within-brain connections, contribute to successful communication, pointing to shift from a within-brain feedback loop to a two-brains feedback loop.

Association between circadian sleep regulation and cortical gyrification in young and older adults.

The circadian system orchestrates sleep timing and structure and is altered with increasing age. Sleep propensity, and particularly REM sleep is under strong circadian control and has been suggested to play an important role in brain plasticity. In this exploratory study, we assessed whether surface-based brain morphometry indices are associated with circadian sleep regulation and whether this link changes with age. Twenty-nine healthy older (55-82 years; 16 men) and 28 young participants (20-32 years; 13 men) underwent both structural magnetic resonance imaging and a 40-h multiple nap protocol to extract sleep parameters over day and night time. Cortical thickness and gyrification indices were estimated from T1-weighted images acquired during a classical waking day. We observed that REM sleep was significantly modulated over the 24-h cycle in both age groups, with older adults exhibiting an overall reduction in REM sleep modulation compared to young individuals. Interestingly, when taking into account the observed overall age-related reduction in REM sleep throughout the circadian cycle, higher day-night differences in REM sleep were associated with increased cortical gyrification in the right inferior frontal and paracentral regions in older adults. Our results suggest that a more distinctive allocation of REM sleep over the 24-h cycle is associated with regional cortical gyrification in aging, and thereby point towards a protective role of circadian REM sleep regulation for age-related changes in brain organization.

An examination of white matter integrity and functional network organization in Subjective Cognitive Decline using Diffusional Kurtosis Imaging‐based tractography resting state fMRI

AbstractBackgroundSubjective Cognitive Decline (SCD) may increase the risk of Alzheimer’s Disease and related dementias (ADRD). Brain network changes may occur at both the functional and structural levels in SCD. To better understand these network changes, diffusional kurtosis imaging (DKI) and functional connectome (FC) measures were examined in SCD and healthy controls (HC). We hypothesized that DKI and FC measures in the medial temporal lobe would be sensitive to differences between SCD and healthy controls (HC).MethodSCD classification was determined by clinician evaluation or Everyday Cognition average item score > 1.6, with objectively healthy cognitive performance (Montreal Cognitive Assessment score > 22). DKI and fMRI images were acquired on a Siemens PRISMA scanner in 33 SCD subjects and 33 age/sex matched HCs (mean age=69.1 yr). DSI Studio’s Automatic fiber tracking extracted the DKI measure mean kurtosis (MK) in 3 bilateral white matter bundles associated with ADRD: inferior longitudinal fasciculus (ILF), cingulum‐parahippocampus (CP), and uncinate fasciculus (UF). FC measure eigenvector centrality (Evc) was calculated in 19 medial temporal nodes using the Brain Connectivity Toolbox. Generalized Linear Models were conducted for each bundle to determine whether EVC in the left ventrolateral amygdala (LAMG) was predicted by MK, Diagnosis or the MK*Diagnosis interaction.ResultIn the left ILF, the effect of MK approached significance (p = .054) as did the MK*Diagnosis interaction (p = .059). Lower MK was associated with higher LAMG EVC in HC (r = ‐.40) but there was no association between MK and EVC for SCD. In the left UF, effect of MK was significant (p = .003), but no effect of Diagnosis and no interaction. Lower MK was associated with higher LAMG EVC (r = ‐.359) in both SCD and HC.ConclusionThis preliminary study showed that in HC, white matter integrity of the ILF was associated with LAMG functional connectivity, but this association was absent in SCD. While present results should be interpreted with caution due to small sample sizes, these findings indicate that healthy aging structural‐functional associations are disrupted in SCD, but future studies should determine whether this is a reliable marker of brain organization changes.

An Adolescent Confronted With Cluttering: The Story of Johan

Purpose: This case study is presented to inform the reader of potential speech, language, cognitive, and emotional characteristics in preadolescent cluttering. Method: This case study describes a 10-year-old boy who started to clutter during preadolescence. The case illustrates that, in some adolescents, cluttering can co-occur with temporary stuttering-like behavior. In this case, signs of disturbances in speech-language production associated with behavioral impulsiveness as a young child were noted. Speech, language, cognitive, and emotional results of the case are reported in detail. Results: The changes in fluency development are reported and discussed within the context of changes in the adolescent brain as well as adolescent cognitive and emotional development. While being unaware of their speech condition before adolescence, during preadolescence, the changes in brain organization lead to an increase in rate and a decrease in speech control. Given that the client had limited understanding of what was occurring, they were at risk of developing negative communication attitudes. Speech-language therapists are strongly advised to monitor children with cluttering signals in the early years of their adolescence. Supplemental Material: https://doi.org/10.23641/asha.21318072

INSP-07. Improving cognitive outcomes for children treated for cancer: moving beyond the cure

With improved survival rates, increasing numbers of childhood cancer survivors are living with long-term cognitive deficits that negatively impact their ability to attain important life milestones. Our collaborative research program has focused on characterizing cognitive outcomes associated with specific treatment modalities to inform modifications in front-line therapy. This line of investigation has demonstrated the negative cognitive impact of high radiation dose/large treatment volume, posterior fossa syndrome, treatment-related ototoxicity, and lower socioeconomic status (SES). Research-informed, treatment approaches under investigation include proton radiotherapy and hippocampal sparing irradiation, molecularly based risk-adapted therapy, surgical approaches with reduced risk of injury, and on-therapy otoprotectants, as well as identification of modifiable factors that are driving the protective effects of higher SES. Our research also strives to improve specification of cognitive deficits following treatment, at the behavioral and neural systems level, to identify targets for intervention. Study findings have revealed disease- and treatment- related alterations in neural systems supporting attention, working memory, and executive functions, as well as genetic factors that increase risk for cognitive late effects. We are now using sophisticated connectivity brain mapping with enhanced sensitivity to behaviorally-relevant changes in brain organization and sensitivity to intervention-based neuroplasticity to guide cognitive intervention development. A primary research focus moving forward is development of empirically validated interventions that prevent or mitigate cognitive late effects among childhood cancer survivors. Our studies have demonstrated the efficacy of stimulant medications, computerized cognitive training, and aerobic exercise for children treated for cancer, as well as the limitations to these approaches. Current investigations include use of neuroprotectant agents during radiotherapy, combining multiple interventions, interventions tailored to children undergoing treatment in infancy, and the use of virtual reality to increase intervention engagement. Our ultimate benchmark of success is ensuring children are not only cured of cancer but also experience a high quality of life.

IMG-14. Cognitive deficits and altered functional network organization in pediatric brain tumor patients

INTRODUCTION: Survivors of pediatric brain tumors experience significant cognitive deficits from their diagnosis and treatment. Mechanisms of cognitive injury are poorly understood, and predictors of long-term outcome are lacking. Large-scale, distributed brain systems provide a window into brain organization and function that may yield insight into these mechanisms and outcomes. Recent evidence suggests that systems-level changes are related to cognitive performance across the lifespan. Here, we evaluated brain network architecture, cognitive performance, and brain-behavior relationships in pediatric brain tumor patients. METHODS: Forty-nine patients (ages 7-18y.o.) with any brain tumor diagnosis underwent resting state functional Magnetic Resonance Imaging (rsfMRI) during regularly scheduled clinical visits. All patients were tested with the NIH Toolbox Cognition Battery. One-hundred thirty-nine age- and sex-matched typically developing children were used as controls. All data were processed to minimize artifactual sources of variance. Functional brain networks were created for each patient via rsfMRI data from 300 regions of interest that sample the whole brain. Multilinear models were implemented to examine brain-behavior relationships, while accounting for demographic and clinical factors. RESULTS: Functional network organization was significantly altered in patients compared to controls (p<0.001). Network organization was more affected in patients who received whole-brain radiation therapy than those who did not (t=2.52, p<0.015). Patients demonstrated significant impairments in multiple domains of cognitive performance, e.g. attention (p<0.0001). Weak relationships were found between cognitive performance and network organization, none of which survived multiple comparison correction. CONCLUSIONS: Brain network architecture is significantly altered in pediatric brain tumor patients. Whole-brain radiation was related to the largest changes. Most network and cognitive changes were significant with large effect sizes, yet brain-behavior relationships were weak. Our results suggest that systems-level changes in brain organization may provide insight into long-term changes in brain function in pediatric brain tumor patients.

Enhancing cortical network-level participation coefficient as a potential mechanism for transfer in cognitive training in aMCI

Effective cognitive training must improve cognition beyond the trained domain (show a transfer effect) and be applicable to dementia-risk populations, e.g., amnesic mild cognitive impairment (aMCI). Theories suggest training should target processes that 1) show robust engagement, 2) are domain-general, and 3) reflect long-lasting changes in brain organization. Brain regions that connect to many different networks (i.e., show high participation coefficient; PC) are known to support integration. This capacity is 1) relatively preserved in aMCI, 2) required across a wide range of cognitive domains, and 3) trait-like. In 49 individuals with aMCI that completed a 6-week visual speed of processing training (VSOP) and 28 active controls, enhancement in PC was significantly more related to transfer to working memory at global and network levels in VSOP compared to controls, particularly in networks with many high-PC nodes. This suggests that enhancing brain integration may provide a target for developing effective cognitive training.

Reconstructing prehistoric languages

This theme issue builds on the surge of interest in the field of language evolution as part of the broader field of human evolution, gathering some of the field's most prominent experts in order to achieve a deeper, richer understanding of human prehistory and the nature of prehistoric languages. Taken together, the contributions to this issue begin to outline a profile of the structural and functional features of prehistoric languages, including the type of sounds, the nature of the earliest grammars, the characteristics of the earliest vocabularies and some preferred uses, like conversation and insult. By also correlating certain specific features of language with the changes in brain organization during prehistory, the contributions to this issue directly engage the genetic and the neuroscientific aspects of human evolution and cognition. This article is part of the theme issue 'Reconstructing prehistoric languages'.

Across-session consistency of context-driven language processing: A magnetoencephalography study.

Changes in brain organization following damage are commonly observed, but they remain poorly understood. These changes are often studied with imaging techniques that overlook the temporal granularity at which language processes occur. By contrast, electrophysiological measures provide excellent temporal resolution. To test the suitability of magnetoencephalography (MEG) to track language‐related neuroplasticity, the present study aimed at establishing the spectro‐temporo‐spatial across‐session consistency of context‐driven picture naming in healthy individuals, using MEG in two test–retest sessions. Spectro‐temporo‐spatial test–retest consistency in a healthy population is a prerequisite for studying neuronal changes in clinical populations over time. For this purpose, 15 healthy speakers were tested with MEG while performing a context‐driven picture‐naming task at two time points. Participants read a sentence missing the final word and named a picture completing the sentence. Sentences were constrained or unconstrained toward the picture, such that participants could either retrieve the picture name through sentence context (constrained sentences), or could only name it after the picture appeared (unconstrained sentences). The context effect (constrained versus unconstrained) in picture‐naming times had a strong effect size and high across‐session consistency. The context MEG results revealed alpha–beta power decreases (10–20 Hz) in the left temporal and inferior parietal lobule that were consistent across both sessions. As robust spectro‐temporo‐spatial findings in a healthy population are required for working toward longitudinal patient studies, we conclude that using context‐driven language production and MEG is a suitable way to examine language‐related neuroplasticity after brain damage.

Oxycodone Exposure: A Magnetic Resonance Imaging Study in Response to Acute and Chronic Oxycodone Treatment in Rats

The present study was designed to use blood-oxygen-level dependent (BOLD) imaging to “fingerprint” the change in activity in response to oxycodone (OXY) in drug naïve rats before and after repeated exposure to OXY. It was hypothesized that repeated exposure to OXY would initiate adaptive changes in brain organization that would be reflected in an altered response to opioid exposure. Male rats exposed to OXY repeatedly showed conditioned place preference, evidence of drug-seeking behavior and putative neuroadaptation. As these studies were done on awake rats we discovered it was not possible to image rats continuously exposed to OXY due to motion artifact judged to be withdrawal while in the scanner. To circumvent this problem manganese-enhanced MRI (MEMRI) was used to map the distributed integrated activity pattern resulting from continuous OXY exposure. Rats were administered OXY (2.5 mg/kg, i.p.) during image acquisition and changes in BOLD signal intensity were recorded and the activation and deactivation of integrated neural circuits involved in olfaction and motivation were identified. Interestingly, the circuitry of the mesencephalic dopaminergic system showed little activity to the first exposure of OXY. In the MEMRI study, rats received OXY treatments (2.5 mg/kg, twice daily) for four consecutive days following intraventricular MnCl2. Under isoflurane anesthesia, T1-weighted images were acquired and subsequently analyzed showing activity in the forebrain limbic system, ventral striatum, accumbens, amygdala and hippocampus. These results show brain activity is markedly different when OXY is presented to drug naïve rats versus rats with prior, repeated exposure to drug.

Event-related brain potential correlates of brain reorganization of episodic memory throughout the adult lifespan.

The main aim of this study was to characterize the age-related evolution of the event-related brain potentials correlates of successful to tackle the neural reorganization patterns associated with this episodic retrieval. We thus examined the evolution of the event-related brain potential old/new effect across the adult lifespan, in five groups, aged 21-70 years (21-30, 31-40, 41-50, 51-60, and 61-70 years), equalized on their memory performance through a word-stem cued-recall task. This procedure makes it possible to examine the evolution of age-related changes in brain organization during adulthood and to specify the age onset of these changes. Results confirm the hypothesis that aging is associated with major changes in brain functioning. These changes appear to be consistent with both the HAROLD and PASA hypotheses, which postulate an age-related reduction in hemispheric asymmetry and a shift from posterior to anterior areas, respectively. What is notable is that these patterns of brain reorganization are not specific to older stages of life as they begin to develop very early in adult life, around the 30s, and the shift toward frontal regions strengthens in the 60s.

Functional Brain Connectivity during Multiple Motor Imagery Tasks in Spinal Cord Injury.

Reciprocal communication of the central and peripheral nervous systems is compromised during spinal cord injury due to neurotrauma of ascending and descending pathways. Changes in brain organization after spinal cord injury have been associated with differences in prognosis. Changes in functional connectivity may also serve as injury biomarkers. Most studies on functional connectivity have focused on chronic complete injury or resting-state condition. In our study, ten right-handed patients with incomplete spinal cord injury and ten age- and gender-matched healthy controls performed multiple visual motor imagery tasks of upper extremities and walking under high-resolution electroencephalography recording. Directed transfer function was used to study connectivity at the cortical source space between sensorimotor nodes. Chronic disruption of reciprocal communication in incomplete injury could result in permanent significant decrease of connectivity in a subset of the sensorimotor network, regardless of positive or negative neurological outcome. Cingulate motor areas consistently contributed the larger outflow (right) and received the higher inflow (left) among all nodes, across all motor imagery categories, in both groups. Injured subjects had higher outflow from left cingulate than healthy subjects and higher inflow in right cingulate than healthy subjects. Alpha networks were less dense, showing less integration and more segregation than beta networks. Spinal cord injury patients showed signs of increased local processing as adaptive mechanism. This trial is registered with NCT02443558.

Predictive assessment of models for dynamic functional connectivity

In neuroimaging, it has become evident that models of dynamic functional connectivity (dFC), which characterize how intrinsic brain organization changes over time, can provide a more detailed representation of brain function than traditional static analyses. Many dFC models in the literature represent functional brain networks as a meta-stable process with a discrete number of states; however, there is a lack of consensus on how to perform model selection and learn the number of states, as well as a lack of understanding of how different modeling assumptions influence the estimated state dynamics. To address these issues, we consider a predictive likelihood approach to model assessment, where models are evaluated based on their predictive performance on held-out test data. Examining several prominent models of dFC (in their probabilistic formulations) we demonstrate our framework on synthetic data, and apply it on two real-world examples: a face recognition EEG experiment and resting-state fMRI. Our results evidence that both EEG and fMRI are better characterized using dynamic modeling approaches than by their static counterparts, but we also demonstrate that one must be cautious when interpreting dFC because parameter settings and modeling assumptions, such as window lengths and emission models, can have a large impact on the estimated states and consequently on the interpretation of the brain dynamics.

Longitudinal Structural and Functional Differences Between Proportional and Poor Motor Recovery After Stroke

Background. Evolution of motor function during the first months after stroke is stereotypically bifurcated, consisting of either recovery to about 70% of maximum possible improvement (“proportional recovery, PROP”) or in little to no improvement (“poor recovery, POOR”). There is currently no evidence that any rehabilitation treatment will prevent POOR and favor PROP. Objective. To perform a longitudinal and multimodal assessment of functional and structural changes in brain organization associated with PROP. Methods. Fugl-Meyer Assessments of the upper extremity and high-density electroencephalography (EEG) were obtained from 63 patients, diffusion tensor imaging from 46 patients, at 2 and 4 weeks (T0) and at 3 months (T1) after stroke onset. Results. We confirmed the presence of 2 distinct recovery patterns (PROP and POOR) in our sample. At T0, PROP patients had greater integrity of the corticospinal tract (CST) and greater EEG functional connectivity (FC) between the affected hemisphere and rest of the brain, in particular between the ventral premotor and the primary motor cortex. POOR patients suffered from degradation of corticocortical and corticofugal fiber tracts in the affected hemisphere between T0 and T1, which was not observed in PROP patients. Better initial CST integrity correlated with greater initial global FC, which was in turn associated with less white matter degradation between T0 and T1. Conclusions. These findings suggest links between initial CST integrity, systems-level cortical network plasticity, reduction of white matter atrophy, and clinical motor recovery after stroke. This identifies candidate treatment targets.

A Systematic Review of Investigations into Functional Brain Connectivity Following Spinal Cord Injury.

Background: Complete or incomplete spinal cord injury (SCI) results in varying degree of motor, sensory and autonomic impairment. Long-lasting, often irreversible disability results from disconnection of efferent and afferent pathways. How does this disconnection affect brain function is not so clear. Changes in brain organization and structure have been associated with SCI and have been extensively studied and reviewed. Yet, our knowledge regarding brain connectivity changes following SCI is overall lacking.Methods: In this study we conduct a systematic review of articles regarding investigations of functional brain networks following SCI, searching on PubMed, Scopus and ScienceDirect according to PRISMA-P 2015 statement standards.Results: Changes in brain connectivity have been shown even during the early stages of the chronic condition and correlate with the degree of neurological impairment. Connectivity changes appear as dynamic post-injury procedures. Sensorimotor networks of patients and healthy individuals share similar patterns but new functional interactions have been identified as unique to SCI networks.Conclusions: Large-scale, multi-modal, longitudinal studies on SCI patients are needed to understand how brain network reorganization is established and progresses through the course of the condition. The expected insight holds clinical relevance in preventing maladaptive plasticity after SCI through individualized neurorehabilitation, as well as the design of connectivity-based brain-computer interfaces and assistive technologies for SCI patients.

Comprehensive transcriptome analysis of neocortical layers in humans, chimpanzees and macaques.

While human cognitive abilities are clearly unique, underlying changes in brain organization and function remain unresolved. Here we characterized the transcriptome of the cortical layers and adjacent white matter in the prefrontal cortexes of humans, chimpanzees and rhesus macaques using unsupervised sectioning followed by RNA sequencing. More than 20% of detected genes were expressed predominantly in one layer, yielding 2,320 human layer markers. While the bulk of the layer markers were conserved among species, 376 switched their expression to another layer in humans. By contrast, only 133 of such changes were detected in the chimpanzee brain, suggesting acceleration of cortical reorganization on the human evolutionary lineage. Immunohistochemistry experiments further showed that human-specific expression changes were not limited to neurons but affected a broad spectrum of cortical cell types. Thus, despite apparent histological conservation, human neocortical organization has undergone substantial changes affecting more than 5% of its transcriptome.

Temporal metastates are associated with differential patterns of time-resolved connectivity, network topology, and attention

Little is currently known about the coordination of neural activity over longitudinal timescales and how these changes relate to behavior. To investigate this issue, we used resting-state fMRI data from a single individual to identify the presence of two distinct temporal states that fluctuated over the course of 18 mo. These temporal states were associated with distinct patterns of time-resolved blood oxygen level dependent (BOLD) connectivity within individual scanning sessions and also related to significant alterations in global efficiency of brain connectivity as well as differences in self-reported attention. These patterns were replicated in a separate longitudinal dataset, providing additional supportive evidence for the presence of fluctuations in functional network topology over time. Together, our results underscore the importance of longitudinal phenotyping in cognitive neuroscience.

Reorganization of brain networks after spinal cord injury: a qualitative synthesis of the literature

Reorganization of brain networks after spinal cord injury: a qualitative synthesis of the literature

Prenatal binge-like alcohol exposure alters brain and systemic responses to reach sodium and water balance

The aim of the present work is to analyze how prenatal binge-like ethanol exposure to a moderate dose (2.0g/kg; group Pre-EtOH) during gestational days (GD) 17–20 affects hydroelectrolyte regulatory responses. This type of exposure has been observed to increase ethanol consumption during adolescence (postnatal day 30–32). In this study we analyzed basal brain neural activity and basal-induced sodium appetite (SA) and renal response stimulated by sodium depletion (SD) as well as voluntary ethanol consumption as a function of vehicle or ethanol during late pregnancy. In adolescent offspring, SD was induced by furosemide and a low-sodium diet treatment (FURO+LSD). Other animals were analyzed in terms of immunohistochemical detection of Fra-like (Fra-LI-ir) protein and serotonin (5HT) and/or vasopressin (AVP). The Pre-EtOH group exhibited heightened voluntary ethanol intake and a reduction in sodium and water intake induced by SD relative to controls. Basal Na and K concentrations in urine were also reduced in Pre-EtOH animals while the induced renal response after FURO treatment was similar across prenatal treatments. However, the correlation between urine volume and water intake induced by FURO significantly varied across these treatments. At the brain level of analysis, the number of basal Fra-LI-ir was significantly increased in AVP magnocellular neurons of the paraventricular nucleus (PVN) and in 5HT neurons in the dorsal raphe nucleus (DRN) in Pre-EtOH pups. In the experimental group, we also observed a significant increase in Fra-LI along the nucleus of the solitary tract (NTS) and in the central extended amygdala nuclei. In summary, moderate Pre-EtOH exposure produces long-lasting changes in brain organization, affecting basal activity of central extended amygdala nuclei, AVP neurons and the inhibitory areas of SA such as the NTS and the 5HT-DRN. These changes possibly modulate the above described variations in basal-induced drinking behaviors and renal regulatory responses.