Anti-HPV Nanoemulsified-Imiquimod: A New and Potent Formulation to Treat Cervical Cancer.

Cervical cancer is associated with the human papilloma virus (HPV) and nowadays is the fourth most frequent cancer among women. One of the treatments for this disease is based on the application of imiquimod. In this study, we postulated that the use of imiquimod in nanoemulsion results in a better antitumoral effect than the drug administered in its nonencapsulated form for the treatment of cervical cancer. Permeability studies using vaginal mucosa, as membrane, and in vitro studies involving cervical cancer cells (viability, clonogenic assay, and cell death analysis) were performed. We showed that low amount of encapsulated imiquimod permeated the vaginal mucosa. However, a higher percentage of cells died after the treatment with low amount (3.0 µmol L-1) of the formulation compared to the free drug. In addition, the innovative formulation presented a combinatory mechanism of cell death involving autophagy and apoptosis. Our results demonstrate that the imiquimod-loaded nanoemulsioncan be an alternative product for the treatment of cervical cancer validating the hypothesis.

Statistical scattering of waves in disordered waveguides: From microscopic potentials to limiting macroscopic statistics.

We study the statistical properties of wave scattering in a disordered waveguide. The statistical properties of a "building block" of length deltaL are derived from a potential model and used to find the evolution with length of the expectation value of physical quantities. In the potential model the scattering units consist of thin potential slices, idealized as delta slices, perpendicular to the longitudinal direction of the waveguide; the variation of the potential in the transverse direction may be arbitrary. The sets of parameters defining a given slice are taken to be statistically independent from those of any other slice and identically distributed. In the dense-weak-scattering limit, in which the potential slices are very weak and their linear density is very large, so that the resulting mean free paths are fixed, the corresponding statistical properties of the full waveguide depend only on the mean free paths and on no other property of the slice distribution. The universality that arises demonstrates the existence of a generalized central-limit theorem. Our final result is a diffusion equation in the space of transfer matrices of our system, which describes the evolution with the length L of the disordered waveguide of the transport properties of interest. In contrast to earlier publications, in the present analysis the energy of the incident particle is fully taken into account. For one propagating mode, N=1 , we have been able to solve the diffusion equation for a number of particular observables, and the solution is in excellent agreement with the results of microscopic calculations. In general, we have not succeeded in finding a solution of the diffusion equation. We have thus developed a numerical simulation, to be called "random walk in the transfer matrix space," in which the universal statistical properties of a "building block" are first implemented numerically, and then the various building blocks are combined to find the statistical properties of the full waveguide. The reported results thus obtained (in which use was made of a "short-wavelength approximation") are in very good agreement with those arising from truly microscopic calculations, for both bulk and surface disorder. Since the paper has a clear pedagogical aim, we have included, for the benefit of experts and non-experts, a number of appendixes that contain the more involved calculations.

Statistical wave scattering through classically chaotic cavities in the presence of surface absorption.

We propose a model to describe the statistical properties of wave scattering through a classically chaotic cavity in the presence of surface absorption. Experimentally, surface absorption could be realized by attaching an "absorbing patch" to the inner wall of the cavity. In our model, the cavity is connected to the outside by a waveguide with N open modes (or channels), while an experimental patch is simulated by an "absorbing mirror" attached to the inside wall of the cavity; the mirror, consisting of a waveguide that supports N(a) channels, with absorption inside and a perfectly reflecting wall at its end, is described by a subunitary scattering matrix S(a). The number of channels N(a) , as a measure of the geometric cross section of the mirror, and the lack of unitarity P(a) = [symbol: see text]N(a) - S(a)(+)S(a) , as a measure of absorption, are under our control: these parameters have an important physical significance for real experiments. The absorption strength in the cavity is quantified by gamma(a) = tr P(a). The statistical distribution of the resulting S matrix for N = 1 open channel and only one absorbing channel, N(a) = 1, is solved analytically for the orthogonal and unitary universality classes, beta = 1 and beta = 2, respectively, and the results are compared with those arising from numerical simulations. The relation with other models existing in the literature, in some of which absorption has a volumetric character, is also studied.

Genetic variation of foot-and-mouth disease virus during persistent infection in cattle.

Genetic variation of foot-and-mouth disease virus O1 Campos has been analyzed in consecutive isolates recovered over a one- or two-year period from four cattle with experimental persistent infection. Comparisons of RNase T1 two-dimensional maps and nucleotide sequences of the VP1-coding region revealed a continual, although irregular, increase in the fixation of mutations as the infection progressed. Most changes were not conserved in consecutive isolates. These results, together with the substantial rates of genomic variation observed between some pairs of strains recovered at close time periods, suggested the coexistence of heterogeneous populations in which variants evolve independently from each other, and predominate at irregular time intervals. Furthermore, non-related patterns of variation were observed in the four animals. Similarly, genetic diversity of representative strains from major serotype O outbreaks in endemic disease regions of southeastern Brazil and central eastern Argentina which occurred between 1958 and 1983, suggested that outbreak strains are also likely to represent fluctuations of heterogeneous populations which evolve independently from each other. The possible role of persistent infections in the introduction of variant populations in the field is discussed.

Adenoviral-mediated gene transfer to the filtering bleb in rabbits.

PURPOSE: To determine if genes can be transferred to fibroblasts in the filtering bleb using adenoviral vectors. MATERIALS AND METHODS: Twelve New Zealand albino rabbits underwent bilateral full-thickness sclerostomies. On postoperative day 1 an adenoviral vector carrying a reporter gene (lacZ) was injected into the right-eye bleb and saline was injected into the left eye bleb of each rabbit. Three rabbits were euthanized on each of the after days (days 3, 7, 14, and 21). The eyes were enucleated and tissue samples from the filtering bleb were processed for beta-galactosidase activity (a marker for lacZ gene expression) and expression of vimentin (a fibroblast marker). The median number of cells per high-power field with both beta-galactosidase activity and vimentin expression on days 3, 7, 14, and 21 in the right and left eyes were counted to determine adenoviral-mediated gene expression in fibroblasts. RESULTS: In the adenoviral vector-treated eyes, the median number of cells per high-power field with both beta-galactosidase and vimentin expression on days 3, 7, 14, and 21 was 83,100, 1, and 0, respectively. No beta-galactosidase activity was noted in the saline-treated eyes. CONCLUSIONS: Adenoviral vectors can transfer genes to fibroblasts in filtering blebs after glaucoma surgery. The peak efficiency of gene transfer to fibroblasts occurred 7 days after glaucoma surgery. These studies show a potential for genetic manipulation of fibroblast activity in filtering blebs after glaucoma surgery.

Influence of keratometric readings on comparative intraocular pressure measurements with Goldmann, Tono-Pen, and noncontact tonometers.

PURPOSE: To evaluate the influence of keratometric measurements on the concordance of intraocular pressure (IOP) readings with three applanation tonometers. PATIENTS AND METHODS: The IOPs of 404 eyes of 202 patients from a general eye clinic were measured by Goldmann, Tono-Pen XL, and noncontact Nidek NT 2000 tonometers. Differences in the IOP measurements between Goldmann and the other two tonometers (deltaIOPG-TP and deltaIOPG-NCT) were analyzed in the overall group and within three subgroups based on keratometry measures: flatter, intermediate, and steeper corneas (quartiles as cut-off points). Regression analysis of deltaIOP and keratometry measures was also performed. Right and left eyes were analyzed separately. RESULTS: The IOP readings taken with the Goldmann tonometer were significantly higher than those taken with either of the other two tonometers within the overall study population. The deltaIOP within the three keratometric subgroups was not statistically significant except for deltaIOPG-TP in the right eyes. Regression analysis showed that keratometry and deltaIOPG-TP and deltaIOPG-NCT had a significant but very weak coefficient of correlation in the right eye, but not in the left. CONCLUSION: Corneal curvature within the normal range does not have a clinically significant influence on the concordance of IOP readings obtained with Goldmann and Tono-Pen or noncontact tonometers.

Electronic transport through ballistic chaotic cavities: reflection symmetry, direct processes, and symmetry breaking.

We extend previous studies on transport through ballistic chaotic cavities with spatial left-right (LR) reflection symmetry to include the presence of direct processes. We first analyze fully LR-symmetric systems in the presence of direct processes and compare the distribution w(T) of the transmission coefficient T with that for an asymmetric cavity with the same "optical" S matrix. We then study the problem of "external mixing" of the symmetry caused by an asymmetric coupling of the cavity to the outside. We first consider the case where symmetry breaking arises because two symmetrically positioned waveguides are coupled to the cavity by means of asymmetric tunnel barriers. Although this system is asymmetric with respect to the LR operation, there is an effect of the symmetry of the cavity it was constructed from. Second, we break LR symmetry in the absence of direct processes by asymmetrically positioning the two waveguides and compare the results with those for the completely asymmetric case.

Pituitary apoplexy. Clinical course, endocrine evaluations and treatment analysis.

BACKGROUND: The purpose was to analyze clinical manifestations, hormonal changes, diagnosis difficulties and treatment of pituitary apoplexy (PA). EXPERIMENTAL DESIGN: A retrospective study of clinical records from patients with pituitary adenomas admitted from January 1980 to June 1996; the purpose was to identify the patients with clinical evidence compatible with PA. SETTING: Neurosurgery unit of an institutional hospital. PATIENTS: Sixteen (12.8%) of 125 patients with pituitary adenomas were analyzed because they had pituitary apoplexy. INTERVENTIONS: Surgical treatment by the trans-sphenoidal or transcranial route or both routes; dexamethasone (DXM) treatment with 16 mg/day i.v. MEASURES: Hormone assays were performed either by radioimmunoassay or by chemical luminescence. RESULTS: Tumors were nonfunctioning in nine patients and functioning in seven. TSH and prolactin basal serum levels were impaired in 55.5% and 10%, respectively; after exogenous TRH 80% of the patients did not show stimulation of TSH and prolactin secretions. LH and FSH levels were low in 63.6% and 54.6% of the patients, respectively; gonadotrophin-releasing hormone (GnRH) testing was abnormal in 75% of the patients evaluated. Cortisol levels were low in 50% of the patients. After insulin-induced hypoglycemia, cortisol and GH failed to rise in 25% and 40% of cases, respectively. Ten patients were submitted to surgical treatment, but none during PA. The average time from the onset of apoplectic symptoms and surgery was 70+/-50 days. Only one patient died two months after surgery. Five patients were treated with dexamethasone (DXM) during the apoplectic symptoms: three patients died; one patient had good quality of life; the other patient was treated initially with DXM with improvement of vision, but after surgery he developed panhypopituirarism. Two other patients did not receive specific treatment for PA. CONCLUSIONS: PA is not a rare pituitary adenoma complication and its prognosis may be poor; baseline hormone levels showed a wide range of abnormalities of pituitary function; surgical treatment was required in the majority of patients and the prognosis was relatively good; on the contrary, the treatment with DXM only had high levels of mortality.

Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in all cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.

Visual field defects in children with congenital glaucoma.

PURPOSE: To determine visual field defects in a cohort of children with congenital glaucoma. METHODS: Monocular visual fields were measured in 24 meridians for targets V4e, I4e, I2e, and I1e, using a Goldmann perimeter in a group of 13 children between the ages of 4 and 14 years with congenital glaucoma and 10 age-matched healthy children. Localized visual field defects (eg, paracentral scotoma, nasal step, and arcuate scotoma) were determined by abnormal findings or shape of the eye in at least one of each of the targets presented. RESULTS: Visual field extent for target 12e was significantly constricted for unilateral and bilateral cases of congenital glaucoma when compared with control eyes. A post-hoc procedure (Tukey Test) showed significant differences between unilateral cases and normal control eyes, and between bilateral cases (best outcome eye) and normal control eyes. Stimuli V4e and 14e results were comparable for patients and normals. Stimulus I1e showed significantly different total extent visual field for bilateral and normal controls. Specific visual field defects were found only in bilateral cases. Paracentral scotoma was found in 1 of 12 eyes with bilateral congenital glaucoma. Nasal steps were found in 6 of 12 eyes with bilateral congenital glaucoma. Arcuate scotoma were found in 4 of 12 eyes with bilateral congenital glaucoma. CONCLUSION: Localized visual fields were found in 37.5% of eyes with congenital glaucoma. Early treatment for congenital glaucoma provided better visual field outcome.

Infectivity assays of foot-and-mouth disease virus: contact transmission between cattle and buffalo (Bubalus bubalis) in the early stages of infection.

No differences were observed between cattle and Indian buffalo (Bubalus bubalis) in terms of temperature, viraemia or virus replication in the pharyngeal area, during the acute phase of foot-and-mouth disease. Like cattle, the Indian buffalo became infected and excreted virus before any clinical signs of foot-and-mouth disease developed. The disease was transmitted from cattle to buffalo and vice versa, during the acute stage of infection, as if the animals had been of the same species, presumably because of their close phylogenetic relationship. There were more tongue lesions in the cattle than in the buffalo. Foot lesions in the buffalo at first had a scaley appearance, but later became vesicular. Anti-virus infection associated antigen and neutralising antibodies were synthesised at the same time in both species and reached similar titres in the same period. Persistent infection in the buffalo during the first 35 days after infection was similar to that in the cattle.

[Recording of the intracranial pressure]. / Registro da pressão intracraniana

A revision of the methodology for recording intracranial pressure and its use in man is made. Experience is presented with animal embolism followed by recording the extradural pressure. Preliminary report is made about the extradural and intraventricular pressure in patients with signs of involvement of the brain stem.

[Acute traumatic subdural haematomas: study of 110 cases]. / Hematoma subdural agudo traumático: estudo de 110 pacientes.

We report a series of 110 patients with acute traumatic subdural hematoma (ASDH) admitted at HBDF emergency within 1994 (January 1st to December 1st). All patients were treated according to the same protocol. There was a predominance of males (79%), with ages ranging from 14 to 70, being car accidents (20%) and car-pedestrian accidents (34%) the most frequent causes. The majority of patients (85.7%) was admitted in very serious condition, with a score of 8 points on the Glasgow Coma Scale (GCS) or lesser, which directly influenced the mortality rates. CT scan was the diagnostic procedure of choice, and it showed contusion and brain swelling to be the most frequent associated intracranial lesions. Surgery was carried out in 45.1% of cases and, in most instances, through an ample fronto-temporo-parietal craniotomy, with hematoma drainage and dural reconstitution. In 54.9% of cases, clinical conditions did not allow surgery and in this group, 69.6%.

[Measurement of arteriovenous oxygen difference in the monitoring of patients with subarachnoid hemorrhage due to cerebral aneurysm]. / Medida da diferença artério-venosa de oxigênio na monitorização de pacientes com hemorragia subaracnóidea por aneurisma cerebral.

The arterious venous oxygen difference (AVDO2) due to the close relationship with cerebral metabolic rate of oxygen and cerebral blood flow shows metabolic alterations that occur in some pathological situations in the brain including subarachnoid haemorrhage. The AVDO2 was calculated by the Fick equation and the results evaluated by the Glasgow outcome scale. Measurements of arteriojugular oxygen difference were carried out in 30 patients with subarachnoid haemorrhage due to rupture of intracranial aneurysms, as an attempt to monitor the relationship between changes in AVDO2, clinical picture, and evolution of the patients. The subarachnoid haemorrhage was diagnosed by CT scan in 17 patients and by lumbar punction in 13 and the diagnosis of arterial vasospasm was carried out by clinical evaluation and confirmed by four vessels angiogram in only eight patients. Eighteen patients were admitted with Hunt & Hess (H&H) I/II, seven with H&H III and five with H&H IV/V. Nineteen patients had AVDO2 normal and this group had three deaths; five patients had AVDO2 continuously low with three deaths; and six patients had AVDO2 continuously high with two deaths. The patients with normal AVDO2 had better prognosis and clinical evolution than the patients with abnormal values of AVDO2. In conclusion, AVDO2 measurements could not be correlated with the diagnosis of vasospasm, but was useful in the early identification of metabolic changes that occur after subarachnoid haemorrhage and could be used as an supplementary monitoring in the clinical evaluation of patients with this pathology.

[Chondromyxoid fibroma of the thoracic spine: a case report and review of the literature]. / Fibroma condromixóide da coluna torácica: relato de caso e revisão da literatura.

A case of chondromyxoid fibroma (CMF) arising from the 5th right costovertebral junction and spreading into spinal canal causing spinal cord compression is presented. A myelotomography revealed a complete block at T5 level. The patient underwent a decompressive laminectomy with removal of an epidural tumor. This specimen was sent for pathological examination and interpreted as a CMF. The patient had a neurological improvement, post operative MRI revealed a spinal cord free of compression, and we decided on the follow up of the case. Two years later there was recurrence of the tumor. A posterolateral access by costotransversectomy was made and the lesion was resected. The patient had a neurological improvement which persists on the follow up (two years, at present). Clinical, radiologic and histologic findings, surgical management and recurrence are discussed. The pertinent literature is reviewed.

Microwave-induced combustion of crude oil for further rare earth elements determination by USN-ICP-MS.

A procedure for light and heavy crude oils digestion by microwave-induced combustion (MIC) is proposed for the first time for further rare earth elements (REE) determination by inductively coupled plasma mass spectrometry (ICP-MS) equipped with an ultrasonic nebulizer (USN). Samples of crude oil (API density of 10.8-23.5, up to 250 mg) were inserted in polycarbonate capsules and combusted using 20 bar of oxygen and 50 µL of 6 mol L(-1) ammonium nitrate as igniter. Nitric acid solutions (1-14.4 mol L(-1)) were evaluated for analyte absorption and a reflux step was applied after combustion (5 min of microwave irradiation at 1400 W) in order to achieve better analyte recoveries. Accuracy was evaluated using a spiked sample and also by comparison of results obtained by microwave-assisted digestion combined to ultraviolet radiation (MW-UV) and by neutron activation analysis (NAA). Using 3 mol L(-1) HNO3, quantitative recoveries (better than 97%) were obtained for all analytes. Blank values were always negligible. Agreement was higher than 96% for La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y by comparison of results with those obtained by MW-UV and by NAA (only for La, Ce, Nd, Sm, and Yb). Residual carbon content in digests using MIC was always below 1%. As an advantage over conventional procedures for crude oil digestion, using MIC, it was possible to use diluted acid as absorbing solution, obtaining better limits of detection and avoiding interferences in REE determination by USN-ICP-MS.

Focused microwave-induced combustion for digestion of botanical samples and metals determination by ICP OES and ICP-MS.

The advantages and shortcomings of focused microwave-induced combustion (FMIC) for digestion of plant samples were studied. The effects of sample mass, absorbing solution, oxygen gas flow-rate, and time of reflux step on recoveries of major, minor and trace metals were systematically evaluated. Afterwards, Al, Ba, Ca, Co, Cr, Cu, Mg, Mn, Ni, Sr, V, and Zn were determined by inductively coupled plasma optical emission spectrometry (ICP OES) and by inductively coupled plasma mass spectrometry (ICP-MS). The main advantages of FMIC when compared to microwave-assisted wet digestion (MAWD) and focused-microwave-assisted wet digestion (FMAWD) are the possibility to digest larger masses of samples (up to 3g) using shorter heating times and diluted nitric acid solution for absorbing all analytes. Using the selected experimental conditions for FMIC, residual carbon content was lower than 0.7% for all samples and relative standard deviation (RSD) varied from 1.5 to 14.1%. Certified reference materials (NIST 1515 apple leaves and NIST 1547 peach leaves) were used for checking accuracy and determined values for all metals were in agreement with certified values at a 95% confidence level. No statistical difference (ANOVA, 95% of confidence level) was observed for results obtained by FMIC, FMAWD, and MAWD. Limits of detection were lower when using FMIC in the range of 0.02-0.15 µg g(-1) for ICP OES and 0.001-0.01 µg g(-1) for ICP-MS, which were about 3 and 6 times lower than the values obtained by FMAWD and MAWD, respectively. It is important to point out that FMIC was a suitable sample preparation method for major, minor and trace metals by both determination techniques (ICP OES and ICP-MS). Additionally, since it allows lower LODs (because up to 3g of sample can be digested) and diluted acid solutions are used (without any further dilution), the use of ICP-MS is not mandatory.

On the lifetime-width relation for a decaying state and the uncertainty principle.

A new formulation of the uncertainty relation of position and momentum, and of energy and time, is presented. The connection between the lifetime of excited states and the energy width of these states, which does not follow from the usual uncertainty relation, is shown to be a consequence of the expression here derived.

Synthesis of elastic microfibrillar components fibrillin-1 and fibrillin-2 by human optic nerve head astrocytes in situ and in vitro.

The purpose of this study was to identify elastic microfibrillar components fibrillin-1 and fibrillin-2 in optic nerve heads of adult normal and glaucomatous subjects, in cultured optic nerve head astrocytes (type 1B astrocytes), as well as fibrillin-1 in fetal optic nerve heads. To characterize synthesis and gene expression of microfibrillar proteins in human optic nerve heads and cultured type 1B astrocytes, light microscopy immunohistochemistry, in situ hybridization, and RT-PCR or Northern blots were performed. Our results demonstrated that fibrillin-1 was associated with blood vessels, astrocytes in the glial columns and cribriform plates, and with astrocyte processes in the nerve bundles in all samples. In glaucomatous optic nerves there was enhanced fibrillin-1 immunoreactivity, especially surrounding blood vessels. Fibrillin-2 was localized primarily to blood vessels in all samples, without qualitative differences between normal and glaucomatous samples. In fetal optic nerve heads fibrillin-1 mRNA was localized to glial cells and to the blood vessel walls. In adult optic nerve heads, there was little fibrillin-1 mRNA as detectable by in situ hybridization and RT-PCR. There was no detectable upregulation of fibrillin-1 mRNA in glaucoma. In cultured type 1B astrocytes, fibrillin-1 staining was mostly pericellular. There was little fibrillin-2 immunoreactivity. In conclusion, astrocytes from the optic nerve head deposit elastic microfibrillar components in situ and in vitro, with a predominance of fibrillin-1. Upregulation of fibrillin-1 mRNA was not observed in glaucoma, suggesting that increased transcription may occur early in the disease process. Cultures of type 1B astrocytes from the optic nerve head provides a useful model to study mechanisms regulating the interactions of elastin and the microfibrils in optic nerve head astrocytes.

Expression of the aphthovirus RNA polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections.

A plasmid has been constructed containing the DNA sequences that direct the expression of the aphthovirus RNA-dependent RNA polymerase (virus infection-associated antigen, VIAA) in its native form. The aphthovirus polypeptide was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant protein has been purified and used in enzyme-linked immunoelectrotransfer blots to detect aphthovirus-specific antibodies in the sera of persistently infected animals. Furthermore, studies were carried out to test the hypothesis that antibodies against other nonstructural antigens appear in the sera of these animals. It was established that antibodies against polypeptides 3A and 3B can serve as complementary markers for late aphthovirus-carrier state detection. The considerable potential of this approach to detect aphthovirus-specific antibodies, when the isolation of infectious virus is not possible, was demonstrated. Negative results were obtained in animals from virus-free areas and in vaccinated cattle. This assay has the added advantage that no infectious or noninfectious virus is involved during antigen production.