Tear Proteins Research Articles

Changes in tear protein profile in dogs with keratoconjunctivitis sicca following topical treatment using cyclosporine A.

Background and Aim:Keratoconjunctivitis sicca (KCS) is a chronic inflammatory ocular disease that occurs in many dog breeds worldwide. This study aimed to investigate the tear protein pattern of healthy dogs, KCS dogs, and KCS dogs after treatment with cyclosporine A (CsA).Materials and Methods:Twenty-eight dogs of any breed were enrolled in the study. The subjects were divided into three groups: Healthy, KCS, and CsA-treated dogs. Tear samples were collected using Schirmer strips. Tear proteins extracted from the strips were analyzed using two-dimensional electrophoresis. For the first dimension, total protein from tears was separated by isoelectric focusing. The second dimension was performed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed and the protein spots of differential expression were manually cut for protein annotation using mass spectrometry.Results:In total, 12 protein spots were excised and subjected to protein identification. Associated with KCS, six protein spots were a downregulated protein, namely, lysozyme. The other six protein spots were upregulated in KCS dogs, consisting of heat shock protein beta-1, protein S100-A12, and keratin type II cytoskeletal 1 and 5. After treatment with CsA for 45 days, the lysozyme protein was still decreasing and the inflammation protein (S100-A12) was not identified.Conclusion:Inflammatory tear proteins and proteins involved in cellular stress were present in KCS dogs and appeared to be reduced in medicated eyes. Treatment with topical CsA in the short term may not improve the activity of antibacterial proteins. Changes in the expression patterns of these four proteins might be useful for disease severity and progression assessment, as well as for exploring a novel method for dry eye management in dogs.

Diagnostic biomarkers in tear fluid: from sampling to preanalytical processing

Tear fluid is receiving growing attention as a source for novel diagnostic biomarkers. Multiple techniques are available for its collection and impact the composition of acquired samples. We sought to provide a direct comparison of two collection methods with regard to implementation, acceptance, and impact on sample composition. Tear fluid was collected from fifteen healthy volunteers with capillary tubes and Schirmer strips and analyzed for total protein and IgG concentrations. Sampling parameters and perception by test persons were compared. The use of capillary tubes was more convenient for the participants while causing more effort for the collector. Tear flow rates as well as the relative and absolute amount of IgG were higher when Schirmer strips were used. Consecutive collections with Schirmer strips significantly influenced tear flow rates, IgG, and protein concentrations. A moderate correlation was observed between tear flow rates and IgG concentrations for both methods. Samples collected with both methods can be analyzed by isoelectric focusing, a potential diagnostic application in the field of neurology. The specific advantages and limitations of tear fluid sampling with either capillary tubes or Schirmer strips demonstrate the need for a thorough investigation of collection methods with regard to the application of interest.

Identification of Differentially Expressed Tear Proteins after Corneal Reconstruction with DPSIS Graft in Dogs Suffering from Keratoconjunctivitis Sicca

Identification of Differentially Expressed Tear Proteins after Corneal Reconstruction with DPSIS Graft in Dogs Suffering from Keratoconjunctivitis Sicca

Critical role of mass spectrometry proteomics in tear biomarker discovery for multifactorial ocular diseases (Review).

The tear film is a layer of body fluid that maintains the homeostasis of the ocular surface. The superior accessibility of tears and the presence of a high concentration of functional proteins make tears a potential medium for the discovery of non-invasive biomarkers in ocular diseases. Recent advances in mass spectrometry (MS) have enabled determination of an in-depth proteome profile, improved sensitivity, faster acquisition speed, proven variety of acquisition methods, and identification of disease biomarkers previously lacking in the field of ophthalmology. The use of MS allows efficient discovery of tear proteins, generation of reproducible results, and, more importantly, determines changes of protein quantity and post-translation modifications in microliter samples. The present review compared techniques for tear collection, sample preparation, and acquisition applied for the discovery of tear protein markers in normal subjects and multifactorial conditions, including dry eye syndrome, diabetic retinopathy, thyroid eye disease and primary open-angle glaucoma, which require an early diagnosis for treatment. It also summarized the contribution of MS to early discovery by means of disease-related protein markers in tear fluid and the potential for transformation of the tear MS-based proteome to antibody-based assay for future clinical application.

Review of Approaches for Increasing Ophthalmic Bioavailability for Eye Drop Formulations.

Ophthalmic diseases represent a significant problem as over 2 billion people worldwide suffer from vison impairment and blindness. Eye drops account for around 90% of ophthalmic medications but are limited in success due to poor patient compliance and low bioavailability. Low bioavailability can be attributed to short retention times in the eye caused by rapid tear turnover and the difficulty of drug diffusion through the multi-layered structure of the eye that includes lipid-rich endothelial and epithelial layers as well as the stroma which is high in water content. In addition, there are barriers such as tight junctional complexes in the corneal epithelium, lacrimal turnover, nasolacrimal drainage, blinking reflexes, efflux transporters, drug metabolism by ocular enzymes, and drug binding to or repulsion from conjunctival mucins, tear proteins, and melanin. In order to maximize transport through the cornea while minimizing drug loss through other pathways, researchers have developed numerous methods to improve eye drop formulations including the addition of viscosity enhancers, permeability enhancers, mucoadhesives, and vasoconstrictors, or using formulations that include puncta occlusion, nanocarriers, or prodrugs. This review explains the mechanism behind each of these methods, examines their history, analyzes previous and current research, evaluates future applications, and discusses the pros and cons of each technique.

Lipocalin-1 is the acceptor protein for phospholipid transfer protein in tears

Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa. Previously, phospholipid transfer protein was found in tears but an acceptor protein was not identified. To search for the acceptor protein(s) in tears a fluorescent phospholipid transfer assay was altered to omit the extrinsic acceptor. Human tears were incubated with fluorescent micelles and showed marked transfer activity verifying a native acceptor protein must be present. Reconstituted tears without tear lipocalin (lipocalin-1) eliminated the transfer of phospholipids. To determine if phospholipid transfer protein is involved in carrying phospholipid to the surface of tears from tear lipocalin, a fraction enriched in phospholipid transfer protein was injected into the subphase of a tear mimicking buffer in which tear lipocalin was present. The addition of phospholipid transfer protein did not increase the thickness of the surface layer regardless of the presence of lipid bearing tear lipocalin. The data show that phospholipid transfer protein transfers phospholipid from micelles to tear lipocalin. Phospholipid transfer protein does not transport the phospholipid. While tear lipocalin has no intrinsic transfer activity from micelles, it is the acceptor protein for phospholipid transfer protein in tears.

Effect of dry eye diseases on the level of Protein and Lipids in Tears of Postmenopausal Women.

The study is aimed at investigating the effect of dry eye disease on the level of protein and lipids in tears of postmenopausal women. The study was carried out on 33 postmenopausal women with dry eye symptoms and 22 postmenopausal women that were asymptomatic. The ocular surface disease index (OSDI) questionnaire classified participants as being symptomatic or asymptomatic of dry eye. Tear stability and tear volume were assessed with Fluorescein Tear Break up Time (FBUT) and Schirmer’s test respectively. Tears were analysed for protein (total protein, albumin, and globulin) and lipid (cholesterol and triglycerides). The mean total protein for Dry Eye (DE) was higher than that for Non-dry eye (NDE). Albumin and globulin were also higher for the DE group when compared to the NDE group. These were however, not statistically significant (p > 0.05). There was also no significant difference in triglycerides and cholesterol between DE and NDE. There was no significant correlation between clinical signs (tear volume and tear breakup time) and total protein, albumin, globulin cholesterol and triglycerides in the NDE and DE eye groups. The study shows that proteins and lipids do not change in postmenopausal women irrespective of the presences of dry eye disease.

Comparative analysis of tear protein in keratoconic scleral lens wearers with varying limbal clearance

Comparative analysis of tear protein in keratoconic scleral lens wearers with varying limbal clearance

Sodium-Sensitive Contact Lens for Diagnostics of Ocular Pathologies.

The ability to measure all the electrolyte concentrations in tears would be valuable in ophthalmology for research and diagnosis of dry eye disease (DED) and other ocular pathologies. However, tear samples are difficult to collect and analyze because the total volume is small and the chemical composition changes rapidly. Measurements of electrolytes in tears is challenging because typical clinical assays for proteins and other biomarkers cannot be used to detect ion concentrations tears. Here, we report the contact lens which is sensitive to sodium ion (Na+), one of the dominant electrolytes in tears. The Na ions in tears is diagnostic for DED. Three sodium-sensitive fluorophores (SG-C16, SG-LPE and SG-PL) were synthesized by derivatizing the sodium green with 1-hexadecyl amine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine or poly-L-lysine, respectively. These probes were bound to modern silicone hydrogel (SiHG) contact lens, Biofinity from Cooper Vision. Doped lenses were tested for sodium ion dependent spectral properties of probes within the contact lens. The probes displayed changes in intensity and lifetime in response to Na+ concentration, were completely reversible, no significant probe wash-out from the lenses, were not affected by proteins in tears and were not removed after repeated washing. These results are the first step to our long-term goal, which is a lens sensitive to all the electrolytes in tears. We presented design, synthesis and implementation of three new sodium sensitive probes within a silicon hydrogel lens. Contact lenses to measure the other electrolytes in tears can be developed using the same approach by synthesis and testing of new ion-sensitive fluorophores.

Tear protein analysis in patients with primary acquired nasolacrimal duct obstruction treated with lacrimal passage intubation.

The pathophysiology of nasolacrimal duct obstruction (NLDO) may involve quantitative as well as qualitative changes in tears. We measured tear protein concentrations in patients with primary acquired NLDO and compared them with the tear proteins in healthy individuals and patients with dry eye disease. Case-control study. Twenty-four patients diagnosed with primary acquired NLDO who underwent endoscopic lacrimal passage intubation were included in the study. Tear fluid was collected with Schirmer's test strips three times: before intubation, after intubation while the nasolacrimal tube was in place, and after extubation. At the same time, 24 age-matched normal controls and 24 patients with dry eye were selected from subjects whose tears were collected during the same study period. We measured the following components of tear fluid: total protein, lactoferrin, albumin, and interleukin 6 (IL-6) levels. Total proteins and albumin levels in tears of the NLDO group were significantly lower than those of the control group (P < 0.001 and P = 0.014, respectively, Mann-Whitney U test). Total proteins, albumin, and lactoferrin levels were significantly increased after extubation of nasolacrimal tube (P < 0.001, P = 0.004, and P = 0.029, respectively, Wilcoxon matched-pairs signed-rank test). There were no statistically significant differences in total protein, albumin, lactoferrin and IL-6 levels between patients with NLDO after lacrimal passage extubation and controls. Our study shows distinct differences in protein composition of tear fluid in NLDO eyes and demonstrates that these can be normalized by nasolacrimal tube placement.

Tear Proteomics Study of Dry Eye Disease: Which Eye Do You Adopt as the Representative Eye for the Study?

Most studies about dry eye disease (DED) chose unilateral eye for investigation and drew conclusions based on monocular results, whereas most studies involving tear proteomics were based on the results of pooling tears from a group of DED patients. Patients with DED were consecutively enrolled for binocular clinical tests, tear biochemical markers of DED, and tear proteome. We found that bilateral eyes of DED patients may have similar but different ocular surface performance and tear proteome. Most ocular surface homeostatic markers and tear biomarkers were not significantly different in the bilateral eyes of DED subjects, and most clinical parameters and tear biomarkers were correlated significantly between bilateral eyes. However, discrepant binocular presentation in the markers of ocular surface homeostasis and the associations with tear proteins suggested that one eye’s performance cannot represent that of the other eye or both eyes. Therefore, in studies for elucidating tear film homeostasis of DED, we may lose some important messages hidden in the fellow eye if we collected clinical and proteomic data only from a unilateral eye. For mechanistic studies, it is recommended that researchers collect tear samples from the eye with more severe DED under sensitive criteria for identifying the more severe eye and evaluating the tear biochemical and proteomic markers with binocular concordance drawn in prior binocular studies.

Невидимый защитник глаза – лактоферрин

The main protective proteins that are synthesized by eye cells are lactoferrin (Lf), lysozyme, immunoglobulin-A, and tear lipocalins. It has been proven that Lf is contained in biological fluids and mucous membranes of various organs; this highlights the importance of this protein in the first line of defense from pathogenic microorganisms. Lf is a non-heme iron-binding chelating glycoprotein from the transferrin family. Lf carries out bactericidal, fungicidal, antiviral, antioxidant and transport functions, prevents the formation of free radicals, inhibits lipid peroxidation, activates enzymes of the antioxidant system. Lf is contained in tears in the highest concentration (about 2 mg/ml, 25% of tear proteins), the average concentration is 1.42 mg/ml. Lf is an important component providing homeostasis of the ocular surface, modulates the activity of T-lymphocytes and macrophages in infections, prevents the multiplication of pathogenic microflora, the development of inflammation, protects the integrity of the cornea, promotes healing from microtraumas, controls the level of iron in the lacrimal fluid, and protects against toxins. These properties of Lf open up prospects for its application in the treatment of chronic diseases of the ocular surface and, in particular, dry eye disease. Lf concentration in tears decreases during sleep, with age, in dry eye disease, keratitis and conjunctivitis, when using contact lenses, that increase the risk of developing eye infections. The first results of the application of ophthalmic drops Lacto (NOVAX® PHARMA) showed good tolerance and therapeutic efficacy in the treatment of inflammatory diseases of the ocular surface. Keywords: lactoferrin, tear, antimicrobial, immunomodulatory, anti-inflammatory properties, ocular surface.

The influence of tobacco exposure in color vision

Cigarette has about four thousand types of substances and its consumption is a risk factor for a variety of diseases, including neuro-ophthalmic disorders. This is a literature survey about the influence of smoking on color vision. Review of literature based in the Google Scholar research platforms, Scielo, Science Direct and the Bank of Dissertations and Theses of CAPES. Smoking articles, smoking, smoking and color vision and color vision in articles titles were adopted for inclusion criteria. Thus, 31 of which were sealed. In addition to cognitive changes, chronic use of tobacco can cause damage to retinal cells, impairing color vision, changes in tear protein patterns, adverse effects on the precorneal tear film and ocular surface. Metabolism is indirectly affected by means of the reduction of plasma levels of antioxidants and increasing the production of free radicals that impair eye health and blood flow in the ocular choroid. The chronic consumption of cigarettes causes changes in color vision / discrimination and such analysis alerts the harmful effects caused by the substances that compose it. In addition, psychophysical vision examination can be used as a screening test for intoxication processes that may be occurring in the subject.

Quantitative Proteomics and Weighted Correlation Network Analysis of Tear Samples in Adults and Children With Diabetes and Dry Eye.

PurposeDiabetics are more prone to suffer from dry eye (DE). The ages of diabetes are decreasing, so ocular surface status in younger generations is worthy of attention. We used tandem mass tag (TMT)–labeled proteomics and weighted correlation network analysis (WGCNA) to identify differentially expressed proteins in the tear proteome of adults and children with diabetic DE.MethodsStudy subjects were divided into six groups of 10, including three groups each for adults and children. The adult groups included diabetics with DE (A), diabetics without DE (B), and normal controls (C); the corresponding groups of children were identified as (D), (E), and (F). DE tests were performed on all subjects. We extracted total proteins and labeled them with TMTs for analysis. WGCNA was used to recognize hub genes.ResultsTear film function was poorer in patients with diabetic DE. In adults, 1922 proteins were identified, and WGCNA analysis revealed three hub genes related to diabetic DE. For children, 2709 proteins were identified, and WGCNA analysis identified one hub gene related to diabetic DE. Kyoto Encyclopedia of Genes and Genomes analysis found similarities among metabolic pathways involved in differential expression of proteins in adult and child tear samples.ConclusionsThe pathogenesis of diabetic DE was highly similar in adults and children. The differentially expressed tear proteins in type 2 diabetes of adults and children was associated with inflammation, immune factors, and lipid metabolism.Translational RelevanceOur findings found high similarities in the pathogenesis of diabetic DE in adults and children.

Electrospun GelMA fibers and p(HEMA) matrix composite for corneal tissue engineering

The development of biocompatible and transparent three-dimensional materials is desirable for corneal tissue engineering. Inspired from the cornea structure, gelatin methacryloyl-poly(2-hydroxymethyl methacrylate) (GelMA-p(HEMA)) composite hydrogel was fabricated. GelMA fibers were produced via electrospinning and covered with a thin layer of p(HEMA) in the presence of N,N′-methylenebisacrylamide (MBA) as cross-linker by drop-casting. The structure of resulting GelMA-p(HEMA) composite was characterized by spectrophotometry, microscopy, and swelling studies. Biocompatibility and biological properties of the both p(HEMA) and GelMA-p(HEMA) composite have been investigated by 3D cell culture, red blood cell hemolysis, and protein adsorption studies (i.e., human serum albumin, human immunoglobulin and egg white lysozyme). The optical transmittance of the GelMA-p(HEMA) composite was found to be approximately 70% at 550 nm. The GelMA-p(HEMA) composite was biocompatible with tear fluid proteins and convenient for cell adhesion and growth. Thus, as prepared hydrogel composite may find extensive applications in future for the development of corneal tissue engineering as well as preparation of stroma of the corneal material.

Impact of diurnal variation, sex, tear collection method, and disease state on tear protein levels in dogs

To investigate the effects of various biological factors on total protein concentration (TPC) and serum albumin levels in canine tears. 10 healthy beagles (5 female, 5 male) were used. Experiments were conducted on separate days, collecting tears with either capillary tubes or Schirmer strips, as follows: (i) Tear collection at 3hours intervals (from 6am to 12am); and (ii) Tear collection before and 20minutes following topical histamine application (1, 10, 375mg/mL) to induce mild, moderate, and severe conjunctivitis, respectively. TPC and serum albumin were measured with infrared spectroscopy and ELISA, respectively. Tear film TPC and serum albumin ranged from 9.7-26.1mg/mL and 6.4-1662.6µg/mL, respectively. Protein levels did not differ significantly among time points (P≥.080). Median coefficient of variation (CV%) was lower with Schirmer strips compared to capillary tubes for both TPC (12% vs 15%, P=.020) and serum albumin (57% vs 78%, P=.232). TPC (P<.001), but not serum albumin was greater in male vs. female dogs. Serum albumin, but not TPC (P≥.099), increased significantly with each grade of conjunctivitis severity (P<.001), with no differences between collection devices (P≥.322); median increase was 106%, 1389%, and 2871% in eyes with mild, moderate, and severe conjunctivitis, respectively. There is no apparent diurnal variation in canine tear protein levels. Blood-tear barrier breakdown with conjunctivitis allows serum albumin to leak into the tear film at high concentrations. Schirmer strips compare well with capillary tubes for bioanalytical purposes in healthy and diseased eyes, and this collection method may offer improved reproducibility for protein quantification.

Early changes in tear film protein profiles after femtosecond LASIK surgery

BackgroundFemtosecond laser-assisted in situ keratomileusis (LASIK) has proven to be an efficacious, predictable, and safe procedure for the correction of refractive errors. We examined the early tear protein changes of patients undergoing LASIK surgery in order to better understand the mechanisms and proteins related to laser corneal surgery and initial recovery.MethodsCorneal flaps were created with Ziemer FEMTO LDV Z6 I femtosecond laser and stroma was ablated using Wavelight EX500 excimer laser. Tear samples were collected preoperatively as well as 1.5 h and 1 month after LASIK treatment using glass microcapillary tubes. Relative quantification of tear proteins was performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS).ResultsSWATH-MS revealed that 158 proteins had altered expression levels 1.5 h after the operation. Two-thirds of these proteins, mostly connected to migration and inflammation response, returned to preoperative levels within the first postoperative month. The other proteins, which did not return to baseline levels, included proteins connected to for example epithelial barrier function. We also identified several proteins, which correlated with surgical variables, such as the amount of correction, flap thickness and flap diameter.ConclusionsThe present study showed that an uneventful femtosecond LASIK refractive surgery induced a significant immune cell migration and inflammation-associated changes in tear proteomics profile quickly after the operation, but the expression of most proteins recovered almost completely to the preoperative levels within the first month. The individual proteins identified in our study are potential targets for the follow-up and modification of LASIK-induced biochemical processes.

A Novel Integrated Active Herbal Formulation Ameliorates Dry Eye Syndrome by Inhibiting Inflammation and Oxidative Stress and Enhancing Glycosylated Phosphoproteins in Rats.

Dry eye syndrome (DES) is a chronic condition of the eye with insufficient production of tears leading to inadequate lubrication of eyes. Symptoms of DES are associated with discomfort and redness of the eye, blurred vision, and tear film instability which leads to the damaged ocular surface. Inflammation and oxidative stress play a significant role in the pathogenesis of the disease. In this study, the protective effect of different doses (100 or 200 mg/kg) of a novel multi-component oral formulation of lutein/zeaxanthin, curcumin, and vitamin D3 (LCD) was evaluated using a rat model with benzalkonium chloride (BAC)-induced dry eye syndrome. The formulation was administered orally to rats for 4 weeks. We observed a significant improvement in tear volume, tear breakup time, tear film integrity, and reduction in overall inflammation in rats fed with the LCD at dose 200 mg/kg performing better than 100 mg/kg. Furthermore, the formulation helped in lowering oxidative stress by increasing antioxidant levels and restored protective tear protein levels including MUC1, MUC4, and MUC5AC with 200 mg of LCD having the most significant effect. The results strongly suggest that the combination of lutein/zeaxanthin, curcumin, and vitamin-D3 is effective in alleviating the symptoms of dry eye condition with a multi-modal mechanism of action.

Comparative analysis of the tear protein profile in herpes simplex virus type 1 epithelial keratitis

BackgroundHerpes simplex virus type 1 (HSV-1) keratitis is a major cause of corneal blindness in the world, and an in-depth understanding of its pathogenesis may help improve existing diagnosis and treatment. The purpose of this study is to compare and analysis the total tear protein profile of HSV-1 epithelial keratitis patients, and to quantify the potential candidate biomarkers of HSV-1 epithelial keratitis.MethodsWe investigated the proteome in tear fluid from three HSV-1 epithelial keratitis patients and three healthy control subjects using nano-scale liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. Functional annotation of differentially expressed proteins was done with the Gene Ontology (GO) analysis. ELISA was done to quantify the potential candidate biomarkers in 26 clinical cases.ResultsTear fluid from three HSV-1 epithelial keratitis patients and three healthy control subjects contained a total of 1275 proteins and 326 proteins were unique to tear fluid of HSV-1 epithelial keratitis patients. Bioinformatics analysis revealed that tear proteins from HSV-1 epithelial keratitis patients may be involved in metabolic processes, antigen presentation, inflammatory response, and in the TNF-mediated and T cell receptor pathways. Furthermore, IL1A, IL12B, DEFB4A, and CAMP, which are associated with the inflammatory response and inhibition of viral infection, were significantly more abundant in the HSV-1 epithelial keratitis patients than in the healthy control subjects.ConclusionsThis study reports the proteomic profile of tears in HSV-1 epithelial keratitis for the first time and identifies a number of unique differentially expressed proteins.

Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.