The role of nutrition and oxidative stress as aging factors in Caenorhabditis elegans.

The molecular mechanism of aging, which has been a "black box" for many years, has been elucidated in recent years, and the nematode C. elegans, which is a model animal for aging research, has played a major role in its elucidation. From the analysis of C. elegans longevity-related mutant genes, many signal transduction systems, with the insulin/insulin-like growth factor signal transduction system at the core, have emerged. It has become clear that this signal transduction system is greatly affected by external nutrients and is involved in the downstream regulation of oxidative stress, which is considered to be one of the main causes of aging.

Streptococcus thermophilus extends lifespan through activation of DAF-16-mediated antioxidant pathway in Caenorhabditis elegans.

Streptococcus thermophilus bacteria, which are widely used as fermented starter for dairy production, exert various beneficial health effects. Nevertheless, even though pro-longevity effects of various probiotics have been reported, no report has described Streptococcus thermophilus effects on longevity. This study was conducted to evaluate Streptococcus thermophilus effects on lifespan extension and to elucidate the Streptococcus thermophilus-mediated longevity mechanism using Caenorhabditis elegans worms as a model animal. They were fed standard food (Escherichia coli OP50) or Streptococcus thermophilus from the young adult stage. Feeding with Streptococcus thermophilus, compared to Escherichia coli OP50, to Caenorhabditis elegans extend the lifespan, reduced lipofuscin accumulation, and maintain vigorous locomotion. Feeding with Streptococcus thermophilus did not alter the worm growth curve or the offspring number, indicating that the Streptococcus thermophilus-mediated lifespan extension is not attributable to caloric restriction. The qRT-PCR data showed that Streptococcus thermophilus increased the expression of daf-16 and some of its downstream antioxidant genes. Furthermore, the pro-longevity effects of Streptococcus thermophilus were decreased in loss-of-function mutant of daf-16. Results show that Streptococcus thermophilus extends the lifespan of Caenorhabditis elegans through DAF-16-mediated antioxidant pathway activation.

Interaction between the ins/IGF-1 and p38 MAPK signaling pathways in molecular compensation of sod genes and modulation related to intracellular ROS levels in C. elegans.

Superoxide dismutases, which catalytically remove intracellular superoxide radicals by the disproportionation of molecular oxygen and hydrogen peroxide, are encoded by the sod-1 to -5 genes in the nematode C. elegans. Expression of the sod genes is mutually compensatory for the modulation of intracellular oxidative stress during aging. Interestingly, several-fold higher expression of the sod-1 to -4 was induced in a sod-5 deletion mutant, despite the low expression levels of sod-5 in wild-type animals. Consequently, this molecular compensation facilitated recovery of lifespan in the sod-5 mutant. In previous reports, two transcription factors DAF-16 and SKN-1 are associated with the compensatory expression of sod genes, which are downstream targets of the ins/IGF-1 and p38 MAPK signaling pathways activated under oxidative and heavy metal stresses, respectively. Here, we show that p38 MAPK signaling regulates induction of not only the direct expression of sod-1, -2 and -4 but also the indirect modulation of DAF-16 targets, such as sod-3 and -5 genes. Moreover, a SKN-1 target, the insulin peptide gene ins-5, partially mediates the expression of DAF-16 targets via p38 MAPK signaling. These findings suggest that the interaction of ins/IGF-1 and p38 MAPK signaling pathways plays an important role in the fine-tuning of molecular compensation among sod genes to protect against mitochondrial oxidative damage during aging.

Relationship between Serum Antioxidative Vitamin Concentrations and Type 2 Diabetes in Japanese Subjects.

A high intake of green leafy vegetables rich in antioxidative nutrients such as vitamin C and ß-carotene may protect against the risk of type 2 diabetes. Measurement of the circulating nutrient concentrations can indicate the nutrient status more directly, and vitamin C and carotenoids are recognized as good biomarkers for the intake of fruits and vegetables. The aim of this study was to investigate the relationships between serum antioxidative vitamin concentrations and type 2 diabetes in Japanese subjects. The study subjects comprised 506 men and 493 women who first underwent anti-aging health checks at Tokai University Tokyo Hospital. Serum concentration of vitamin (V) A, VC, α-tocoferol, ß-carotene, VB12, folate, ferritin and homocysteine, and fasting plasma glucose and HbA1c were used for analysis. Low levels of ß-carotene and VC were significantly associated with dysglycemia. Diabetic subjects showed significantly decreased ß-carotene and VC levels, and multivariate analyses suggested that low levels of ß-carotene and VC were factors related to diabetes. Low levels of ß-carotene and VC are significantly related to dysglycemia/type 2 diabetes, and encouraging people at a higher risk of diabetes to take more green vegetables may be useful as a dietary intervention to improve the antioxidative vitamin status and dysglycemia.

Isolation of lactic acid bacteria capable of reducing environmental alkyl and fatty acid hydroperoxides, and the effect of their oral administration on oxidative-stressed nematodes and rats.

Reinforcement of the hydroperoxide-eliminating activity in the small and large intestines should prevent associated diseases. We previously isolated a lactic acid bacterium, Pediococcus pentosaceus Be1 that facilitates a 2-electron reduction of hydrogen peroxide to water. In this study, we successfully isolated an alternative lactic acid bacterium, Lactobacillus plantarum P1-2, that can efficiently reduce environmental alkyl hydroperoxides and fatty acid hydroperoxides to their corresponding hydroxyl derivatives through a 2-electron reduction. Each strain exhibited a wide concentration range with regard to the environmental reducing activity for each hydroperoxide. Given this, the two lactic acid bacteria were orally administered to an oxygen-sensitive short-lived nematode mutant, and this resulted in a significant expansion of its lifespan. This observation suggests that P. pentosaceus Be1 and L. plantarum P1-2 inhibit internal oxidative stress. To determine the specific organs involved in this response, we performed a similar experiment in rats, involving induced lipid peroxidation by iron-overloading. We observed that only L. plantarum P1-2 inhibited colonic mucosa lipid peroxidation in rats with induced oxidative stress.

Uric acid, ferritin and γ-glutamyltransferase can be informative in prediction of the oxidative stress.

The anti-oxidant system is affected not only by aging but also many lifestyle factors. We aimed to clarify the determinants of medical check-up items affecting the anti-oxidant system. We studied 959 Japanese individuals who underwent anti-aging health check-ups (mean age: 61.1 years) at Tokai University from 2006 to 2016. As parameters of oxidative stress, we measured serum total anti-oxidant status, 8-hydroxy-2'-deoxyguanosine, and isoprostane. Anti-aging health check-up data and lifestyle information were collected from participants in this study. Step-wise multiple regression analyses were conducted to identify determinants that influence serum total anti-oxidant status, 8-hydroxy-2'-deoxyguanosine, and isoprostane, respectively. Serum total anti-oxidant status was significantly correlated with uric acid, vitamin A, folate, and valine. 8-hydroxy-2'-deoxyguanosine was significantly correlated with age, ferritin, drinking habit, and vitamin Eα. Isoprostane was significantly correlated with vitamin Eα, γ-glutamyltransferase, ferritin, and smoking habit. The strong antioxidant powers of uric acid and vitamins were confirmed. It was suggested that branched-chain amino acids themselves such as valine or peptides containing them may possess antioxidant ability because of its strong correlation. Uric acid, ferritin, and γ-glutamyltransferase, which are common items measured in medical checkups, can be informative in predicting the oxidative stress situation in a general medical examination.

Metabolic Biomarkers in Nematode C. elegans During Aging.

Changes in energy metabolism occur not only in diseases such as cancer but also in the normal development and aging processes of various organisms. These metabolic changes result to lead to imbalances in energy metabolism related to cellular and tissue homeostasis. In the model organism C. elegans, which is used to study aging, an imbalance in age-related energy metabolism exists between mitochondrial oxidative phosphorylation and aerobic glycolysis. Cellular lactate and pyruvate are key intermediates in intracellular energy metabolic pathways and can indicate age-related imbalances in energy metabolism. Thus, the cellular lactate/pyruvate ratio can be monitored as a biomarker during aging. Moreover, recent studies have proposed a candidate novel biomarker for aging and age-related declines in the nematode C. elegans.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Long-term administration of pDC stimulative lactic acid bacteria, Lactococcus lactis strain Plasma, prevents immune-senescence and decelerates individual senescence.

Aging is accompanied by the decline in immune function, resulting in increasing susceptibility to infectious diseases and tumorigenesis. In our previous reports, we showed that Lactococcus lactis subsp. lactis strain Plasma (LC-Plasma) stimulated plasmacytoid dendritic cells (pDCs), which play an important role in viral infection, and oral administration of LC-Plasma showed prophylactic effects against viral infection both in mice and humans. However, the effects of long-term administration of LC-Plasma are not known. In this study, we investigated the effect of long-term oral administration of LC-Plasma on IFN-α induction activity and individual senescence in the senescence-accelerated mice strains Prone 1 (SAMP1) and Prone 10 (SAMP10). LC-Plasma administration promoted IFN-α induction activity and increased the naïve T cell ratio in SAMP1 mice. In SAMP10 mice, in addition to preventing a decrease in the naïve T cell ratio, aging-associated skin thinning was suppressed histologically and the expression of representative tight junction genes, such as Claudin-1 and Zo-1, was increased. Furthermore, age-related muscle weight loss tended to be suppressed in the LC-Plasma group and expression of the muscle degeneration gene FoxO-1 was significantly suppressed. Related to these phenotypes, the senescence score in the LC-Plasma group was significantly decreased at 47 weeks of age compared with that in the control group. Taken together, long-term oral administration of LC-Plasma could prevent immune-senescence and other senescence phenotypes at the organ level. Therefore, LC-Plasma is suggested to be a useful functional food material for decelerating individual senescence.

Impaired p53/CEP-1 is associated with lifespan extension through an age-related imbalance in the energy metabolism of C. elegans.

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP-1 mediates the stress response, activates germ line apoptosis and regulates meiotic chromosome segregation. A reduction in its expression, which frequently occurs in mammalian cancer cells, extends lifespan and induces an adaptive response in C. elegans. However, these effects do not involve an increase in oxidative stress resistance. Here, we showed that intermittent exposure to hyperoxia, which induces oxidative stress resistance and lowers the production of ROS derived from mitochondrial respiration in C. elegans, slightly improved the lifespan extension of cep-1 mutant. Interestingly, ATP levels were increased without an increase in oxygen consumption in cep-1 mutant during aging. In the wild-type, lactate levels and consequentially the lactate/pyruvate ratio decreased during aging in adults. Furthermore, the expression levels of mitochondrial respiration-related sco-1, which is a target of p53/CEP-1, as well as those of gluconeogenesis regulation and mammalian sirtuin ortholog genes, were also increased in the aged and adaptive conditioned wild-type animals. In contrast, the lactate/pyruvate ratio increased in cells of the cep-1 mutant and was amplified by intermittent hyperoxia. These results suggest that impaired p53/CEP-1 leads to an imbalance in the age-related energy metabolic alteration between mitochondrial oxidative phosphorylation and aerobic glycolysis and plays an important role in the extension of both intact and adaptive lifespans.

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain.

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle-aged Tet-mev-1 mice with the SDHCV69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle-aged Tet-mev-1 mice overproduced MitoSOX Red-detectable mitochondrial ROS compared to age-matched wild-type C57BL/6J mice, only young adult Tet-mev-1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn- and Cu/Zn-SODs) activities to eliminate the MitoSOX Red-detectable mitochondrial ROS. In contrast, middle-aged Tet-mev-1 mice accumulated both MitoSOX Red-detectable mitochondrial ROS and CM-H2 DCFDA-detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle-aged Tet-mev-1 mice relative to age-matched wild-type C57BL/6J mice. Furthermore, only middle-aged Tet-mev-1 mice showed JNK/SAPK activation and Ca2+ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100ß in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age-dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.

Preparation of Clinically Useful Sennoside-reduced Rhubarb.

OBJECTIVE: The aim of this study was to develop a method of removing sennoside to reduce the cathartic effect of rhubarb while conserving its other pharmacological activities. METHODS: Rhubarb powder was steam autoclaved at 121°C and 0.14 MPa for 20, 60, or 120 minutes, and HPLC analysis was conducted to determine levels of rhubarb components. Mice were fed non-autoclaved or 20-minute-autoclaved rhubarb extracts. Feces were collected and weighed over a 24-hour period. India ink was orally administered to determine the distance of fecal migration through the intestinal tract. RESULTS: Autoclaving 20, 60, and 120 minutes decreased sennoside A and B to trace levels but only autoclaving 20 minutes conserved most of the (+)-catechin, (-)-epicatechin, and (-)-epicatechin gallate contents (i.e., 69%, 90%, 88%, respectively). Therefore only rhubarb autoclaved for 20 minutes was used in subsequent experiments. Fecal output (in g) in mice treated with water (control), autoclaved rhubarb, and non-autoclaved rhubarb was 2.78 ± 0.07, 3.30 ± 0.13 (p = 0.348), and 3.81 ± 0.07 (p = 0.005). India ink migration was far less in mice treated with autoclaved rhubarb vs non-autoclaved rhubarb. CONCLUSION: Steam autoclaving the rhubarb for 20 minutes reduces sennoside levels and its cathartic activity while conserving its other pharmacological activities.

Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models.

Oxidative stress is associated with some forms of both male and female infertility. However, there is insufficient knowledge of the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. There are a number of animal models for understanding age-dependent decrease of reproductive ability and diabetic embryopathy, especially abnormal spermatogenesis, oogenesis and embryogenesis with mitochondrial dysfunctions. Several important processes occur in mitochondria, including ATP synthesis, calcium ion storage, induction of apoptosis and production of reactive oxygen species (ROS). These events have different effects on the several aspects of reproductive function. Tet-mev-1 conditional transgenic mice, developed after studies with the mev-1 mutant of the nematode C. elegans, offer the ability to carefully regulate expression of doxycycline-induced mutated SDHC(V69E) levels and hence modulate endogenous oxidative stress. The mev-1 models have served to illuminate the effects of complex II deficiency-dependent mitochondrial ROS production, although interestingly they maintain normal mitochondrial and intracellular ATP levels. In this review, the reproductive dysfunctions are presented focusing on fertility potentials in each gamete, early embryogenesis, maternal conditions with placental function and neonatal development.

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice.

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.

Association between insulin resistance and plasma amino acid profile in non-diabetic Japanese subjects.

AIMS/INTRODUCTION: Elevation of the branched-chain amino acids (BCAAs), valine, leucine and isoleucine; and the aromatic amino acids, tyrosine and phenylalanine, has been observed in obesity-related insulin resistance. However, there have been few studies on Asians, who are generally less obese and less insulin-resistant than Caucasian or African-Americans. In the present study, we investigated the relationship between homeostasis model assessment of insulin resistance (HOMA-IR) and plasma amino acid concentration in non-diabetic Japanese participants. MATERIALS AND METHODS: A total of 94 healthy men and women were enrolled, and plasma amino acid concentration was measured by liquid chromatography/mass spectrometry after overnight fasting. The associations between HOMA-IR and 20 amino acid concentrations, and anthropometric and clinical parameters of lifestyle-related diseases were evaluated. RESULTS: The mean age and body mass index were 40.1 ± 9.6 years and 22.7 ± 3.9, respectively. Significantly positive correlations were observed between HOMA-IR and valine, isoleucine, leucine, tyrosine, phenylalanine and total BCAA concentration. Compared with the HOMA-IR ≤ 1.6 group, the HOMA-IR > 1.6 group showed significantly exacerbated anthropometric and clinical parameters, and significantly elevated levels of valine, isoleucine, leucine, tyrosine, phenylalanine and BCAA. CONCLUSIONS: The present study shows that the insulin resistance-related change in amino acid profile is also observed in non-diabetic Japanese subjects. These amino acids include BCAAs (valine, isoleucine and leucine) and aromatic amino acids (tyrosine and phenylalanine), in agreement with previous studies carried out using different ethnic groups with different degrees of obesity and insulin resistance.

Genetically induced oxidative stress in mice causes thrombocytosis, splenomegaly and placental angiodysplasia that leads to recurrent abortion.

Historical data in the 1950s suggests that 7%, 11%, 33%, and 87% of couples were infertile by ages 30, 35, 40 and 45, respectively. Up to 22.3% of infertile couples have unexplained infertility. Oxidative stress is associated with male and female infertility. However, there is insufficient evidence relating to the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. Recently, we have established Tet-mev-1 conditional transgenic mice, which can express the doxycycline-induced mutant SDHC(V69E) transgene and experience mitochondrial respiratory chain dysfunction leading to intracellular oxidative stress. In this report, we demonstrate that this kind of abnormal mitochondrial respiratory chain-induced chronic oxidative stress affects fertility, pregnancy and delivery rates as well as causes recurrent abortions, occasionally resulting in maternal death. Despite this, spermatogenesis and early embryogenesis are completely normal, indicating the mutation's effects to be rather subtle. Female Tet-mev-1 mice exhibit thrombocytosis and splenomegaly in both non-pregnant and pregnant mice as well as placental angiodysplasia with reduced Flt-1 protein leading to hypoxic conditions, which could contribute to placental inflammation and fetal abnormal angiogenesis. Collectively these data strongly suggest that chronic oxidative stress caused by mitochondrial mutations provokes spontaneous abortions and recurrent miscarriage resulting in age-related female infertility.

Survey of Attitudes of Japanese Women Toward Genetic/Genomic Research.

Previous surveys have suggested that elderly Japanese women have the lowest scientific interest and literacy within the Japanese population and among populations across Western countries. Because recent tremendous advances in genome analysis are likely to be incorporated into standard biomedical assessments throughout the world, we conducted surveys to investigate the attitudes toward genetic/genomic research of Japanese women aged between 55 and 65 years. Current surveys indicate that obtaining adequate informed consent from elderly Japanese women is complicated. The limitation is especially relevant to participants' literacy in genetics and genomic studies. Results of the surveys also indicate that even after the informed consent is obtained, researchers must continue to supply updated study information to the study subjects, which enables them to obtain additional information on the use of their samples and genetic/genomic information. Failure to consider these obligations may lead to a loss of the public's trust and thus affect research progress on medical genomics.

Ethyl methanesulfonate induces mutations in Caenorhabditis elegans embryos at a high frequency.

Mutagenesis protocols typically call for exposure of late-stage larvae or adults to a mutagen with the intention of inducing mutations in a robust germ line. Instead, ca. 16,000 CB665 [unc-58(e665)] one- to four-cell embryos of the nematode Caenorhabditis elegans were hand selected and exposed to ethyl methanesulfonate (EMS) for 50min. Twenty-one reversion mutants were recovered, of which 17 were intragenic suppressors of the e665 mutation. The mutation frequency was 6.5-fold higher than when CB665 adults were similarly mutagenized, which was predicted given that cell-cycle checkpoints are muted in C. elegans embryos. The mutation spectrum was similar to that obtained after standard EMS mutagenesis.

Haploinsufficiency of akt1 prolongs the lifespan of mice.

There is increasing evidence that nutrient-sensing machinery is critically involved in the regulation of aging. The insulin/insulin-like growth factor-1 signaling pathway is the best-characterized pathway with an influence on longevity in a variety of organisms, ranging from yeast to rodents. Reduced expression of the receptor for this pathway has been reported to prolong the lifespan; however, the underlying mechanisms are largely unknown. Here we show that haploinsufficiency of Akt1 leads to an increase of the lifespan in mice. Akt1 (+/-) mice had a lower body weight than their littermates with less fat mass and normal glucose metabolism. Ribosomal biogenesis and the mitochondrial DNA content were significantly reduced in these mice, along with a decrease of oxidative stress. Consistent with the results obtained in mice, inhibition of Akt-1 promoted longevity in nematodes (Caenorhabditis elegans), whereas activation of Akt-1 shortened the lifespan. Inhibition of Akt-1 led to a decrease of ribosomal gene expression and the mitochondrial DNA content in both human cells and nematodes. Moreover, deletion of ribosomal gene expression resulted in a decrease of the mitochondrial DNA content and normalized the lifespan shortened by Akt-1 activation in nematodes. These results suggest that an increase of mitochondrial amount and energy expenditure associated with enhanced protein synthesis accelerates both aging and the onset of age-associated diseases.