Hybrid MR-PET of brain tumours using amino acid PET and chemical exchange saturation transfer MRI.

PURPOSE: PET using radiolabelled amino acids has become a promising tool in the diagnostics of gliomas and brain metastasis. Current research is focused on the evaluation of amide proton transfer (APT) chemical exchange saturation transfer (CEST) MR imaging for brain tumour imaging. In this hybrid MR-PET study, brain tumours were compared using 3D data derived from APT-CEST MRI and amino acid PET using O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). METHODS: Eight patients with gliomas were investigated simultaneously with 18F-FET PET and APT-CEST MRI using a 3-T MR-BrainPET scanner. CEST imaging was based on a steady-state approach using a B1 average power of 1µT. B0 field inhomogeneities were corrected a Prametric images of magnetisation transfer ratio asymmetry (MTRasym) and differences to the extrapolated semi-solid magnetisation transfer reference method, APT# and nuclear Overhauser effect (NOE#), were calculated. Statistical analysis of the tumour-to-brain ratio of the CEST data was performed against PET data using the non-parametric Wilcoxon test. RESULTS: A tumour-to-brain ratio derived from APT# and 18F-FET presented no significant differences, and no correlation was found between APT# and 18F-FET PET data. The distance between local hot spot APT# and 18F-FET were different (average 20 ± 13 mm, range 4-45 mm). CONCLUSION: For the first time, CEST images were compared with 18F-FET in a simultaneous MR-PET measurement. Imaging findings derived from18F-FET PET and APT CEST MRI seem to provide different biological information. The validation of these imaging findings by histological confirmation is necessary, ideally using stereotactic biopsy.

Methods for molecular imaging of brain tumours in a hybrid MR-PET context: Water content, T2∗, diffusion indices and FET-PET.

The aim of this study is to present and evaluate a multiparametric and multi-modality imaging protocol applied to brain tumours and investigate correlations between these different imaging measures. In particular, we describe a method for rapid, non-invasive, quantitative imaging of water content of brain tissue, based on a single multiple-echo gradient-echo (mGRE) acquisition. We include in the processing a method for noise reduction of the multi-contrast data based on Principal Component Analysis (PCA). Noise reduction is a key ingredient to obtaining high-precision water content and transverse relaxation T2∗ values. The quantitative method is applied to brain tumour patients in a hybrid MR-PET environment. Active tumour tissue is identified by means of FET-PET; oedema, white and grey-matter segmentation is performed based on MRI contrasts. Water content information is not only relevant by itself, but also as a basis for correlations with other quantitative measures of water behaviour in tissue and interpreting the microenvironment of water. Water content in active tumour tissue (84%) and oedema (79%) regions is found to be higher than that of normal WM (69%) and close to that of normal GM (83%). Consistent with literature reports, mean kurtosis is measured to be lower in tumour and oedema regions than in normal WM and GM, whereas mean diffusivity is increased. Voxel-based correlations between water content and diffusion indices obtained with diffusion kurtosis tensor imaging, and between quantitative MRI and FET-PET are reported for 8 brain tumour patients. The effective transverse relaxation time T2∗ is found to be the MR parameter showing the strongest correlations with other MR indices derived here and with FET-PET.

Multimodal Fingerprints of Resting State Networks as assessed by Simultaneous Trimodal MR-PET-EEG Imaging.

Simultaneous MR-PET-EEG (magnetic resonance imaging - positron emission tomography - electroencephalography), a new tool for the investigation of neuronal networks in the human brain, is presented here for the first time. It enables the assessment of molecular metabolic information with high spatial and temporal resolution in a given brain simultaneously. Here, we characterize the brain's default mode network (DMN) in healthy male subjects using multimodal fingerprinting by quantifying energy metabolism via 2- [18F]fluoro-2-desoxy-D-glucose PET (FDG-PET), the inhibition - excitation balance of neuronal activation via magnetic resonance spectroscopy (MRS), its functional connectivity via fMRI and its electrophysiological signature via EEG. The trimodal approach reveals a complementary fingerprint. Neuronal activation within the DMN as assessed with fMRI is positively correlated with the mean standard uptake value of FDG. Electrical source localization of EEG signals shows a significant difference between the dorsal DMN and sensorimotor network in the frequency range of δ, θ, α and ß-1, but not with ß-2 and ß-3. In addition to basic neuroscience questions addressing neurovascular-metabolic coupling, this new methodology lays the foundation for individual physiological and pathological fingerprints for a wide research field addressing healthy aging, gender effects, plasticity and different psychiatric and neurological diseases.

Bone regeneration induced by a 3D architectured hydrogel in a rat critical-size calvarial defect.

Bone regeneration can be stimulated by implantation of biomaterials, which is especially important for larger bone defects. Here, healing potency of the porous ArcGel was evaluated in a critical-size calvarial bone defect in rats in comparison with clinical standard autologous bone and Bio-Oss® Collagen (BioOss), a bone graft material frequently used in clinics. Bone healing and metabolic processes involved were monitored longitudinally by [18F]-fluoride and [18F]-FDG µ-PET/CT 1d, 3d, 3w, 6w, and 12w post implantation. Differences in quality of bone healing were assessed by ex vivo µ-CT, mechanical tests and histomorphometry. The amount of bone formed after implantation of ArcGel was comparable to autologous bone and superior to BioOss (histomorphometry). Furthermore, microarchitecture of newly formed bone was more physiological and better functional in case of ArcGel (push-out tests). [18F]-FDG uptake increased until 3d after implantation, and decreased until 12w for both ArcGel and BioOss. [18F]-fluoride uptake increased until 3w post implantation for all materials, but persisted significantly longer at higher levels for BioOss, which indicates a prolonged remodelling phase. The study demonstrates the potential of ArcGel to induce restitutio ad integrum comparable with clinical standard autologous bone and better bone regeneration in large defects compared to a commercial state-of-the-art biomaterial.

Multimodal Imaging in Malignant Brain Tumors: Enhancing the Preoperative Risk Evaluation for Motor Deficits with a Combined Hybrid MRI-PET and Navigated Transcranial Magnetic Stimulation Approach.

BACKGROUND AND PURPOSE: Motor deficits in patients with brain tumors are caused mainly by irreversible infiltration of the motor network or by indirect mass effects; these deficits are potentially reversible on tumor removal. Here we used a novel multimodal imaging approach consisting of structural, functional, and metabolic neuroimaging to better distinguish these underlying causes in a preoperative setting and determine the predictive value of this approach. MATERIALS AND METHODS: Thirty patients with malignant brain tumors involving the central region underwent a hybrid O-(2-[(18)F]fluoroethyl)-L-tyrosine-PET-MR imaging and motor mapping by neuronavigated transcranial magnetic stimulation. The functional maps served as localizers for DTI tractography of the corticospinal tract. The spatial relationship between functional tissue (motor cortex and corticospinal tract) and lesion volumes as depicted by structural and metabolic imaging was analyzed. RESULTS: Motor impairment was found in nearly all patients in whom the contrast-enhanced T1WI or PET lesion overlapped functional tissue. All patients who functionally deteriorated after the operation showed such overlap on presurgical maps, while the absence of overlap predicted a favorable motor outcome. PET was superior to contrast-enhanced T1WI for revealing a motor deficit before the operation. However, the best correlation with clinical impairment was found for T2WI lesion overlap with functional tissue maps, but the prognostic value for motor recovery was not significant. CONCLUSIONS: Overlapping contrast-enhanced T1WI or PET-positive signals with motor functional tissue were highly indicative of motor impairment and predictive for surgery-associated functional outcome. Such a multimodal diagnostic approach may contribute to the risk evaluation of operation-associated motor deficits in patients with brain tumors.

Applications of PET imaging of neurological tumors with radiolabeled amino acids.

Routine diagnostics and treatment monitoring of brain tumors is usually based on contrast-enhanced magnetic resonance imaging (MRI). However, the capacity of structural MRI to differentiate neoplastic tissue from non-specific treatment changes may be limited especially after therapeutic interventions such as neurosurgical resection, radio- and chemotherapy. Metabolic imaging using PET may provide relevant additional information on tumor metabolism, which allows for more accurate diagnostics especially in clinically equivocal situations. In contrast to the widely used ¹8F-2-fluoro-2-deoxy-D-glucose, which exhibits a poor tumor-to-background contrast within the brain, amino acid tracers provide high sensitivity to detect primary tumors, recurrent or residual gliomas, including most low-grade gliomas. The method improves targeting of biopsy and provides additional information of tumor extent, which is helpful for planning neurosurgery and radiotherapy. In the further course of the disease, amino acid positron-emission tomography (PET) allows a sensitive monitoring of treatment response, the early detection of tumor recurrence, and an improved differentiation of tumor recurrence from treatment-related changes. In the past, the method had only limited availability due to the use of radiopharmaceuticals with a short half-life. In recent years, however, novel amino acid tracers labeled with positron emitters with a longer half-life have been developed and clinically validated which allow a more efficient and cost-effective application. These developments and the well-documented diagnostic performance of PET using radiolabeled amino acids suggest that its application continues to spread and that the method may be available as a routine diagnostic technique for certain indications in the near future.

Meningioangiomatosis in a patient with progressive focal neurological deficit-case report and review of literature.

Meningioangiomatosis (MA) represents a vascular hamartoma accompanied by meningothelial cell proliferation. It generally becomes symptomatic with difficult to control seizures, though in some patients it may be asymptomatic. We present the case of a 41-year-old male patient with a newly developed central distal monoparesis of the left leg. Cranial magnetic resonance imaging (MRI) and further diagnostic characterization via (18)F-Fluoro-Ethyl-Tyrosine positron emission tomography ((18)F-FET-PET) indicated a low-grade glioma. Histopathological diagnosis revealed a meningioangiomatosis. The clinical, radiological and neuropathological findings of this rare constellation are described and discussed with the actual literature.

A randomised comparison of the LMA SupremeTM and LMAProSealTM in children.

We conducted a randomised trial comparing the size-2 LMA Supreme™ with the LMA ProSeal™ in 60 children undergoing surgery. The outcomes measured were airway leak pressure, ease and time for insertion, fibreoptic examination, incidence of gastric insufflation, ease of gastric tube placement, quality of the airway during anaesthetic maintenance and complications. There were no statistically significant differences between the LMA Supreme and LMA ProSeal in median (IQR [range]) insertion time (12 (10-15 [7-18]) s vs 12 (10-13 [8-25]) s; p = 0.90), airway leak pressures (19 (16-21 [12-30]) cmH(2) O vs 18 (16-24 [10-34]) cmH(2) O; p = 0.55), fibreoptic position of the airway or drain tube, ease of gastric access and complications. Both devices provided effective ventilation requiring minimal airway manipulation. The LMA Supreme can be a useful alternative to the LMA ProSeal when single-use supraglottic devices with gastric access capabilities are required.

Integrated boost IMRT with FET-PET-adapted local dose escalation in glioblastomas. Results of a prospective phase II study.

PURPOSE: Dose escalations above 60 Gy based on MRI have not led to prognostic benefits in glioblastoma patients yet. With positron emission tomography (PET) using [(18)F]fluorethyl-L-tyrosine (FET), tumor coverage can be optimized with the option of regional dose escalation in the area of viable tumor tissue. METHODS AND MATERIALS: In a prospective phase II study (January 2008 to December 2009), 22 patients (median age 55 years) received radiochemotherapy after surgery. The radiotherapy was performed as an MRI and FET-PET-based integrated-boost intensity-modulated radiotherapy (IMRT). The prescribed dose was 72 and 60 Gy (single dose 2.4 and 2.0 Gy, respectively) for the FET-PET- and MR-based PTV-FET((72 Gy)) and PTV-MR((60 Gy)). FET-PET and MRI were performed routinely for follow-up. Quality of life and cognitive aspects were recorded by the EORTC-QLQ-C30/QLQ Brain20 and Mini-Mental Status Examination (MMSE), while the therapy-related toxicity was recorded using the CTC3.0 and RTOG scores. RESULTS: Median overall survival (OS) and disease-free survival (DFS) were 14.8 and 7.8 months, respectively. All local relapses were detected at least partly within the 95% dose volume of PTV-MR((60 Gy)). No relevant radiotherapy-related side effects were observed (excepted alopecia). In 2 patients, a pseudoprogression was observed in the MRI. Tumor progression could be excluded by FET-PET and was confirmed in further MRI and FET-PET imaging. No significant changes were observed in MMSE scores and in the EORTC QLQ-C30/QLQ-Brain20 questionnaires. CONCLUSION: Our dose escalation concept with a total dose of 72 Gy, based on FET-PET, did not lead to a survival benefit. Acute and late toxicity were not increased, compared with historical controls and published dose-escalation studies.

SU-E-J-201: What is the Importance of Dose Recalculation for Adaptive Radiotherapy Dose Assessment?

PURPOSE: To quantify the effect of using the planned dose distributions in lieu of performing dose recalculations on daily in-room images for adaptive radiotherapy (ART) dose assessment of head and neck cancer patients. METHODS: 16 patients with cancers of the head and neck were treated using the TomoTherapy Hi-Art II (Accuray Inc., Sunnyvale, CA). Images of all patients were acquired prior to each treatment using the megavoltage CT (MVCT) capability of the TomoTherapy unit. Overall, images from 528 fractions were evaluated. For every image set, the delivered dose was estimated by both recalculating the dose distribution using the acquired MVCT and also by simply overlaying the planned distribution on the new images. ART dose assessment was performed using deformable image registration (DIR) to deform contours from the treatment plan to the images acquired during each fraction and to accumulate the estimated dose delivered during each fraction back to the reference treatment plan. The same DIR maps were applied to both dose estimation methods. Dosimetric endpoints were then compared between the DVHs computed using the recalculated or planned dose distributions. RESULTS: The mean PTV D95% and D05% endpoints were 0.6±0.5% and 1.4±0.8% lower using the planned dose distributions compared to the recalculated distributions, respectively, across all patients. The mean parotid D50% was 2.4±1.5% greater using the planned distributions compared to the recalculated distributions. The parotid D50% from the planned distributions was also highly correlated with the parotid D50% from the recalculated distributions for each patient (mean r2=0.97±0.09). CONCLUSIONS: For this treatment modality and site, dosimetric differences observed between overlaying the planned dose distributions and recalculating distributions on daily images were typically well within ±5%. This indicates that the dose distribution itself is robust against anatomic variations. This study was funded, in part, by a grant from Accuray Inc.

[Use of amino acid PET in the Diagnostic and Treatment Management of cerebral gliomas]. / Anwendung der Aminosäure-PET in der Diagnostik und Therapie von zerebralen Gliomen.

Structural as well as functional imaging methods are of special importance in neurooncology. Improvements of radionuclide and magnetic resonance-based imaging modalities over the past decade have enabled clinicians to non-invasively assess the dynamics of disease-specific processes at the molecular level in patients with malignant gliomas. To date, a range of complementary imaging parameters have been established in the diagnostic work-up of patients with brain tumours. Magnetic resonance imaging (MRI) provides morphological information as well as functional information such as vascular permeability, cell density, tumour perfusion, and metabolic information by using magnetic resonance spectroscopy. The use of radiolabelled amino acids for positron emission tomography (PET) allows a better delineation of tumour margins and improves targeting of biopsy and radiotherapy, and planning surgery. In addition, amino acid imaging appears useful in distinguishing tumour recurrence from non-specific post-therapeutic scar tissue, in predicting prognosis in low-grade gliomas, and in monitoring metabolic response during treatment. Taken together, MRI and PET provide complementary information about tumour biology and activity, thereby resulting in an improved understanding of the kinetics of tumour growth and therefore allow new insights into the pathophysiology of malignant brain tumours. However, multimodal imaging studies comparing the value of amino acid PET and functional methods of MRI (e. g., perfusion and diffusion weighted imaging) are needed. From these studies, surrogate MRI and PET imaging techniques need to be derived to gain complementary structural and functional information of brain tumours that can be placed into common clinical practice which will optimise the clinical management of patients with malignant gliomas.

A randomised trial comparing the laryngeal mask airway Supreme™ with the laryngeal mask airway Unique™ in children.

We conducted a randomised controlled trial comparing the laryngeal mask airway Supreme(™) with the laryngeal mask airway Unique(™) in children. Fifty children presenting for elective surgery were randomly assigned to receive either the laryngeal mask airway Supreme or laryngeal mask airway Unique. The outcomes measured were airway leak pressure, ease and time for insertion, insertion success rate, fibreoptic examination, incidence of gastric insufflation, ease of gastric tube placement through the laryngeal mask airway Supreme, quality of airway during anaesthetic maintenance and complications. Median (IQR [range]) time to successful device placement was shorter with the laryngeal mask airway Unique, 14.5 [13.5-16.3 (10.0-23.6)] s than with the laryngeal mask airway Supreme, 17.4 [14.8-19.8 (11.5-29.2)] s; p = 0.007. Median (IQR [range]) airway leak pressures for the laryngeal mask airway Supreme and laryngeal mask airway Unique were 20 [16-21 (12-22)] cmH(2)O and 15 [14-18 (10-24)] cmH(2)O, respectively (p = 0.001). The incidence of gastric insufflation was lower with the laryngeal mask airway Supreme (zero vs six patients), p = 0.01. In conclusion, the laryngeal mask airway Supreme performed as well as the laryngeal mask airway Unique and is a useful alternative for airway maintenance, particularly in children who require evacuation of gastric contents during anaesthesia.

The effect of temporal HU variations on the uncertainty of dose recalculations performed on MVCT images.

Over the course of radiation therapy, a patient's anatomy may change substantially. The relatively recent addition of frequent in-room imaging to assist with patient localization has provided a database of images that may be used to recalculate dose distributions for adaptive radiotherapy purposes. The TomoTherapy Hi-Art II unit (Accuray Inc., Sunnyvale, CA, USA) uses a helical scanning geometry and a megavoltage (MV) beam to acquire volumetric patient images. This study evaluated the uncertainty of dose calculations performed on megavoltage CT (MVCT) images as a function of temporal Hounsfield Unit (HU) variations observed in the imaging system over three years on two machines. A baseline error between dose calculations performed on kVCT and MVCT images was established using a series of phantoms. This baseline error ranged from -1.4% to 0.6%. Materials of differing densities were imaged and MVCT numbers were measured periodically. The MVCT number of solid water varied from 5 to 103 HU and consistently increased prior to target replacement. Finally, the dosimetric uncertainty of the temporal HU variation was assessed using MVCT images of typical head and neck, lung and prostate cancer patients. Worst-case MVCT recalculation errors could approach 5%, 7% and 10% for the head and neck, lung and prostate images, respectively. However, if a tolerance of ±30 HU were maintained for the MVCT number of solid water, dosimetric errors were limited to ±2.5%, ±3% and ±4%, respectively.

[German guidelines for brain tumour imaging by PET and SPECT using labelled amino acids]. / PET- und SPECT-Untersuchungen von Hirntumoren mit radioaktiv markierten Aminosäuren.

For the primary diagnosis of brain tumours, morphological imaging by means of magnetic resonance imaging (MRI) is the current method of choice. The complementary use of functional imaging by positron emitting tomography (PET) and single photon emitting computerized tomography (SPECT) with labelled amino acids can provide significant information on some clinically relevant questions, which are beyond the capacity of MRI. These diagnostic issues affect in particular the improvement of biopsy targeting and tumour delineation for surgery and radiotherapy planning. In addition, amino acid labelled PET and SPECT tracers are helpful for the differentiation between tumour recurrence and non-specific post-therapeutic tissue changes, in predicting prognosis of low grade gliomas, and for metabolic monitoring of treatment response. The application of dynamic PET examination protocols for the assessment of amino acid kinetics has been shown to enable an improved non-invasive tumour grading. The purpose of this guideline is to provide practical assistance for indication, examination procedure and image analysis of brain PET/SPECT with labelled amino acids in order to allow for a high quality standard of the method. After a short introduction on pathobiochemistry and radiopharmacy of amino acid labelled tracers, concrete and detailed information is given on the several indications, patient preparation and examination protocols as well as on data reconstruction, visual and quantitative image analysis and interpretation. In addition, possible pitfalls are described, and the relevant original publications are listed for further information.

High resolution BrainPET combined with simultaneous MRI.

UNLABELLED: After the successful clinical introduction of PET/CT, a novel hybrid imaging technology combining PET with the versatile attributes of MRI is emerging. At the Forschungszentrum Jülich, one of four prototypes available worldwide combining a commercial 3T MRI with a newly developed BrainPET insert has been installed, allowing simultaneous data acquisition with PET and MRI. The BrainPET is equipped with LSO crystals of 2.5 mm width and Avalanche photodiodes (APD) as readout electronics. Here we report on some performance characteristics obtained by phantom studies and also on the initial BrainPET studies on various patients as compared with a conventional HR+ PET-only scanner. MATERIAL, METHODS: The radiotracers [18F]-fluoro-ethyl-tyrosine (FET), [11C]-flumazenil and [18F]-FP-CIT were applied. RESULTS: Comparing the PET data obtained with the BrainPET to those of the HR+ scanner demonstrated the high image quality and the superior resolution capability of the BrainPET. Furthermore, it is shown that various MR images of excellent quality could be acquired simultaneously with BrainPET scans without any relevant artefacts. DISCUSSION, CONCLUSION: Initial experiences with the hybrid MRI/BrainPET indicate a promising basis for further developments of this unique technique allowing simultaneous PET imaging combined with both anatomical and functional MRI.

Cain and Abel reloaded? Kin recognition and male-male aggression in three-spined sticklebacks Gasterosteus aculeatus L.

The influence of relatedness on male-male aggression was tested in three-spined sticklebacks Gasterosteus aculeatus. The intensity of aggression against brothers and non-kin males did not differ significantly, indicating that kin recognition plays at most a minor role in aggressive interactions between male G. aculeatus.

Performance evaluation of an improved optical computed tomography polymer gel dosimeter system for 3D dose verification of static and dynamic phantom deliveries.

The performance of a next-generation optical computed tomography scanner (OCTOPUS-5X) is characterized in the context of three-dimensional gel dosimetry. Large-volume (2.2 L), muscle-equivalent, radiation-sensitive polymer gel dosimeters (BANG-3) were used. Improvements in scanner design leading to shorter acquisition times are discussed. The spatial resolution, detectable absorbance range, and reproducibility are assessed. An efficient method for calibrating gel dosimeters using the depth-dose relationship is applied, with photon- and electron-based deliveries yielding equivalent results. A procedure involving a preirradiation scan was used to reduce the edge artifacts in reconstructed images, thereby increasing the useful cross-sectional area of the dosimeter by nearly a factor of 2. Dose distributions derived from optical density measurements using the calibration coefficient show good agreement with the treatment planning system simulations and radiographic film measurements. The feasibility of use for motion (four-dimensional) dosimetry is demonstrated on an example comparing dose distributions from static and dynamic delivery of a single-field photon plan. The capability to visualize three-dimensional dose distributions is also illustrated.

Determination of neutron energy spectra inside a water phantom irradiated by 64 MeV neutrons.

A NE230 deuterated liquid scintillator detector (25 mm diameter x 25 mm) has been used to investigate neutron energy spectra as a function of position in a water phantom under irradiation by a quasi-monoenergetic 64 MeV neutron beam. Neutron energy spectra are obtained from measurements of pulse height spectra by the NE230 detector using the Bayesian unfolding code MAXED. The experimentally measured energy spectra are compared with spectra calculated by Monte Carlo simulation using the code MCNPX.

Evaluation of a diode array for QA measurements on a helical tomotherapy unit.

A helical tomotherapy system is used in our clinic to deliver intensity-modulated radiation therapy (IMRT) treatments. Since this machine is designed to deliver IMRT treatments, the traditional field flatness requirements are no longer applicable. This allows the unit to operate without a field flatness filter and consequently the 400 mm wide fan beam is highly inhomogeneous in intensity. The shape of this beam profile is mapped during machine commissioning and for quality assurance purposes the shape of the beam profile needs to be monitored. The use of a commercial diode array for quality assurance measurements is investigated. Central axis beam profiles were acquired at different depths using solid water built-up material. These profiles were compared with ion chamber scans taken in a water tank to test the accuracy of the diode array measurements. The sensitivity of the diode array to variations in the beam profile was checked. Over a seven week period, beam profiles were repeatedly measured. The observed variations are compared with those observed with an on-board beam profile monitor. The diode measurements were in agreement with the ion chamber scans. In the high dose, low gradient region the average ratio between the diode and ion chamber readings was 1.000 +/- 0.005 (+/- 1 standard deviation). In the penumbra region the agreement was poorer but all diodes passed the distance to agreement (DTA) requirement of 2 mm. The trend in the beam profile variations that was measured with the diode array device was in agreement with the on-board monitor. While the calculated amount of variation differs between the devices, both were sensitive to subtle variations in the beam profile. The diode array is a valuable tool to quickly and accurately monitor the beam profile on a helical tomotherapy unit.