The evaluation of whitening efficacy of cosmetic products using a human skin pigmentation spot model.

BACKGROUND: To establish a pigmentation spot model on human skin and to assess whitening efficacy for whitening products by this established pigmentation spot model. METHODS: Twenty subjects between 20 and 45 years old with skin phototype III or IV were selected. Three consecutive daily UV exposures were performed on buttocks of the subjects as follows: Day 1=1 minimal erythema dose (MED), Day 2=0.5 MED and Day 3=0.5 MED. After the first UV exposure, a selected whitening product was applied to the subjects twice a day on UV exposure area. The application of the whitening product to subjects on the exposed areas was continued till Day 27. CM2500d chromameter, Maxmeter MX18 and visual evaluation were used to assess changes of skin color. RESULTS: A pigmentation spot model after UV exposure was established. The measurement of the pigment spot showed that L* value declined abruptly at Day 3 and then slowly reached to a lowest point at Day 6. L* value of the pigment spot almost remained at the same level until Day 20, thereafter increased slowly. The a* value showed an abrupt increase at Day 3 and slowly reached to a maximal level at Day 6. The a* value slowly declined toward its baseline level. Likewise, the erythema index also increased significantly at Day 3, and reached to a maximal level at Day 6 and then slowly declined. However, L*, a* and erythema indices did not return to their baseline levels during the 27-day period of this study. On the other hand, b* value started to increase from Day 3 and such increase was observed continuously to Day 27. Melanin index also showed a slow increase during the first 3 days. It started to increase rapidly from Day 3 and a to maximal level at Day 9 and maintain at a plateau till Day 27 (with an exception at Day 13). To assess the whitening product by this pigmentation spot model, DeltaL, Deltab*, and DeltaM values were analyzed. It showed that absolute DeltaL value and Deltab*value of whitening products were lower than those values of the vehicle of the whitening product at each checkpoint, while DeltaM value of the whitening product was lower only at Day 9 and Day 20, although no statistically significant differences was found. The visual results also strongly supported that the whitening product enhanced the decrease of pigmentation. CONCLUSION: This study showed that repeated UV exposure was able to induce a long extensive period of pigment formation. The resulted pigmentation spot was able to maintain at an elevated level till Day 20. Clinical subjective evaluations together with combined objective instrument measurements were still important to assess whitening and spot-removing ability of a material due to the instrument limitation for color differentiations. This kind of pigmentation spot model can be used to assess whitening efficacy for whitening or spot-removing products. In addition, the combinations of subjective and objective methods were able to serve as advisable references to assess the whitening efficacy of products.

Cloning and characterization of the T-box gene Tbx6 in Xenopus laevis.

Tbx6 is a member of the T-box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6-expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

Pollen tube attraction by the synergid cell.

In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

Detection of antipituitary antibodies in patients with autoimmune thyroid disease.

The aim of the present study was to investigate the prevalence of antipituitary antibodies (APA) in patients with autoimmune thyroid disease as determined by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Results by Western blot analysis showed positivity for APA in serum of 22.4% of patients with Graves' disease (n=143, p<0.05) and 18.5% of patients with Hashimoto's thyroiditis (n=54, p<0.05), which were significantly higher than 6.2% in healthy controls (n=97). Similar results were obtained with ELISA. The titers of APA measured by ELISA (APA/ELISA) remained unchanged before and after therapy with antithyroid drug for Graves' disease, while thyrotropin-binding inhibitor immunoglobulins (TBII) decreased significantly. Similarly, no changes in APA by Western blot analysis were observed after therapy. In patients with Graves' disease, APA were not associated with thyroid status. There was no difference in APA between patients with positive and negative thyroid autoantibodies. A significant but weak positive correlation between APA/ELISA and anti-human GH measured by ELISA (anti-hGH/ELISA) was observed in patients with Graves' disease (r=0.601 p<0.001) and Hashimoto's thyroiditis (r=0.428 p<0.005). These findings have demonstrated the existence of APA detected by Western blot analysis and ELISA in some cases of autoimmune thyroid disease. The present results suggest that hGH and other antigens may be involved in APA in patients with Graves' disease and Hashimoto's thyroiditis.

Antipituitary antibodies as pathogenetic factors in patients with pituitary disorders.

Our previous reports showed detection of antipituitary antibodies (APA) by immunoblot analysis and enzyme linked immunosorbent assay (ELISA) by using rat pituitary tissue as antigen in patients with certain endocrine disorders. In the present report, we evaluated APA by using our immunoblot and ELISA technique in 76 patients with various pituitary disorders. The prevalence of a 22 kDa band of APA detected by immunoblot was found to be significantly higher (P<0.01) in patients with pituitary disorders (20 of 76, 26%) than in the controls (3 of 209, 1%). APA levels detected by ELISA were significantly higher in patients with GH deficiency, isolated ACTH deficiency, acromegaly, and idiopathic panhypopituitarism compared with control (mean +/- SD; 2.40+/-2.66, 2.36+/-1.87, 2.09+/-1.87, 3.10+/-1.96 versus 1.42+/-0.64 (C.I.) P<0.05, respectively). APA levels detected by ELISA in 7 patients with GH deficiency showed a statistically significant decrease (p<0.05) after administration of GH replacement therapy. APA detection by immunoblot is useful in examining as pathogenesis, while ELISA may be useful as an objective index of pathological state in patients with autoimmune-related pituitary disorders.

Practical protective tools for occupational exposure: 1) double focus spectacles for the aged with highly refracted glass lens 2) remodeled barrier for radiation protection.

SUMMARY: Two practical protective tools for occupational exposure for neurointerventional radiologists are presented. The first purpose of this study was to investigate the effectiveness of double focus spectacles for the aged with a highly refracted glass lens (special spectacles for the aged) for radiation protection of the crystalline lens of the eye in comparison with other spectacles on the market, based on the measurement of film density which was obtained by exposure of X-ray through those spectacles. As a result of the film densitometry mentioned above, the effectiveness of special spectacles for the aged in radiation protection was nearly equal to the effectiveness of a goggle type shield which is made with a 0.07 mm lead-equivalent plastic lens. The second purpose of this study was to investigate the effectiveness of the protective barrier, which we remodeled for cerebral angiography or neuroendovascular therapy, for radiation exposure, based on the measurement in a simulated study with a head phantom, and on the measurement of radiation exposure in operaters during procedures of clinical cases. In the experimental study radiation exposure in supposed position of the crystalline lens was reduced to about one third and radiation exposure in supposed position of the gonadal glands was reduced to about one seventh, compared to radiation exposure without employing the barrier. The radiation exposure was monitored at the left breast of three radiologists, in 215 cases of cerebral angiography. Employing the barrier in cerebral angiography, average equivalent dose at the left breast measured 1.49mu Sv during 10 min of fluoroscopy. In three kinds of neuroendovascular therapy in 40 cases, radiation exposure in an operator was monitored in the same fashion and the dose was recorded less than the result reported in previous papers in which any protective barrier have not been employed in the procedure (1,2). As a result, the two above mentioned protective tools are considered practical in clinical usage and very effective to reduce radiation exposure in an operator of interventional neuroradiolgy which may sometimes require many hours to complete the therapy under extended fluoroscopic time. 1) The first topic of this report is double focus spectacles for the aged with a highly refracted glass lens (special spectacles for the aged).

Laboratory diagnosis of dengue virus infection by reverse transcriptase polymerase chain reaction (RT-PCR) and IgM-capture enzyme-linked immunosorbent assay (ELISA).

Dengue virus infections are a major public health problem in most tropical and sub-tropical countries of the world. Dengue is occasionally imported by travelers who visit tropical areas and become infected with dengue virus. Laboratory diagnosis is essential for confirming the diagnosis of this virus. For purposes of confirmation, detection of specific IgM by IgM-capture enzyme-linked immunosorbent assay (ELISA) and of dengue virus genome by reverse transcriptase polymerase chain reaction (RT-PCR) have recently been used. In the present study, we tested serum specimens from dengue-suspected Japanese cases, by IgM-capture ELISA, RT-PCR, HI, and virus isolation. Serum samples collected before or on the day of defervescence were positive by RT-PCR, though no PCR-positive samples were obtained after fever day 1. IgM-capture ELISA was positive as early as disease day 4, and all samples but one were IgM-positive when collected on disease day 5 or later. In light of these findings, we recommend that both RT-PCR and IgM-capture ELISA be performed, irrespective of the stage of dengue illness. Combination of RT-PCR and IgM-capture ELISA increases the ability to diagnose dengue virus infection, even in the only that a single serum specimen from the patient is available.

[Current status of Japanese encephalitis in Japan].

Japanese encephalitis (JE) cases confirmed by serological and/or virological tests, and clinically typical fatal JE cases have been collected using individual report cards in Japan since 1965. A total of 324 confirmed cases (male: 167, female: 157) reported between 1982 and 1996 were analyzed. Annual incidence of JE cases was less than one hundred cases from 1982 to 1991, and less than 10 cases after 1992. Age distribution of the patients was: 0-9 years old, 43 cases (13%); 10-39 years old, 30 cases (9%); 40-69 years old, 161 cases (50%); over 70 years old, 90 cases (29%). The highest incidence in male cases was observed in the age group of 50-59 years, and that in female was in 60-69 years. Most of the patients (95%) were living in the western parts of Japan. No patient was reported from Okinawa, southern-most prefecture of Japan, and from Tohoku-Hokkaido area, northern parts of Japan. The date of onset were distributed from the end of July to the end of October, and the highest incidence was observed in the end of August. Prognosis of the patients were: complete recovery, 99 cases (31%); sequelae, 156 cases (48%); total, 56 cases (17%); unknown, 13 cases (4%). Vaccination histories of patients were reported as vaccinee, 4 cases (1%); incomplete vaccinee, 14 cases (4%); non-vaccinee, 181 cases (56%); unknown, 125 cases (39%). Spread of JE virus in Japan has not been diminished as shown by the data of antibody acquisition of young swine in every summer in Japan. Based on these observations we conclude that JE vaccination has contributed to maintaining the low JE incidence among children in Japan.

Portal blood flow and liver regeneration in auxiliary partial orthotopic liver transplantation in a canine model.

Functional competition has been shown to lead to a detrimental outcome in auxiliary liver transplantation. We evaluated the interaction in auxiliary partial orthotopic liver transplantation between the native liver and the graft in terms of portal flow and regeneration. The need for diversion of the portal flow to the graft was also assessed. Reduced-size liver grafts were transplanted orthotopically after partial hepatectomy in beagles. There were two groups: the preserved group, where portal inflow to the native liver was preserved, and the ligated group, where it was interrupted. Portal flow was measured serially and liver regeneration was evaluated on postoperative day 5. Functional competition was not observed in the preserved group. On the other hand, ligation of the native liver portal vein had no obviously detrimental effects on the remnant native liver. This leads to the conclusion that the portal vein to the native liver can be safely ligated to prevent functional competition.

Studies on circulating anti-pituitary antibodies in NIDDM patients.

The clinical significance of anti-pituitary antibodies (APA) was examined by enzyme-linked immunosorbent assay (ELISA) in individuals with non-insulin dependent diabetes mellitus (NIDDM). Serum samples were obtained from 150 NIDDM patients and 45 normal subjects. Urinary C-peptide (U-CPR) was also measured for the NIDDM patients. APA-positive serum was incubated with porcine pancreas, liver, kidney, or spleen powder and analyzed by immunoblot. The prevalence of APA was found to be significantly (P<0.05) higher in NIDDM patients (24.7%) than in the controls (6.7%) by ELISA. The index values for APA were inversely related to the levels of U-CPR (P<0.005). The levels of U-CPR were significantly (P<0.0001) lower and the prevalence of insulin deficiency was significantly (P<0.05) higher in NIDDM patients who were APA positive than in those who were APA negative. The presence of APA may therefore be related to reduced secretion of insulin in NIDDM patients. In Western blot analysis, preincubation of APA-positive sera with porcine pancreas powder prevented recognition of the 22-kD protein (APA). The possibility of a common autoantigenicity in the pancreas and the pituitary was indicated.

Auxiliary partial orthotopic liver transplantation from living donors: significance of portal blood flow.

BACKGROUND: Auxiliary liver transplantation has several advantages over standard orthotopic liver transplantation. However, functional competition has been reported even in auxiliary partial orthotopic liver transplantation (APOLT). We evaluated herein the interaction in APOLT between the native liver and the graft in terms of portal blood flow and regeneration. The need for diversion of the portal blood flow to the graft was also assessed. METHODS: A total of 15 patients received APOLT from living donors. Portal blood flow to the native liver was preserved in 6 patients, and the portal vein to the native liver was preemptively transected at the time of transplantation in 9 patients. RESULTS: Of the patients with preservation of the portal blood flow to the native liver, two showed inadequate graft portal blood flow just after operation, and in the other three patients the graft portal blood flow decreased or the graft atrophied after deterioration of the graft function. In the patients with preemptive transection of the portal vein to the native liver, optimal graft portal blood flow was obtained, and the native liver, supplied only by arterial inflow, supported a small-for-size graft until the graft regenerated. The damage to the native liver was minimal. CONCLUSIONS: Functional competition may occur in APOLT with preservation of the portal blood flow to the native liver, whereas preemptive transection of the native liver portal vein is a safe procedure and effectively prevents the portal steal phenomenon.

Determination of antipituitary antibody in patients with endocrine disorders by enzyme-linked immunosorbent assay and Western blot analysis.

The identification of pituitary antigens recognized by human antipituitary antibodies (APAs) is important in evaluating the pathophysiology of multiendocrine disorders linked to autoimmune factors. However, there is no convenient method for the quantitative analysis of circulating APAs. This study reports the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of APAs. APAs were measured by ELISA and confirmed by Western blot analysis in sera from patients with endocrine disorders. APAs were detected frequently in patients with autoimmune thyroiditis, insulin-dependent diabetes mellitus (IDDM), or pituitary dwarfism. Circulating APAs were detected in 18% of patients with autoimmune thyroiditis. Confirmation by Western blot revealed positivity for APAs in the serum of 36% of patients with Hashimoto disease and in 29% of patients with Graves disease. Notably, 39% of patients with IDDM were also positive for APAs by ELISA. The identification of APAs by ELISA may be useful in evaluating autoimmune mechanisms involved in patients with multiendocrine disorders.

A case of autoimmune hypophysitis associated with asymptomatic primary biliary cirrhosis.

We report a 61-year old male patient with panhypopituitarism complicated with asymptomatic primary biliary cirrhosis (PBC). T1-weighted magnetic resonance imaging demonstrated high intensity of the anterior pituitary gland. There was no mass lesion or enlargement of the pituitary gland or the stalk. Immunoblot analysis of the patient's sera with rat pituitary antigens revealed a band with a molecular size of 22 kD. Anti-M2 mitochondrial antibody has been consistently positive for five years. Liver biopsy revealed portal hepatitis with periportal infiltration of the inflammatory cells. This is the first case report of autoimmune hypophysitis complicated with asymptomatic PBC.

Hepatic artery thrombosis in living related liver transplantation.

BACKGROUND: Hepatic artery thrombosis (HAT) after orthotopic liver transplantation remains a significant cause of graft loss in pediatric patients. We previously reported that the microsurgical techniques for arterial anastomosis can reduce the incidence of HAT in living related liver transplantation (LRLT). The purpose of this study is to analyze the risk factors for HAT after LRLT. A total of 245 patients received 250 liver transplants. METHODS: Eight arteries in eight patients, reconstructed with the use of loupe magnification (HAT; 1/8, 12.5%), were excluded from this study. We observed HAT in 4 patients of the 242 transplants (1.7%, HAT group). Seventeen factors were compared between the HAT and the control group (those without HAT). RESULTS: HAT occurred in 3 of 33 grafts (9%) from ABO-incompatible donors, whereas it occurred in 1 of 209 grafts (0.5%) from identical or compatible donors (P=0.008). The corrected volume of fresh-frozen plasma intraoperatively transfused in the HAT group (46.9+/-30.3 ml/kg) was significantly (P=0.015) different from that in the control group (10.2+/-1.9 ml/mg). In all four patients with HAT, emergent revisions of the anastomosis were performed. Two patients with ABO-incompatible grafts died of hepatic failure and sepsis. CONCLUSIONS: Although microsurgical techniques can minimize the surgical risk factors for HAT, overtransfusion of fresh-frozen plasma in high-risk patients (ABO incompatible) may be a critical factor in the development of HAT in LRLT.

Presence of anti-pituitary antibodies and GAD antibodies in NIDDM and IDDM.

OBJECTIVE: To evaluate the clinical significance of the presence of anti-pituitary antibodies (APAs) in patients with NIDDM or IDDM and to examine the relationship of APAs to GAD antibodies (GADAs). RESEARCH DESIGN AND METHODS: Serum samples were obtained from patients with NIDDM and IDDM. APAs, determined by Western blot analysis, and GADAs, determined by radioimmunoassay, were detected in the patients' sera and control sera. Urinary levels of C-peptide (U-CPR) were measured. RESULTS: The prevalence of APAs was significantly higher in patients with NIDDM (24.2%) or IDDM (56.8%) than in healthy control subjects (6%). In patients with NIDDM, the levels of U-CPR were significantly lower, and the prevalence of insulin deficiency was higher in APA+ patients than in APA- patients. CONCLUSIONS: This is the first study to demonstrate that the prevalence of APAs is increased in patients with NIDDM and IDDM. The presence of APAs may be related to reduced secretion of insulin in NIDDM patients.

Coexistence of a graft with the preserved native liver in auxiliary partial orthotopic liver transplantation from a living donor for ornithine transcarbamylase deficiency.

BACKGROUND: Auxiliary partial orthotopic liver transplantation (APOLT) has recently been performed in patients with noncirrhotic metabolic liver diseases. However, long-term outcomes for the preserved native liver and the transplanted liver graft have not been clearly established yet. METHODS: The recipient was a 36-month-old girl with ornithine transcarbamylase deficiency. She underwent APOLT, using her father's left lateral segment. RESULTS: Liver function was normalized soon after APOLT and the patient was able to ingest a normal diet without medication. Coexistence of the well-functioning native liver and graft was demonstrated in a computed tomography scan, Doppler ultrasonography, scintigraphy, and histological examination, during a relatively long-term follow-up period. CONCLUSIONS: APOLT seems to be most useful for the treatment of noncirrhotic metabolic liver diseases.

Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR.

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Saikosaponin homologues from Verbascum spp. The structures of mulleinsaponins I-VII.

From the aerial parts of Verbascum (V.) sinaiticum, V. thapsiforme, V. fruticulosum and Celsia roripifolia, seven new saikosaponin homologues, called mulleinsaponins I-VII, having 13,28-epoxy-olean-11-ene skelton were isolated together with eight known saikosaponin homologues, 3-O-beta-D-fucopyranosyl saikogenin F, saikosaponin a, desrhamnosylverbascosaponin, songarosaponins C, D, mimengoside A and buddlejasaponins I, IV. The structures of mulleinsaponins I-VII were characterized as 3-O-beta-D-glucopyronosyl-(1-->3)-beta-D-fucopyranosyl-6-deoxy- saikogenin F, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)-beta-D - fucopyranosyl-16-deoxysaikogenin F, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)-beta-D - fucopyranosyl-saikogenin F, 3-O-alpha-L-rhamnopyranosyl- (1-->4)-beta-D-glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-be ta -D-fucopyranosyl-21 beta-hydroxysaikogenin F, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)-[beta- D- glucopyranosyl-(1-->2)]-beta-D-fucopyranosyl-21 beta-acetoxysaikogenin F, 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D- glucopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D- fucopyranosyl-16 beta-acetoxysaikogenin F and 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl- (1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranosylsaikogeni n F 16-O-beta-D-glucopyranoside, respectively, from chemical and spectroscopic evidence.

[Laboratory virological diagnosis of imported dengue cases].

Suspected dengue cases in Japan during the period of 1985-1995, 173 cases with unknown febrile illness entering or returning from mostly southeast Asia, were tested by serology and RT-PCR gene amplification. Seventy seven cases were confirmed by a significant rise of dengue 2 (Den 2) HI antibody in paired sera or by detection of HI antibody titer higher than 1:320 in single serum. The other 3 cases with antibody levels not higher than 1:80 in paired sera and 12 cases with an antibody 1:160 in single sera were positively suspected of dengue infection but were not confirmed. Countries of origin of confirmed cases were Thailand (39 cases), Philippinse (15), India (13), and Indonesia (9). Sera of dengue cases showed high degrees of cross reactivity of Japanese encephalitis virus (JEV) in HI test but not in IgM capture ELISA. Sera of confirmed JEV-infected cases, however, showed practically no cross reactivity to Den 1 4 in HI test, suggesting unilateral cross reactivity of HI antibody. RT-PCR detected the Den 1 genome in sera of 3 cases obtained within 3 days after onset and the Den 2 genome in serum of case 4 days after onset. Although the number is limited, 92 (53%) out of 173 cases of febrile illness of unknown etiology entering Japan from tropical countries were either confirmed or positively suspected to be dengue fever. Considering possibilities of under reporting, importations of tropical viral infections should be bigger in number and will necessitate our intensified alertness.