Tau positron emission tomography imaging in C9orf72 repeat expansion carriers.

BACKGROUND AND PURPOSE: AV-1451 (18 F-AV-1451, flortaucipir) positron emission tomography was performed in C9orf72 expansion carriers to assess tau accumulation and disease manifestation. METHODS: Nine clinically characterized C9orf72 expansion carriers and 18 age- and gender- matched cognitively normal individuals were psychometrically evaluated and underwent tau positron emission tomography imaging. The regional AV-1451 standard uptake value ratios from multiple brain regions were analyzed. Spearman correlation was performed to relate the AV-1451 standard uptake value ratio to clinical, psychometric and cerebrospinal fluid measures. RESULTS: C9orf72 expansion carriers had increased AV-1451 binding in the entorhinal cortex compared to controls. Primary age-related tauopathy was observed postmortem in one patient. AV-1451 uptake did not correlate with clinical severity, disease duration, psychometric performance or cerebrospinal fluid markers. CONCLUSION: C9orf72 expansion carriers exhibited increased AV-1451 uptake in entorhinal cortex compared to cognitively normal controls, suggesting a propensity for primary age-related tauopathy. However, AV-1451 accumulation was not associated with psychometric performance in our cohort.

Structural signature of sporadic Creutzfeldt-Jakob disease.

BACKGROUND AND PURPOSE: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapidly progressive neurodegenerative disease caused by an abnormal isoform of the human prion protein. Structural magnetic resonance imaging in patients with pathologically confirmed sCJD was compared with cognitively normal individuals to identify a cortical thickness signature of sCJD. METHODS: This retrospective cross-sectional study compared patients with autopsy-confirmed sCJD with dementia (n = 11) with age- and sex-matched cognitively normal individuals (n = 22). We identified regions of interest (ROIs) in which cortical thickness was most affected by sCJD. Within patients with sCJD, the relationship between ROI cortical thickness and clinical measures (disease duration, cerebrospinal fluid tau and diffusion-weighted imaging abnormalities) was evaluated. RESULTS: Compared with cognitively normal individuals, patients with sCJD had significantly reduced cortical thickness in multiple ROIs, including the fusiform gyrus, precentral gyrus, precuneus and superior temporal gyrus bilaterally; the caudal middle frontal gyrus, superior frontal gyrus, postcentral gyrus, inferior temporal gyrus and transverse temporal gyrus in the left hemisphere; and the superior parietal lobule in the right hemisphere. Only one patient with sCJD had co-pathology consistent with Alzheimer's disease. Reduced cortical thickness did not correlate with disease duration, presence of diffusion restriction or elevated cerebrospinal fluid tau. CONCLUSION: Cortical signature changes in sCJD may reflect brain changes not captured by standard clinical measures. This information may be used with clinical measures to inform the progression of sCJD and patterns of prion protein spread throughout the brain. These results may have implications for prediction of symptomatic progression and plausibly for development of therapeutic strategies.

The impact of human immune deficiency virus and hepatitis C coinfection on white matter microstructural integrity.

The purpose of the present study is to examine the integrity of white matter microstructure among individuals coinfected with HIV and HCV using diffusion tensor imaging (DTI). Twenty-five HIV+ patients, 21 HIV+/HCV+ patients, and 25 HIV- controls were included in this study. All HIV+ individuals were stable on combination antiretroviral therapy (cART; ≥3 months). All participants completed MRI and neuropsychological measures. Clinical variables including liver function, HIV-viral load, and CD4 count were collected from the patient groups. DTI metrics including mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), and fractional anisotropy (FA) from five subregions of the corpus callosum were compared across groups. The HIV+/HCV+ group and HIV+ group were similar in terms of HIV clinical variables. None of the participants met criteria for cirrhosis or fibrosis. Within the anterior corpus callosum, significant differences were observed between both HIV+ groups compared to HIV- controls on DTI measures. HIV+ and HIV+/HCV+ groups had significantly lower FA values and higher MD and RD values compared to HIV- controls; however, no differences were present between the HIV+ and HIV+/HCV+ groups. Duration of HIV infection was significantly related to DTI metrics in total corpus callosum FA only, but not other markers of HIV disease burden or neurocognitive function. Both HIV+ and HIV+/HCV+ individuals had significant alterations in white matter integrity within the corpus callosum; however, there was no evidence for an additive effect of HCV coinfection. The association between DTI metrics and duration of HIV infection suggests that HIV may continue to negatively impact white matter integrity even in well-controlled disease.

Brain morphometric correlates of metabolic variables in HIV: the CHARTER study.

Obesity and other metabolic variables are associated with abnormal brain structural volumes and cognitive dysfunction in HIV-uninfected populations. Since individuals with HIV infection on combined antiretroviral therapy (CART) often have systemic metabolic abnormalities and changes in brain morphology and function, we examined associations among brain volumes and metabolic factors in the multisite CNS HIV AntiRetroviral Therapy Effects Research (CHARTER) cohort, cross-sectional study of 222 HIV-infected individuals. Metabolic variables included body mass index (BMI), total blood cholesterol (C), low- and high-density lipoprotein C (LDL-C and HDL-C), blood pressure, random blood glucose, and diabetes. MRI measured volumes of cerebral white matter, abnormal white matter, cortical and subcortical gray matter, and ventricular and sulcal CSF. Multiple linear regression models allowed us to examine metabolic variables separately and in combination to predict each regional volume. Greater BMI was associated with smaller cortical gray and larger white matter volumes. Higher total cholesterol (C) levels were associated with smaller cortex volumes; higher LDL-C was associated with larger cerebral white matter volumes, while higher HDL-C levels were associated with larger sulci. Higher blood glucose levels and diabetes were associated with more abnormal white matter. Multiple atherogenic metabolic factors contribute to regional brain volumes in HIV-infected, CART-treated patients, reflecting associations similar to those found in HIV-uninfected individuals. These risk factors may accelerate cerebral atherosclerosis and consequent brain alterations and cognitive dysfunction.

Relationship of amyloid beta and neurofibrillary tau deposition in Neurodegeneration in Aging Down Syndrome (NiAD) study at baseline.

IMPORTANCE: Adults with Down syndrome (DS) are at high-risk of revealing Alzheimer's disease (AD) pathology, in part due to the triplication of chromosome 21 encoding the amyloid precursor protein. Adults with DS are uniformly affected by AD pathology by their 30's and have a 70% to 80% chance of clinical dementia by their 60's. Our previous studies have assessed longitudinal changes in amyloid beta (Aß) accumulation in DS. OBJECTIVE: The goal of the present study was to assess the presence of brain tau using [18F]AV-1451 positron emission tomography (PET) in DS and to assess the relationship of brain tau pathology to Aß using Pittsburgh Compound B (PiB)-PET. DESIGN: Cohort study. SETTING: Multi-center study. PARTICIPANTS: Participants consisted of a sample of individuals with DS and sibling controls recruited from the community; exclusion criteria included contraindications for magnetic resonance imaging (MRI) and/or a medical or psychiatric condition that impaired cognitive functioning. EXPOSURES: PET brain scans to assess Aß ([11C]PiB) and tau ([18F]AV-1451) burden. MAIN OUTCOMES AND MEASURES: Multiple linear regression models (adjusted for chronological age, sex and performance site) were used to examine associations between regional [18F]AV-1451 standard uptake value ratio (SUVR) (based on regions associated with Braak stages 1-6) and global [11C]PiB SUVR (as both a continuous and dichotomous variable). RESULTS: A cohort of 156 participants (mean age = 39.05, SD(8.4)) were examined. These results revealed a significant relationship between in vivo Aß and tau pathology in DS. As a dichotomous variable, [18F]AV-1451 retention was higher in each Braak region in PiB(+) participants. We also found, based on our statistical models, starting with the Braak 3 region of interest (ROI), an acceleration of [18F]AV-1451 SUVR deposition with [11C]PiB SUVR increases.

Coupling of neural activation to blood flow in the somatosensory cortex of rats is time-intensity separable, but not linear.

Changes in cerebral blood flow (CBF) because of functional activation are used as a surrogate for neural activity in many functional neuroimaging studies. In these studies, it is often assumed that the CBF response is a linear-time invariant (LTI) transform of the underlying neural activity. By using a previously developed animal model system of electrical forepaw stimulation in rats (n = 11), laser Doppler measurements of CBF, and somatosensory evoked potentials, measurements of neural activity were obtained when the stimulus duration and intensity were separately varied. These two sets of time series data were used to assess the LTI assumption. The CBF data were modeled as a transform of neural activity (N1-P2 amplitude of the somatosensory evoked potential) by using first-order (linear) and second-order (nonlinear) components. Although a pure LTI model explained a large amount of the variance in the data for changes in stimulus duration, our results demonstrated that the second-order kernel (i.e., a nonlinear component) contributed an explanatory component that is both statistically significant and appreciable in magnitude. For variations in stimulus intensity, a pure LTI model explained almost all of the variance in the CBF data. In particular, the shape of the CBF response did not depend on intensity of neural activity when duration was held constant (time-intensity separability). These results have important implications for the analysis and interpretation of neuroimaging data.

Effects of variations in interstimulus interval on activation-flow coupling response and somatosensory evoked potentials with forepaw stimulation in the rat.

In functional neuroimaging studies, the hemodynamic response to functional activation is used as a surrogate marker for neuronal activity, typically in response to task paradigms that use periodic stimuli. With use of a model system of electrical forepaw stimulation in rats (n = 14) with laser-Doppler (LD) monitoring of cerebral blood flow (CBF) changes in the somatosensory cortex, the effects of variations in the interstimulus interval (ISI) on the hemodynamic response to periodic stimuli were examined. A characteristic peak flow response was seen for 4-second stimuli and a peak and plateau response were seen for all 8-second stimuli regardless of ISI. However, both the amplitude of the LD(CBF) response and the integrated response were significantly reduced for shorter ISIs, whereas the baseline flow was not altered. Somatosensory evoked potential responses were also recorded in some rats (n = 8) and remained unchanged for the various ISIs for a particular stimulus duration. These results suggest that the decrease in the LD(CBF) responses observed with shorter ISIs likely represents a refractoriness of the hemodynamic response and not neuronal function. These results may have important implications for the optimization and interpretation of functional activation paradigms that use periodic stimuli.

Dynamic changes in cerebral blood flow, O2 tension, and calculated cerebral metabolic rate of O2 during functional activation using oxygen phosphorescence quenching.

Changes in cerebral blood flow (CBF) using laser-Doppler and microvascular O2 oxygen tension using oxygen-dependent phosphorescence quenching in the rat somatosensory cortex were obtained during electrical forepaw stimulation. The signal-averaged CBF response resulting from electrical forepaw stimulation consisted of an initial peak (t = 3.1 +/- 0.8 seconds after onset of stimulation), followed by a plateau phase that was maintained throughout the length of the stimulus. In contrast, microvascular O2 tension changes were delayed, reached a plateau level (t = 23.5 +/- 1.7 seconds after the onset of stimulation) that remained for the length of the stimulus and for several seconds after stimulus termination, and then returned to baseline. Using Fick's equation and these dynamic measurements, changes in the calculated cerebral metabolic rate of oxygen (CMRO2) during functional stimulation were determined. The calculated CMRO2 response initially was comparable with the CBF, but with protracted stimulation, CMRO2 changes were approximately one-third that of CBF changes. These results suggest that a complex relation exists, with comparable changes in CBF and CMRO2 initially occurring after stimulation but excessive changes in CBF compared with CMRO2 arising with protracted stimulation.

Neuroimaging of recovery of function after stroke: implications for rehabilitation.

Stroke is a leading cause of morbidity and mortality in individuals. Many patients have good functional recovery after stroke. The mechanisms of recovery remain largely unknown. Neuroimaging of patients recovering from stroke may provide important insight into the mechanisms of recovery as well as assist in the development of new rehabilitation techniques. The first part of this article reviews previous neuroimaging studies that have monitored the reorganization within the motor and language areas after stroke. In the second section, a unifying theory based on John Hughlings Jackson's "Principles of Compensation" is presented as a possible theory for recovery of function. In the final portion of the article, possible implications and future applications of neuroimaging studies for rehabilitation are presented.

Activation-flow coupling with forepaw stimulation in female and male rats.

Activation-flow coupling (AFC), the coupling of changes in cerebral blood flow (CBF) with neuronal function, is the basis for many functional neuroimaging techniques. Prior studies have shown that females have higher cerebral blood flow levels than males and that estrogen may affect the mediators involved in AFC. No studies have compared AFC responses between males and females. We assessed the AFC responses to forepaw stimulation using signal-averaged laser Doppler (LD) measurements of CBF in alpha-chloralose anesthetized female and male rats. Results for various stimulus parameters were similar for both sexes except at 2 Hz where females had a higher blood flow response. These results suggest that the AFC responses in males and females are similar, but require further validation in humans.

Signal averaged laser Doppler measurements of activation-flow coupling in the rat forepaw somatosensory cortex.

Regional alterations in cerebral blood flow (CBF) are widely used as a surrogate for neuronal function based on an intact coupling between changes in regional CBF and metabolism, activation-flow coupling (AFC). To further investigate parameters affecting AFC, we have implemented a rat model with electrical forepaw stimulation under alpha-chloralose anesthesia using laser Doppler (LD) measurements of flow parameters through thinned skull over contralateral somatosensory cortex. Signal averaging of the LD response was used to improve reproducibility. A characteristic flow response to electrical forepaw stimulation was reliably recorded from the somatosensory cortex using signal averaging. Stimulation at 5 Hz maximized the LD response, and constant current stimulation up to 1 mA did not induce changes in systemic blood pressure. The shape of the flow response consisted of an initial peak followed by a steady state plateau phase which was observed for stimulation durations longer than 4 s. When individual LD parameters of velocity, red blood cell concentration (CRBC), and cerebral blood flow (CBF) were compared, changes in LDCBF were primarily attributable to changes in LDvelocity rather than LDCRBC. This finding was also observed during hypercapnia. Characterization of AFC in the model provides a background for future studies of the effects of pharmacological manipulation or pathophysiological states.

The effects of graded hypercapnia on the activation flow coupling response due to forepaw stimulation in alpha-chloralose anesthetized rats.

Activation flow coupling (AFC), changes in cerebral blood flow (CBF) due to changes in neural activity with functional stimulation, provides the physiological basis of many neuroimaging techniques. Hypercapnia leads to an increase in CBF while neural activity remains unaffected. Laser Doppler (LD) flowmetry was used to measure CBF changes (LD(CBF)) in the somatosensory cortex due to periodic electrical forepaw stimulation (4 s in duration) before and during graded hypercapnia (3% CO(2), 5% CO(2) and 10% CO(2)). With increasing CO(2) concentrations, the baseline LD(CBF) progressively increased. The peak height (PH) of the LD(CBF) response, expressed as a percent change from the observed baseline for each hypercapnic state, significantly decreased (P<0.05) with increasing CO(2) concentrations. However, the absolute magnitude of the LD(CBF) change was independent of CO(2) concentration. The temporal dynamics of the LD(CBF) response during hypercapnia were significantly prolonged compared to baseline conditions (P<0.05).

Transcranial laser doppler mapping of activation flow coupling of the rat somatosensory cortex.

Signal averaged laser Doppler (LD) through a thinned skull over the rat somatosensory cortex was used to map the spatial and temporal characteristics of activation-flow coupling, the change in regional cerebral blood flow (CBF) due to neuronal activation, in response to electrical forepaw stimulation. The location of maximal changes in amplitude of the LD response was reproducibly recorded at 4-5 mm lateral to Bregma. This location is very similar but slightly posterior and lateral to results obtained from electrophysiological, magnetic resonance imaging (MRI), and optical imaging studies. The latency of the activation-flow coupling (AFC) response was inversely correlated to response amplitude, with shorter latencies at positions of maximal amplitude.

Sex differences in the cerebral blood flow response after brief hypercapnia in the rat.

Hypercapnia primarily affects cerebral blood flow (CBF) and not cerebral metabolism. We compared the CBF responses due to electrical forepaw stimulation before and after brief hypercapnia in male, non-ovarectomized female, and ovarectomized female rats. Prior to hypercapnia the CBF responses were similar for all three groups. Seven minutes after brief hypercapnic exposure the CBF responses to forepaw stimulation were augmented in all groups. However, both 30 and 60 min after hypercapnia, the magnitude of the CBF responses to forepaw stimulation remained elevated for males and ovarectomized females, but not for non-ovarectomized females. These results suggest that estrogen may modulate the upregulation of the CBF response observed after transient hypercapnia.

Temporal dynamics of the partial pressure of brain tissue oxygen during functional forepaw stimulation in rats.

The partial pressure of tissue oxygen (pO2) was measured in rat somatosensory cortex during periodic electrical forepaw stimulation of either 1 min or 4 s in duration, and correlated with simultaneous laser Doppler flowmetry. For both stimulus durations, a transient decrease in tissue pO2 preceded blood flow changes, followed by a peak in blood flow and an overshoot in tissue pO2. With protracted stimulation, tissue pO2 remained only slightly above pre-stimulus baseline, while blood flow was maintained at a reduced plateau phase. A sustained post-stimulus undershoot in tissue pO2 was observed only for the 1 min stimulus. These findings suggest a complex dynamic relationship between oxygen utilization and blood flow.

Role of obesity, metabolic variables, and diabetes in HIV-associated neurocognitive disorder.

OBJECTIVE: To evaluate relationships between HIV-associated neurocognitive disorder and metabolic variables in a subgroup of HIV+ participants examined in a prospective, observational, multicenter cohort study. METHODS: In a cross-sectional substudy of the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort, 130 HIV+ participants provided fasting blood samples. Neurocognitive impairment (NCI) was defined by performance on neuropsychological tests adjusting for age, education, gender, and race/ethnicity. Global ratings and global deficit scores were determined. Demographics, biomarkers of HIV disease, metabolic variables, combination antiretroviral therapy (CART) history, other drug exposures, and self-reported diabetes were examined in multivariate models predicting NCI. Separate models were used for body mass index (BMI) alone (n = 90) and BMI and waist circumference (WC) together (n = 55). RESULTS: NCI (global impairment rating ≥5) was diagnosed in 40%. In univariate analyses, age, longer duration of HIV infection, obesity, and WC, but not BMI, were associated with NCI. Self-reported diabetes was associated with NCI in the substudy and in those >55 in the entire CHARTER cohort. Multivariate logistic regression analyses demonstrated that central obesity (as measured by WC) increased the risk of NCI and that greater body mass may be protective if the deleterious effect of central obesity is accounted for. CONCLUSIONS: As in HIV-uninfected persons, central obesity, but not more generalized increases in body mass (BMI), was associated with a higher prevalence of NCI in HIV+ persons. Diabetes appeared to be associated with NCI only in older patients. Avoidance of antiretroviral drugs that induce central obesity might protect from or help to reverse neurocognitive impairment in HIV-infected persons.

Cognitively unimpaired HIV-positive subjects do not have increased 11C-PiB: a case-control study.

OBJECTIVES: Diagnostic challenges exist for differentiating HIV dementia from Alzheimer disease (AD) in older HIV-infected (HIV+) individuals. Similar abnormalities in brain amyloid-beta42 (Alphabeta42) metabolism may be involved in HIV-associated neuropathology and AD. We evaluated the amyloid-binding agent (11)C-Pittsburgh compound B ((11)C-PiB), a biomarker for Alphabeta42 deposition, in cognitively unimpaired HIV+ (n = 10) participants and matched community controls without dementia (n = 20). METHODS: In this case-control study, all participants had an (11)C-PiB scan within 2 years of concomitant CSF studies and neuropsychometric testing. Statistical differences between HIV+ and community controls for demographic and clinical values were assessed by chi(2) tests. Participants were further divided into either low (<500 pg/mL) or normal (>or=500 pg/mL) CSF Alphabeta42 groups with Student t tests performed to determine if regional differences in fibrillar amyloid plaque deposition varied with CSF Alphabeta42. RESULTS: Regardless of CSF Alphabeta42 level, none of the HIV+ participants had fibrillar amyloid plaques as assessed by increased (11)C-PiB mean cortical binding potential (MCBP) or binding potential within 4 cortical regions. In contrast, some community controls with low CSF Alphabeta42 (<500 pg/mL) had high (11)C-PiB MCBP with elevated binding potentials (>0.18 arbitrary units) within cortical regions. CONCLUSIONS: Cognitively unimpaired HIV+ participants, even with low CSF Alphabeta42 (<500 pg/mL), do not have (11)C-PiB parameters suggesting brain fibrillar amyloid deposition. The dissimilarity between unimpaired HIV+ and preclinical AD may reflect differences in Abeta42 production and/or formation of diffuse plaques. Future longitudinal studies of HIV+ participants with low CSF Abeta42 and normal (11)C-PiB are required.

Resting cerebral blood flow: a potential biomarker of the effects of HIV in the brain.

OBJECTIVE: HIV enters the brain soon after infection causing neuronal damage and microglial/astrocyte dysfunction leading to neuropsychological impairment. We examined the impact of HIV on resting cerebral blood flow (rCBF) within the lenticular nuclei (LN) and visual cortex (VC). METHODS: This cross-sectional study used arterial spin labeling MRI (ASL-MRI) to measure rCBF within 33 HIV+ and 26 HIV- subjects. Nonparametric Wilcoxon rank sum test assessed rCBF differences due to HIV serostatus. Classification and regression tree (CART) analysis determined optimal rCBF cutoffs for differentiating HIV serostatus. The effects of neuropsychological impairment and infection duration on rCBF were evaluated. RESULTS: rCBF within the LN and VC were significantly reduced for HIV+ compared to HIV- subjects. A 2-tiered CART approach using either LN rCBF < or =50.09 mL/100 mL/min or LN rCBF >50.09 mL/100 mL/min but VC rCBF < or =37.05 mL/100 mL/min yielded an 88% (29/33) sensitivity and an 88% (23/26) specificity for differentiating by HIV serostatus. HIV+ subjects, including neuropsychologically unimpaired, had reduced rCBF within the LN (p = 0.02) and VC (p = 0.001) compared to HIV- controls. A temporal progression of brain involvement occurred with LN rCBF significantly reduced for both acute/early (<1 year of seroconversion) and chronic HIV-infected subjects, whereas rCBF in the VC was diminished for only chronic HIV-infected subjects. CONCLUSION: Resting cerebral blood flow (rCBF) using arterial spin labeling MRI has the potential to be a noninvasive neuroimaging biomarker for assessing HIV in the brain. rCBF reductions that occur soon after seroconversion possibly reflect neuronal or vascular injury among HIV+ individuals not yet expressing neuropsychological impairment.

Caudate blood flow and volume are reduced in HIV+ neurocognitively impaired patients.

OBJECTIVE: To evaluate the effects of HIV-associated neurocognitive impairment on caudate blood flow and volume. METHODS: The authors performed continuous arterial spin labeled MRI on 42 HIV+ patients (23 subsyndromic and 19 HIV neurosymptomatic) on highly active antiretroviral therapy and 17 seronegative controls. They compared caudate blood flow and volume among groups. RESULTS: A stepwise decrease in both caudate blood flow and volume was observed with increasing HIV-associated neurocognitive impairment. Compared with seronegative controls, baseline caudate blood flow was reduced in HIV+ neurosymptomatic patients (p = 0.001) with a similar decreasing trend for subsyndromic HIV+ patients (p = 0.070). Differences in caudate volume were observed only for neurosymptomatic HIV+ patients compared with controls (p = 0.010). A Jonckheere-Terpstra test for trends was significant for both caudate blood flow and volume for each of the three subgroups. Pearson product moment correlation coefficients were not significant between caudate blood flow and volume for each group. CONCLUSIONS: Decreasing trends in caudate blood flow and volume were associated with significantly increasing HIV-associated neurocognitive impairment (HNCI), with the greatest decreases observed for more severely impaired patients. However, reductions in caudate blood flow and volume were poorly correlated. Changes in residual caudate blood flow may act as a surrogate biomarker for classifying the degree of HNCI.

Perfusion changes with photic stimulation during two phases of the menstrual cycle: a pilot study comparing controls and true menstrual migraine patients.

This pilot study investigated the effect of menstrual cycle phase (late luteal and mid-follicular) on cerebral perfusion changes during photic stimulation in both controls (n = 5) and true menstrual migraine patients (n = 5). No significant differences in resting baseline perfusion were observed between the two groups during either phase of the menstrual cycle. During the late luteal phase, changes in perfusion within the occipital lobe due to photic stimulation were similar for both groups. However, during the mid-follicular phase, occipital perfusion during visual stimulation decreased for controls but significantly increased for true menstrual migraine patients (P < 0.05). A two way repeated measures anova also demonstrated a significant difference between menstrual migraine patients and controls for photic activation (P < 0.05).