Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (SeO). A laboratory strain of Bacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na2SeO3). After harvest, the washed, lyophilized B. Subtilis and M. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of SeO. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray SeO had a bioavailability of 2% of selenite, but that the bioavailability of red SeO depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray SeO and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3-4%). These findings suggest that aerobic bacteria such as B. subtilis and M. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Liver selenium and testis phospholipid hydroperoxide glutathione peroxidase are associated with growth during selenium repletion of second-generation Se-deficient male rats

We have previously shown that changes in glutathione peroxidase-1 (GPX1; H2O2:oxidoreductase, EC 1.11.1.9), plasma thyroid hormone and glutathione-S-transferase were not associated with changes in growth observed in second-generation (F2) severely Se-deficient rats; we also found that liver phospholipid hydroperoxide glutathione peroxidase (GPX4; EC 1.11.1.12) activity falls in first-generation (F1) Se-deficient rats to 41% of levels in Se-adequate rats. The purposes of this study were to determine the effect of F2 Se deficiency on GPX4 and to detect early changes in Se parameters associated with growth after single, small Se injections. Se-deficient male F2 weanling rats were randomly divided into two groups and fed a Se-deficient crystalline amino acid (0.003 microg Se/g diet; -Se) diet or that diet supplemented for 14 d with 0.2 microg Se/g diet (+Se) as Na2SeO3. Growth of -Se rats was 55% of the rate of +Se rats. Liver Se, GPX1 activity, GPX4 activity and testis GPX4 activity in -Se rats at 14 d were 1, 2, 23 and 13%, respectively, of levels in +Se rats. In a series of experiments, additional F2 male weanling rats were fed the -Se diet for 14 d and then were given an intraperitoneal single saline injection of 0, 1 or 5 microg Se/100 g body weight (BW) as Na2SeO3 and killed 1 or 7 d later. Rats injected with 1 or 5 microg Se/100 g BW grew 36 or 48%, respectively, above the rate of saline-injected rats. Liver Se concentration increased 367% and testis GPX4 activity doubled in rats 1 d after injection of 1 microg Se/100 g BW compared with saline-injected rats; these parameters were further elevated with 5 microg Se/100 g BW injections. Increases in liver Se and testis GPX4 activity were the parameters best associated with improved growth after Se injection, but the molecular role for Se in growth remains unclear.     

Beneficial effect of selenium supplementation during murine infection with Trypanosoma cruzi

Selenium (Se) has been shown to function as an antioxidant that may enhance immunity during microbial infection. To investigate the effect of elevated levels of Se on the course of experimental Chagas' disease, 5 groups of C3HeB/FeJ mice were infected with 10(3) bloodform trypomastigotes of a Brazil strain of Trypanosoma cruzi while receiving supplements of 0 ppm, 2 ppm, 4 ppm, 8 ppm, or 16 ppm Se as sodium selenate in drinking water. After 64 days of infection, survival ranged from 0 to 60%, with groups receiving 4 ppm and 8 ppm Se exhibiting 60% survival and the group without Se exhibiting 0% survival. In addition, parasitemia levels of mice supplemented with Se were significantly lower (P<0.01) than in nonsupplemented mice. The results of the present study suggest that Se supplementation does have a beneficial effect during murine infection with T. cruzi, resulting in decreased parasitemias and increased longevity.     

Modulation of embryonic glutathione peroxidase activity and phenytoin teratogenicity by dietary deprivation of selenium in CD-1 mice

Selenium (Se)-dependent and -independent glutathione (GSH) peroxidases detoxify H2O2 and lipid hydroperoxides, which may mediate the teratogenicity of phenytoin and related xenobiotics. To test this hypothesis, CD-1 mice were placed on Se-deficient diets for 15, 25 or 40 days and bred so that the day of analysis corresponded to gestational day 11. In Se-replete control animals, embryonic peroxidase activities were only 5% of activities in maternal liver (P < .05). After 15 days of Se deprivation, maternal activities for H2O2 (reflecting Se-dependent peroxidase) and cumene hydroperoxide (CmOOH) (reflecting both Se-dependent and -independent peroxidases) were reduced to 20% (P < .05) and 35% of controls, respectively. At this time, the incidence of fetal cleft palates initiated by phenytoin (55 mg/kg intraperitoneally on gestational days 11, 12 and 13) was doubled, from 12% to 25% (P < .05). Selenite rescue (Na2SeO3, 350 micrograms/kg intraperitoneally on day 9) restored maternal and embryonic peroxidase activities and completely inhibited phenytoin-initiated postpartum lethality and fetal resorptions in animals that had been Se depleted for 15 days. After 40 days of Se deprivation, maternal and embryonic peroxidase/H2O2 activities were reduced to < 1% and 27% of Se-replete controls, respectively. In contrast, maternal peroxidase/CmOOH activity was increased to 70% of controls, reflecting induction of Se-independent peroxidase, compared with that with 15 days' depletion. Phenytoin-initiated cleft palates with 40 days' depletion appeared to be reduced (16%) compared with Se-replete controls (24%) (P < .07). In 40-day Se-depleted animals given selenite rescue, the 10% incidence of cleft palates was significantly lower than that in the 40-day Se-replete group (24%) but not the Se-depleted group (16%). This is the first demonstration of reduced Se-dependent GSH peroxidase activities in embryonic tissues with dietary Se-deprivation. The results implicate reactive oxygen species and lipid hydroperoxides in the mechanism of phenytoin teratogenicity and suggest that GSH peroxidases are important embryoprotective enzymes.     

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.     

高镍铜阳极泥中硒、碲、铜的顺序脱除

针对高含镍铜阳极泥,采用直接添加氢氧化钠焙烧-碱浸-酸浸流程进行Se、Te、Cu的脱除试验研究,并对过程的反应机理进行了分析。研究发现,加碱氧化焙烧过程中硒化物和碲化物中的Cu变成Cu O和Cu3Te O6;Se、Te分别转变成在碱性溶液中易溶的Na2Se O3和不溶的Ag2Te O3、Cu3Te O6,为Se、Te、Cu的选择性脱除奠定了基础。试验结果表明,最佳焙烧-碱浸的条件为:Na OH剂量为阳极泥的10%,焙烧时间1.5h,焙烧温度500℃。碱浸时间1.0h、Na OH浓度20g/L、碱浸温度80℃、液固比5∶1。在此条件下Se的浸出率为95.50%,碱浸渣中Se的含量从3.93%下降到0.23%。碱浸渣酸浸除铜碲的最佳条件为:H2SO4浓度为90g/L、酸浸温度70℃、酸浸时间1.0h、液固比20∶1;在此条件下,Cu、Te的脱除率分别为96.18%、98.48%。     

Efficacy of chlorine and calcinated calcium treatment of alfalfa seeds and sprouts to eliminate Salmonella

The efficacy of a 20,000 ppm calcium hypochlorite treatment of alfalfa seeds artificially contaminated with Salmonella was studied. Salmonella populations reached >7.0 log on sprouts grown from seeds artificially contaminated with Salmonella and then treated with 20,000 ppm Ca(OCl)(2). The efficacy of spray application of chlorine (100 ppm) to eliminate Salmonella during germination and growth of alfalfa was assessed. Alfalfa seed artificially contaminated with Salmonella was treated at germination, on day 2 or day 4, or for the duration of the growth period. Spray application of 100 ppm chlorine at germination, day 2, or day 4 of growth was minimally effective resulting in approximately a 0.5-log decrease in population of Salmonella. Treatment on each of the 4 days of growth reduced populations of Salmonella by only 1.5 log. Combined treatment of seeds with 20,000 ppm Ca(OCl)(2) and followed by 100 ppm chlorine or calcinated calcium during germination and sprout growth did not eliminate Salmonella.     

Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution

Ethane evolution was monitored from vitamin E and selenium (Se)-deficient rats to determine if lipid peroxidation occurs in vivo when these rats develop fatal organ lesions. Weanling rats were fed a vitamin E and Se-deficient, or supplemented, diet for 40 to 90 days. Each was then prefasted for 4 hours and fasting was continued for 24 to 40 hours while ethane was collected. Approximately 50% of the doubly-deficient rats died as a result of fasting. Pathological signs included hematuria, lung hemorrhage, and liver necrosis. Ethane evolution increased exponentially 10 to 20 hours before death and then declined 2 hours before death. Rats that survived (at least 5 days after ethane collection) evolved 7.4+/-1.3 nmoles ethane/100 g body weight/24 hours compared to 100+/-6 for rats that died. Supplementation of the basal diet with vitamin E (200 IU/kg), Se (0.2 ppm, as Na2SeO3), or both, completely prevented mortality and reduced ethane evolution values to 0.4+/-0.2, 3.1+/-0.4, or 0.2+/-0.2, respectively. These experiments indicate that lipid peroxidation occurs in vivo as a result of vitamin E and Se deficiency, and the peroxidation process greatly accelerates during the terminal phase of the fatal disease.     

Interaction of estradiol, progesterone and corticosterone on uterine connective tissue degrading enzymes

The impact of ovarian hormones and corticosterone acetate on uterine connective tissue degrading enzymes were studied in mature albino rats. Ovariectomy resulted in a significant increase in the activities of alpha- and beta-galactosidases and glucosidases in the uterus. Administration of estradiol to ovariectomized rats brought back the activities of alpha-galactosidase and alpha-glucosidase to normalcy. While beta-galactosidase and beta-glucosidase were significantly decreased. Administration of progesterone to ovariectomized rats resulted in the increase of alpha- and beta-galactosidases and glucosidases. Administration of corticosterone to ovariectomized rats produced a further increase in alpha- and beta-galactosidases and glucosidases in the uterus. Adrenalectomy in ovary intact rats produced a decrease in alpha-galactosidase however, beta-glucosidase was significantly increased. Administration of corticosterone to ovary intact rats significantly increased the activities of alpha- and beta-galactosidases, while alpha- and beta-glucosidases were found to be decreased. Ovariectomy resulted in a significant increase in the activities of cathepsin-D and cathepsin-E. Administration of estradiol to ovariectomized rats brought back the activity of cathepsin-D to normalcy, whereas cathepsin-E was significantly increased. Administration of progesterone as well as estradiol to ovariectomized rats significantly increased the levels of cathepsin-E, however, cathepsin-D was brought back to normalcy. Administration of corticosterone to ovariectomized rats as well as ovariectomy + adrenalectomy significantly increased the activity of cathepsin-D and cathepsin-E. Adrenalectomy significantly decreased the activity of cathepsin-D, while administration of corticosterone increased the cathepsin-D and cathepsin-E in the uterus. Therefore, these results suggest that estradiol is a potent ovarian steroid protecting the extra cellular matrix components. The effect of progesterone appears to modulate and act hand in hand with estradiol. Corticosterone appears to have an opposite effect to that of estradiol.     

Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene

A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity. The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available. Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria. The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.     

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.     

LD 50 and selenium concentration in the organs of rabbits following oral administration of sodium selenite and testing of the toxicity of Ursoselevit-Pr?mix

LD50/24hr was established in the first of a series of experiments on 72 rabbits for orally applied sodium selenite. The dosage was 8.62 mg/kg live weight, the confidence interval being (1 - alpha = 0.95) +/- 0.13 mg/kg. The value was four times as high following intravenous application. Complete lethality was recorded from 15 mg Na2SeO3/kg live weight within 21 hours. Thirty-six animals were involved in the second experiment of the series. They had 50 or 100 per cent Ursoselevit-Pr?mix (30 ppm Se) in their rations. Body mass development of the test animals was superior to that recorded from the controls in the first 50 days, after which limit the former declined strongly in a few days. Their general condition worsened. Postmortem findings, following slaughter, included catarrhal enteritis, toxic liver dystrophy, scattered pulpous tumours in the spleen, and interstitial nephritis. In the third experiment (50 per cent Ursoselevit-Pr?mix with 60 ppm Se in the rations), the test animals developed better than the controls during the first two months, after which point they exhibited the same clinical symptoms as those observed in the second experiment, stopped to put on weight, and eventually turned cachectic. The pathomorphological findings were identical with those obtained from the second experiment. The selenium concentrations in the organs of the test animals all were much higher than those of the controls. Their amounts in excess to base values were up to eleven times in the blood, nine times in the liver, twelve times in the kidneys, and 13 times in the muscles.     

Changes in T-cell phenotype and adhesion molecules expression in psoriatic lesions after low-dose cyclosporin therapy

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).     

稀土在杜仲上的应用研究

选用易于观察研究的杜仲幼苗、幼林、种子、扦插用的枝条作为试验材料,研究了稀土在杜仲上的应用效果和施用技术,并研制了一种高效价廉的林用促长剂     

Effects of methylation of the beta-galactosidase genome upon in vitro synthesis of beta-galactosidase

A template DNA from phage lambdah80dlacp5 coding for the in vitro synthesis of beta-galactosidase was used to study the effect of DNA methylation by the alkylating agent, dimethyl sulfate (DMS). Increasing the levels of DMS up to 50 mM concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37 degrees C, 3-4% Of nucleotides were methylated. The increase was linear to about 0.6% nucleotide methylation level. A higher yield was obtained at 37 degrees C incubation temperature than at 20 degrees C. Methylation of lambdah80dlacp5 DNA alone without methylation of other factors in the incubation mixture caused inhibition of the synthesis of beta-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protein and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nucleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protein and RNA synthetic activities were found to be 72% and 44%, respectively.     

Evaluating the efficacy of selenium-enriched yeast and sodium selenite on tissue selenium retention and serum glutathione peroxidase activity in grower and finisher swine

Three experiments conducted with grower-finisher pigs evaluated sodium selenite and a Se-enriched yeast source at various dietary Se levels on Se retention, tissue and serum Se concentrations, and serum glutathione peroxidase (GSH-Px) activity. Experiment 1 was a balance trial conducted in a 2 x 3 factorial arrangement in a randomized complete block (RCB) design in six replicates. Both Se sources were added at .1, .3, or .5 ppm Se. Crossbred barrows (n = 36) averaging 35.9 kg BW were placed in individual metabolism crates and fed their treatment diets, with feces and urine collected for a 7-d test period. Selenium retention increased as dietary Se levels increased, particularly when the Se-enriched yeast was provided, resulting in a Se source x Se level interaction (P < .01). As dietary Se levels increased, urinary Se increased more when pigs were fed sodium selenite, whereas fecal Se increased more when the Se-enriched yeast was fed; both excretion routes resulting in Se level x Se source interaction responses (P < .01). Experiments 2 and 3 were conducted as RCB involving grower (n = 210) and finisher (n = 266) pigs, respectively, and evaluated the two Se sources each at .1, .3, or .5 ppm Se with a non-Se-fortified basal diet serving as a negative control. In Exp. 2, pigs were fed their treatment diets from 22.2 to 60 kg BW in five replicates, whereas in Exp. 3 diets were fed from 65.8 to 105 kg BW in six replicates. Grower pigs fed sodium selenite had serum GSH-Px activity that reached a plateau at .1 ppm Se and .3 ppm when the Se-enriched yeast source was fed, but the interaction response was not significant (P < .15). During the finisher period, serum GSH-Px activity reached a plateau at .1 ppm Se for both Se sources. Serum Se concentrations were lower at .1 ppm Se when the Se-enriched yeast source was fed, resulting in a source x level interaction response for both grower (P < .05) and finisher (P < .01) periods. Loin Se contents were higher in grower and finisher pigs as dietary Se levels increased when the Se-enriched yeast was fed, resulting in a Se source x Se level interaction (P < .01). The results suggest that more Se was retained in muscle tissue when the Se-enriched yeast source was fed, that serum GSH-Px activity reached a plateau at approximately .1 ppm Se, and that sodium selenite may be more biologically available for GSH-Px activity than the Se-enriched yeast source.     

中和法测定工业二氧化硒中SeO_2的含量

:SeO2 溶于水生成亚硒酸 ,属中等二元强酸。采用中和法测定SeO2 含量 ,分析流程短 ,操作简便。测定结果的RSD为 0 11%。利用误差修正法对测定值进行修正 ,可获得准确的结果。     

The association of yogurt starters with Lactobacillus casei DN 114.001 in fermented milk alters the composition and metabolism of intestinal microflora in germ-free rats and in human flora-associated rats

The aim of this study was to compare the effects of milk and of various fermented milks on the composition and metabolic activities of the intestinal microflora. Groups of eight rats were fed for 6 wk a diet containing 30% nonfermented milk (M), yogurt (Y), milk fermented with Lactobacillus casei (LcFM) or milk fermented with the association of L. casei DN 114.001 and yogurt starters (LcYFM). In the first study, the survival of the lactic acid bacteria from the fermented milks was assessed by bacterial enumeration in feces of germ-free rats (GF rats) fed milk or fermented milks. The metabolic activities of the lactic acid bacteria were studied in these rats by the measurement of glycolytic activities and products of bacterial fermentation, i.e., acetate and lactate (isoforms L and D). In a second study, the effects of fermented milks on the composition and metabolism [gas, glycolytic activities, short-chain fatty acids (SCFA), alcohol and ammonia] of human flora were studied using human flora-associated rats (HF rats). In GF rats, the survival of L. casei in the feces did not differ between those fed the LcFM and LcYFM diets. L. bulgaricus was detected in the feces of the rats fed Y, whereas Streptoccus thermophilus was found in the feces of the LcYFM group. In HF rats, fecal concentration of Bifidobacteria was greater in the LcFM group than in the others. beta-Glucuronidase (EC 3.2.1.31) activity was lower in rats fed LcFM and Y than in those fed M and LcYFM, whereas beta-galactosidase (3.2.1.23), alpha-glucosidase (EC 3.2.1 20) and beta-glucosidase (EC 3.2.1.21) activities were higher in the LcYFM group compared with the others. Methane excretion was higher in rats fed Y than in other groups. Cecal SCFA concentrations did not differ in LcFM, Y and M groups, but total SCFA, acetate, propionate and butyrate were significantly greater in the LcYFM group. These results suggest that milk fermented with the combination of L. casei and yogurt starters leads to specific effects that are different from the simple addition of the effects found with yogurt and milk fermented with L. casei. These specific effects are potentially beneficial to human health.