Quantitative Assessment of Pathological Tau Burden in Essential Tremor: A Postmortem Study.

Essential tremor (ET) patients develop more cognitive impairment and dementia than controls, although there are surprisingly few data on the neuropathological basis for cognitive changes in ET. In this postmortem study, we assessed tau and other pathologies in 26 ET cases and 73 controls (non-ET) (1:3 matching). The mean age = 88.6 years; 55% were cognitively normal, 24% had mild cognitive impairment (MCI), and 20% had dementia. We found similar burdens of pathology using Braak, ß-amyloid and Lewy body assessments in ET and controls. In contrast, among cognitively normal subjects, ET cases had a higher number of NFT-positive neurons in the neocortex than controls (p < 0.001); the number of NFT-positive neurons in the medial temporal lobe was similar in these 2 groups (p = 0.22). Among subjects with MCI, ET cases also had higher numbers of NFT-positive neurons in the neocortex than controls (p < 0.001) but again, not in the medial temporal lobe (p = 0.55). Among subjects with dementia, the number of NFT-positive neurons was similar in ET cases and controls. Cognitive function correlated with quantitative neurofibrillary tangle counts in ET cases and controls. In the context of ET, pre-dementia tau burden is higher than in the absence of ET, suggesting a predisposition to tau pathology.

Device-Embedded Cameras for Eye Tracking-Based Cognitive Assessment: Validation With Paper-Pencil and Computerized Cognitive Composites.

BACKGROUND: As eye tracking-based assessment of cognition becomes more widely used in older adults, particularly those at risk for dementia, reliable and scalable methods to collect high-quality data are required. Eye tracking-based cognitive tests that utilize device-embedded cameras have the potential to reach large numbers of people as a screening tool for preclinical cognitive decline. However, to fully validate this approach, more empirical evidence about the comparability of eyetracking-based paradigms to existing cognitive batteries is needed. OBJECTIVE: Using a population of clinically normal older adults, we examined the relationship between a 30-minute Visual Paired Comparison (VPC) recognition memory task and cognitive composite indices sensitive to a subtle decline in domains associated with Alzheimer disease. Additionally, the scoring accuracy between software used with a commercial grade eye tracking camera at 60 frames per second (FPS) and a manually scored procedure used with a laptop-embedded web camera (3 FPS) on the VPC task was compared, as well as the relationship between VPC task performance and domain-specific cognitive function. METHODS: A group of 49 clinically normal older adults completed a 30-min VPC recognition memory task with simultaneous recording of eye movements by a commercial-grade eye-tracking camera and a laptop-embedded camera. Relationships between webcam VPC performance and the Preclinical Alzheimer Cognitive Composite (PACC) and National Institutes of Health Toolbox Cognitive Battery (NIHTB-CB) were examined. Inter-rater reliability for manually scored tests was analyzed using Krippendorff's kappa formula, and we used Spearman's Rho correlations to investigate the relationship between VPC performance scores with both cameras. We also examined the relationship between VPC performance with the device-embedded camera and domain-specific cognitive performance. RESULTS: Modest relationships were seen between mean VPC novelty preference and the PACC (r=.39, P=.007) and NIHTB-CB (r=.35, P=.03) composite scores, and additional individual neurocognitive task scores including letter fluency (r=.33, P=.02), category fluency (r=.36, P=.01), and Trail Making Test A (-.40, P=.006). Robust relationships were observed between the 60 FPS eye tracker and 3 FPS webcam on both trial-level VPC novelty preference (r=.82, P<.001) and overall mean VPC novelty preference (r=.92 P<.001). Inter-rater agreement of manually scored web camera data was high (kappa=.84). CONCLUSIONS: In a sample of clinically normal older adults, performance on a 30-minute VPC task correlated modestly with computerized and paper-pencil based cognitive composites that serve as preclinical Alzheimer disease cognitive indices. The strength of these relationships did not differ between camera devices. We suggest that using a device-embedded camera is a reliable and valid way to assess performance on VPC tasks accurately and that these tasks correlate with existing cognitive composites.

Longitudinal assessment of chemotherapy-induced changes in brain and cognitive functioning: A systematic review.

In addition to the burden of a life-threatening diagnosis, cancer patients are struggling with adverse side-effects from cancer treatment. Chemotherapy has been linked to an array of cognitive impairments and alterations in brain structure and function ("chemobrain"). In this review, we summarized the existing evidence that evaluate the changes in cognitive functioning and brain with chemotherapy, as assessed using structural and functional MRI-based techniques in a longitudinal design. This review followed the latest PRISMA guidelines using Embase, Medline, PsychINFO, Scopus, and Web of Science databases with date restrictions from 2012 to 2017. Fourteen research articles met the key inclusion criteria: (i) the studies involved adult cancer patients (mean age ≥ 18); (ii) the use of chemotherapy in the treatment of cancer; (iii) pre-post assessment of behavioral and brain-based outcomes; and (iv) abstracts written in English. Effect sizes of subjective and objective cognitive impairments from the reviewed studies were estimated using Cohen's d or z-scores. We calculated percentage of mean change or effect sizes for main neuroimaging findings when data were available. Strength of the correlations between brain alterations and cognitive changes was obtained using squared correlation coefficients. Small to medium effect sizes were shown? on individual tests of attention, processing speed, verbal memory, and executive control; and medium effect sizes on self-report questionnaires. Neuroimaging data showed reduced grey matter density in cancer patients in frontal, parietal, and temporal regions. Changes in brain function (brain activation and cerebral blood flow) were observed with cancer across functional networks involving (pre)frontal, parietal, occipital, temporal, and cerebellar regions. Data from diffusion-weighted MRI suggested reduced white matter integrity involving the superior longitudinal fasciculus, corpus callosum, forceps major, and corona radiate, and altered structural connectivity across the whole brain network. Finally, we observed moderate-to-strong correlations between worsening cognitive function and morphological changes in frontal brain regions. While MRI is a powerful tool for detection of longitudinal brain changes in the 'chemobrain', the underlying biological mechanisms are still unclear. Continued work in this field will hopefully detect MRI metrics to be used as biomarkers to help guide cognitive treatment at the individual cancer patient level.

Modified Pfeffer Questionnaire for Functional Assessment in Parkinson Disease.

INTRODUCTION: It was proposed to modify the Pfeffer questionnaire (PQ) for functional assessment in patients with Parkinson disease (PD). AIM: To determine the cutoff score for diagnosis of functional impairment in patients with PD by modified PQ (mPQ). METHODS: A total of 110 patients with PD were enrolled into the study, and a neuropsychological test battery was performed to assess their cognitive status. Regarding functional assessment, the mPQ has been applied, and their results were compared to the functional assessment by Informant Questionnaire on Cognitive Decline in the Elderly adapted for use in Brazil (IQCODE-BR). The statistical analysis was accomplished through receiver operating characteristic (ROC) curve with evaluation of the area under the curve, sensitivity, and specificity of the new cutoff point. RESULTS: Eighty-nine patients with PD were evaluated with a mean age of 63.69 ± 9.14 years. Cognitive status categorization was 28.10% as normal, 44.94% as mild cognitive impairment, and 26.96% of patients as dementia associated with PD. The average score on PQ was 3.49 ± 4.79 and on the mPQ 2.56 ± 3.49. In IQCODE-BR, the average score was 6.75 ± 32.72. The ROC curve for the new cutoff point presented 47.4% sensitivity, 88.10% specificity, and 0.757 of area under the curve, with a standard deviation of 0.055 (95% confidence interval: 0.650-0.864). CONCLUSION: 3.5 is proposed as the cutoff point to define functional impairment in patients with PD by mPQ.

Financial Exploitation Is Associated With Structural and Functional Brain Differences in Healthy Older Adults.

BACKGROUND: Age-related brain changes leading to altered socioemotional functioning may increase vulnerability to financial exploitation. If confirmed, this would suggest a novel mechanism leading to heightened financial exploitation risk in older adults. Development of predictive neural markers could facilitate increased vigilance and prevention. In this preliminary study, we sought to identify structural and functional brain differences associated with financial exploitation in older adults. METHODS: Financially exploited older adults (n = 13, 7 female) and a matched cohort of older adults who had been exposed to, but avoided, a potentially exploitative situation (n = 13, 7 female) were evaluated. Using magnetic resonance imaging, we examined cortical thickness and resting state functional connectivity. Behavioral data were collected using standardized cognitive assessments, self-report measures of mood and social functioning. RESULTS: The exploited group showed cortical thinning in anterior insula and posterior superior temporal cortices, regions associated with processing affective and social information, respectively. Functional connectivity encompassing these regions, within default and salience networks, was reduced, while between network connectivity was increased. Self-reported anger and hostility was higher for the exploited group. CONCLUSIONS: We observed financial exploitation associated with brain differences in regions involved in socioemotional functioning. These exploratory and preliminary findings suggest that alterations in brain regions implicated in socioemotional functioning may be a marker of financial exploitation risk. Large-scale, prospective studies are necessary to validate this neural mechanism, and develop predictive markers for use in clinical practice.

Cognitive assessment in Amyotrophic Lateral Sclerosis by means of P300-Brain Computer Interface: a preliminary study.

OBJECTIVE: To investigate the use of P300-based Brain Computer Interface (BCI) technology for the administration of motor-verbal free cognitive tests in Amyotrophic Lateral Sclerosis (ALS). METHODS: We recruited 15 ALS patients and 15 age- and education-matched healthy subjects. All participants underwent a BCI-based neuropsychological assessment, together with two standard cognitive screening tools (FAB, MoCA), two psychological questionnaires (BDI, STAI-Y) and a usability questionnaire. For patients, clinical and respiratory examinations were also performed, together with a behavioural assessment (FBI). RESULTS: Correlations were observed between standard cognitive and BCI-based neuropsychological assessment, mainly concerning execution times in the ALS group. Moreover, patients provided positive rates concerning the BCI perceived usability and subjective experience. Finally, execution times at the BCI-based neuropsychological assessment were useful to discriminate patients from controls, with patients achieving lower processing speed than controls regarding executive functions. CONCLUSIONS: The developed motor-verbal free neuropsychological battery represents an innovative approach, that could provide relevant information for clinical practice and ethical issues. Its use for cognitive evaluation throughout the course of ALS, currently not available by means of standard assessment, must be addressed in further longitudinal validation studies. Further work will be aimed at refining the developed system and enlarging the cognitive spectrum investigated.

Effect of common neuropathologies on progression of late life cognitive impairment.

Brain pathologies of Alzheimer's (AD), cerebrovascular, and Lewy body diseases are common in old age, but the relationship of these pathologies with progression from normal cognitive function to the various stages of cognitive impairment is unknown. In this study, we fit latent Markov models from longitudinal cognitive data to empirically derive 3 latent stages corresponding to no impairment, mild impairment, and moderate impairment; then, we examined the associations of common neuropathologies with the rates of transition among these stages. Cognitive and neuropathological data were available from 653 autopsied participants in 2 ongoing cohort studies of aging who were cognitively healthy at baseline (mean baseline age 79.1 years) and had longitudinal cognitive data. On average, participants in these analyses developed mild impairment 5 years after enrollment, progressed to moderate impairment after an additional 3.4 years, and stayed impaired for 2.8 years until death. AD and chronic macroscopic infarcts were associated with a higher risk of progression to mild impairment and subsequently to moderate impairment. By contrast, Lewy bodies were associated only with progression from mild to moderate impairment. The 5-year probability of progression to mild or moderate impairment was 20% for persons without any of these 3 pathologies, 38% for AD only, 51% for AD and macroscopic infarcts, and 56% for AD, infarcts, and Lewy bodies. Thus, the presence of AD pathology alone nearly doubles the risk of developing cognitive impairment in late life, and the presence of multiple pathologies further increases this risk over multiple years before death.

Design and methods of the NiCK study: neurocognitive assessment and magnetic resonance imaging analysis of children and young adults with chronic kidney disease.

BACKGROUND: Chronic kidney disease is strongly linked to neurocognitive deficits in adults and children, but the pathophysiologic processes leading to these deficits remain poorly understood. The NiCK study (Neurocognitive Assessment and Magnetic Resonance Imaging Analysis of Children and Young Adults with Chronic Kidney Disease) seeks to address critical gaps in our understanding of the biological basis for neurologic abnormalities in chronic kidney disease. In this report, we describe the objectives, design, and methods of the NiCK study. DESIGN/METHODS: The NiCK Study is a cross-sectional cohort study in which neurocognitive and neuroimaging phenotyping is performed in children and young adults, aged 8 to 25 years, with chronic kidney disease compared to healthy controls. Assessments include (1) comprehensive neurocognitive testing (using traditional and computerized methods); (2) detailed clinical phenotyping; and (3) multimodal magnetic resonance imaging (MRI) to assess brain structure (using T1-weighted MRI, T2-weighted MRI, and diffusion tensor imaging), functional connectivity (using functional MRI), and blood flow (using arterial spin labeled MRI). Primary analyses will examine group differences in neurocognitive testing and neuroimaging between subjects with chronic kidney disease and healthy controls. Mechanisms responsible for neurocognitive dysfunction resulting from kidney disease will be explored by examining associations between neurocognitive testing and regional changes in brain structure, functional connectivity, or blood flow. In addition, the neurologic impact of kidney disease comorbidities such as anemia and hypertension will be explored. We highlight aspects of our analytical approach that illustrate the challenges and opportunities posed by data of this scope. DISCUSSION: The NiCK study provides a unique opportunity to address key questions about the biological basis of neurocognitive deficits in chronic kidney disease. Understanding these mechanisms could have great public health impact by guiding screening strategies, delivery of health information, and targeted treatment strategies for chronic kidney disease and its related comorbidities.

Structural and functional assessment of the brain in European Americans with mild-to-moderate kidney disease: Diabetes Heart Study-MIND.

BACKGROUND: Advanced chronic kidney disease (CKD) is associated with altered cerebral structure and function. Relationships between mild-to-moderate CKD and brain morphology and cognitive performance were evaluated in European Americans (EAs). METHODS: A total of 478 EAs with estimated glomerular filtration rate (eGFR) >45 mL/min/1.73 m(2) and urine albumin:creatinine ratio (UACR) < 300 mg/g, most with type 2 diabetes (T2D), were included. Measures of total intracranial volume (TICV), cerebrospinal fluid volume, total white matter volume (TWMV), total gray matter volume (TGMV), total white matter lesion volume (TWMLV), hippocampal white matter volume (HWMV) and hippocampal gray matter volume (HGMV) were obtained with magnetic resonance imaging. Cognitive testing included memory (Rey Auditory Visual Learning Test), global cognition (Modified Mini-Mental State Examination) and executive function (Stroop Task, Semantic Fluency, Digit Symbol Substitution Test). Associations with CKD were assessed using log-transformed eGFR and UACR, adjusted for age, sex, body mass index, smoking, hemoglobin A1c, blood pressure, diabetes duration, cardiovascular disease and education. RESULTS: Participants were 55.2% female, 78.2% had T2D; mean ± SD age 67.6 ± 9.0 years, T2D duration 16.4 ± 6.5 years, eGFR 92.0 ± 22.3 mL/min/1.73 m(2) and UACR 23.8 ± 39.6 mg/g. In adjusted models, eGFR was negatively associated with TICV only in participants with T2D [parameter estimate (ß): -72.2, P = 0.002]. In non-diabetic participants, inverse relationships were observed between eGFR and HGMV (ß: -1.0, P = 0.03) and UACR and normalized TWMLV (ß: -0.2, P = 0.03). Kidney function and albuminuria did not correlate with cognitive testing. CONCLUSIONS: In EAs with mild CKD enriched for T2D, brain structure and cognitive performance were generally not impacted. Longitudinal studies are necessary to determine when cerebral structural changes and cognitive dysfunction develop with progressive CKD in EAs.

Magnetic resonance imaging of the preterm infant brain.

Despite improvements in neonatal care, survivors of preterm birth are still at a significantly increased risk of developing life-long neurological difficulties including cerebral palsy and cognitive difficulties. Cranial ultrasound is routinely used in neonatal practice, but has a low sensitivity for identifying later neurodevelopmental difficulties. Magnetic Resonance Imaging (MRI) can be used to identify intracranial abnormalities with greater diagnostic accuracy in preterm infants, and theoretically might improve the planning and targeting of long-term neurodevelopmental care; reducing parental stress and unplanned healthcare utilisation; and ultimately may improve healthcare cost effectiveness. Furthermore, MR imaging offers the advantage of allowing the quantitative assessment of the integrity, growth and function of intracranial structures, thereby providing the means to develop sensitive biomarkers which may be predictive of later neurological impairment. However further work is needed to define the accuracy and value of diagnosis by MR and the techniques's precise role in care pathways for preterm infants.

Advanced magnetic resonance imaging techniques in the preterm brain: methods and applications.

Brain development and brain injury in preterm infants are areas of active research. Magnetic resonance imaging (MRI), a non-invasive tool applicable to both animal models and human infants, provides a wealth of information on this process by bridging the gap between histology (available from animal studies) and developmental outcome (available from clinical studies). Moreover, MRI also offers information regarding diagnosis and prognosis in the clinical setting. Recent advances in MR methods - diffusion tensor imaging, volumetric segmentation, surface based analysis, functional MRI, and quantitative metrics - further increase the sophistication of information available regarding both brain structure and function. In this review, we discuss the basics of these newer methods as well as their application to the study of premature infants.

Impaired cognitive executive dysfunction in adult treated hypertensives with a confirmed diagnosis of poorly controlled blood pressure.

BACKGROUND: High blood pressure (BP) poses a major risk for cognitive decline. Aim of the study was to highlight the relationship between cognitive assessment scores and an effective therapeutic BP control. METHODS: By medical visit and ambulatory BP monitoring (ABPM), we studied 302 treated hypertensives, subdivided according to office/daytime BP values into 120 with good (GC) and 98 poor (PC) BP control, 40 with "white coat hypertension" (WCH) and 44 a "masked-hypertension" phenomenon (MH). Patients underwent neuropsychological assessment to evaluate global cognitive scores at the Mini Mental State Examination (MMSE) and Frontal Assessment Battery (FAB) and attention/executive functions (Delayed Recall, Digit Span Forwards, Digit Span Backwards, Selective Attention, Verbal Fluency, Stroop Test and Clock Drawing). Carotid intima-media thickness (IMT) served as the index of vascular damage. RESULTS: There were no differences among the groups in terms of gender, age, education, metabolic assessment, clinical history and hypertension treatment. GC presented lower office and ambulatory BP values and IMT. PC performed worse than GC on global executive and attention functions, especially executive functions. In PC, office systolic BP (SBP) was significantly associated to the MMSE and FAB scores and, in particular, to Verbal Fluency, Stroop Errors and Clock Drawing tests. Office diastolic BP (DBP) was associated to Selective attention, nocturnal SBP to Digit Span backwards and Verbal Fluency. Worse cognitive assessment scores were obtained in WCH than GC. CONCLUSIONS: The findings showed that in adult treated hypertensives, a poor BP control, as both doctor's office and daytime scores, is associated to impaired global cognitive and especially executive/attention functions.

Cognitive impairment in temporal lobe epilepsy: role of online and offline processing of single cell information.

Cognitive impairment is a common comorbidity in temporal lobe epilepsy (TLE) and is often considered more detrimental to quality of life than seizures. While it has been previously shown that the encoding of memory during behavior is impaired in the pilocarpine model of TLE in rats, how this information is consolidated during the subsequent sleep period remains unknown. In this study, we first report marked deficits in spatial memory performance and severe cell loss in the CA1 layer of the hippocampus lower spatial coherence of firing in TLE rats. We then present the first evidence that the reactivation of behavior-driven patterns of activity of CA1 place cells in the hippocampus is intact in TLE rats. Using a template-matching method, we discovered that real-time (3-5 s) reactivation structure was intact in TLE rats. Furthermore, we estimated the entropy rate of short time scale (∼250 ms) bursting activity using block entropies and found that significant, extended temporal correlations exist in both TLE and control rats. Fitting a first-order Markov Chain model to these bursting time series, we found that long sequences derived from behavior were significantly enriched in the Markov model over corresponding models fit on randomized data confirming the presence of replay in shorter time scales. We propose that the persistent consolidation of poor spatial information in both real time and during bursting activity may contribute to memory impairments in TLE rats.

Assessment of regional MR diffusion changes in dementia with Lewy bodies and Alzheimer's disease.

BACKGROUND: Dementia with Lewy bodies (DLB) and Alzheimer's disease (AD) are common forms of dementia, yet diagnosis is often difficult. Diffusion tensor imaging (DTI) is an MR technique used to assess neuronal microstructural integrity that may help develop a better understanding of the differences between the conditions. METHODS: We recruited subjects with DLB (n = 35), AD (n = 36), and similar aged healthy controls (n = 35). T1 weighted anatomical and diffusion MR images were acquired at 3 Tesla. Region of interest (ROI) analysis was used to measure fractional anisotropy (FA) and mean diffusivity (MD) in five structures: precuneus, thalamus, pons, midbrain, and amygdala. Where appropriate diffusivity measures (FA, MD) were correlated with selected clinical measures. RESULTS: Compared to controls, DLB subjects were characterized by reduced FA (p = 0.016) and increased MD (p = 0.007) in the precuneus. Amygdala diffusivity was positively correlated with UPDRS-III score in DLB (p = 0.003). In AD, reduced FA in the precuneus was also observed compared to controls (p = 0.026), and was associated with impaired global cognition (MMSE score) (p = 0.03). CONCLUSIONS: Our findings highlight the potential importance of the precuneus in the pathogenesis of DLB as well as AD. Diffusion tensor MRI may shed new light on the different neurobiological changes underpinning the key clinical features of DLB and AD.

Cognitive reserve: implications for assessment and intervention.

The concept of reserve is used to explain the observation that some individuals function better than others in the presence of brain pathology. This article reviews the concept of reserve from its theoretical basis to the implication of reserve for clinical practice. A distinction between brain reserve, referring to individual differences in the anatomic substrate, and cognitive reserve, referring to differences in the flexibility or adaptivity of cognitive networks, is useful. Epidemiologic evidence indicates that a set of life exposures including higher educational and occupational attainment, and engaging in leisure activities is associated with a lower risk of incident dementia, suggesting that these life exposures may enhance cognitive reserve. This provides a basis for controlled clinical studies that can test specific exposures that may enhance reserve. The concept of cognitive reserve also has important implications for clinical practice in terms of diagnosis and prognosis.

Predictors of costs in dementia in a longitudinal perspective.

OBJECTIVE: To analyse predictors of costs in dementia from a societal perspective in a longitudinal setting. METHOD: Healthcare resource use and costs were assessed retrospectively using a questionnaire in four waves at 6-month intervals in a sample of dementia patients (N = 175). Sociodemographic data, dementia severity and comorbidity at baseline, cognitive impairment and impairment in basic and instrumental activities of daily living were also recorded. Linear mixed regression models with random intercepts for individuals were used to analyse predictors of total and sector-specific costs. RESULTS: Impairment in activities of daily living significantly predicted total costs in dementia patients, with associations between basic activities of daily living and formal care costs on the one and instrumental activities of daily living and informal care costs on the other hand. Nursing home residence was associated with lower total costs than residence in the community. There was no effect of cognition on total or sector-specific costs. CONCLUSION: Cognitive deficits in dementia are associated with costs only via their effect on the patients' capacity for activities of daily living. Transition into a nursing home may reduce total costs from a societal perspective, owing to the fact that a high amount of informal care required by severely demented patients prior to transition into a nursing home may cause higher costs than inpatient nursing care.

Visual assessment of brain magnetic resonance imaging detects injury to cognitive regulatory sites in patients with heart failure.

BACKGROUND: Heart failure (HF) patients exhibit depression and executive function impairments that contribute to HF mortality. Using specialized magnetic resonance imaging (MRI) analysis procedures, brain changes appear in areas regulating these functions (mammillary bodies, hippocampi, and frontal cortex). However, specialized MRI procedures are not part of standard clinical assessment for HF (which is usually a visual evaluation), and it is unclear whether visual MRI examination can detect changes in these structures. METHODS AND RESULTS: Using brain MRI, we visually examined the mammillary bodies and frontal cortex for global and hippocampi for global and regional tissue changes in 17 HF and 50 control subjects. Significantly global changes emerged in the right mammillary body (HF 1.18 ± 1.13 vs control 0.52 ± 0.74; P = .024), right hippocampus (HF 1.53 ± 0.94 vs control 0.80 ± 0.86; P = .005), and left frontal cortex (HF 1.76 ± 1.03 vs control 1.24 ± 0.77; P = .034). Comparison of the visual method with specialized MRI techniques corroborates right hippocampal and left frontal cortical, but not mammillary body, tissue changes. CONCLUSIONS: Visual examination of brain MRI can detect damage in HF in areas regulating depression and executive function, including the right hippocampus and left frontal cortex. Visual MRI assessment in HF may facilitate evaluation of injury to these structures and the assessment of the impact of potential treatments for this damage.