Effect of prothymosin α on neuroplasticity following cerebral ischemia­reperfusion injury.

Prothymosin α (ProT), a highly acidic nuclear protein with multiple cellular functions, has shown potential neuroprotective properties attributed to its anti­necrotic and anti­apoptotic activities. The present study aimed to investigate the beneficial effect of ProT on neuroplasticity after ischemia­reperfusion injury and elucidate its underlying mechanism of action. Primary cortical neurons were either treated with ProT or overexpressing ProT by gene transfection and exposed to oxygen­glucose deprivation for 2 h in vitro. Immunofluorescence staining for ProT and MAP­2 was performed to quantify ProT protein expression and assess neuronal arborization. Mice treated with vehicle or ProT (100 µg/kg) and ProT overexpression in transgenic mice received middle cerebral artery occlusion for 50 min to evaluate the effect of ProT on neuroplasticity­associated protein following ischemia­reperfusion injury. The results demonstrated that in cultured neurons ProT significantly increased neurite lengths and the number of branches, accompanied by an upregulation mRNA level of brain­derived neurotrophic factor. Furthermore, ProT administration improved the protein expressions of synaptosomal­associated protein, 25 kDa and postsynaptic density protein 95 after ischemic­reperfusion injury in vivo. These findings suggested that ProT can potentially induce neuroplasticity effects following ischemia­reperfusion injury.

Nicotinamide Deteriorates Post-Stroke Immunodepression Following Cerebral Ischemia-Reperfusion Injury in Mice.

(1) Background: Inducing experimental stroke leads to biphasic immune responses, where the early activation of immune functions is followed by severe immunosuppression accompanied by spleen and thymus atrophy. Nicotinamide, a water-soluble B-group vitamin, is a known neuroprotectant against brain ischemia in animal models. We examined the effect of nicotinamide on the central and peripheral immune response in experimental stroke models. (2) Methods: Nicotinamide (500 mg/kg) or saline was intravenously administered to C57BL/6 mice during reperfusion after transiently occluding the middle cerebral artery or after LPS injection. On day 3, the animals were examined for behavioral performance and were then sacrificed to assess brain infarction, blood-brain barrier (BBB) integrity, and the composition of immune cells in the brain, thymus, spleen, and blood using flow cytometry. (3) Results: Nicotinamide reduced brain infarction and microglia/macrophage activation following MCAo (p < 0.05). Similarly, in LPS-injected mice, microglia/macrophage activation was decreased upon treatment with nicotinamide (p < 0.05), suggesting a direct inhibitory effect of nicotinamide on microglia/macrophage activation. Nicotinamide decreased the infiltration of neutrophils into the brain parenchyma and ameliorated Evans blue leakage (p < 0.05), suggesting that a decreased infiltration of neutrophils could, at least partially, be the result of a more integrated BBB structure following nicotinamide treatment. Our studies also revealed that administering nicotinamide led to retarded B-cell maturation in the spleen and subsequently decreased circulating B cells in the thymus and bloodstream (p < 0.05). (4) Conclusions: Cumulatively, nicotinamide decreased brain inflammation caused by ischemia-reperfusion injury, which was mediated by a direct anti-inflammatory effect of nicotinamide and an indirect protective effect on BBB integrity. Administering nicotinamide following brain ischemia resulted in a decrease in circulating B cells. This warrants attention with respect to future clinical applications.

Lithium upregulates growth-associated protein-43 (GAP-43) and postsynaptic density-95 (PSD-95) in cultured neurons exposed to oxygen-glucose deprivation and improves electrophysiological outcomes in rats subjected to transient focal cerebral ischemia following a long-term recovery period.

OBJECTIVES: Lithium has numerous neuroplastic and neuroprotective effects in patients with stroke. Here, we evaluated whether delayed and short-term lithium treatment reduces brain infarction volume and improves electrophysiological and neurobehavioral outcomes following long-term recovery after cerebral ischemia and the possible contributions of lithium-mediated mechanisms of neuroplasticity. METHODS: Male Sprague Dawley rats were subjected to right middle cerebral artery occlusion for 90 min, followed by 28 days of recovery. Lithium chloride (1 mEq/kg) or vehicle was administered via intraperitoneal infusion once per day at 24 h after reperfusion onset. Neurobehavioral outcomes and somatosensory evoked potentials (SSEPs) were examined before and 28 days after ischemia-reperfusion. Brain infarction was assessed using Nissl staining. Primary cortical neuron cultures were exposed to oxygen-glucose deprivation (OGD) and treated with 2 or 20 µM lithium for 24 or 48 h; subsequent brain-derived neurotrophic factor (BDNF), growth-associated protein-43 (GAP-43), postsynaptic density-95 (PSD-95), and synaptosomal-associated protein-25 (SNAP-25) levels were analyzed using western blotting. RESULTS: Compared to controls, lithium significantly reduced infarction volume in the ischemic brain and improved electrophysiological and neurobehavioral outcomes at 28 days post-insult. In cultured cortical neurons, BDNF, GAP-43, and PSD-95 expression were enhanced by 24- and 48-h treatment with lithium after OGD. CONCLUSION: Lithium upregulates BDNF, GAP-43, and PSD-95, which partly accounts for its improvement of neuroplasticity and provision of long-term neuroprotection in the ischemic brain.Abbreviations: BDNF: brain-derived neurotrophic factor; ECM: extracellular matrix; EDTA: ethylenediaminetetraacetic acid; GAP-43: growth-associated protein-43; GSK-3ß: glycogen synthase kinase-3ß; HBSS: Hank's balanced salt solution; LCBF: local cortical blood perfusion; LDF: laser-Doppler flowmetry; MCAO: middle cerebral artery occlusion; MMP: matrix metalloproteinase; NMDA: N-methyl-D-aspartate; NMDAR: N-methyl-D-aspartate receptor; OCT: optimal cutting temperature compound; OGD: oxygen-glucose deprivation; PSD-95: postsynaptic density-95; SDS: sodium dodecyl sulfate; SNAP-25: synaptosomal-associated protein-25; SSEP: somatosensory evoked potential.

Short-term lithium treatment protects the brain against ischemia-reperfusion injury by enhancing the neuroplasticity of cortical neurons.

OBJECTIVES: Lithium exerts a broad neuroprotective effect on the brain. This study examined whether lithium exerts therapeutic effects on stroke by restoring neural connections at the ischemic core of cortices post brain insult. METHODS: We treated rats with lithium or vehicle (saline) every 24 h for the first 72 h, starting at the beginning of reperfusion after inducing middle cerebral artery occlusion (MCAO) in rats. Somatosensory evoked potential (SSEP) recording and behavioral testing were employed to evaluate the beneficial effects of lithium treatment. To examine the effects of lithium-induced neuroplasticity, we evaluated the dendritic morphology in cortex pyramidal cells and the primary neuronal cell culture that underwent brain insults and oxygen and glucose deprivation (OGD), respectively. RESULTS: The results demonstrated that rats subjected to MCAO had prolonged N1 latency and a decreased N1/P1 amplitude at the ipsilateral cortex. Four doses of lithium reduced the brain infarction volume and enhanced the SSEP amplitude. The results of neurobehavioral tests demonstrated that lithium treatment improved sensory function, as demonstrated by improved 28-point clinical scale scores. In vitro study results showed that lithium treatment increased the dendritic lengths and branches of cultured neurons and reversed the suppressive effects of OGD. The in vivo study results indicated that lithium treatment increased cortical spine density in various layers and resulted in the development of the dendritic structure in the contralateral hemisphere. CONCLUSION: Our study confirmed that neuroplasticity in cortical neurons is crucial for lithium-induced brain function 50 recovery after brain ischemia.

Cinnamophilin enhances temozolomide-induced cytotoxicity against malignant glioma: the roles of ROS and cell cycle arrest.

BACKGROUND: Temozolomide (TMZ) has been widely used to treat glioblastoma multiforme (GBM). However, many mechanisms are known to quickly adapt GBM cells to chemotherapy with TMZ, leading to drug resistance and expansion of tumor cell populations. METHODS: We subjected human glioblastoma cell lines and an animal model of glioblastoma xenografts with TMZ-based adjuvant treatments to evaluate the synergistic effect of cinnamophilin (CINN), a free radical scavenger. RESULTS: Our results showed that the combined treatment of CINN and TMZ potentiated the anticancer effect and apoptotic cell death in glioma cell lines and enhanced antitumor action in glioma xenografts. TMZ induced reactive oxygen species (ROS) burst and elevated G2 arrest in glioma cells. The CINN-suppressed ROS burst in TMZ-treated glioma cells might be associated with increased apoptosis, as indicated by the upregulation of TUNEL-positive glioma cells. CINN-pretreated glioma cells exhibited increased cyclin B expression and reduced phosphorylation of Cdk1, suggesting reduced G2 arrest in the combined treatment group. Moreover, CINN lowered the protein level of LC3, a hallmark of autophagy, in TMZ-treated cells. CONCLUSIONS: These findings suggest that CINN may restore TMZ toxicity in glioma cancer by suppressing the ROS/G2 arrest pathway.

3D-Customized Guiding Template for Posterior Fixation in Complex Atlantoaxial Instability-Preliminary Experiences of National Cheng Kung University Hospital.

Objective Atlantoaxial fixation is technically demanding and challenging, especially in cases with anatomical abnormality. The purpose of this study is to report the effectiveness of the three-dimensional (3D)-customized guiding template for placement of C1 and C2 screws in cases with abnormalities. Method Two patients with anatomical abnormality and one without were included. The preoperative computed tomography (CT) image was analyzed using our software. The entry point, trajectory, and depth of the screws were designed based on these images. Templates with screw guiding cylinders and cervical spine model were created. In operation, guiding templates were applied directly to the laminae. Drilling, tapping, and screwing were performed through the cylinders. To evaluate the accuracy, deviation of the screw axis from the preplanned trajectory was measured on postoperative CT. A classification system was taking to evaluate the pedicle screw insertion. Results In complex cases, one of C2 screws has grade 2 deviation, and two has grade 1. There was no deviation in screws of C1. All patients achieved symptoms free after 6 months follow-up. Conclusion Although 3D-printed template for atlantoaxial fixation still has limitation in complex cases, it has been proved usefulness and makes the most difficult and dangerous spinal posterior fixation easy to achieve.

Chemical constituents from the stems of Machilus philippinensis Merr. and the neuroprotective activity of cinnamophilin.

The Machilus genus (Lauraceae) had been extensively utilized in folk medicine due to its broad range of bioactivities. In the present study, a series of chromatographic separations of the methanol extract of stems of M. philippinensis led to the identification of thirty eight compounds totally. Among these, biscinnamophilin (1), machilupins A-C (2-4), machilutone A (5), and machilusoxide A (6) were new compounds reported for the first time. In addition, 5 was characterized with a unprecedented carbon skeleton. Other known compounds, including the major compounds cinnamophilin (7) and meso-dihydroguaiaretic acid (8), are identified by comparison of their physical and spectroscopic data with reported values. One of the reported compounds, cinnamophilin A (10), should be revised as dehydroguaiaretic acid (9) after careful comparison of all the 1H and 13C NMR data. Moreover, the neuroprotective activity of cinnamophilin (7) was examined in a primary cortical neuron culture and the results indicated that 7 was effective against glutamate induced excitotoxicity.

YC­1 reduces inflammatory responses by inhibiting nuclear factor­κB translocation in mice subjected to transient focal cerebral ischemia.

3­(5­hydroxymethyl­2­furyl)­1­benzyl­indazole (YC­1) is understood to protect against ischemic stroke, but the molecular basis for its neuroprotection remains to be fully characterized. The present study investigated the influence of YC­1 on inflammatory responses following experimental stroke. Previous studies indicated that nuclear factor (NF)­κB­driven signals serve a pivotal role in mediating inflammatory responses following stroke. Ischemic stroke results in activation of NF­κB to induce gene expression of factors including inducible nitric oxide synthase, interleukin (IL)­1ß, IL­6 and matrix metalloproteinases (MMPs). The results of the present study demonstrated that YC­1 effectively reduced brain infarction and brain edema, and improved blood­brain barrier leakage. Additionally, animals treated with YC­1 exhibited significant reductions in neutrophil and macrophage infiltration into the ischemic brain. Furthermore, YC­1 effectively inhibited NF­κB translocation and binding activity, and the activity and expression of MMP­9 following ischemic stroke. In conclusion, YC­1 may effectively attenuate NF­κB­induced inflammatory damage following cerebral ischemia­reperfusion.

Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR­like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10-100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

Therapeutic window for YC­1 following glutamate­induced neuronal damage and transient focal cerebral ischemia.

3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), has been demonstrated to inhibit platelet aggregation, vascular contraction and hypoxia­inducible factor 1 activity in vitro and in vivo. The present study investigated the neuroprotective efficacy of YC­1 in cultured neurons exposed to glutamate­induced excitotoxicity and in an animal model of stroke. In a cortical neuronal culture model, YC­1 demonstrated neurotoxicity at a concentration >100 µM, and YC­1 (10­30 µM) achieved potent cytoprotection against glutamate­induced neuronal damage. Additionally, YC­1 (30 µM) effectively attenuated the increase in intracellular Ca2+ levels. Delayed treatment of YC­1 (30 µM) also protected against glutamate­induced neuronal damage and cell swelling in cultured neurons, though only at 4 h post­treatment. In addition, immediate treatment of YC­1 (30 µM) following the exposure of cortical neurons to glutamate (300 µM) produced a marked reduction in intracellular pH. Delayed treatment of YC­1 (25 mg/kg) protected against ischemic brain damage in vivo, though only when administered at 3 h post­insult. Thus, YC­1 exhibited neuroprotection against glutamate-induced neuronal damage and in mice subjected to transient focal cerebral ischemia. This neuroprotection may be mediated via its ability to limit the glutamate­induced excitotoxicity. However, the neuroprotective therapeutic window of YC­1 is only at 3 h in vivo and 4 h in vitro, which may, at least in part, be attributed to its ability to reduce the intracellular pH in the early phase of ischemic stroke. Although YC­1 provided the potential for clinical therapy, the treatment time point must be carefully evaluated following ischemia.

Magnolol protects against ischemic-reperfusion brain damage following oxygen-glucose deprivation and transient focal cerebral ischemia.

In the present study, the neuroprotective potential of magnolol against ischemia-reperfusion brain injury was examined via in vivo and in vitro experiments. Magnolol exhibited strong radical scavenging and antioxidant activity, and significantly inhibited the production of interleukin­6, tumor necrosis factor­a and nitrite/nitrate (NOX) in lipopolysaccharide-stimulated BV2 and RAW 264.7 cells when applied at concentrations of 10 and 50 µM, respectively. Magnolol (100 µM) also significantly attenuated oxygen­glucose deprivation­induced damage in neonatal rat hippocampal slice cultures, when administered up to 4 h following the insult. In a rat model of stable ischemia, compared with a vehicle­treated ischemic control, pretreatment with magnolol (0.01­1 mg/kg, intravenously) significantly reduced brain infarction following ischemic stroke, and post­treatment with magnolol (1 mg/kg) remained effective and significantly reduced infarction when administered 2 h following the onset of ischemia. Additionally, magnolol (0.3 and 1 mg/kg) significantly reduced the accumulation of superoxide anions at the border zones of infarction and reduced oxidative damage in the ischemic brain. This was assessed by measuring the levels of NOX, malondialdehyde and myeloperoxidase, the ratio of glutathione/oxidized glutathione and the immunoreactions of 8­hydroxy­2'­deoxyguanosine and 4­hydroxynonenal. Thus, magnolol was revealed to protect against ischemia­reperfusion brain damage. This may be partly attributed to its antioxidant, radical scavenging and anti­inflammatory effects.

Taiwan Neurosurgical Spine Society: The New Shining Star.

As spine surgery flourished in Taiwan and neurosurgeons became more involved in spine surgery towards the end of the 20th century, the Taiwan Neurosurgical Spine Society (TNSS), earlier named the Taiwan Neurospinal Society, was established on March 11, 2001. As its main founder, Dr. Chun-I Huang was elected as the first president of the TNSS. The goals of the TNSS were to promote research, to hold academic seminars, to participate in international conferences, and to exchange clinical experiences. The mission of the TNSS was successful, and the profession of spine surgery in Taiwan advanced during the first decade of the 21st century, culminating in the TNSS joining ASIA SPINE in 2010. Since its establishment, the TNSS has always been supportive of collaboration and communication with the Korean Spinal Neurosurgery Society and the Neurospinal Society of Japan. Through periodical meetings, supported by the TNSS, surgeons worldwide have enjoyed a platform of sharing and mutual learning. To further promote academic research, the TNSS has officially supported the journal Neurospine since 2018. With extensive efforts from local and international surgeons, the TNSS will continue to adhere to its mission and to advance the profession of spine surgery.

Neuroanatomical and electrophysiological recovery in the contralateral intact cortex following transient focal cerebral ischemia in rats.

Objectives Focal cerebral ischemia may induce synaptic, electrophysiological, and metabolic dysfunction in remote areas. We have shown that the remote dendritic spine density changes and electrophysiological diaschisis in the acute and subacute stages after stroke previously. Here, we further evaluated electrophysiological outcomes and synapto-dendritic plasticity in long-term recovery in the contralateral cortex following focal cerebral ischemia. Methods Male Sprague-Dawley rats were subjected to intraluminal suture occlusion for 90 min or sham-occlusion. Somatosensory electrophysiological recordings (SSEPs) and neurobehavioral tests were recorded each day for 28 days. Postmortem brains were sectioned and subjected to Nissl staining and Golgi-Cox impregnation through a 28-day period following ischemic stroke. Results In the ipsilateral cortex, infarct size in the cortex and striatum was decreased after the subacute stage; the brains showed reduced swelling in the cortex and stratum 3 days after ischemic insults. Dendritic spine density and SSEP amplitude decreased significantly during a 28-day recovery period. In the contralateral cortex, dendritic spine density and SSEP amplitude decreased significantly for 21 days after ischemic stroke, but recovered to baseline by day 28. The deterioration of the dendritic spine (density reduction) in the ischemic cortex was observed; however, this increased neuroplasticity in the contralateral cortex in the subacute stage. Discussion Focal cerebral ischemia-reperfusion induces time-dependent reduction of dendritic spine density and electrophysiological depression in both the ipsilateral and contralateral cortices and intact brain. This neuroanatomical and electrophysiological evidence suggests that neuroplasticity and functional re-organization in the contralateral cortex is possible following focal cerebral ischemia.

Anti-inflammatory Flavan-3-ol-dihydroretrochalcones from Daemonorops draco.

Four A-type flavan-3-ol-dihydroretrochalcone dimers, dragonins A-D (1-4), were characterized from the traditional Chinese medicine Sanguis Draconis. The structures of 1-4 were elucidated by spectroscopic and spectrometric analyses. Compounds 1 and 2 exhibited significant inhibition of fMLP/CB-induced superoxide anion and elastase. The signaling pathways accounting for the inhibitory effects of compound 2 were also elucidated. These purified A-type flavan-3-ol-dihydroretrochalcones are new potential leads for the development of anti-inflammatory drugs.

Chemical Constituents and Anti-inflammatory Principles from the Fruits of Forsythia suspensa.

Fifty compounds were isolated from the fruits of Forsythia suspensa, including 13 new compounds characterized as eight new diterpenoids (1-8), three new lignans (9-11), a new iridoid (12), and a new triterpenoid (13). Their structures were established on the basis of spectroscopic and spectrometric analysis. Most of the isolated compounds were examined for their anti-inflammatory activity in vitro. The results showed that several compounds displayed significant inhibition of fMLP/CB-induced superoxide anion generation and elastase release, with IC50 values ranging from 0.6 ± 0.1 to 8.6 ± 0.8 µg/mL and from 0.8 ± 0.3 to 7.3 ± 1.1 µg/mL, respectively.

Comparison of fresh/frozen isolation method in DNA preparation of meningiomas for flow cytometry.

The level of G2/M-phase can represent tumor grading to discriminate benign from atypical meningiomas using flow cytometric analysis. In this study, we compare two tumor DNA prepared methods using fresh and frozen samples for flow cytometric analysis. The specimens were obtained from tumoral tissues of 28 microsurgically resected meningiomas as approved by the institutional review board. Single-cell suspensions were prepared from fresh and frozen tumor tissues using fresh and frozen isolation methods. The coefficient of variation (CV) of DNA and the level of G2/M-phase were assessed by flow cytometric analysis. For fresh samples prepared using the fresh isolation method, atypical meningiomas had significantly higher G2/M-phase levels than those of benign meningiomas. In contrast, for fresh samples prepared using the frozen isolation method, the levels of G2/M-phase in benign and atypical meningiomas were severely interfered. Benign meningiomas could not be discriminated from atypical meningiomas based on the level of G2/M-phase. Additionally, frozen samples prepared using the frozen isolation method had significantly higher values of G2/M-phase in benign and atypical meningioma than those of fresh samples using fresh isolation method. CV was used to estimate the quality of DNA fixation. The diploid G0/G1 peak determined from fresh samples obtained using the fresh isolation method had a smaller CV than those for frozen samples obtained using the frozen isolation method. DNA prepared from fresh samples obtained using fresh isolation method is more suitable for discriminating benign from atypical meningiomas using flow cytometric analysis.

Application of flow cytometry for evaluating clinical prognosis and histopathological grade of human glioma.

OBJECTIVES: Flow cytometry was applied to predict the biological parameters of tumor behavior based on the DNA content distribution of tumors. We used flow cytometry to determine the number of cell cycles for the characterization of intracranial gliomas and its possible prognostic role. METHODS: Flow cytometric analysis of the DNA content was performed for 37 fresh operative glioma specimens. The expression of Ki-67 in glioma specimens was detected using immunohistochemistry staining. The check points of G2/M-phase fractions, cyclin B, and pCdk1 (Y15) were analyzed using Western immunoblotting. RESULTS: Compared to low-grade (grade I/II) gliomas, significant differences in the Ki-67, cyclin B, G2/M-phase, and S+G2/M-phase expressions were found in high-grade (grade III/IV) gliomas. Furthermore, receiver operating characteristic (ROC) analysis indicated optimal cutoff points for the G2/M-phase and S+G2/M-phase fractions of 13.47 and 17.26%, respectively, which can be used to differentiate cases with low- and high-grade gliomas. Additionally, both G2/M-phase and S+G2/M-phase fractions had significant association with the expression of Ki-67 in the gliomas. The gliomas were classified by the DNA content. We found that patients with high-grade glioma had worse survival rate than patients with low-grade glioma. Meanwhile, ROC curve analysis gave cutoffs for G2/M-phase of 9.4% and for S+G2/M-phase fractions of 15.04% as best predicting survival. The patients with glioma had poor survival when the levels of G2/M-phase and S+G2/M-phase were more than 9.4 and 15.04%, respectively. In contrast, no significant association between the DNA content of glioma patients and their age, tumor recurrence, and tumor size was found. DISCUSSION: Our results indicate that flow cytometry analysis for G2/M-phase and S+G2/M-phase fractions can be used for tumor grading for rapidly differentiating low- from high-grade gliomas.