Idebenone Antagonizes P53-Mediated Neuronal Oxidative Stress Injury by Regulating CD38-SIRT3 Protein Level.

Idebenone, an antioxidant used in treating oxidative damage-related diseases, has unclear neuroprotective mechanisms. Oxidative stress affects cell and mitochondrial membranes, altering Adp-ribosyl cyclase (CD38) and Silent message regulator 3 (SIRT3) protein expression and possibly impacting SIRT3's ability to deacetylate Tumor protein p53 (P53). This study explores the relationship between CD38, SIRT3, and P53 in H2O2-injured HT22 cells treated with Idebenone. Apoptosis was detected using flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining after determining appropriate H2O2 and Idebenone concentrations.In this study, Idebenone was found to reduce apoptosis and decrease P53 and Caspase3 expression in H2O2-injured HT22 cells by detecting apoptosis-related protein expression. Through bioinformatics methods, CD38 was identified as the target of Idebenone, and it further demonstrated that Idebenone decreased the expression of CD38 and increased the level of SIRT3. An increased NAD+/NADH ratio was detected, suggesting Idebenone induces SIRT3 expression and protects HT22 cells by decreasing apoptosis-related proteins. Knocking down SIRT3 downregulated acetylated P53 (P53Ac), indicating SIRT3's importance in P53 deacetylation.These results supported that CD38 was used as a target of Idebenone to up-regulate SIRT3 to deacetylate activated P53, thereby protecting HT22 cells from oxidative stress injury. Thus, Idebenone is a drug that may show great potential in protecting against reactive oxygen species (ROS) induced diseases such as Parkinson's disease, and Alzheimer's disease. And it might be able to compensate for some of the defects associated with CD38-related diseases.

CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression.

Non-coding RNAs (ncRNAs) induced competing endogenous RNAs (ceRNA) play crucial roles in various biological process by regulating target gene expression. However, the studies of ceRNA networks in the regulation of ovarian ovulation processing of chicken remains deficient compared to that in mammals. Our present study revealed that circEML1 was differential expressed in hen's ovarian tissues at different ages (15 W/20 W/30 W/68 W) and identified as a loop structure from EML1 pre-mRNA, which promoted the expressions of CYP19A1/StAR and E2/P4 secretion in follicular granulosa cells (GCs). Furthermore, circEML1 could serve as a sponge of gga-miR-449a and also found that IGF2BP3 was targeted by gga-miR-449a to co-participate in the steroidogenesis, which possibly act the regulatory role via mTOR/p38MAPK pathways. Meanwhile, in the rescue experiment, gga-miR-449a could reverse the promoting role of circEML1 to IGF2BP3 and steroidogenesis. Eventually, this study suggested that circEML1/gga-miR-449a/IGF2BP3 axis exerted an important role in the steroidogenesis in GCs of chicken.

Bifunctional Fluorescent Probes for the Detection of Mustard Gas and Phosgene.

Mustard gas [sulfur mustard (SM)] and phosgene are the most frequently used chemical warfare agents (CWAs), which pose a serious threat to human health and national security, and their rapid and accurate detection is essential to respond to terrorist attacks and industrial accidents. Herein, we developed a fluorescent probe with o-hydroxythioketone as two sensing sites, AQso, which can detect and distinguish mustard gas and phosgene. The dual-sensing-site probe AQso reacts with mustard gas to form a cyclic product with high sensitivity [limit of detection (LOD) = 70 nM] and is highly selective to SM over phosgene, SM analogues, active alkylhalides, acylhalides, and nerve agent mimics, in ethanol solutions. When encountering phosgene, AQso rapidly converts to cyclic carbonate, which is sensitive (LOD = 14 nM) and highly selective. Their sensing mechanisms of AQso to mustard gas and phosgene were well demonstrated by separation and characterization of the sensing products. Furthermore, a facile test strip with the probe was prepared to distinguish 2-chloroethyl ethyl sulfide (CEES) and phosgene in the gas phase by different fluorescence colors and response rates. Not using the complicated instrument, the qualitative and quantitative detection of CEES or phosgene can be achieved only by measuring the red-green-blue (RGB) channel intensity of the test strip after being exposed to CEES or phosgene gas by the smartphone with an RGB color application.

Nutrients strengthen density dependence of per-capita growth and mortality rates in the soil bacterial community.

Density dependence in an ecological community has been observed in many macro-organismal ecosystems and is hypothesized to maintain biodiversity but is poorly understood in microbial ecosystems. Here, we analyze data from an experiment using quantitative stable isotope probing (qSIP) to estimate per-capita growth and mortality rates of bacterial populations in soils from several ecosystems along an elevation gradient which were subject to nutrient addition of either carbon alone (glucose; C) or carbon with nitrogen (glucose + ammonium-sulfate; C + N). Across all ecosystems, we found that higher population densities, quantified by the abundance of genomes per gram of soil, had lower per-capita growth rates in C + N-amended soils. Similarly, bacterial mortality rates in C + N-amended soils increased at a significantly higher rate with increasing population size than mortality rates in control and C-amended soils. In contrast to the hypothesis that density dependence would promote or maintain diversity, we observed significantly lower bacterial diversity in soils with stronger negative density-dependent growth. Here, density dependence was significantly but weakly responsive to nutrients and was not associated with higher bacterial diversity.

Intravenous immunoglobulin for interstitial lung diseases of anti-melanoma differentiation-associated gene 5-positive dermatomyositis.

OBJECTIVE: Rapidly progressive interstitial lung disease (RP-ILD) in DM patients positive for anti-melanoma differentiation-associated gene 5 (anti-MDA5) autoantibody (MDA5-DM) often have a poor prognosis, frequently fatal. As there is a scarcity of data regarding the effect of intravenous immunoglobulin (IVIG) on RP-ILD in MDA5-DM patients (MDA5-RPILD), we conducted this study to determine the efficacy of a IVIG add-on initial treatment. METHODS: Patients with newly-onset MDA5-RPILD from September 2018 to June 2020 were retrospectively reviewed for 6 months in the First Affiliated Hospital of Zhengzhou University. They were divided into two groups: IVIG and non-IVIG groups. The major measurement of treatment outcome was the difference in the mortality in 3-month and 6-month between two group patients. Other relevant indicators were also recorded, including the incidence of infection, the dosages of GCs, the remission rate and the variables in laboratory data. RESULTS: The IVIG group (n = 31) showed significantly lower 6-month mortality rate than the non-IVIG group (n = 17) (22.6% vs 52.9%; P =0.033). The IVIG group patients had a higher remission rate at 3 months (71.0% vs 41.2%; P =0.044). Gradual reduction was observed in the first 3 months with regard to the titre of anti-MDA5 autoantibody, the serum level of ferritin and the ground glass opacification GGO scores. CONCLUSION: IVIG adjunct therapy is a very effective first-line treatment for patients with MDA5-RPILD. IVIG may increase the survival and remission rate by lowering ferritin concentration, anti-MDA5 titre and GGO score.

SPAG1 promotes the development of AML by activating the ERK/MAPK signaling pathway and affects the chemotherapy sensitivity of venetoclax.

Sperm-associated antigen 1 (SPAG1) is considered to be associated with infertility and tumorigenesis. However, its function in acute myeloid leukemia (AML) remains unclear. In this study, we evaluated the expression level of SPAG1 and explored its clinical prognostic value in patients with AML, as well as its biological function in AML cells. SPAG1 is widely expressed in AML patients, resulting in a poor prognosis. However, its expression was not associated with Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Utilizing the RNA interference knockdown tests, we found that SPAG1 could promote the proliferation and survival of AML cells and regulate the expression of structural maintenance of chromosomes protein 3 (SMC3), activating the ERK/MAPK signaling pathway. Furthermore, we discovered that inhibiting SPAG1 impacted AML cell susceptibility to venetoclax. In conclusion, SPAG1 may serve as a potential therapeutic target in AML.

Nonsequential double ionization of alkaline-earth metal atoms by intense mid-infrared femtosecond pulses.

A systematic study of nonsequential double ionization (NSDI) of alkaline-earth metal atoms with mid-infrared femtosecond pulses is reported. We find that the measured NSDI yield shows a strong target dependence and it is more suppressed for alkaline-earth metal with higher ionization potential. The observation is attributed to the differences in the recollision induced excitation and ionization cross sections of alkaline-earth metals. This work indicates that NSDI of alkaline-earth metals can be generally understood within recollision picture and sheds light on ultrafast control of electron correlation and dynamics of ionic excited states during NSDI of atoms with complex structures.

From pools to flow: The PROMISE framework for new insights on soil carbon cycling in a changing world.

Soils represent the largest terrestrial reservoir of organic carbon, and the balance between soil organic carbon (SOC) formation and loss will drive powerful carbon-climate feedbacks over the coming century. To date, efforts to predict SOC dynamics have rested on pool-based models, which assume classes of SOC with internally homogenous physicochemical properties. However, emerging evidence suggests that soil carbon turnover is not dominantly controlled by the chemistry of carbon inputs, but rather by restrictions on microbial access to organic matter in the spatially heterogeneous soil environment. The dynamic processes that control the physicochemical protection of carbon translate poorly to pool-based SOC models; as a result, we are challenged to mechanistically predict how environmental change will impact movement of carbon between soils and the atmosphere. Here, we propose a novel conceptual framework to explore controls on belowground carbon cycling: Probabilistic Representation of Organic Matter Interactions within the Soil Environment (PROMISE). In contrast to traditional model frameworks, PROMISE does not attempt to define carbon pools united by common thermodynamic or functional attributes. Rather, the PROMISE concept considers how SOC cycling rates are governed by the stochastic processes that influence the proximity between microbial decomposers and organic matter, with emphasis on their physical location in the soil matrix. We illustrate the applications of this framework with a new biogeochemical simulation model that traces the fate of individual carbon atoms as they interact with their environment, undergoing biochemical transformations and moving through the soil pore space. We also discuss how the PROMISE framework reshapes dialogue around issues related to SOC management in a changing world. We intend the PROMISE framework to spur the development of new hypotheses, analytical tools, and model structures across disciplines that will illuminate mechanistic controls on the flow of carbon between plant, soil, and atmospheric pools.

Estrogen Abolishes the Repression Role of gga-miR-221-5p Targeting ELOVL6 and SQLE to Promote Lipid Synthesis in Chicken Liver.

Few studies have been conducted regarding the biological function and regulation role of gga-miR-221-5p in the liver. We compared the conservation of miR-221-5p among species and investigated the expression pattern of gga-miR-221-5p, validating the direct target genes of gga-miR-221-5p by dual luciferase reporter assay, the biological function of gga-miR-221-5p in the liver was studied by gga-miR-221-5p overexpression and inhibition. Furthermore, we explored the regulation of gga-miR-221-5p and its target genes by treatment with estrogen and estrogen antagonists in vivo and in vitro. The results showed that miR-221-5p was highly conserved among species, expressed in all tested tissues and significantly downregulated in peak-laying hen liver compared to pre-laying hen liver. Gga-miR-221-5p could directly target the expression of elongase of very long chain fatty acids 6 (ELOVL6) and squalene epoxidase (SQLE) genes to affect triglyceride and total cholesterol content in the liver. 17ß-estradiol could significantly inhibit the expression of gga-miR-221-5p but promote the expression of ELOVL6 and SQLE genes. In conclusion, the highly conservative gga-miR-221-5p could directly target ELOVL6 and SQLE mRNAs to affect the level of intracellular triglyceride and total cholesterol. Meanwhile, 17ß-estradiol could repress the expression of gga-miR-221-5p but increase the expression of ELOVL6 and SQLE, therefore promoting the synthesis of intracellular triglyceride and cholesterol levels in the liver of egg-laying chicken.

A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii.

Proteinase inhibitors with the ability to inhibit specific proteinases are usually closely connected with the immune system. Interestingly, proteinase inhibitors are also a common ingredient in the organic matrix of mollusk shells. However, the molecular mechanism that underlies the role of proteinase inhibitors in immune system and shell mineralization is poorly known. In this study, a Kunitz serine proteinase inhibitor (HcKuPI) was isolated from the mussel Hyriopsis cumingii. HcKuPI was specifically expressed in dorsal epithelial cells of the mantle pallium and HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer. These results indicated that HcKuPI plays a vital role in shell nacreous layer biomineralization. Moreover, the expression pattern of HcKuPI during LPS challenge and pearl formation indicated its involvement in the antimicrobial process during pearl sac formation and nacre tablets accumulation during pearl formation. In the in vitro calcium carbonate crystallization assay, the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate. Taken together, these results indicate that HcKuPI is involved in antimicrobial process during pearl formation, and participates in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation. These findings extend our knowledge of the role of proteinase inhibitors in immune system and shell biomineralization.

The solvent-controlled chemoselective construction of C-S/S-S bonds via the Michael reaction/thiol coupling of quinoline-2-thiones.

K2CO3-catalyzed thio-Michael addition using quinoline-2-thiones and α,ß-unsaturated carbonyl compounds was used to assess the chemoselective construction of C-S and S-S bonds under mild reaction conditions in different solvents. The C-S bond showed a better chemoselective construction in EtOH whereas the S-S bond showed a better chemoselective construction in 1,4-dioxane. The corresponding products, generated from the reaction, presented a significant solvent-controlling effect.

Identification of nacre matrix protein genes hic14 and hic19 and their roles in crystal growth and pearl formation in the mussel Hyriopsis cumingii.

Biological mineralization is a highly programmed process in which inorganic minerals reassociate under the strict control of the extracellular matrix to form minerals with special functions and patterns. Shells are biominerals, and their synthesis is finely regulated by organic matrix including matrix proteins, polysaccharides, lipids, pigments, free amino acids, and small peptides. In this study, two matrix protein genes, hic14 and hic19, were isolated from the mantle of the mussel Hyriopsis cumingii. Tissue expression analysis showed that both proteins were expressed mainly in the mantle, and in situ hybridization of mantle tissues showed that they were specifically expressed in the dorsal epithelial cells of mantle pallial. Therefore, hic14 and hic19 were both nacreous layer matrix proteins. In the pearl insertion experiment, hic14 and hic19 kept low expression during pearl sac formation and disordered calcium carbonate deposition, and increased significantly during pearl nacre accumulation, which showed that both proteins participated in the mineralization of pearl nacre. In the RNA interference experiment, shell nacre tablet growth was inhibited after crystal nucleation due to the decreased expression of hic14, and crystal morphology and arrangement of nacre were highly modified after expression of hic19 was inhibited. These results provided further evidence that both hic14 and hic19 participated in nacreous layer biomineralization.

Expression profile analysis of microRNAs during hair follicle development in the sheep foetus.

MicroRNAs (miRNAs) regulate the development and growth cycle of hair follicles (HFs). The molecular mechanism by which miRNAs determine the development of HFs in the sheep foetus remains elusive. In this study, the expression profiles of miRNAs at 11 development periods (45, 55, 65, 75, 85, 95, 105, 115, 125, 135 and 145 d) in sheep foetus skin were analysed by high-throughput sequencing and bioinformatics analysis. A total of 72 conserved miRNAs, 44 novel miRNAs and 32 known miRNAs were significantly differentially expressed. qRT-PCR results for 18 miRNAs were consistent with the sequencing data. 85 d of foetal development was the starting point for secondary hair follicle (SF) development according to tissue morphology and cluster analysis. In SF development, the prolactin signalling pathway and platelet activation played important roles, and 10 miRNAs were potential candidate miRNAs in SF initiation.

Adjusting synchronizability of coupled oscillatory power networks via feedback control schemes.

In this paper, the reliable synchronization of oscillatory power networks with different topologies is investigated by presenting two simple control strategies, namely, phase feedback control and frequency feedback control. The power networks are modeled by the coupled second-order Kuramoto oscillators that represent both consumers and generators. Through the simulations on the power networks with control, it is found that both the proposed control strategies can effectively enhance the synchronizability of the power networks, except for the case when the frequency feedback strategy is adopted for a coupled power network with the WS structure. In this case, it is observed that the critical coupling strength reaches the smallest value when the feedback control strength is equal to one and a sudden drop of order parameter occurs as the control strength further increases. This work provides innovative ideas for constructing a cost-effective power system.

Acoustic radiation forces on three-layered drug particles in focused Gaussian beams.

Drug delivery by acoustic waves is a crucial technology for targeted therapy. Recently, a three-layered drug micro-particle was proposed and fabricated, the second shell of which greatly improves both the encapsulation of the drug and the flexibility in its release rate. In this work, the acoustic radiation force (ARF) of an acoustic focused Gaussian beam on a three-layered particle comprising an inner drug core (D), a middle layer of poly(lactide-co-glycolide) (PLGA), and an outer chitosan shell (CS) is investigated. A three-layered elastic shell (TES) mimics the D-PLGA-CS structure, and the acoustic scattering from and ARF of the D-PLGA-CS are studied using Mie theory. This paper focuses on how the geometry and acoustic parameters of the outer shell influence the ARF, finding that the Poisson's ratio of the outer shell affects the ARF more than does the density or Young's modulus. In addition, this paper finds that the choice of the inner drug has little effect on the ARF acting on the D-PLGA-CS particle. The present work may benefit the acoustic manipulation of both TESs and three-layered drugs.

miR-34a-5p Increases Hepatic Triglycerides and Total Cholesterol Levels by Regulating ACSL1 Protein Expression in Laying Hens.

Accumulating evidence has shown that miR-34a serves as a posttranscriptional regulatory molecule of lipid metabolism in mammals. However, little studies about miR-34a on lipid metabolism in poultry have been reported until now. To gain insight into the biological functions and action mechanisms of miR-34a on hepatic lipid metabolism in poultry, we firstly investigated the expression pattern of miR-34a-5p, a member of miR-34a family, in liver of chicken, and determined its function in hepatocyte lipid metabolism by miR-34a-5p overexpression and inhibition, respectively. We then validated the interaction between miR-34a-5p and its target using dual-luciferase reporter assay, and explored the action mechanism of miR-34a-5p on its target by qPCR and Western blotting. Additionally, we looked into the function of the target gene on hepatocyte lipid metabolism by gain- and loss-of-function experiments. Our results indicated that miR-34a-5p showed a significantly higher expression level in livers in peak-laying hens than that in pre-laying hens. miR-34a-5p could increase the intracellular levels of triglycerides and total cholesterol in hepatocyte. Furthermore, miR-34a-5p functioned by inhibiting the translation of its target gene, long-chain acyl-CoA synthetase 1 (ACSL1), which negatively regulates hepatocyte lipid content. In conclusion, miR-34a-5p could increase intracellular lipid content by reducing the protein level, without influencing mRNA stability of the ACSL1 gene in chickens.

Genome-Wide Analysis of the FABP Gene Family in Liver of Chicken (Gallus gallus): Identification,Dynamic Expression Profile, and RegulatoryMechanism.

The fatty acid-binding protein (FABP) gene family, which encodes a group of fatty acid-trafficking molecules that affect cellular functions, has been studied extensively in mammals. However, little is known about the gene structure, expression profile, and regulatory mechanism of the gene family in chickens. In the present study, bioinformatics-based methods were used to identify the family members and investigate their evolutionary history and features of gene structure. Real-time PCR combined with in vivo and in vitro experiments were used to examine the spatiotemporal expression pattern, and explore the regulatory mechanism of FABP genes. The results show that nine members of the FABP gene family, which branched into two clusters and shared a conserved FATTYACIDBP domain, exist in the genome of chickens. Of these, seven FABP genes, including FABP1, FABP3-7, and FABP10 were abundantly expressed in the liver of hens. The expression levels of FABP1, FABP3, and FABP10 were significantly increased, FABP5 and FABP7 were significantly decreased, and FABP4 and FABP6 remained unchanged in hens at the peak laying stage in comparison to those at the pre-laying stage. Transcription of FABP1 and FABP3 were activated by estrogen via estrogen receptor (ER) α, whilst FABP10 was activated by estrogen via ERß. Meanwhile, the expression of FABP1 was regulated by peroxisome proliferator activated receptor (PPAR) isoforms, of which tested PPARα and PPARß agonists significantly inhibited the expression of FABP1, while tested PPARγ agonists significantly increased the expression of FABP1, but downregulated it when the concentration of the PPARγ agonist reached 100 nM. The expression of FABP3 was upregulated via tested PPARß and PPARγ agonists, and the expression of FABP7 was selectively promoted via PPARγ. The expression of FABP10 was activated by all of the three tested PPAR agonists, but the expression of FABP4-6 was not affected by any of the PPAR agonists. In conclusion, members of the FABP gene family in chickens shared similar functional domains, gene structures, and evolutionary histories with mammalian species, but exhibited varying expression profiles and regulatory mechanisms. The results provide a valuable resource for better understanding the biological functions of individual FABP genes in chickens.

Integrated Analysis of MiRNA and Genes Associated with Meat Quality Reveals that Gga-MiR-140-5p Affects Intramuscular Fat Deposition in Chickens.

BACKGROUND/AIMS: Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects the tenderness, juiciness, and favor of chicken. An increasing number of studies are focusing on the functions of microRNAs (miRNAs) during the adipogenic process. However, little is known about miRNAs associated with poultry IMF deposition, especially intramuscular adipocyte differentiation. METHODS: The IMF content of two physiological stages was measured, and miRNA-Seq and RNA-Seq data were integrated and analyzed. A chicken intramuscular adipocyte cell differentiation model was constructed. A luciferase reporter assay, miRNA overexpression, and Oil Red O staining were used to confirm the targets of gga-miR-140-5p. RESULTS: Our results showed that late-laying-period hens, which had a higher IMF content, exhibited lower global expression levels of miRNAs than juvenile hens. A total of 104 differentially expressed (DE) miRNAs were identified between the two groups. Integrated analysis of differentially expressed genes and DE miRNAs identified a total of 378 miRNA-mRNA pairs. Functional enrichment analysis revealed that these intersecting genes are involved in ubiquitin-mediated proteolysis, the peroxisome proliferator-activated receptor signaling pathway, glycerophospholipid metabolism, and fatty acid elongation and degradation pathways. Furthermore, we demonstrated that gga-miR-140-5p promoted intramuscular adipocyte differentiation via targeting retinoid X receptor gamma. CONCLUSION: Our findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

MicroRNA expression profile and functional analysis reveal their roles in contact inhibition and its disruption switch of rat vascular smooth muscle cells.

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.