The aim of this study was to retrospectively assess the diagnostic performance of multidetector CT (MDCT) for the diagnosis of acute cholangitis using a new scoring method.
Of 80 patients with suspected biliary disease who underwent biphasic CT and endoscopic retrograde cholangiography, 39 were diagnosed as having acute cholangitis (Group 1) and 41 patients were classified as suspected biliary disease (Group 2). 100 age-matched patients without evidence of biliary disease were selected randomly as a control group (Group 3). Each patient's axial scan was scored by two independent radiologists for the extent of transient hepatic attenuation difference, the presence of biliary dilatation and identification of a biliary obstructive lesion. The difference in the scores among the three groups was evaluated and the optimal cut-off score for the diagnosis of acute cholangitis was determined. Interobserver agreement was also evaluated.
The total scores (mean±standard deviation) for Groups 1, 2 and 3 were 7.0±2.0, 4.4±2.4 and 0.9±1.2, respectively, for Reviewer 1 and 7.2±1.8, 4.3 ±2.7 and 0.7±1.1, respectively, for Reviewer 2. Significant differences were found for the subscores and the total scores among the three groups (p<0.001). Using a cut-off score of ≥5, the sensitivity and specificity for diagnosing acute cholangitis were 84.6% and 83.7%, respectively, for Reviewer 1 and 89.7% and 83.7%, respectively, for Reviewer 2. Agreement for the subscores between readers was good to excellent (κ=0.74–0.86).
Based on dynamic MDCT and the described CT scoring method, the diagnosis of acute choangitis can be made with high sensitivity and specificity.
Perforation usually requires emergency surgery and may affect the prognosis of patients with gastrointestinal tumours. Accurate pre-operative diagnosis of these conditions is important because proper management such as curative surgical treatment may be needed. The aims of this article are to illustrate CT appearances of perforated tumours of the gastrointestinal tract and to discuss their impact on clinical management.
The purpose of this study was to compare the diagnostic accuracy of time-resolved MR angiography (TR-MRA) with that of conventional venography for the detection and grading of ovarian venous reflux, which aid in the diagnosis of pelvic venous congestion.
We performed a retrospective analysis of 19 consecutive patients who underwent TR-MRA and conventional venography. The images were analysed by two radiologists in a randomised “blinded” manner. With the use of conventional venography as a gold standard, the images were reviewed to determine if differences in the detection and grading of ovarian venous reflux were seen between TR-MRA and conventional venography; the sensitivity, specificity and accuracy of TR-MRA compared with that of conventional venography were evaluated. The McNemar test was performed to determine the significance of any differences. Interobserver agreement was analysed using generalised κ statistics.
There was no significant difference between TR-MRA and conventional venography for grading ovarian venous reflux (p>0.05). The sensitivity, specificity and diagnostic accuracy of TR-MRA were found to be 66.7%, 100% and 78.9%, and 75%, 100% and 84.2%, respectively, for the two observers. The weighted κ-values indicated excellent agreement between the two observers for grading ovarian venous reflux on TR-MRA (κ=0.894).
TR-MRA is an accurate method for accessing pelvic venous congestion.
Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand
found in Panax ginseng. Gintonin induces transient
through G protein-coupled LPA receptors. Large-conductance Ca2+-activated
channels are expressed in blood vessels and neurons and
play important roles in blood vessel relaxation and attenuation of
neuronal excitability. BKCa channels are activated by transient
and are regulated by various Ca2+-dependent kinases. We
investigated the molecular mechanisms of BKCa channel activation
by gintonin. BKCa channels are heterologously expressed in
Xenopus oocytes. Gintonin treatment induced BKCa channel activation in
oocytes expressing the BKCa channel α subunit in a
concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL).
Gintonin-mediated BKCa channel activation was blocked by a PKC
inhibitor, calphostin, and by the calmodulin inhibitor,
calmidazolium. Site-directed mutations in BKCa channels targeting
CaM kinase II or PKC phosphorylation sites but not PKA
phosphorylation sites attenuated gintonin action. Mutations in the
Ca2+ bowl and the regulator of K+ conductance (RCK) site also
blocked gintonin action. These results indicate that
gintonin-mediated BKCa channel activations are achieved through
LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase
II pathways and calcium binding to the Ca2+ bowl and RCK domain.
Gintonin could be a novel contributor against blood vessel
constriction and over-excitation of neurons.
The aim of this study was to evaluate the relationships between the severity of appendicitis as depicted on CT and blood inflammatory markers of serum white blood cell (WBC) count and C-reactive protein (CRP).
CT images in 128 patients (109 surgically proven and 19 with clinically excluded appendicitis) were retrospectively reviewed. Two radiologists by consensus evaluated and scored (using a 0, 1 or 2 point scale) severities based on CT-determined appendiceal diameters, appendiceal wall changes, caecal changes, periappendiceal inflammatory stranding and phlegmon or abscess formation. We investigated whether CT findings were significantly related to elevated WBC counts or CRP levels and performed the correlations of WBC counts and CRP levels with CT severity scores. Patients were also subjectively classified using four grades from normal (Grade I) to perforated appendicitis (Grade IV) on the basis of CT findings to evaluate differences in WBC counts and CRP levels between grades.
Only appendiceal wall changes and the phlegmon or abscess formation were related to elevated WBC counts and CRP levels, respectively (p<0.05). CT severity scores were found to be more strongly correlated with CRP levels (r = 0.669) than with WBC counts (r = 0.222). On the basis of CT grades, the WBC counts in Grade I were significantly lower than in other grades (p<0.001), whereas CRP levels in Grade IV were significantly higher than in other grades (p<0.001).
CRP levels were found to correlate with CT-determined acute appendicitis severity and could be a useful predictor for perforated appendicitis, whereas WBC counts might be useful to detect early acute appendicitis.
The purpose of this study was to evaluate the use of diffusion-weighted imaging (DWI) for the detection and characterisation of focal hepatic lesions compared with the use of T2 weighted imaging.
45 patients with 97 hepatic lesions (51 malignant lesions and 46 benign lesions) were included in this retrospective study. Malignant hepatic lesions included 12 hepatocellular carcinomas, 26 metastases and 13 intrahepatic cholangiocarcinomas. Benign hepatic lesions included 19 haemangiomas and 27 cysts. The MRI protocol for the upper abdomen included T2 weighted images, in- and opposed-phase T1 weighted images and dynamic T1 weighted images. Breath-hold fat-suppressed single-shot echo planar DWI was performed with the following parameters: 1338/66; b factors, 0, 50 and 800 s mm–2. Two independent observers reviewed the T2 weighted images and the DWI to detect and to characterise the hepatic lesions.
For detection of malignant hepatic lesions, the use of DWI showed a significantly higher detection rate than the use of T2 weighted images (p<0.05). However, there was no significant difference between the use of DWI and T2 weighted images for benign hepatic lesions. For the differentiation between malignant and benign hepatic lesions, there was no significant difference in sensitivity, specificity and accuracy between the use of T2 weighted images and the use of DWI.
The use of DWI was better for the detection of malignant hepatic lesions than the use of T2 weighted images. However, for detection of benign hepatic lesions and characterisation of hepatic lesions, the use of DWI was equivalent to the use of T2 weighted images.
It has been recognized that Gastrodia elata Bl (GE), an oriental herb medicine, ameliorates various neurological disorders, that GE modulates the monoaminergic and GABAergic systems, and that GE possess antioxidant activities. We examined whether GE affects methamphetamine (MA)-induced striatal dopaminergic toxicity in mice. Treatment with MA (7.5 mg/kg, i.p. × 4) resulted in significant decreases in behavioural activity (as shown by locomotor activity and rota rod performance), dopamine level, tyrosine hydroxylase (TH) activity, and TH protein expression (as evaluated by immunocytochemistry and western blot analysis). In addition, MA treatment showed significant increases in lipid peroxidation [as evaluated by 4-hydroxy-2-nonenal (4-HNE) expression and malondialdehyde formation], protein oxidation (as shown by protein carbonyl expression and its formation), and reactive oxygen species (ROS) formation. Treatment with GE significantly attenuates MA-induced behavioural and dopaminergic impairments, and oxidative stresses in a dose-dependent manner. Our results suggest that GE treatment shows anti-dopaminergic effects in response to MA insult via, at least in part, inhibiting oxidative stresses in the striatum of the mice.
Gastrodia elata Bl; methamphetamine; dopamine; oxidative stress.
It has been demonstrated that 5-HT1A receptors play an important role in the pathophysiology of schizophrenia. Because Gastrodia elata Bl (GE) modulates the serotonergic system, we examined whether GE could affect phencyclidine (PCP)-induced abnormal behavior in mice. Repeated treatment with PCP increased immobility time, while it decreased social interaction time and recognition memory. PCP-induced abnormal behaviors were significantly attenuated by GE, and these effects were comparable to those of 8-OH-DPAT, a 5-HT1A receptor agonist. Furthermore, GE-mediated effects were counteracted by WAY 100635, a 5-HT1A receptor antagonist. Our results suggest that the antipsychotic effects of GE are, at least in part, mediated via activation of 5-HT1A in mice.
Gastrodia elata Bl; phencyclidine; schizophrenia; 5-HT1A receptors.
It has been suggested that GABAergic neurotransmission can modulate cocaine dependence and seizure activity. Since Gastrodia elata Bl (GE), an oriental herb agent, has been shown to enhance GABAergic transmission, we examined whether GE affects cocaine-induced seizures, conditioned place preference (CPP), and behavioral sensitization in mice. Treatment with GE (500 or 1000 mg/kg, p.o.) significantly delayed seizure onset time and significantly shortened seizure duration induced by cocaine (90 mg/kg, i.p.). In addition, cocaine (15 mg/kg, i.p.)-induced CPP was significantly attenuated by GE in a dose-dependent manner. However, GE did not significantly alter behavioral sensitization induced by cocaine (15 mg/kg, i.p.). In order to understand whether GABAergic receptors are implicated in GE-mediated pharmacological action in response to cocaine, GABAA receptor antagonist bicuculline and GABAB receptor antagonist SCH 50911 were employed in the present study. GE-mediated attenuations on the cocaine-induced seizures and CPP were significantly reversed by bicuculline (0.25 or 0.5 mg/kg, i.p.), but not by SCH 50911 (1.5 or 3.0 mg/kg, i.p.). Therefore, our results suggest that GE attenuates cocaine-induced seizures and CPP via, at least in part, GABAA receptor activation.
Gastrodia elata Bl; cocaine; seizure; conditioned place preference; GABAA receptors.
Cocaine, as an indirect dopamine agonist, induces selective behavioral and physiological events such as hyperlocomotion and dopamine release. These changes are considered as consequences of cocaine-induced molecular adaptation such as CREB and c-Fos. Recently, methanolic extracts from licorice was reported to decrease cocaine-induced dopamine release and c-Fos expression in the nucleus accumbens. In the present study, we investigated the effects of liquiritigenin (LQ), a main compound of licorice, on acute cocaine-induced behavioral and molecular changes in rats. LQ attenuated acute cocaine-induced hyperlocomotion in dose-dependent manner. In addition, LQ inhibited CREB phosphorylation and c-Fos expression in the striatum and the nucleus accumbens induced by acute cocaine. Results provide strong evidence that LQ effectively attenuates the acute behavioral effects of cocaine exposure and prevents the induction of selective neuroadaptive changes in dopaminergic signaling pathways. Further investigation of LQ from licorice extract might provide a novel therapeutic strategy for the treatment of cocaine addiction.
Liquiritigenin; cocaine; hyperlocomotion; CREB; c-Fos; nucleus accumbens; striatum.
To determine whether the tumor cell contamination of peripheral blood stem cells influences clinical impacts on high-dose chemotherapy in patients with metastatic breast cancer, we analyzed carcinoembryonic antigen (CEA) mRNA in the apheresis products by nested RT-PCR (reverse transcriptase-polymerase chain reaction). A total of 38 metastatic breast cancer patients and ten normal healthy subjects as a negative control were included. Twenty out of 38 (51.3%) apheresis products from patients with metastatic breast cancer were positive for CEA mRNA. CEA mRNA was noted in 54.8% (17/31) of patients mobilized with chemotherapy plus G-CSF and 42.8% (3/7) of patients with G-CSF alone. There was no significant difference in age, estrogen receptor, menopausal status, mobilization method, disease free interval, or number of metastasis sites (1 vs > or = 2) between positive and negative groups. The presence of CEA mRNA in apheresis products did not influence the time to progression and overall survival in both groups. However, both the univariate and the multivariate analysis disclosed that the number of metastasis was associated with survival significantly. We suggest that the tumor cell contamination does not predict poor treatment outcome in patients with metastatic breast cancer.
Background: Although the adenoma to carcinoma pathway in colorectal cancer is well described, the mechanisms of carcinogenesis in the small intestine remain unclear.
Aims: The aim of this study was to investigate candidate genes in the genetic pathway of adenocarcinoma of the small intestine.
Subjects and methods: A total of 21 non-familial, non-ampullary adenocarcinomas of the small intestine were analysed. DNA was extracted from formalin fixed paraffin wax embedded tissue using standard techniques. The replication error (RER) status was determined by amplification of BAT26. The mutation cluster region (MCR) of the adenomatous polyposis coli (APC) gene was screened using polymerase chain reaction single strand conformational polymorphism and direct sequencing. Immunohistochemistry was performed on formalin fixed paraffin wax embedded tissue using monoclonal antibodies for hMLH1, hMSH2, β-catenin, E-cadherin, and p53.
Results: Fourteen male and seven female patients with a median age of 64 years (range 21–85) presented with adenocarcinoma of the duodenum (10), jejunum (7), and ileum (4). One cancer (5%) was found to be RER+, and all tumours stained positive for hMLH1 and hMSH2. No mutations were detected in the MCR of the APC gene. β-Catenin showed increased nuclear expression with loss of membranous staining in 10 cancers (48%). Absent or decreased membrane expression of E-cadherin was found in eight cancers (38%). Strong staining of p53 was found in the nucleus of five cancers (24%).
Conclusion: We did not detect mutations in the MCR of the APC gene, and this suggests that adenocarcinoma of the small intestine may follow a different genetic pathway to colorectal cancer. Abnormal expression of E-cadherin and β-catenin was common and reflects an early alternative to APC in this pathway in which mutations may be found in adenocarcinoma of the small intestine.
small intestine; adenocarcinoma; replication error; adenomatous polyposis coli; β-catenin; E-cadherin; p53
Both 5-fluorouracil and doxorubicin are commonly used agents in chemotherapy of gastric cancer in adjuvant setting as well as metastatic disease. In a variety of malignancies, high expression of multidrug resistance-associated protein1 and P-glycoprotein has been associated with resistance to doxorubicin, whereas 5-fluorouracil resistance has correlated with the level of thymidylate synthase expression. We evaluated the expression of multidrug resistance-associated protein1, P-glycoprotein, and thymidylate synthase using immunohistochemistry in 103 locally advanced gastric cancer patients (stage IB-IV) who underwent 5-fluorouracil and doxorubicin-based adjuvant chemotherapy after curative resection and investigated the association between their expression and clinicopathologic characteristics including prognosis of the patients. While high expression (⩾5% of tumour cells positive) of multidrug resistance-associated protein1 and P-glycoprotein was observed in 70 patients (68%) and 42 patients (41%), respectively, 65 patients (63%) had primary tumours with high expression (⩾25% of tumour cells positive) of thymidylate synthase. There was a significant association between multidrug resistance-associated protein1 and P-glycoprotein expression (P<0.0001) as well as P-glycoprotein and thymidylate synthase expression (P<0.0001). High multidrug resistance-associated protein1 and P-glycoprotein expressions were associated with well and moderately differentiated histology (P<0.0001 and P=0.03, respectively) and intestinal type (P<0.0001 and P=0.009, respectively). High multidrug resistance-associated protein1 expression correlated with lymph node metastasis (P=0.037), advanced stage (P=0.015), and older age (P=0.021). Five-year disease-free survival and overall survival of total patients were 55.2% and 56.2%, respectively, with a median follow-up of 68 months. There were no significant differences in disease-free survival and overall survival according to the expression of multidrug resistance-associated protein1 (P=0.902 and P=0.975, respectively), P-glycoprotein (P=0.987 and P=0.955, respectively), and thymidylate synthase (P=0.604 and P=0.802, respectively). Concurrent high expression of these proteins (high multidrug resistance-associated protein1/P-glycoprotein, high multidrug resistance-associated protein1/thymidylate synthase, high P-glycoprotein/thymidylate synthase) did not correlate with disease-free survival or overall survival. Even high expression of all three proteins was not associated with poor disease-free survival (P=0.919) and overall survival (P=0.852). In conclusion, high expression of multidrug resistance-associated protein1, P-glycoprotein, and thymidylate synthase did not predict poor prognosis of gastric cancer patients treated with 5-fluorouracil and doxorubicin-based adjuvant chemotherapy. A larger study including patients treated with surgical resection alone would be necessary.
British Journal of Cancer (2002) 86, 1578–1585. DOI: 10.1038/sj/bjc/6600305 www.bjcancer.com
© 2002 Cancer Research UK
gastric cancer; multidrug resistance-associated protein1; P-glycoprotein; thymidylate synthase; adjuvant chemotherapy; prognosis
We evaluated the expression of thymidylate synthase (TS) in locally advanced gastric cancer patients treated with adjuvant chemotherapy after curative resection and investigated the association between TS expression and clinicopathologic characteristics including prognosis of the patients. TS expression was evaluated by immunohistochemical staining using TS106 monoclonal antibody in 103 locally advanced gastric cancer patients (stage IB–IV) who underwent 5-fluorouracil (5-FU) and doxorubicin-based adjuvant chemotherapy after curative resection. 65 patients (63%) had primary tumours with high TS expression (≥ 25% of tumour cells positive), and 38 patients (37%) demonstrated low TS expression (< 25% of tumour cells positive or no staining). High TS expression was associated with male gender (P = 0.002), poorly differentiated histology (P = 0.015), and mixed type in Lauren’s classification (P = 0.027). There were no statistically significant differences in 4-year disease-free survival (60.0% vs 57.2%, P = 0.548) and overall survival (59.6% vs 59.3%, P = 0.792) between high-TS group and low-TS group. In conclusion, although high TS expression was associated with poorly differentiated histology and mixed type in Lauren's classification, it did not predict poor disease-free and overall survival in gastric cancer patients treated with 5-FU and doxorubicin-based adjuvant chemotherapy after curative resection. Further prospective studies including the evaluation of other biological markers associated with the resistance to 5-FU and doxorubicin are necessary. © 2001 Cancer Research Campaign http://www.bjcancer.com
gastric cancer; thymidylate synthase; adjuvant chemotherapy; drug resistance; prognosis
There has been no standard therapy for patients with metastatic colorectal cancer who have failed to first-line fluorouracil-based treatment. The present study was designed to assess the efficacy and toxicities of a combination of oxaliplatin, 5-fluorouracil (5-FU) and leucovorin in fluoropyrimidine-pretreated patients with metastatic colorectal cancer. Chemotherapy consisted of oxaliplatin 85 mg/m2 on day 1, followed by leucovorin 20 mg/m2 and 5-FU 1,200 mg/m2 on days 1 and 2. Treatment courses were repeated every two weeks. Thirty-nine patients were enrolled in this study. All patients previously received fluoropyrimidine-based chemotherapy. Thirty-one patients were assessable for response and 33 for treatment toxicity. Six patients required dose reduction of 5-FU due to grade III/IV cytopenia. Nausea/vomiting and peripheral neuropathy were common non-hematologic toxicities. Overall response rate was 42.0% including 3 complete response and 10 partial response. The median response duration was 91 days (range, 28-224+). The median duration of progression-free survival was 132 days (range, 40-308). A combination of oxaliplatin, 5-FU, and leucovorin showed high response rate in fluoropyrimidine-pretreated patients with metastatic colorectal cancer, but the duration of response was relatively short. It may be worthwhile to explore its therapeutic potential in the first-line treatment setting.
Isolated splenic metastasis arising from colorectal carcinoma is very rare and there has been only 6 cases reported in the English literature. A new case is presented, and its possible pathogenesis was considered with previously reported cases. A 65-year-old male patient had received a right hemicolectomy for ascending colon cancer 36 months earlier. He was followed up regularly with serial measurement of serum carcinoembryonic antigen (CEA). Rising serum CEA was discovered from 33 months postoperatively and CT revealed an isolated splenic metastasis. He therefore underwent splenectomy, which was proven to be a metastatic adenocarcinoma with similar histological feature to the original tumor. As all reported cases showed elevated serum CEA at the time of metastasis, isolated splenic metastasis might be associated with CEA in regard to its biological functions of immunosuppression and adhesion.
The aim of this study was to determine the effect of partial liquid ventilation (PLV) using a perfluorocarbon (PFC) on gas exchange and lung inflammatory response in a canine acute lung injury model. After inducing severe lung injury by oleic acid infusion, beagle dogs were randomized to receive either gas ventilation only (control group, n = 6) or PLV (PLV group, n = 7) by sequential instillation of 10 mL/kg of perfluorodecalin (PFC) at 30 min intervals till functional residual capacity was attained. Measurements were made every 30 min till 210 min. Then the lungs were removed and bronchoalveolar lavage (BAL) (35 mL/kg) was performed on the right lung and the left lung was submitted for histologic analysis. There was significant improvement in PaO2 and PaCO2 in the PLV group compared to the control group (p < 0.05) which was associated with a significant decrease in shunt (p < 0.05). There was no significant difference in parameters of lung mechanics and hemodynamics. There was a significant decrease in cell count and neutrophil percentage in BAL fluid and significantly less inflammation and exudate scores in histology in the PLV group (p < 0.05). We conclude that PLV with perfluorodecalin improves gas exchange and decreases inflammatory response in the acutely-injured lung.
Extramammary Paget's disease (EMPD) is an intraepithelial neoplastic disorder which is included as a rare malignant condition. However, it sometimes shows aggressive behavior of local recurrence and coexisting malignancy. We had experienced nine cases of EMPD involving the scrotum for seven years. Two cases of them presented metastasis. The first case presented extensive inguinal lymph node metastasis with underlying adnexal adenocarcinoma one year after wide local excision. The second case initially presented multiple metastasis to the liver and in the lymph node. The latter, showing fulminant progression with liver metastasis, may be only the second case reported in English literature. EMPD is considered as a malignant neoplasm with aggressive behavior from initial presentation. Because wide local excision of the lesion alone may be occasionally insufficient, a careful follow-up must be done to detect recurrence or internal malignancy.
We report a case of myelitis after plasma-derived hepatitis B vaccination. The patient was a 31-year-old man who presented with progressive sensory symptoms in extremities that developed 2 weeks after a third vaccination. MRI of the cervicothoracic region revealed swelling and T2 high signal at the level of C4 to C5 cord, and isolated enhancement in the posterior columns between C4 and C5 cord. The significance of MRI findings and HLA haplotype of the patient will be briefly discussed.
It has been known that the vestibular system is concerned with feelings of dizziness or vertigo. The vestibulo-thalamic pathway has also been described previously. However, there has been no confirmative report so far regarding the pathway through the internal capsule to the cortex. We have experienced 13 patients with symptoms of dizziness and/or vertigo whose lesions are located only around the internal capsule, mainly at the posterior limb and/or the genu. It is suggestive that fibers with dizziness may pass through a part of the internal capsule, probably through some part of the posterior limb and/or the genu.
Chronic mucocutaneous candidiasis is an immunodeficiency disease characterized by T-cell dysregulation and chronic superficial candidal infections. We report on three patients with chronic mucocutaneous candidiasis who developed autoantibodies to erythrocytes. Our first patient, a 19-year-old female, developed autoimmune hemolytic anemia (AIHA) that required multiple courses of treatment, including corticosteroids, intravenous immunoglobulin, and danazol. During the last exacerbation of AIHA, intensive treatment with corticosteroids and intravenous immunoglobulin failed and yet the patient responded to plasmapheresis. Our second patient, a 21-year-old male, developed AIHA which responded to oral corticosteroid therapy. Our third patient, a 6-year-old female without evidence of hemolysis, was found to have erythrocyte autoantibodies on routine screening. These three patients had positive direct antiglobulin tests, and the first patient had both immunoglobulin G (IgG) and IgM erythrocyte autoantibodies, while the remaining two patients had only IgG autoantibody. This is the first report of the association of AIHA with chronic mucocutaneous candidiasis. We suggest that all patients with chronic mucocutaneous candidiasis be screened periodically for erythrocyte autoantibodies. Plasmapheresis, a safe ancillary procedure in the management of AIHA, may be life-saving in some cases. The occurrence of erythrocyte autoantibodies in mucocutaneous candidiasis may be related to immunoregulatory disorders in this disease.