17 Interestingly, no alterations had been located in the expression of mRNA coding for another Fn splice variant, Fn extracellular domain B in uninjured lungs. We then assessed the expression of MMP 2 and MMP 9 and their inhibitors tissue inhibitor of metalloproteinases one and TIMP two, respectively. In Figure 7A, we noticed elevated mRNA expression of MMP 2, MMP 9 and TIMP 2, but not TIMP 1 with age. Gelatin zymography showed enhanced gelatinolytic activity linked to MMP 9 in previous lungs compared with young lungs. Gelatinolytic action of MMP 2 was not altered. Main Fibroblasts Harvested From Previous Lungs Demonstrate Decreased Expression of Thy 1 We had been thinking about evaluating lung fibroblasts obtained from uninjured youthful versus previous lungs for his or her expression of Thy one. Thy 1 is a surface protein that regulates TGF B1 activation and Fn expression.
18 Moreover, selleck inhibitor Thy 1 null mice show enhanced fibrosis in response selleck chemicals to bleomycin induced lung injury compared with wild kind mice. eight We located that fibroblasts harvested from both younger and outdated lungs have related morphology that has a slight grow in rectangle/round cells within the lungs of previous mice, and that is suggestive of Thy one detrimental fibroblasts 19. Nonetheless, fibroblasts from aged lungs showed a significant lessen in Thy one mRNA expression. Constant with this particular, fibroblasts isolated from outdated lungs were submitted to movement cytometry evaluation, which showed a lower from the % of Thy one good cells and imply fluorescence intensity for Thy 1 expression per cell. To determine regardless of whether epigenetic mechanisms were accountable to the reduction of Thy one expression with age, we treated lung fibroblasts isolated in the lungs of old mice with AZA, a DNA methyltransferase inhibitor. sixteen We located no major transform in Thy one mRNA expression in lung fibroblasts soon after treatment method with AZA.
DISCUSSION Aged or outdated lungs display enhanced susceptibility to injury and development of fibrosis, however the mechanisms accountable for this remain poorly understood. We hypothesize that aging is linked to a profibrotic phenotype that leads to enhanced susceptibility to disrepair and fibrosis after lung injury. We noticed that previous mice build additional pronounced fibrosis just after bleomycin induced

lung injury in contrast with youthful mice. Interestingly, this susceptibility to fibrosis in old lungs was connected to elevated expression on the profibrotic development component TGF B1, the matrix glycoprotein Fn EDA and MMPs and activation within the TGF B1/ Smad3 pathway with increased Smad3 phosphorylation and DNA binding. Additionally, these adjustments were associated with alterations in lung fibroblast phenotype as highlighted by a lessen in Thy one expression.

We replaced Y116 with alanine or glutamic acid and observed a reduction of function in IFN signaling assays. The conservation of these residues amid these viruses underlines the significance of the tyrosine at this posi tion. Moreover, Caignard et al. not too long ago reported that the minimum region demanded for that interaction of measles virus V protein with STAT1 is residues 110 to 120, which contains Y110 and is really similar in position on the NiV P, V, or W STAT1 binding domain that we dene here. Even so, alignment in the NiV and measles virus P genes displays incredibly little identity outside the hexapeptide sequence. The perform ality of our phenylalanine substitution and lack of rescue by substitute with phosphomimetic glutamic acid in NiV P sug gest that tyrosine phosphorylation at this web-site is simply not crucial for protein perform and level extra to a structural selleck chemicals relevance for this residue.
Making use of a newly established reverse genetics method, we suc cessfully created viruses through which C protein expression is expected to get eradicated, due to the mutation of two probable initiator AUG codons, too since the introduction of the down stream, in frame, halt codon. The G121E mutation, predicted to abrogate inhibition of STAT1 by P, CCI-779 V, and W, was created into this Cko background because the mutation would otherwise also result in a C protein amino acid modify. The Cko virus is attenuated compared to the WT in each in the cell lines examined, plus the G121E mutant displays related development kinetics, sug gesting that the stage mutation in P, V, and W doesn’t even further have an effect on replication. Interestingly, the addition with the G121E mu tation to a Cko background did not more impair replication, even in 293T cells, that are, contrary to Vero cells, capable of making IFN.
This may possibly be explained through the capability of NiV V and W to block the induction of IFN, which

is not really anticipated to be dependent on an interaction with STAT1. Potential experiments will determine the quantities of IFN pro duced by IFN competent cells for the duration of infection. Examination of the standing of STAT1 all through WT or Cko NiV infection uncovered a striking phenotype in that all STAT1 seems for being nuclear but not tyrosine phos phorylated. Given the W protein has become found to direct nonphosphorylated STAT1 to the nucleus, whilst the P and V proteins sustain STAT1 during the cytoplasm, it appears that W is a dominant aspect that controls STAT1 activation in NiV contaminated cells. In long term studies, it will be of curiosity to find out regardless of whether viruses that lack W expression but retain WT P and V expression hold the ability to correctly protect against STAT1 activation. Introduction of your G121E muta tion thoroughly reversed the inhibition viewed while in the Cko virus contaminated cells. As viewed in uninfected cells, STAT1 was not phosphory lated and was cytoplasmic prior to IFN addition.

This confirms the presence of glutamate synthase in mosquitoes, and suggests the enzyme contributes to your production of glutamate for your synthesis of proline. Quite a few vital enzymes linked to ammonia metabolism showed activity in homogenates of mosquito excess fat entire body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamate synthase, glutamine synthetase, pyrroline five carboxylate synthetase, and pyrroline five carboxylate reductase were cloned and sequenced. The mRNA expression patterns of these genes have been examined by true time reverse transcriptase polymerase chain reaction selleckchem in excess fat physique and midgut just before and following a blood meal. The results display that female mosquitoes have evolved productive mechanisms to detoxify huge load of ammonia. Kinetic of incorporation of 15N from labeled ammonia into amino acids in Aedes aegypti females P. Y. Scaraffia1, Q. Zhang2, V. H. Wysocki2, J.
Isoe1 and M. A. Wells1 1 Department of Biochemistry Molecular Biophysics and Center for Insect Science. Department of Chemistry, University of Arizona, Tucson, AZ, USA. We’ve got not too long ago demonstrated that Aedes aegypti females selleck chemicals WP1130 can detoxify ammonia primarily as a result of the synthesis of glutamine and proline in conjunction with the ammonia, uric acid and urea excretion. Now, we’ve got established a protocol to research the kinetics of incorporation of 15 N from labeled ammonia into glutamine, glutamic acid, alanine and proline in Ae. aegypti. Mosquitoes have been fed 3% sucrose answers containing either 80 mM 15 NH4Cl or 80 mM glutamine labeled with 15N in both the amide nitrogen or in the two amide and amine nitrogens. In some experiments, distinct inhibitors of glutamine synthetase or glutamate synthase had been added to the feeding answers. At numerous times publish feeding which varied between 0 and 96 hours, entire mosquitoes were immersed in liquid nitrogen.
Total bodies of ten insects had been homogenized in water. The suspension was centrifuged as well as the supernatant collected. The samples plus deuterium labeled inner specifications were derivatized as dimethylformamidine isobutyl esters or isobutyl esters. PS-341 The quantification of 15N labeled and unlabeled amino acids was performed at a series of different neutral losses by carrying out multiple reaction monitoring scans inside a triple quadrupole mass spectrometer. The outcomes showed that the rate of incorporation of 15N from labeled ammonia into amino acids was rapid and that the label very first appeared during the amide side chain of Gln and then while in the amino group of Gln, Glu, Ala and Professional. The addition of inhibitors of crucial enzymes within the ammonia metabolism pathway confirmed that mosquitoes effectively metabolize ammonia by a metabolic route that largely will involve glutamine synthetase and glutamate synthase. Also, a full deduced amio acid sequence for GltS of Ae. n

Genes that had been downregulated through the primary infestation have been enriched for GO terms selleck that fell into two clusters. nucleotide metabolism/transcription and Very similar Expres sion to Fibroblast Development Factor and IL 17Rs or SEFIR. The former cate gory contained terms related to gene expression and metabolic processes even though the SEFIR group includes domains recognized to participate in IL 17RA signaling independently of classic Toll/IL 1R structures together with MyD88 and TRIF. In contrast with the major infestation, upregulated transcripts throughout secondary exposure have been enriched for GO terms linked to a wide array of categories as proven in Table 3. From this data, 4 main categories are evi dent. cytokine, chemotaxis, immune cell signaling and activation, and leukocyte adhesion.
Other groups of enriched terms included T cell regulation and cell sur face molecules whilst the remaining terms clustered into a amount of small classes associated with putative secreted, hematopoietic lineage, irritation, protein protein interactions, activation peptide, and tyrosine kinase phosphorylation. On the flip side, only GO terms PHA793887 negative regulation of cell proliferation and SEFIR were considerably enriched from genes downregulated in the secondary publicity. Modulation of gene expression all through principal infestations Although gene ontology allows assessment of inapparent biological processes inside a list of genes, it does not permit direct comparison in between time factors or infestations in the gene degree. To facilitate this, all 233 genes measured have been divided into individual groups depending on shared qualities from the translated protein. These groups along with the genes in each group is often accessed in additional file three. Genes modulated during the main infestation are shown in Figure 2.
Upregulated genes that were constantly expressed during the course of tick feeding integrated cytokines IL ten, IL six, and IL 1b, chemokines CCL2, 7, CXCL1, 2, and 5, pattern recognition receptor CLEC7a, modulator of irritation prostaglandin endoperoxide synthase two, extracellular matrix proteases MMP9, 10, and 13, and the adhesion molecules L selectin, and b two integrin. Amid downregulated genes, probably the most notable had been Roscovitine members within the IL 17 receptor household, which had been regularly downregulated during the entire feeding method while the pro inflammatory cyto kines IL 17a, c, d, and f were not expressed. Other downregulated genes had been T cell molecules CD28, GATA3, and retinoic acid related orphan nuclear hor mone receptor C, DNA repair molecule telo merase reverse transcriptase, basement membrane/ECM structural elements HAPLN1 and LAMA1, neural adhesion molecule NCAM2, mitogenic pathway member JUN, WNT pathway members JUN and LEF1, hedgehog pathway members BMP2 and 4, and retinoic acid pathway member HOXA1.

Moreover, IFN induction could be stimulated by TLR3 signaling and viral infection and has an tiviral exercise, just like IFN / and IFN. Though we didn’t observe any manufacturing of IFN in our experiments, because it is made within a tissue specic fashion, it performs functions just like these of IFN / albeit on dif ferent cell forms. The exact same is genuine for IFN. it had been not created from the cells used in our experiments and thus won’t deliver a level of redundancy in broblasts. Yet, in the whole animal method, IFN / signaling recruits T and NK cells, which develop IFN to elicit antiviral results. Thus, to utilize MEFs to review the function of IFN or IFN while in the absence of IFN receptors, specialized immune cells would must be isolated from your mutant mice for in vitro experi mentation. Our success indicate that when the IFN / receptor is required to curb viral replication, it’s dispensable for that in duction of sure inammatory and apoptotic genes.
We iden tify likely pathways, via IRF3 or IL 1 activation or Hoxa13, Polr2a, Nr4a1, or Ing1 induction, that may contribute to this redundancy. Even further experimentation is needed to in terrogate these prospective mechanisms and the way the proteins encoded by every single gene may well elicit inammatory or apoptotic selleck responses during the absence in the IFN / receptor. Of particu lar interest could be the mechanism of IL one activation from the absence from the IFN / receptor, due to the fact latest studies have proven that this selleck chemical Mocetinostat molecule is central to inammasome signaling. With each other, our research and people described above illustrate methods in which the host has established overlapping mechanisms to respond to viral infections and that redundancies come about inside of host signaling mechanisms, which possible produced through the coevolution of pathogen and host.
The mouse autonomously replicating parvovirus Minute vi rus of mice is a little icosahedral nonenveloped lytic virus containing a single stranded DNA genome of about 5. one kb. Though infection of grownup or neo natal mice with MVMp is asymptomatic, virus injected in utero into creating embryos mounts an aggressive infection which eventually kills the host. The MVMp life cycle is ideal supported in vivo as well as in vitro by broblastic cells, espe cially transformed derivatives like the mouse A9 line. The MVMp genome consists of two overlapping transcription units encoding two nonstructural and two structural professional teins whose expression is driven through the P4 and P38 pro moters, respectively. Between the parvoviral merchandise, the NS1 polypeptide will be the big cytotoxic issue. For 2 decades, MVMp has attracted signicant awareness because of its onco tropic and oncolytic properties, displayed in the two rodent and human cells. The parvoviral oncotropism has become up to now ascribed to your dependency from the virus life cycle on host cell aspects current through the S phase from the cell cycle and/or Corresponding writer.

PE is really a multisystem condition, the subclassifications of PE are frequently defined as. mild, moderate, and significant, also as early and late. The idea of early and late PE is more modern day, and it really is widely accepted that these two entities have unique etiologies and should really be thought to be numerous varieties with the selleckchem ailment. In our hospital, we generally select conservative remedy, not cesarean delivery, for your pregnant female with early preeclamp sia, so there may be a limitation in getting the placenta tis sue of those patients, even so, in our long term study, we count on that we could receive variety of specimens includ ing the mom blood and placenta of early and late PE, and do some exploration on the Wnt signaling within the early and late PE. Clinical chance things for intrauterine fetal development con tain serious PE, cardiopulmonary strategy problems, um bilical cord and placenta dysfunction, and so on.
On this examine, we also noticed the B catenin expression was de creased, plus the DKK1expression was increased from the significant PE group who had an IUGR fetus in contrast with those with an AGA fetus. However, the outcomes advised that there is no distinction within the expression of B catenin and DKK1 among sufferers with preterm and term PE. Prospective factors for this locating may perhaps be the premature birth consists of spontaneous premature Rutoside de livery and therapeutic premature delivery, the influence aspects are complicated. Conclusions In summary, the pathogenesis of PE is highly complex, along with the final results of this study indicated that B catenin and DKK1 are expressed in human third trimester placentas, decreased B catenin expression, also as DKK1 over expression may be involved in the system of your pathogenesis of PE. Even further research would elucidate their actual roles from the pathogenesis of PE.
Moreover, the

ex pression of B catenin and DKK1 during the first and sec ond trimester, too since the likely roles in the other associated proteins in Wnt signaling pathway in PE, awaits further examine. Discovery of Gab docking proteins Ten years on Using the escalating isolation and cloning of protein tyro sine kinase substrates and association partners in the mid 1990s, a sizable variety of proteins without intrin sic enzymatic activity had been described and termed as adap tor, scaffold or docking proteins. While these terms are often applied interchangeably, adaptor proteins are usu ally smaller in dimension and often perform as an inter or intra molecular bridge between two proteins or within a single protein, respectively, and thereby play an important part within the assembly of bigger protein complexes or even the stabili sation of specified conformational states.

Nonetheless, these tis sues also showed similar above expression of energetic pSTAT3/STAT3. Vast majority of HPV precancer, cancer and standard tissues lacked expression of STAT3 and pSTAT3 when only a tiny variety of HPV16 precan cers and cancers had no or lower STAT3 expression. Interestingly, immunohis tochemical examination of precancer lesions specifically of LSILs showed a focal positivity of STAT3 and when these circumstances have been analyzed with respect to their HPV16 standing they showed a lower background staining without any nuclear positivity for STAT3 likewise as pSTAT3 in HPV damaging LSIL sections. In contrast, HPV16 positive LSIL sections revealed a powerful focal positivity and nuclear localization of each STAT3 and pSTAT3 in basal and suprabasal cell layers of cer vical epithelium. Differential expression and activation of STAT3 in diverse histopathological grades of your HPV16 constructive cancer lesions Considering the fact that STAT3 expression/activation was localized in HPV16 precancer lesions.
To determine its correlation we examined STAT3 expression in HPV16 cervical cancer situations with unique histopathological grades. Forty five cancer biopsies with confirmed histo pathology and HPV16 positivity had been re evaluated for STAT3 and pSTAT3 expression. As proven in Figure 5A and 5B, a comparative immunoblotting and immunohis tochemical analysis exposed a decrease expression of STAT3 and pSTAT3 in WDSCC instances in comparison to MDSCC Seliciclib CDK inhibitor and PDSCC that had substantial STAT3 and pSTAT3 expression. Elevated level of pSTAT3 in MDSCC and PDSCC have been also corroborated with extreme nuclear positivity of STAT3 in histologi cally innovative cancer tissues and was observed in as large as 78% and 88% of cancer cells in MDSCC and PDSCC respectively. In contrast, only in 53% of cells in WDSCC sections showed nuclear localization of STAT3 proteins.
Collectively, these selleck findings indicate that con stitutive activation of STAT3 is usually a frequent occurrence in substantial grade malignant cervical carcinomas and positively correlated with poorer histopathological grades. Discussion In the present study, we demonstrate aberrantly expressed and constitutively lively STAT3 the two in cervi cal cancer cell lines and in cervical precancer and cancer lesions. Expression of STAT3 was elevated at transcript level and was discovered related to simultaneous maximize in phosphorylation at each, Tyr705 and Ser727, that are recognized to manage STAT3 dimerization, nuclear transport, DNA binding and transactivation. Dually phosphorylated STAT3 present in cervical precancer and cancer

lesions was observed to localize for the nuclei and possessed a functional DNA binding exercise. Our immu noblotting, IHC and DNA binding assays unveiled that aberrant STAT3 exercise increases as a perform of sever ity with the disorder from precancer to cancer in the course of cervi cal carcinogenesis and was uncovered connected with HPV16 favourable lesions.

As previously reported, leptin can stimulate tube like structures in vitro. To investigate the mechanism of this result, we applied Aca1, a potent ObR antagonist, produced in our labora tories and confirmed to inhibit leptin signaling in LN18 and LN229 cells. Treatment method of HUVEC with a hundred ng/mL leptin for 8 h made 80% increase in ES formation compared with untreated cells. Addition of Aca1 consistently counteracted this leptin dependent effect. In the lowest concentration applied Aca1 completely reverted the leptin induced ES increase, whereas a slight reduction from the ES quantity vs. manage was observed during the presence of Aca1 at 25 and 50 nM concentrations. Notably, Aca1 alone did not have an effect on the quantity of ES relative to con trol, except to get a slight decrease at the highest concen tration, suggesting its precise exercise in direction of ObR in presence of leptin.
In parallel, we taken care of HUVEC with selleck 50 ng/mL VEGF, both alone or in presence of SU1498, a potent inhibitor of VEGFR2. VEGF enhanced by 60% the amount of ES, and this impact was antagonized by SU1498 in a dose dependent manner, using the most effective response mentioned at five uM. Following, we assessed the proliferative response of HUVEC to leptin within the presence or absence of ObR antagonist. MGCD265 Leptin at 200 ng/mL improved the growth of HUVEC by 25% relative to control. The addition of Aca1 interfered with leptin induced prolifera tion within a dose dependent method. In particular, Aca1 at 25 nM absolutely and drastically abolished leptin mito genic effects, whilst the antagonist on the substantial est concentration developed cytotoxic effects, appreciably a lot more pronounced within the absence of leptin. Having said that, no wonderful influence on cell development was detected in HUVEC handled with Aca1 alone at ten and 25 nM.
The parallel experiments with VEGF demonstrated that 50 ng/mL VEGF stimulated HUVEC proliferation by 27% relative to untreated cells. SU1498 reduced this impact in the dose dependent method. 5 uM SU1498 totally blocked VEGF effects, whereas greater concentrations from the inhibitor were cytotoxic. To investigate the mechanism

of Aca1 and SU1498 interference with leptin or VEGF results on HUVEC, we studied should the antagonists are able to inhibit ligand induced intracellular STAT3 signaling. The induction of STAT3 by leptin or VEGF in HUVEC was previously reported. We confirmed that leptin activates STAT3 in these cells and discovered that Aca1 is in a position to sig nificantly cut down leptin dependent STAT3 phosphoryla tion. Similarly, VEGF activated STAT3, and SU1498 lowered STAT3 phosphorylation in VEGF trea ted HUVEC. These above data propose that Aca1 and SU1498 are suitable to evaluate the distinct contributions of leptin and VEGF in angiogenic and mitogenic effects of CM derived from GBM cell cultures.

Furthermore, current research have demonstrated that Kif3a is important for Shh dependent growth of cerebellar progenitors. Conditional ablation within the gene in cells derived from Cre expressing cells under the human glial fibrillary acidic protein pro moter resulted in reduction of primary cilia in cerebellar granule cell precursors. On this animal model, GCPs were specified, but a extreme defect in late embryonic and early postnatal expansion of GCPs resulted in atrophied cere bella. Exactly the same animal model was analyzed for professional duction of grownup neural stem cells while in the hippocampus and also revealed the absence of primary cilia in the devel oping dentate gyrus. Main cilia and Shh signaling are very important for that growth and establishment of granule neuron precursors while in the post natal dentate gyrus.
Mutant mice for Stumpy lack cilia and also have evident abnor malities in post natal developing brain regions, including a hypoplasic hippocampus characterized you can find out more by a major deficiency in astrocyte like neural precursors. Cobblestone is a hypomorphic allele with the IFT gene Ift88. Cobblestone mutants display both severe defects inside the for mation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, Smad inhibitor as well as a rest of both dorsal ventral and rostral caudal compartmental boundaries. In this animal model, Gli3 proteolytic processing is diminished and an upregulation of canonical Wnt signaling from the neocortex and within the cau dal forebrain is observed. These success indicate a crucial purpose for ciliary function during the producing forebrain. Furthermore, the inactivation of Ift172 revealed that it’s required inside the patterning in the mammalian brain, and it plays a important function in principal cilia formation dur ing growth.
Over the basis of every one of these studies, the function of cilia

in devel opment is vital in defining the framework of the organism. In fact, impairment in cilia perform prospects to structural defects. A number of ciliopathies this kind of as Bardet Biedl, Almstrm syndrome, Joubert and oral facial digital syndrome type I are pleiotropic ailments, which include things like limb abnormalities, renal cystic illness, CNS abnormali ties like mental retardation, and/or obesity. In sev eral circumstances, nevertheless, mental retardation isn’t linked with CNS structural abnormalities. Obesity and mental retardation, not related with structural defects, might be regarded behavioral defects. This observation suggests that cilia could have a significant role in organ mainte nance and perform, still to become defined, in addition to the properly established function in the course of advancement.

Yet another instance of this intimate relation was demonstrated in human colon cancer cell lines that vary only within their p21 check out point status. Cells with wild variety p21, when irradiated with radiation, underwent a cell cycle development arrest fol lowed by clonogenic survival, the place as cells lacking p21, when irradiated with radiation, did not undergo a cell cycle development arrest and in addition proceeded to apopto sis. Cells that undergo a development arrest could be pro tected from apoptosis and may perhaps for this reason be in the long run resistant for the cytotoxic agent. Curcumin the curry for cure. our hypothesis Cell cycle progression is an important biological event possessing controlled regulation in ordinary cells, which almost universally turns into aberrant or deregulated in transformed and neoplastic cells.
In this regard, focusing on deregulated cell cycle progression and its modulation by numerous all-natural and inhibitor SP600125 synthetic agents are gaining wide spread attention lately to control the unchecked development and proliferation in cancer cells. The truth is, a vast number of experimental research convincingly display that numerous phytochemicals halt uncontrolled cell cycle professional gression in cancer cells. Amid these phytochemicals, curcumin continues to be identified as one within the main pure anticancer agents exerting anti neoplastic activity in vari ous kinds of cancer cells. Right here we hypothesize that curcu min asserts its anti tumor activity in cancer cells by altering the de regulated cell cycle via cyclin depend ent, p53 dependent and p53 independent path strategies. In the crossroads of substitute and primary stream medication Turmeric is implemented for thousands of many years in Ayurvedic and common Chinese medication.
In modern times, curcumin, the yellow pigment on the spice turmeric, continues to get implemented as an alternative medicinal agent in many parts of South East Chelerythrine Asia to the treatment of com mon ailments such as stomachic upset, flatulence, jaun dice, arthritis, sprains, wounds and skin infections between numerous others. Curcumin and turmeric solutions have already been characterized as risk-free by

overall health authorities this kind of because the Food and Drug Administration in United states of america of America, Food and Agriculture Organization/World Well being Organization. Curcumin has entered scientific clinical trials at the phase I and II clinical trial degree only while in the final ten 15 many years. A phase III research of gem citabine, curcumin and celecoxib is due to open to recruit ment with the Tel Aviv Sourasky Healthcare Center for individuals with metastatic colorectal cancer. Why curcumin Curcumin is known as a element of turmeric, the yellow spice derived from your roots of your plant Curcuma longa. Curcuma longa is a brief stemmed perennial, which grows to about a hundred cm in height. It has curved leaves and oblong, ovate or cylindrical rhizomes.