ABSTRACT
Cationic antimicrobial peptide PGLa gets into close contact with the anionic bacterial cell membrane, facilitating cross-membrane transport phenomena and membrane disruption depending on the concentration. The mechanisms of action are closely associated with the tilted insertion geometry of PGLa. Therefore, we aimed to understand the interaction between the transmembrane potential (TMP) and the orientation of the membrane-bound PGLa helix. Molecular dynamics simulations were performed with TMP, and we found that the PGLa tilt angle relative to the membrane is coupled with the TMP. Elevated TMP increases the population of the tilted state. We observed positive feedback between the tilt angle and the TMP, which occurs due to the electrostatic interaction between the peptidic helix and the Na+ cations at the membrane-water interface. These TMP coupled phenomena can contribute to understanding the direct antimicrobial and adjuvant effects of PGLa in combination with regular antibiotics.
Subject(s)
Anti-Infective Agents , Antimicrobial Peptides , Membrane Potentials , Anti-Infective Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Bacterial Agents/pharmacology , Water , Lipid Bilayers/chemistryABSTRACT
The negative membrane potential of bacterial cells influences crucial cellular processes. Inspired by the molecular scaffold of the antimicrobial peptide PGLa, we have developed antimicrobial foldamers with a computer-guided design strategy. The novel PGLa analogues induce sustained membrane hyperpolarization. When co-administered as an adjuvant, the resulting compounds - PGLb1 and PGLb2 - have substantially reduced the level of antibiotic resistance of multi-drug resistant Escherichia coli, Klebsiella pneumoniae and Shigella flexneri clinical isolates. The observed antibiotic potentiation was mediated by hyperpolarization of the bacterial membrane caused by the alteration of cellular ion transport. Specifically, PGLb1 and PGLb2 are selective ionophores that enhance the Goldman-Hodgkin-Katz potential across the bacterial membrane. These findings indicate that manipulating bacterial membrane electrophysiology could be a valuable tool to overcome antimicrobial resistance.
ABSTRACT
Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.Communicated by Ramaswamy H. Sarma.
Subject(s)
Molecular Dynamics Simulation , Receptor, Metabotropic Glutamate 5 , Allosteric Regulation , Allosteric Site , Binding Sites , LigandsABSTRACT
EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.
Subject(s)
Edetic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Pyrophosphatases/antagonists & inhibitors , Taq Polymerase/antagonists & inhibitors , Binding Sites/drug effects , Enzyme Inhibitors/chemistry , Escherichia coli Proteins/metabolism , Humans , Molecular Docking Simulation , Mycobacterium tuberculosis/enzymology , Pyrophosphatases/metabolism , Taq Polymerase/metabolismABSTRACT
1,4-Dioxane is a cytotoxic B2-type human carcinogen, a serious water pollutant produced solely by industrial activity. The effect of 1,4-dioxane on phospholipid membrane models composed of dipalmitoyl-phosphatidylcholine (DPPC) and its branched isomer (isodipalmitoyl-phosphatidylcholine, IPPC) was investigated using MD simulations. Clear and polluted membranes were compared by membrane parameters such as area per lipid (APL), volume per lipid (VPL), compressibility modulus, membrane thickness, and orderliness of lipid tails. While neat systems significantly differ from each other, the presence of the pollutant has the same effect on both types of lipid membranes. High density of dioxane appears in the vicinity of ester groups, which pushes away lipid headgroups from each other, leading to an overall change in lipid structure: APL and VPL grows, while the orderliness of lipid tails, membrane thickness, and compressibility modulus decrease. Orientational preferences of water and dioxane molecules were also investigated and different membrane regions have been specified according to the stance of water molecules. Free-energy profile for 1,4-dioxane penetration mechanism into DPPC membranes was carried out using metadynamics for two different concentrations of the pollutant (c1 = 7.51 g/dm3, c2 = 75.10 g/dm3), which showed that the higher the concentration is, the lower the free energy of penetration gets. Only a small free-energy barrier was found in the headgroup region and accumulation of dioxane is thermodynamically unfavored in the middle of the bilayer. The penetration mechanism has been described in detail based on the orientational preference of 1,4-dioxane molecules and the free-energy profiles.
Subject(s)
Dioxanes/chemistry , Lipid Bilayers/chemistry , Molecular Dynamics Simulation , Phospholipids/chemistry , ThermodynamicsABSTRACT
In order to study the effects of peptide exposure to oxidative attack, we chose a model reaction in which the hydroxyl radical discretely abstracts a hydrogen atom from the α-carbon of each residue of a highly amyloidogenic region in the human calcitonin hormone, hCT15-19. Based on a combined Molecular Mechanics / Quantum Mechanics approach, the extended and folded L- and D-configuration and radical intermediate hCT15-19 peptides were optimized to obtain their compactness, secondary structure and relative thermodynamic data. The results suggest that the epimerization of residues is generally an exergonic process that can explain the cumulative nature of molecular aging. Moreover, the configurational inversion induced conformational changes can cause protein dysfunction. The epimerization of the central residue to the D-configuration induced a hairpin structure in hCT15-19, concomitant with a possible oligomerization of human calcitonin into Aß(1-42)-like amyloid fibrils present in patients suffering from Alzheimer's disease.
Subject(s)
Amyloidogenic Proteins/chemistry , Calcitonin/chemistry , Peptide Fragments/chemistry , Amyloid beta-Peptides/chemistry , Density Functional Theory , Humans , Hydrogen Bonding , Models, Chemical , Molecular Dynamics Simulation , Oxidation-Reduction , Protein Structure, Secondary , Stereoisomerism , ThermodynamicsABSTRACT
Computer simulations of four lipid membranes of different compositions, namely neat DPPC and PSM, and equimolar DPPC-cholesterol and PSM-cholesterol mixtures, are performed in the presence and absence of the general anesthetics diethylether and sevoflurane both at 1 and 600â¯bar. The results are analyzed in order to identify membrane properties that are potentially related to the molecular mechanism of anesthesia, namely that change in the same way in any membrane with any anesthetics, and change oppositely with increasing pressure. We find that the lateral lipid density satisfies both criteria: it is decreased by anesthetics and increased by pressure. This anesthetic-induced swelling is attributed to only those anesthetic molecules that are located close to the boundary of the apolar phase. This lateral expansion is found to lead to increased lateral mobility of the lipids, an effect often thought to be related to general anesthesia; to an increased fraction of the free volume around the outer preferred position of anesthetics; and to the decrease of the lateral pressure in the nearby range of the ester and amide groups, a region into which anesthetic molecules already cannot penetrate. All these changes are reverted by the increase of pressure. Another important finding of this study is that cholesterol has an opposite effect on the membrane properties than anesthetics, and, correspondingly, these changes are less marked in the presence of cholesterol. Therefore, changes in the membrane that can lead to general anesthesia are expected to occur in the membrane domains of low cholesterol content.
Subject(s)
Anesthetics, General/pharmacology , Lipid Bilayers/chemistry , Membrane Lipids/chemistry , Membranes/drug effects , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Cholesterol/chemistry , Cholesterol/metabolism , Computer Simulation , Lipid Bilayers/metabolism , Membrane Lipids/metabolism , Membranes/metabolism , Models, Molecular , Molecular Dynamics Simulation , PressureABSTRACT
DJ-1 (PARK7) is a multifunctional protein linked to the onset and progression of a number of diseases, most of which are associated with high oxidative stress. The Cys106 of DJ-1 is unusually reactive and thus sensitive to oxidation, and due to high oxidative stress it was observed to be in various oxidized states in disease condition. The oxidation state of Cys106 of DJ-1 is believed to determine the specific functions of the protein in normal and disease conditions. Here we report molecular dynamics simulation and biophysical experimental studies on DJ-1 in reduced (Cys106, S-), oxidized (Cys106, SO2-), and over-oxidized (Cys106, SO3-) states. To simulate the different oxidation states of Cys106 in DJ-1, AMBER related force field parameters were developed and reported for 3-sulfinoalanine and cysteine sulfonic acid. Our studies found that the overall structure of DJ-1 in different oxidation states was similar globally, while it differed locally significantly, which have implications on its stability, function and its link to disease on-set. Importantly, the results suggest that over-oxidation may trigger loss of functions due to local structural modification in the Cys106 containing pocket of DJ-1 and structurally destabilize the dimeric state of DJ-1, which is believed to be its bioactive conformation. Such loss of functions would result in reduced ability of DJ-1 to protect from oxidative stress insults and may lead to increased progression of disease.
Subject(s)
Cysteine/metabolism , Oxidative Stress/genetics , Protein Conformation , Protein Deglycase DJ-1/metabolism , Biophysical Phenomena , Cysteine/genetics , Humans , Molecular Dynamics Simulation , Mutation , Oxidation-Reduction , Protein Deglycase DJ-1/chemistry , Protein Deglycase DJ-1/geneticsABSTRACT
dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent.
Subject(s)
Bacterial Proteins/chemistry , Molecular Dynamics Simulation , Mycobacterium tuberculosis/enzymology , Pyrophosphatases/chemistry , Catalytic DomainABSTRACT
In order to identify molecular models of the human 5-HT6 receptor suitable for virtual screening, homology modeling and membrane-embedded molecular dynamics simulations were performed. Structural requirements for robust enrichment were assessed by an unbiased chemometric analysis of enrichments from retrospective virtual screening studies. The two main structural features affecting enrichment are the outward movement of the second extracellular loop and the formation of a hydrophobic cavity deep in the binding site. These features appear transiently in the trajectories and furthermore the stretches of uniformly high enrichment may only last 4-10 ps. The formation of the inner hydrophobic cavity was also linked to the active-like to inactive-like transition of the receptor, especially the so-called connector region. The best structural models provided significant and robust enrichment over three independent ligand sets.
Subject(s)
Drug Design , Receptors, Serotonin/metabolism , Binding Sites , Computer-Aided Design , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Receptors, Serotonin/chemistryABSTRACT
The energetics of peptide bond formation is an important factor not only in the design of chemical peptide synthesis, but it also has a role in protein biosynthesis. In this work, quantum chemical calculations at 10 different levels of theory including G3MP2B3 were performed on the energetics of glutathione formation. The strength of the peptide bond is found to be closely related to the acid strength of the to-be N-terminal and the basicity of the to-be C-terminal amino acid. It is shown that the formation of the first peptide activates the amino acid for the next condensation step, manifested in bacterial protein synthesis where the first step is the formation of an N-formylmethionine dipeptide. The possible role of glutathione in prebiotic molecular evolution is also analyzed. The implications of the thermodynamics of peptide bond formation in prebiotic peptide formation as well as in the preference of α- instead of ß- or γ-amino acids are discussed. An empirical correction is proposed for the compensation of the error due to the incapability of continuum solvation models in describing the change of the first solvation shell when a peptide bond is formed from two zwitterions accompanied by the disappearance of one ion pair.
Subject(s)
Glutathione/chemistry , Peptides/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Molecular Dynamics Simulation , Peptides/metabolism , Quantum Theory , ThermodynamicsABSTRACT
Micellisation process for sodium dodecyl sulphate and sodium cholate in 1â¶1 molar ratio was investigated in a combined approach, including several experimental methods and coarse grained molecular dynamics simulation. The critical micelle concentration (cmc) of mixed micelle was determined by spectrofluorimetric and surface tension measurements in the temperature range of 0-50°C and the values obtained agreed with each other within the statistical error of the measurements. In range of 0-25°C the cmc values obtained are temperature independent while cmc values were increased at higher temperature, which can be explained by the intensive motion of the monomers due to increased temperature. The evidence of existing synergistic effect among different constituent units of the micelle is indicated clearly by the interaction parameter (ß1,2) calculated from cmc values according to Rubingh. As the results of the conductivity measurements showed the negative surface charges of the SDS-NaCA micelle are not neutralized by counterions. Applying a 10 µs long coarse-grained molecular dynamics simulation for system including 30-30 SDS and CA (with appropriate number of Na+ cations and water molecules) we obtained semi-quantitative agreement with the experimental results. Spontaneous aggregation of the surfactant molecules was obtained and the key steps of the micelle formation are identified: First a stable SDS core was formed and thereafter due to the entering CA molecules the size of the micelle increased and the SDS content decreased. In addition the size distribution and composition as well as the shape and structure of micelles are also discussed.
Subject(s)
Sodium Cholate/chemistry , Sodium Dodecyl Sulfate/chemistry , Surface-Active Agents/chemistry , Micelles , Molecular Conformation , Molecular Dynamics Simulation , TemperatureABSTRACT
The epimerization of amino acid residues increases with age in living organisms. In the present study, the structural consequences and thermodynamic functions of the epimerization of thymopentin (TP-5), the active site of the thymic hormone thymopoietin, were studied using molecular dynamics and density functional theory methods. The results show that free radical-initiated D-amino acid formation is energetically favoured (-130 kJmol(-1)) for each residue and induces significant changes to the peptide structure. In comparison to the wild-type (each residue in the L-configuration), the radius of gyration of the D-Asp(3) epimer of the peptide decreased by 0.5 Å, and disrupted the intramolecular hydrogen bonding of the native peptide. Beyond establishing important structural, energetic and thermodynamic benchmarks and reference data for the structure of TP-5, these results disseminate the understanding of molecular ageing, the epimerization of amino acid residues.
Subject(s)
Free Radicals/chemistry , Peptides/chemistry , Thymopentin/chemistry , Amino Acids/chemistry , Hydrogen Bonding , Peptide Fragments/chemistry , Thymopentin/metabolismABSTRACT
In spite of recent investigations into the potential pharmaceutical importance of bile acids as drug carriers, the structure of bile acid aggregates is largely unknown. Here, we used global optimization techniques to find the lowest energy configurations for clusters composed between 2 and 10 cholate molecules, and evaluated the relative stabilities of the global minima. We found that the energetically most preferred geometries for small aggregates are in fact reverse micellar arrangements, and the classical micellar behaviour (efficient burial of hydrophobic parts) is achieved only in systems containing more than five cholate units. Hydrogen bonding plays a very important part in keeping together the monomers, and among the size range considered, the most stable structure was found to be the decamer, having 17 hydrogen bonds. Molecular dynamics simulations showed that the decamer has the lowest dissociation propensity among the studied aggregation numbers.
Subject(s)
Bile Acids and Salts/chemistry , Cholic Acid/chemistry , Drug Carriers , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Micelles , Molecular Dynamics SimulationABSTRACT
We report the identification of novel histamine H4 receptor ligands by ensemble docking on homology model conformers derived from molecular dynamics simulations. Selected receptor models from the trajectories demonstrated superior virtual screening performance compared to the initial models. The ensemble of the best models was able to retrieve a diverse set of known H4 ligands. Prospective virtual screening against these models and subsequent in vitro experimental validation identified novel H4 ligands. Compound 3 showing highest affinity and ligand efficiency represents an interesting scaffold for further medicinal chemistry exploration.
ABSTRACT
The formation of ligand-protein complexes requires simultaneous adaptation of the binding partners. In structure based virtual screening, high throughput docking approaches typically consider the ligand flexibility, but the conformational freedom of the protein is usually taken into account in a limited way. The goal of this study is to elaborate a methodology for incorporating protein flexibility to improve the virtual screening enrichments on GPCRs. Explicit-solvated molecular dynamics simulations (MD) were carried out in lipid bilayers to generate an ensemble of protein conformations for the X-ray structures and homology models of both aminergic and peptidergic GPCRs including the chemokine CXCR4, dopamine D3, histamine H4, and serotonin 5HT6 holo receptor complexes. The quality of the receptor models was assessed by enrichment studies to compare X-ray structures, homology models, and snapshots from the MD trajectory. According to our results, selected frames from the MD trajectory can outperform X-ray structures and homology models in terms of enrichment factor and AUC values. Significant changes were observed considering EF1% values: comparing the original CXCR4, D3, and H4 targets and the additional 5HT6 initial models to that of the best MD frame resulted in 0 to 6.7, 0.32 to 3.5 (10×), 13.3 to 26.7 (2×), and 0 to 14.1 improvements, respectively. It is worth noting that rank-average based ensemble evaluation calculated for different ensemble sizes could not improve the results further. We propose here that MD simulation can capture protein conformations representing the key interacting points of the receptor but less biased toward one specific chemotype. These conformations are useful for the identification of a "consensus" binding site with improved performance in virtual screening.
Subject(s)
Molecular Docking Simulation , Molecular Dynamics Simulation , Receptors, CXCR4/chemistry , Receptors, Dopamine D3/chemistry , Receptors, G-Protein-Coupled/chemistry , Receptors, Histamine/chemistry , Receptors, Serotonin/chemistry , Area Under Curve , Binding Sites , Crystallography, X-Ray , High-Throughput Screening Assays , Humans , Ligands , Lipid Bilayers/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Histamine H4 , Structural Homology, Protein , Thermodynamics , User-Computer InterfaceABSTRACT
Non-reactive, comparative (2 × 1.2 µs) molecular dynamics simulations were carried out to characterize the interactions between glutathione (GSH, host molecule) and hydroxyl radical (OH(â¢), guest molecule). From this analysis, two distinct steps were identified in the recognition process of hydroxyl radical by glutathione: catching and steering, based on the interactions between the host-guest molecules. Over 78% of all interactions are related to the catching mechanism via complex formation between anionic carboxyl groups and the OH radical, hence both terminal residues of GSH serve as recognition sites. The glycine residue has an additional role in the recognition of OH radical, namely the steering. The flexibility of the Gly residue enables the formation of further interactions of other parts of glutathione (e.g. thiol, α- and ß-carbons) with the lone electron pair of the hydroxyl radical. Moreover, quantum chemical calculations were carried out on selected GSH/OH(â¢) complexes and on appropriate GSH conformers to describe the energy profile of the recognition process. The relative enthalpy and the free energy changes of the radical recognition of the strongest complexes varied from -42.4 to -27.8 kJ/mol and from -21.3 to 9.8 kJ/mol, respectively. These complexes, containing two or more intermolecular interactions, would be the starting configurations for the hydrogen atom migration to quench the hydroxyl radical via different reaction channels.
Subject(s)
Free Radicals/chemistry , Glutathione/chemistry , Hydroxyl Radical/chemistry , Models, Theoretical , Free Radicals/metabolism , Glutathione/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydroxyl Radical/metabolism , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , ThermodynamicsABSTRACT
Penicillin, travels through bodily fluids, targeting and acylatively inactivating enzymes responsible for cell-wall synthesis in gram-positive bacteria. Somehow, it avoids metabolic degradation remaining inactive en route. To resolve this ability to switch from a non-active, to a highly reactive form, we investigated the dynamic structure-activity relationship of penicillin by inelastic neutron spectroscopy, reaction kinetics, NMR and multi-scale theoretical modelling (QM/MM and post-HF ab initio). Results show that by a self-activating physiological pH-dependent two-step proton-mediated process, penicillin changes geometry to activate its irreversibly reactive acylation, facilitated by systemic intramolecular energy management and cooperative vibrations. This dynamic mechanism is confirmed by the first ever reported characterisation of an antibiotic by neutrons, achieved on the TOSCA instrument (ISIS facility, RAL, UK).
Subject(s)
Penicillins/chemistry , Quantum Theory , Acylation , Catalysis , Electrons , Hydrogen-Ion Concentration , Kinetics , Neutrons , Structure-Activity Relationship , ThermodynamicsABSTRACT
Recent studies using ab initio calculations have shown that Cα-centered radical formation by H-abstraction from the backbone of peptide residues has dramatic effects on peptide structure and have suggested that this reaction may contribute to the protein misfolding observed in Alzheimer's and Parkinson's diseases. To enable the effects of Cα-centered radicals to be studied in longer peptides and proteins over longer time intervals, force-field parameters for the Cα-centered Ala radical were developed for use with the OPLS force field by minimizing the sum of squares deviation between the quantum chemical and OPLS-AA energy hypersurfaces. These parameters were used to determine the effect of the Cα-centered Ala radical on the structure of a hepta-alanyl peptide in molecular dynamics (MD) simulations. A negligible sum-of-squares energy deviation was observed in the stretching parameters, and the newly developed OPLS-AA torsional parameters showed a good agreement with the LMP2/cc-pVTZ(-f) hypersurface. The parametrization also demonstrated that derived force-field bond length and bond angle parameters can deviate from the quantum chemical equilibrium values, and that the improper torsional parameters should be developed explicitly with respect to the coupled torsional parameters. The MD simulations showed planar conformations of the Cα-containing residue (Alr) are preferred and these conformations increase the formation of γ-, α-, and π-turn structures depending on the position in the turn occupied by the Alr residue. Higher-ordered structures are destabilized by Alr except when this residue occupies position "i + 1" of the 310-helix. These results offer new insight into the protein-misfolding mechanisms initiated by H-abstraction from the Cα of peptide and protein residues.