Metabolism and Functions of Amino Acids in the Skin.

Amino acids are the building blocks of all proteins, including the most abundant fibrous proteins in the skin, as keratins, collagen and elastin. Sagging and wrinkled skin are features of chronic sun-damaged and aged uncared skin, and they are mainly associated with the deterioration of collagen and elastic fibers. The maintenance of skin structures by self-repair processes is essential to skin health. Thus, amino acids significantly impact the appearance of the skin. Amino acids are important nutrients required for (a) wound healing promotion and repair of the damaged skin; (b) acid-base balance and water retention in cellular layers, such as stratum corneum; (c) protection against sunlight damage; (d) maintenance of an appropriate skin microbiome. This review highlights the contribution of all proteinogenic amino acids and some related metabolites to the skin structures as constituents of the main cutaneous proteins or as signaling molecules for the regulation and determination of skin physiology.

Impact of anaemia on health-related quality of life and cardiac remodelling in patients with lower risk myelodysplastic syndromes. Results of GlobQoL study.

The aim of this study was to analyse the eventual changes in health-related quality of life (HRQoL) and left ventricular function (LVF) over a 1-year follow-up period in a cohort of patients with lower risk myelodysplastic syndromes (MDS) receiving standard supportive treatment, in order to identify potential clues for early clinical intervention, as well as to analyse how they relate to haemoglobin levels and other aspects of the disease. A total of 39 adult anaemic patients with lower risk MDS were included in a prospective, observational, multi-centre study. Changes in performance status, functional capacity and HRQoL were collected by using standardised measures (ECOG scale; SPPB, Short Physical Performance Battery; SF-36, Short-Form 36 questionnaire; QLQ-C30, Quality of Life Core Questionnaire; FACT-An, Functional Assessment of Cancer Therapy-Anaemia scale questionnaires respectively). Need for transfusion (Linear Analogue Scale Assessment), as perceived independently by the patient and the haematologist, was also recorded. No changes in HRQoL (or LVF) were found, except for slight reductions in SF-36 physical function (P = 0.034), SPPB gait speed (P = 0.038) and FACT-An score (P = 0.029), all without apparent immediate clinical relevance for HRQoL, that were unrelated to changes in haemoglobin level. Periodical evaluation of gait speed may assist the clinician in early detection of patient's occult functional decline before it becomes clinically relevant.

Diagnosing pulmonary embolisms: the clinician's point of view. / Diagnóstico de la embolia pulmonar. El punto de vista del médico clínico.

Pulmonary thromboembolism is common and potentially severe. To ensure the correct approach to the diagnostic workup of pulmonary thromboembolism, it is essential to know the basic concepts governing the use of the different tests available. The diagnostic approach to pulmonary thromboembolism is an example of the application of the conditional probabilities of Bayes' theorem in daily practice. To interpret the available diagnostic tests correctly, it is necessary to analyze different concepts that are fundamental for decision making. Thus, it is necessary to know what the likelihood ratios, 95% confidence intervals, and decision thresholds mean. Whether to determine the D-dimer concentration or to do CT angiography or other imaging tests depends on their capacity to modify the pretest probability of having the disease to a posttest probability that is higher or lower than the thresholds for action. This review aims to clarify the diagnostic sequence of thromboembolic pulmonary disease, analyzing the main diagnostic tools (clinical examination, laboratory tests, and imaging tests), placing special emphasis on the principles that govern evidence-based medicine.

Low transformation growth factor-ß1 production and collagen synthesis correlate with the lack of hepatic periportal fibrosis development in undernourished mice infected with Schistosoma mansoni.

Undernourished mice infected (UI) submitted to low and long-lasting infections by Schistosoma mansoni are unable to develop the hepatic periportal fibrosis that is equivalent to Symmers' fibrosis in humans. In this report, the effects of the host's nutritional status on parasite (worm load, egg viability and maturation) and host (growth curves, biology, collagen synthesis and characteristics of the immunological response) were studied and these are considered as interdependent factors influencing the amount and distribution of fibrous tissue in hepatic periovular granulomas and portal spaces. The nutritional status of the host influenced the low body weight and low parasite burden detected in UI mice as well as the number, viability and maturation of released eggs. The reduced oviposition and increased number of degenerated or dead eggs were associated with low protein synthesis detected in deficient hosts, which likely induced the observed decrease in transformation growth factor (TGF)-ß1 and liver collagen. Despite the reduced number of mature eggs in UI mice, the activation of TGF-ß1 and hepatic stellate cells occurred regardless of the unviability of most miracidia, due to stimulation by fibrogenic proteins and eggshell glycoproteins. However, changes in the repair mechanisms influenced by the nutritional status in deficient animals may account for the decreased liver collagen detected in the present study.

MDR1 C3435T polymorphism in Mexican patients with breast cancer.

We investigated whether the MDR1 C3435T polymorphism is associated with fibrocystic changes (FCC), infiltrating ductal breast cancer (IDBC), and/or clinical-pathological features of IDBC in Mexican patients. Samples from women who received surgical treatment in 2007 at the Centro Médico de Occidente (México) were included in the analysis. Genotyping was performed by polymerase chain reaction-restricted fragment length polymorphisms in 64 paraffin-embedded breast samples with IDBC, 64 samples with FCC, and 183 peripheral blood samples of healthy females designated as the healthy group (HG). The frequency of the T allele was 41, 45, and 52% for the FCC, IDBC, and HG samples, respectively. Significant differences were only found between the FCC and HG samples [odds ratio (OR) = 0.64, 95% confidence interval (CI) = 0.43-0.96; P = 0.032]. The prevalence of the T/T genotype was 8, 13, and 24% for FCC, IDBC, and HG samples, respectively. Again, statistical differences were only found between FCC and HG samples for the T/T genotype (OR = 0.28, 95%CI = 0.106-0.77; P = 0.009). Although the T allele and the T/T genotype were less frequent in the IDBC group than in the HG, the differences were not significant. Furthermore, no associations were found between the C3435T polymorphism and clinical-pathological features of the IDBC group. Both the FCC and IDBC groups had a high frequency of the C allele relative to the HG in this sample of women from Western Mexico.

Betacyanin and other antioxidants production during growth of Opuntia stricta (Haw.) fruits.

Mature cactus pears from Opuntia stricta have a dark purple color due to high betacyanin concentration, whose biosynthesis is initiated with the amino acid L-tyrosine as a primary precursor. This study followed the maturation and ripening processes of Opuntia stricta fruits to harvest them at high betacyanin and other antioxidant concentrations. Fruits lasted 9 months for final ripening. Physical and compositional changes at different maturation and ripening stages have been determined. Thus, ripe fruits were around 4.72 ± 0.10 cm length, 2.94 ± 0.05 cm diameter and 22.71 ± 0.20 g weight; moisture and pH were maintained at 87.05 ± 0.19 % and 3.37 ± 0.12, respectively. Purple pigment production started in the ovary of immature fruits four months after anthesis (MAA). Concentration of all analyzed metabolites increased from immature (4 MAA) until ripe (9 MAA) stage. In ripe fruits, reducing sugars were 4.72 ± 0.54 g/100 g ff and total phenols 135.17 ± 0.68 mg gallic acid/100 g ff. Metabolites identified by HPLC were the betacyanins: betanin (60.17 ± 1.08 mg/100 g ff), isobetanin (7.58 ± 0.94 mg/100 g ff) and betanidin (13.48 ± 0.87 mg/100 g ff). Also, L-ascorbic acid (35.03 ± 1.06 mg/100 g ff) and L-tyrosine (4.43 ± 0.73 mg/100 g ff) were determined. Furthermore, the addition of L-tyrosine or L-dopa to fruit pulp of moderately ripe fruits, increased betacyanin concentrations 17 (103.3 ± 3.8 mg/100 g) and 32 % (114.3 ± 4.1 mg/100 g), respectively.

Long-term outcomes in patients with schizophrenia treated with risperidone long-acting injection or oral antipsychotics in Spain: results from the electronic Schizophrenia Treatment Adherence Registry (e-STAR).

BACKGROUND: The electronic Schizophrenia Treatment Adherence Registry (e-STAR) is a prospective, observational study of patients with schizophrenia designed to evaluate long-term treatment outcomes in routine clinical practice. METHODS: Parameters were assessed at baseline and at 3 month intervals for 2 years in patients initiated on risperidone long-acting injection (RLAI) (n=1345) or a new oral antipsychotic (AP) (n=277; 35.7% and 36.5% on risperidone and olanzapine, respectively) in Spain. Hospitalization prior to therapy was assessed by a retrospective chart review. RESULTS: At 24 months, treatment retention (81.8% for RLAI versus 63.4% for oral APs, p<0.0001) and reduction in Clinical Global Impression Severity scores (-1.14 for RLAI versus -0.94 for APs, p=0.0165) were significantly higher with RLAI. Compared to the pre-switch period, RLAI patients had greater reductions in the number (reduction of 0.37 stays per patient versus 0.2, p<0.05) and days (18.74 versus 13.02, p<0.01) of hospitalizations at 24 months than oral AP patients. CONCLUSIONS: This 2 year, prospective, observational study showed that, compared to oral antipsychotics, RLAI was associated with better treatment retention, greater improvement in clinical symptoms and functioning, and greater reduction in hospital stays and days in hospital in patients with schizophrenia. Improved treatment adherence, increased efficacy and reduced hospitalization with RLAI offer the opportunity of substantial therapeutic improvement in schizophrenia.

Highly skewed inactivation of the wild-type X-chromosome in asymptomatic female carriers of spinal and bulbar muscular atrophy (Kennedy's disease).

We examined families with a history of spinal and bulbar muscular atrophy (SBMA) and found that six out of eight female carriers had a skewed inactivation of the wild-type chromosome. Under these genetic conditions, disease manifestations should be expected and therefore we sought neurological and other symptoms of subclinical SBMA. We did not find either clinical symptoms or electrophysiological signs of mutated AR gene in female carriers, despite skewed methylation of the wild-type allele. These findings suggest that skewed methylation of AR genes are not necessarily associated to clinical manifestations in female carriers of the expanded SBMA allele.

RegulonDB (version 3.2): transcriptional regulation and operon organization in Escherichia coli K-12.

RegulonDB is a database on mechanisms of transcription regulation and operon organization in Escherichia coli K-12. The current version has considerably increased numbers of regulatory elements such as promoters, binding sites and terminators. The complete repertoire of known and predicted DNA-binding transcriptional regulators can be considered to be included in this version. The database now distinguishes different allosteric conformations of regulatory proteins indicating the one active in binding and regulating the different promoters. A new set of operon predictions has been incorporated. The relational design has been modified accordingly. Furthermore, a major improvement is a graphic display enabling browsing of the database with a Java-based graphic user interface with three zoom-levels connected to properties of each chromosomal element. The purpose of these modifications is to make RegulonDB a useful tool and control set for transcriptome experiments. RegulonDB can be accessed on the web at the URL: http://www.cifn.unam.mx/Computational_Biology/++ +regulondb/

Antimelanoma effect of 4-S-cysteaminylcatechol, an activated form of 4-S-cysteaminylphenol.

Rational chemotherapy of malignant melanoma could be developed by taking advantage of the presence of melanogenic enzymes in melanoma cells. 4-S-Cysteaminylphenol (4-S-CAP) has been evaluated for melanocytotoxicity and antimelanoma effect. Although 4-S-CAP is selectively toxic to pigmented melanoma cells, it is not potent enough when applied as a single agent. To increase the efficacy of 4-S-CAP, we synthesized 4-S-cysteaminylcatechol (4-S-CAC), an activated form of 4-S-CAP, and compared its biochemical properties and antimelanoma effects with those of the isomers 3-S-cysteaminylcatechol (3-S-CAC) and 2-S-cysteaminyl-hydroquinone (2-S-CAH). 4-S-CAC was found to be a better substrate for melanoma tyrosinase than was L-3,4-dihydroxyphenylalanine, the natural catecholic substrate. 3-S-CAC was a poor substrate, whereas 2-S-CAH was not a substrate. 4-S-CAC was the most cytotoxic to three lines of melanoma cells in vitro, followed by 2-S-CAH and 3-S-CAC. When applied i.p. for 9 days at a dose of 100 mg/kg, 4-S-CAC.HCl, increased by 46-52% the life span of C57BL/6 mice inoculated i.p. with B16 melanoma; this effect was comparable to that of a 50 mg/kg dose of 5-(3,3-dimethyltriazenyl)-1H-imidazole-4-carboxamide. 3-S-CAC was marginally effective, whereas 2-S-CAH was toxic to the host. This systemic toxicity of 2-S-CAH reflected its susceptibility to autoxidation. Growth of B16 melanoma cells inoculated s.c. was significantly inhibited by i.p. administration of 4-S-CAC.HCl (200 mg/kg) for 5 days (P < 0.05). These results suggest that 4-S-CAC is a potent antimelanoma agent, the effect of which is mostly mediated through tyrosinase oxidation.

Comparison of the effects of mesquite pod and Leucaena extracts with phytoestrogens on the reproductive physiology and sexual behavior in the male rat.

Mesquite (Prosopis sp.) and Leucaena leucocephala are widespread legumes, widely used to feed several livestock species and as food source for human populations in several countries. Both mesquite and Leucaena contain several phytoestrogens which might have potential estrogenic effects. Thus, the aim of this study was to evaluate the effects of mesquite pod and Leucaena extracts on several aspects of behavior and reproductive physiology of the male rat. The effects of the extracts were compared with those of estradiol (E2) and of two isoflavones: daidzein (DAI) and genistein (GEN). The following treatments were given to groups of intact male rats: vehicle; mesquite pod extract; Leucaena extract; E2; DAI; GEN. The results indicate that mesquite pod and Leucaena extracts disrupt male sexual behavior in a similar way to DAI and GEN, but less than E2. The main disruptor of sexual behavior was E2, however after 40 and 50days of administration, both extracts and phytoestrogens disrupted sexual behavior in a similar way to E2. The extracts also increased testicular germ cell apoptosis, decreased sperm quality, testicular weight, and testosterone levels, as phytoestrogens did, although these effects were less than those caused by estradiol. The number of seminiferous tubules with TUNEL-positive germ cells increased in extracts treated groups in a similar way to phytoestrogens groups, and E2 caused the greatest effect. The number of TUNEL-positive cells per tubule increased only in Leucaena extract and E2 groups, but not in mesquite- and phytoestrogens-treated groups. Spermatocytes and round spermatids were the TUNEL-positive cells observed in all experimental groups. This effect was associated with smaller testicular weights without atrophy in experimental groups compared with control. Testicular atrophy was only observed in estradiol-treated males. Testosterone decreased in males of all experimental groups, compared with control, this androgen was undetectable in E2 treated males. These results suggest that mesquite pod and Leucaena extracts cause effects similar to those of phytoestrogens in male rat reproduction, these effects were lower than those caused by E2.

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells.

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.

Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins.

Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside the melanosome. Their solubilization is usually carried out by treatment of melanosomal preparations with non-ionic detergents, but, so far, no comparative study of the effect of the detergents employed on the properties of the solubilized proteins has been reported. We have compared the effect of the detergents Brij-35, Nonidet P-40, Tween-20, sodium deoxycholate and Triton X-114 on several properties of the melanogenic enzymes, including the solubilization yield, stability, electrophoretic behaviour and accessibility of epitopes located in the carboxyl tail to specific antibodies. Our data indicate that not only the total amount of enzymes solubilized, but also their relative proportions in the solubilized preparations depend on the detergent used. The non-ionic detergents apparently interact strongly with the melanogenic enzymes, affecting their mobility in SDS-PAGE, and might induce different conformations of the carboxyl tail. Complete replacement of lipids by the detergents results in a decreased stability that can be partially reversed by the addition of endogenous lipids. This treatment also produces a noticeable activation of the tyrosinase isoenzymes, which is higher for TRP1 than for tyrosinase. Taken together, these data show that the transmembrane and carboxyl fragments of the proteins of the tyrosinase family might modulate the stability and activity of the melanogenic enzymes.

Regulation of mammalian melanogenesis. I: Partial purification and characterization of a dopachrome converting factor: dopachrome tautomerase.

A protein that catalyzes the decoloration of dopachrome has been partially purified from B16 mouse melanoma tumors. The enzyme is preferentially associated to the melanosomes, but it is also found in the microsomal and cytosolic fractions of cellular homogenates. The protein is clearly different from tyrosinase, and should be related to the dopachrome oxidoreductase (Barber et al. (1984) J. Invest. Dermatol. 83, 145-149) and the dopachrome conversion factor (Korner and Pawelek (1980) J. Invest. Dermatol. 75, 192-195) since the reaction product of dopachrome conversion is 5,6-dihydroxyindole-2-carboxylic acid. The protein appears to have an oligomeric structure, with a molecular mass slightly higher than 300 kDa estimated by gel filtration, whereas the molecular mass of the monomer might be approx. 46 kDa estimated by SDS-PAGE electrophoresis. Its Km for dopachrome is around 100 microM. The enzyme is competitively inhibited by indoles and is unaffected by metal chelators. It also has the ability to increase the amount of melanin formed from L-tyrosine by melanoma tyrosinase, and therefore, cannot be considered an 'indole blocking factor' as was suggested for the related dopachrome oxidoreductase. Since the reaction catalyzed by the enzyme is a tautomeric shift on dopachrome, we would propose dopachrome tautomerase (EC 5.3.2.3) as the most precise and informative name.

Dopachrome tautomerase decreases the binding of indolic melanogenesis intermediates to proteins.

Dopachrome tautomerase (DCT) is a recently characterized enzyme contributing to the control of melanogenesis in mammals. The enzyme catalyzes the rearrangement of L-Dopachrome (L-DC) to 5,6-dihydroxyindole 2-carboxylic acid (DHICA), while the spontaneous rearrangement of L-DC leads to 5,6-dihydroxyindole (DHI). Due to the lower reactivity of DHICA in comparison to DHI, DCT could provide a protective mechanism against the cytotoxicity of decarboxylated indolic melanogenic intermediates by limiting the formation of these highly reactive decarboxylated species within melanocytes. We have followed the binding of radioactive melanogenic precursors to a model protein, bovine serum albumin (BSA). Using L-DC as initial melanin precursor, this binding was decreased by DCT in a concentration-dependent manner. In the presence of tyrosinase, the binding of L-Dopa-derived intermediates to BSA was also decreased by DCT and the percentage of decrease was even higher than using L-DC as initial melanin precursor. SDS-PAGE followed by fluorographic detection of radioactive bands showed the formation of covalent adducts between BSA and melanin precursors, as well as of aggregated forms of this protein. This aggregation was also diminished by DCT. These data indicate that DCT could play a protective role against the cytotoxic action of decarboxylated indoles within mammalian melanocytes.

Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome.

A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.

Biochemical characterization of the melanogenic system in the eye of adult rodents.

The melanogenic activities in the eye of the adult gerbil (Meriones unguiculatus) have been investigated and compared to those found in the B16 mouse melanoma model. Eye extracts contain tyrosine hydroxylase, DOPA oxidase, DOPAchrome tautomerase and DHICA oxidase activities. The subcellular distribution of these activities was investigated by differential centrifugation and detergent solubilization of the particulate fractions. The distribution pattern closely resembled the one found for mouse melanoma, with a higher percentage of activity associated to the particulate fractions but a substantial proportion in the cytosolic fraction. The tyrosine hydroxylase activity was characterized by a KM of 62 microM for L-tyrosine and a stringent requirement for the co-factor L-DOPA (Ka 10.3 microM). The KM for L-DOPA was 0.41 mM. The sensitivity of the eye and mouse melanoma tyrosinase activity to a variety of substrate analogs and metal chelators was found to be identical. In keeping with these kinetic similarities, eye tyrosinase displayed some structural properties resembling those of the melanoma enzyme. The molecular weight of the enzyme, determined by SDS-PAGE and DOPA oxidase activity stain, was 75 kDa for the eye enzyme and 66.2 kDa for melanoma tyrosinase, and both enzymes were apparently dimeric in non ionic detergent solution. Immunoprecipitation with specific antibodies proved that at least 80% of the total tyrosinase activity could be immunoprecipitated with the specific anti-tyrosinase antibody alpha PEP7, while the anti-TRP-1 monoclonal antibody TMH-1 precipitated little, if any, tyrosinase activity. Taken together, these observations provide the first vis-à-vis comparison of an extracutaneous melanogenic system and the melanogenic system of melanoma. Our results prove that, at least in rodents, the melanogenic system in the eye is similar, but not identical, to the melanin biosynthesis machinery of epidermal melanocytes.

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway.

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.