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Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.
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Antibacterianos , Inhibidores Enzimáticos , Escherichia coli , Exorribonucleasas , Antibacterianos/farmacología , Antibacterianos/química , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Dominio Catalítico , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Bacterias/enzimologíaRESUMEN
Background: HIV self-testing (HIVST) is a practical and effective way to provide HIV testing services to at-risk and underserved populations, particularly men. Utilizing Village Health Teams (VHTs) could enhance community-based delivery of oral HIVST to reach the last un-tested individuals who may be at-risk of infection. However, little is known about what VHTs and facility-based healthcare workers think about facilitating oral HIVST and delivery of subsequent HIV services. We investigated the views of health providers on oral HIVST delivered by VHTs among men in rural communities in Central Uganda. Methods: We conducted a qualitative study in Mpigi district, interviewing 27 health providers who facilitated oral HIV self-testing among men. The providers consisting of 15 VHTs and 12 facility-based health workers were purposively selected. All interviews were audio-recorded, transcribed verbatim, and translated to English for a hybrid inductive-deductive thematic analysis. We used the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) Implementation Science framework to generate and categorize open codes. Results: In terms of reaching men with HIV testing services, the providers considered HIVST to be a fast and convenient method, which could boost HIV testing. However, they also had concerns about its accuracy. In terms of effectiveness, HIVST was perceived as a reliable, user-friendly, and efficient approach to HIV testing. However, it depended on the user's preference for testing algorithms. Regarding adoption, HIVST was considered to enhance autonomy, well-suited for use in the community, and offered opportunities for linkage and re-linkage into care. However, at times HIVST faced hesitance. As for Implementation, VHTs had various support roles in HIVST but had concerns about social insecurities and delays in seeking subsequent facility-based services after HIVST. Regarding Maintenance, providers recommended several ways to improve oral HIVST including; optimizing tracking of HIVST distribution and use, improving linkage and retention in care after HIVST, diversifying HIVST for combined HIV prevention packages and including more languages, broadening sensitization among potential HIVST users and health providers, differentiating distribution models, and prioritizing targeted HIVST efforts. Conclusion: HIVST has the potential to increase testing rates and engagement of men in HIV services. However, for it to be implemented on a population-wide scale, continuous sensitization of potential users and health providers is necessary, along with streamlined structures for tracking kit distribution, use, and reporting of results. Further implementation research may be necessary to optimize the role of health providers in facilitating HIVST.
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Developing drug resistance in the malaria parasite is a reason for apprehension compelling the scientific community to focus on identifying new molecular targets that can be exploited for developing new anti-malarial compounds. Despite the availability of the Plasmodium genome, many protein-coding genes in Plasmodium are still not characterized or very less information is available about their functions. DMAP1 protein is known to be essential for growth and plays an important role in maintaining genomic integrity and transcriptional repression in vertebrate organisms. In this study, we have identified a homolog of DMAP1 in P. falciparum. Our sequence and structural analysis showed that although PfDMAP1 possesses a conserved SANT domain, parasite protein displays significant structural dissimilarities from human homolog at full-length protein level as well as within its SANT domain. PPIN analysis of PfDMAP1 revealed it to be vital for parasite and virtual High-throughput screening of various pharmacophore libraries using BIOVIA platform-identified compounds that pass ADMET profiling and showed specific binding with PfDMAP1. Based on MD simulations and protein-ligand interaction studies two best hits were identified that could be novel potent inhibitors of PfDMAP1 protein.Communicated by Ramaswamy H. Sarma.
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Acinetobacter baumannii is an ESKAPE pathogen that causes endocarditis, pneumonia, blood infections, urinary tract infections, and several other illnesses. In addition, it is mainly responsible for nosocomial infection-related mortality. Gram-negative A. baumannii bacterium (AYE Strain) has high MDR and XDR levels. Due to its function in synthesizing purines and amino acids, folic acid is a significant molecule necessary for the growth of bacteria. The metabolic pathway of folate production is therefore a potential therapeutic target to inhibit bacterial growth. In the current study, the three-dimensional model of 6-Hydroxy-methyl dihydropterinpyrophosphokinase (HPPK) was predicted and subsequently processed through a virtual high throughput screening (vHTS) against compounds from Enamine HTSC library, that could bind to its active site. Three lead candidates (Z73322064, Z354558542, and Z906123504) and a control molecule (7,8 dihydro-7,7-dimethyl-6-hydroxymethlypterin; Accession Number: DB02278) were identified using several screening criteria namely estimated binding affinity, estimated inhibition constant, drug-like properties, ADME properties, mode of binding, and interaction patterns of the screened compounds. The physiological behavior of ligand binding on the HPPK enzyme was then studied using molecular dynamics simulations of apo and ligand bound complexes. This study proposed the following three molecules: Z73322064, Z354338542, and Z906123504 as promising lead candidates against the substrate-binding site of the HPPK enzyme from A. baumannii using global, essential dynamics studies along with MM/PBSA based binding free energy analysis.Communicated by Ramaswamy H. Sarma.
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Human epidermal growth factor receptors (EGFR), namely ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3, and ErbB4/HER4, the trans-membrane family of tyrosine kinase receptors, are overexpressed in many types of cancers. These receptors play an important role in cell proliferation, differentiation, invasion, metastasis and angiogenesis including unregulated activation of cancer cells. Overexpression of ErbB1 and ErbB2 that occurs in several types of cancers is associated with poor prognosis leading to resistance to ErbB1-directed therapies. In this connection, promising strategy to overcome the disadvantages of the existing chemotherapeutic drugs is the use of short peptides as anticancer agents. In the present study, we have performed virtual high throughput screening of natural peptides against ErbB1 and ErbB2 to identify potential dual inhibitors and identified five inhibitors based on their binding affinities, ADMET analysis, MD simulation studies and calculation of free energy of binding. These natural peptides could be further exploited for developing drugs for treating cancer.Communicated by Ramaswamy H. Sarma.
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Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds (τb = 0.70) better than the predicted affinities for the static AlphaFold-predicted structure (τb = 0.42). Taken together, these results suggest that targeting the cryptic pocket is a good strategy for drugging PPM1D and, more generally, that conformations selected from simulation can improve virtual screening when limited structural data is available.
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PPARγ full agonists, thiazolidinediones (TZDs), have been known as a class of most effective drugs for the treatment of type 2 diabetes mellitus (T2DM). However, recently their therapeutic benefits have been compromised by several undesirable side effects. In this study, a host-based repurposing strategy and in combination with comprehensive biological evaluations were synergistically employed to seek for potent PPARγ ligands, which led to the identification of an anti-thrombotic drug, dicoumarol (Dic), as the novel and safer selectively PPARγ modulator (SPPARγM) with advantages over current TZD drugs. The results in vitro showed that Dic had a potent binding affinity and weakly agonistic activity for PPARγ and its downstream key genes. Moreover, in diabetic model, it significantly reduced blood glucose without leading to the weight gain of both body and main organ tissues. Further mechanistic investigations revealed that Dic possessed such desired pharmacological properties mainly through effectively inhibiting the phosphorylation of PPARγ-Ser273 and selectively regulating the expressions of insulin-sensitive and resistance genes. Finally, the docking studies on the analysis of the potent binding mode of Dic with PPARγ revealed a remarkable difference on interaction region compared with other developed PPARγ agonists, which not only gave a proof of concept for the abovementioned mechanism but also provided the molecular basis for the discrimination of Dic from other PPARγ ligands, especially TZD drugs. Taken together, our findings suggested that Dic could serve as a new and promising candidate with good therapeutic index for treating T2DM, especially for those T2DM patients with thrombosis.
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Anticoagulantes , Diabetes Mellitus Tipo 2 , Dicumarol , Hipoglucemiantes , PPAR gamma , Trombosis , Humanos , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dicumarol/química , Dicumarol/farmacología , Dicumarol/uso terapéutico , Hipoglucemiantes/química , Ligandos , PPAR gamma/agonistas , Tiazolidinedionas/efectos adversos , Tiazolidinedionas/farmacología , Trombosis/tratamiento farmacológico , Trombosis/etiología , Anticoagulantes/química , Anticoagulantes/farmacologíaRESUMEN
Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043-DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA.
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PPARγ is well-known as the target receptor of TZD anti-diabetic drugs. However, recently the therapeutic benefits of these TZD drugs have been compromised by many severe side effects because of their full PPARγ agonistic action to lock the AF-2 helix. Herein, we conducted a virtual screening in the combination with structure-based design, synthesis and biological evaluation both in vitro and in vivo, leading to the identification of a potent candidate YG-C-20 as the SPPARγM with improved and safer anti-diabetic therapeutics. Mechanistically, this compound presented such desired pharmacological profiles (e.g., preferable anti-diabetic efficiencies and minimized side effects) mainly via selectively inhibiting the CDK5-mediated phosphorylation of PPARγ-Ser273 and up-regulating the expression of insulin-sensitive genes Adiponectin and Glut4, yet lacking the classical full agonism to induce the adipogenesis and the expression of key adipogenic genes including PPARγ, aP2, CD36, LPL, C/EBPα and FASN. Further validation led to the final recognition of its (R)-configured isomer as the potential conformational form. Subsequent molecular docking studies revealed a unique hydrogen-bonding network of (R)-YG-C-20 with three full PPARγ agonism-unrelated residues, especially with PPARγ-Ser273 phosphorylation-associated site Ser342, which not only gives a clear verification for our structure-based design but also provides a proof of concept for the abovementioned molecular mechanism.
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PPAR gamma , PPAR gamma/metabolismo , Simulación del Acoplamiento MolecularRESUMEN
Vibrio cholerae causes the diarrheal disease cholera which affects millions of people globally. The outer membrane protein U (OmpU) is the outer membrane protein that is most prevalent in V. cholerae and has already been recognized as a critical component of pathogenicity involved in host cell contact and as being necessary for the survival of pathogenic V. cholerae in the host body. Computational approaches were used in this study to screen a total of 37,709 natural compounds from the traditional Chinese medicine (TCM) database against the active site of OmpU. Following a sequential screening of the TCM database, we report three lead compounds-ZINC06494587, ZINC85510056, and ZINC95910434-that bind strongly to OmpU, with binding affinity values of -8.92, -8.12, and -8.78 kcal/mol, which were higher than the control ligand (-7.0 kcal/mol). To optimize the interaction, several 100 ns molecular dynamics simulations were performed, and the resulting complexes were shown to be stable in their vicinity. Additionally, these compounds were predicted to have good drug-like properties based on physicochemical properties and ADMET assessments. This study suggests that further research be conducted on these compounds to determine their potential use as cholera disease treatment.
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Antibacterianos/química , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/química , Productos Biológicos/química , Productos Biológicos/farmacología , Vibrio cholerae/efectos de los fármacos , Sitios de Unión , Humanos , Enlace de Hidrógeno , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fitoquímicos/química , Fitoquímicos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
In this study, SB-VHTS of the old drug library was conducted to seek for novel PPARγ ligand. In the end, an antifungal drug, FN, was identified in vitro and in vivo as a new and potent PPARγ-modulating ligand to demonstrate significantly anti-diabetic and anti-NAFLD efficacies with minimized side effects induced by PPARγ full agonists TZDs drugs. Further mechanistic investigations revealed that FN showed such desired pharmacological properties mainly through selectively activating the expressions of Adiponectin and GLUT4, effectively promoting the Akt Ser473 phosphorylation, inhibiting the expressions of proinflammatory genes including TNF-α, IL-1ß and IL-6 and blocking the PPARγ Ser273 phosphorylation mediated by CDK5 without leading to adipogenesis and increasing the expressions of key adipogenic genes CD36, AP2, LPL, C/EBPα, FASN and PPARγ. Subsequently, a molecular docking study revealed an interesting binding mode between FN and PPARγ LBD including the hydrogen-bonding network among oxygen atom, sulfur atom and nitrogen atom in FN respectively with the PPARγ residues Cys285, Tyr327 and Ser342, which gave proof of concept for the above anti-diabetic action mechanism. Taken together, our findings not only suggest that FN can serve as the new, safe and highly efficacious anti-diabetic and anti-NAFLD agents for clinical use, they can also provide a molecular basis for the future development of PPARγ modulators with a high therapeutic index and the possibility to explore new uses of old drugs for immediate drug discovery.
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Antifúngicos/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Imidazoles/uso terapéutico , PPAR gamma/metabolismo , Células 3T3-L1 , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Antifúngicos/química , Antifúngicos/farmacología , Glucemia/efectos de los fármacos , Células COS , Chlorocebus aethiops , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/metabolismo , Expresión Génica/efectos de los fármacos , Hipoglucemiantes/farmacología , Imidazoles/química , Imidazoles/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Triglicéridos/metabolismoRESUMEN
The inhibition of cyclooxygenase-1 (COX-1) enzyme by Nonsteroidal anti-inflammatory drugs (NSAIDs) exposes the gastrointestinal mucosa to peptic injuries. Selective inhibition of COX-2 generates surpassing anti-inflammatory drug candidates with reduced side effects over current NSAIDs. Phytosterols consumption is reported to decrease the risk of cardiovascular problems. Reports on the selective inhibition of COX-2 by phytosterols are scarce. The present study assesses the anti-inflammatory potentials of phytosterols from Nicotiana tabacum (of the family Solanaceae) through selective inhibition of COX-1 and/or COX-2. Virtual High Throughput Screening (vHTS) and Molecular Docking of phytochemicals from Nicotiana tabacum against the catalytic pockets of COX-1 and COX-2 were used to identify the lead bioactive(s) components of the plant. The hit phytosterols were isolated, histopathological examination of the stomach, in-vivo COX-1/COX-2 mRNAs expression patterns in the liver through reverse transcription-polymerase chain reactions, and enzymes activities of Nicotiana tabacum phytosterol isolates (NTPI) in HCl/ethanol-induced inflammation in Wistar rats were all investigated. Formation of hydrogen bonds favour selective inhibition of COX-2 while hydrophobic interactions favour selective inhibition of COX-1. NTPI demonstrates inhibition of COX-2 by down-regulate the expression of COX-2 mRNA and were ineffective against the expression COX-1 mRNA. NTPI demonstrates hepatoprotective abilities by improving the antioxidant defense system of the liver. Histopathological analyses show NTPI at 50 mg/kg bodyweight regenerates the parietal cells and maintain the gastrointestinal architecture. Drug likeness prediction and ADME toxicity screening show that phytosterols possess good oral bioavailability with no side effects. Phytosterols are selective inhibitors of COX-2, they are hepatoprotective, regenerate parietal cells, and non-toxic.
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Antiinflamatorios/uso terapéutico , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Ensayos Analíticos de Alto Rendimiento/métodos , Simulación del Acoplamiento Molecular/métodos , Nicotiana/química , Fitosteroles/uso terapéutico , Animales , Antiinflamatorios/farmacología , Simulación por Computador , Humanos , Masculino , Ratones , Fitosteroles/farmacología , Ratas WistarRESUMEN
Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.
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Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E. Engagement of the Mnk pathway is critical in acute myeloid leukemia (AML) leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo, suggesting that targeting Mnk kinases may provide a novel approach for treating AML. Here, we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors. The Mnk1 structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk1 crystal structure while carrying out docking simulations of known inhibitors. The hit compounds display activity in Mnk biochemical and cellular assays, including acute myeloid leukemia progenitors. This approach will enable further rational structure-based drug design of new Mnk inhibitors and potentially novel ways of therapeutically targeting this kinase.
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Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.