E3 ubiquitin ligase RNF148 functions as an oncogene in colorectal cancer by ubiquitination-mediated degradation of CHAC2.

We previously reported that RNF148 was involved in the ubiquitination-mediated degradation of CHAC2. However, its molecular mechanism was not determined. In this study, we investigated the role and mechanism of RNF148 in the progression of colorectal cancer (CRC), especially in the process of ubiquitination-mediated degradation of CHAC2. Our results revealed that RNF148 was upregulated in most CRC tissues, and its expression significantly correlated with the 3-year overall survival rate and most clinicopathological parameters of CRC patients. Furthermore, RNF148 served as an independent prognostic biomarker of CRC and promoted CRC cell proliferation and migration while inhibiting cell apoptosis and sensitivity to 5-FU. Mechanistically, RNF148 used its protease-associated domain to bind to the CHAC domain of CHAC2 and target it for degradation. In addition, we identified two phosphorylation and three ubiquitination residues of CHAC2 and identified Y118 and K102 as the critical phosphorylation and ubiquitination residues, respectively. We also identified CHAC2's and RNF148's interacting proteins and discovered their potential interaction network. In conclusion, our current study unveiled the role of RNF148 in CRC and the mechanism of RNF148 in the ubiquitination-mediated degradation of CHAC2, which shed light on providing potential prognostic biomarkers and molecular targets for CRC patients.

Functions and Mechanisms of Brassinosteroids in Regulating Crop Agronomic Traits.

Brassinosteroids (BRs) perform crucial functions controlling plant growth and developmental processes, encompassing many agronomic traits in crops. Studies of BR-related genes involved in agronomic traits have suggested that BRs could serve as a potential target for crop breeding. Given the pleiotropic effect of BRs, a systematic understanding of their functions and molecular mechanisms is conducive for application in crop improvement. Here, we summarize the functions and underlying mechanisms by which BRs regulate the several major crop agronomic traits, including plant architecture, grain size, as well as the specific trait of symbiotic nitrogen fixation in legume crops. For plant architecture, we discuss the roles of BRs in plant height, branching number, and leaf erectness and propose how progress in these fields may contribute to designing crops with optimal agronomic traits and improved grain yield by accurately modifying BR levels and signaling pathways.

Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II.

IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.

Molecular generation strategy and optimization based on A2C reinforcement learning in de novo drug design.

MOTIVATION: In the field of pharmacochemistry, it is a time-consuming and expensive process for the new drug development. The existing drug design methods face a significant challenge in terms of generation efficiency and quality. RESULTS: In this paper, we proposed a novel molecular generation strategy and optimization based on A2C reinforcement learning. In molecular generation strategy, we adopted transformer-DNN to retain the scaffolds advantages, while accounting for the generated molecules' similarity and internal diversity by dynamic parameter adjustment, further improving the overall quality of molecule generation. In molecular optimization, we introduced heterogeneous parallel supercomputing for large-scale molecular docking based on message passing interface communication technology to rapidly obtain bioactive information, thereby enhancing the efficiency of drug design. Experiments show that our model can generate high-quality molecules with multi-objective properties at a high generation efficiency, with effectiveness and novelty close to 100%. Moreover, we used our method to assist shandong university school of pharmacy to find several candidate drugs molecules of anti-PEDV. AVAILABILITY AND IMPLEMENTATION: The datasets involved in this method and the source code are freely available to academic users at https://github.com/wq-sunshine/MomdTDSRL.git.

Forkhead box E1, frequently downregulted by promoter methylation, inhibits colorectal cancer cell growth and migration.

Forkhead box E1 (FOXE1), also known as thyroid transcription factor 2 (TTF-2), belongs to a large family of forkhead transcription factors. It plays important roles in embryogenesis, cell growth, and differentiation. Cancer-specific FOXE1 hypermethylation events have been identified in several cancers. However, the expression and function of FOXE1 in the tumorigenesis of colorectal cancer remain still unknown. In this study, we examined FOXE1 expression and methylation in normal colon mucosa, colorectal cancer (CRC) cell lines, and primary tumors by immunohistochemistry, semi-quantitative RT-PCR, methylation-specific PCR, and bisulfite genomic sequencing. We found that FOXE1 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXE1 expression was significantly correlated with lymph node metastasis and advanced TNM stages, indicating its potential as a tumor marker. Subsequently, we established colon cancer cell lines with stable FOXE1 expression to observe the biological effect on colorectal cancer, including cell growth, migration, actin cytoskeleton, and growth of human colorectal xenografts in nude mice. Ectopic expression of FOXE1 could suppress tumor cell growth and migration and affect the organization of the actin cytoskeleton together with suppressing tumorigenicity in vivo. FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression, and FOXE1 plays a suppressive role in the development and progression of colorectal cancer.

Large-Scale Molecular Dynamics Simulation Based on Heterogeneous Many-Core Architecture.

As the application of molecular dynamics (MD) simulations continues to evolve, the demand for accelerating large-scale simulation systems and handling of enormous simulation tasks is steadily increasing. We propose a parallel acceleration method for large-scale MD simulations based on Sunway heterogeneous many-core processors. This method integrates task scheduling, simulation calculations, and data storage, effectively tackling issues related to large-scale simulations and numerous simulation tasks. The task scheduling strategy flexibly handles tasks on various scales and enables parallel execution of multiple tasks. During the simulation calculations, we ported GROMACS to the Sunway architecture and accelerated the calculation of short-range forces through a heterogeneous processor. Our method achieves approximately 10-fold acceleration and 90% scalability when executing a single simulation task. When handling numerous simulation tasks, our method achieves parallel execution of all of the tasks with 90% scalability. By employing our method, we carried out 50 ns simulations on over 3000 distinct conotoxin structures individually within just 5 h. Additionally, we evaluated more than 200 protein-ligand complexes, and the simulation efficiency significantly exceeded that of midsized to small GPU clusters.

Interaction between plant-based dietary pattern and frailty on cognitive decline: a longitudinal analysis of the Chinese Longitudinal Healthy Longevity Survey cohort.

OBJECTIVES: Frailty is a risk factor for faster cognitive decline, while plant-based dietary patterns are associated with decreased risk of cognitive decline. We aimed to explore their interaction with cognitive function among older adults. METHODS: We used data from the Chinese Longitudinal Healthy Longevity Survey between 2008 and 2018. Frailty was evaluated based on the frailty index (FI), and the plant-based diet index (PDI) was calculated using food frequency questionnaire at baseline. Repeated measures of the Mini-Mental State Examination (MMSE) were utilised to assess cognitive function. We used linear mixed models to estimate regression coefficients (ß) and 95% confidence intervals (CI). RESULTS: We included 7,166 participants with a median follow-up of 5.8 years. Participants in pre-frail (ß = -0.18, 95% CI: -0.24, -0.13) and frail (ß = -0.39, 95% CI: -0.48, -0.30) groups experienced an accelerated decline in MMSE score compared with the robust group. The PDI modified the above association, with corresponding associations with frailty being much more pronounced among participants with a lower PDI (frail vs. robust ß = -0.44, 95% CI: -0.56, -0.32), compared with those with a higher PDI (frail vs. robust ß = -0.27, 95% CI: -0.40, -0.13). In addition, A combination of frailty and a low PDI was strongly associated with a faster decline in MMSE score (ß = -0.52, 95% CI: -0.63, -0.41). CONCLUSION: Adherence to plant-based dietary patterns attenuates the association between frailty and cognitive decline. If the observed association is causal, promoting plant-based dietary patterns may be a strategy to reduce the effects of frailty on neurological health.

Strigolactone signaling regulates cambial activity through repression of WOX4 by transcription factor BES1.

During secondary growth, meristematic cells in the cambium can either proliferate to maintain the stem cell population or differentiate into xylem or phloem. The balance between these two developmental trajectories is tightly regulated by many environmental and endogenous cues. Strigolactones (SLs), a class of plant hormones, were previously reported to regulate secondary growth by promoting cambium activity. However, the underlying molecular mechanisms of SL action in plant secondary growth are not well understood. We performed histological, genetic, and biochemical analyses using genetic materials in Arabidopsis (Arabidopsis thaliana) with altered activity of the transcription factors BRI1-EMS-SUPPRESSOR1 (BES1) or WUSCHEL-related HOMEOBOX4 (WOX4) or lacking MORE AXILLARY SHOOT2 (MAX2), a key positive component in the SL signaling pathway. We found that BES1, a downstream regulator in the SL signaling pathway that promotes shoot branching and xylem differentiation, also inhibits WOX4 expression, a key regulator of cambium cell division in the intercellular TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)-TDIF RECEPTOR (TDR) signaling pathway. The antagonistic roles of BES1 and WOX4 in the regulation of cambium activity may integrate intercellular TDIF signals to efficiently and bidirectionally modulate cambium cell proliferation and differentiation. As both BES1 and WOX4 are widely involved in various endogenous signals and responses to environmental stimuli, these findings may provide insight into the dynamic regulation of cambium development.

Momordica charantia polysaccharide ameliorates D-galactose-induced aging through the Nrf2/ß-Catenin signaling pathway.

Aging is widely thought to be associated with oxidative stress. Momordica charantia (MC) is a classic vegetable and traditional herbal medicine widely consumed in Asia, and M. charantia polysaccharide (MCP) is the main bioactive ingredient of MC. We previously reported an antioxidative and neuroprotective effect of MCP in models of cerebral ischemia/reperfusion and hemorrhage injury. However, the role played by MCP in neurodegenerative diseases, especially during aging, remains unknown. In this study, we investigated the protective effect of MCP against oxidative stress and brain damage in a D-galactose-induced aging model (DGAM). The Morris water maze test was performed to evaluate the spatial memory function of model rats. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were measured and telomerase activity was determined. The results showed that MCP treatment attenuated spatial memory dysfunction induced by D-galactose. In addition, MCP increased antioxidant capacity by decreasing MDA and increasing SOD and GSH levels. MCP treatment also improved telomerase activity in aging rats. Mechanistically, MCP promoted the entry of both Nrf2 and ß-Catenin into the nucleus, which is the hallmark of antioxidation signaling pathway activation. This study highlights a role played by MCP in ameliorating aging-induced oxidative stress injury and reversing the decline in learning and memory capacity. Our work provides evidence that MCP administration might be a potential antiaging strategy.

FOXD3, frequently methylated in colorectal cancer, acts as a tumor suppressor and induces tumor cell apoptosis under ER stress via p53.

Forkhead box D3 (FOXD3), an important member of the forkhead box transcription factor family, has many biological functions. However, the role and signaling pathways of FOXD3 in colorectal cancer (CRC) are still unclear. We examined FOXD3 expression and methylation in normal colon mucosa, CRC cell lines and primary tumors by reverse transcription-polymerase chain reaction, methylation-specific PCR and bisulfite genomic sequencing. We also evaluated its tumor-suppressive function by examining its modulation of apoptosis under endoplasmic reticulum (ER) stress in CRC cells. The FOXD3 target signal pathway was identified by western blotting, immunofluorescence and chromatin immunoprecipitation. We found that FOXD3 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXD3 protein expression was significantly correlated with poor histopathological grading, lymph node metastasis and poor prognosis of patients, indicating its potential as a tumor marker that may be of potential value as a therapeutic target for CRC. Moreover, restoration of FOXD3 expression inhibited the proliferation and migration of tumor cells. FOXD3 also increased mitochondrial apoptosis through the unfolded protein response under ER stress. Furthermore, we found that FOXD3 could bind directly to the promoter of p53 and enhance its expression. Knockdown of p53 impaired the effect of apoptosis induced by FOXD3. In conclusion, we showed for the first time that FOXD3, which is frequently methylated in CRC, acted as a tumor suppressor inducing tumor cell apoptosis under ER stress via p53.

eIF4EBP3 was downregulated by methylation and acted as a tumor suppressor by targeting eIF4E/ß-catenin in gastric cancer.

BACKGROUND: Epigenetic aberrations of tumor suppressor genes (TSGs), particularly DNA methylation, are frequently involved in the pathogenesis of gastric cancer (GC). Through a methylome study, we identified eIF4EBP3 as a methylated gene in GC. However, the role of eIF4EBP3 in GC progression has not been explored. METHODS: The expression and promoter region methylation of eIF4EBP3 in GC and healthy tissues were analyzed in public datasets. eIF4EBP3 expression in GC was detected by semi-quantitative RT-PCR, western blot and immunohistochemistry. We also studied epigenetic alterations and functions in GC. The effects of eIF4EBP3 on cell proliferation, migration and invasion were conducted by functional experiments in vitro and in vivo. Label-free proteomic analysis was applied to identify targets of eIF4EBP3. RESULTS: The expression level of eIF4EBP3 was downregulated in gastric cancer due to promoter region methylation, and was associated with poor survival and tumor progression. Ectopic expression of eIF4EBP3 significantly inhibited tumor cell growth, migration and invasion both in vitro and in vivo. Label-free proteomic analysis indicated eIF4EBP3 downregulated the protein level of ß-catenin, which was confirmed by western blot. Overexpression of ß-catenin reversed the inhibitory effects of eIF4EBP3 on cell growth and migration, indicating that eIF4EBP3 acts on GC cells by targeting the eIF4E/ß-catenin axis. CONCLUSION: These results suggest that eIF4EBP3 is a novel TSG methylated in gastric cancer that may play important roles in GC development and liver metastasis and indicate eIF4EBP3 as a potential metastasis and survival biomarker for GC.

Hepatitis A Outbreaks in Developed Countries: Detection, Control, and Prevention.

The incidence of hepatitis A virus (HAV) infection has been low in developed countries for decades; however, many adults in these countries are susceptible to HAV infection. In recent years, the global trade of food products originating from HAV-endemic countries resulted in HAV outbreaks associated with imported foods in developed countries. This article aims to review the characteristics of selected HAV outbreaks associated with imported food in developed countries during 2012-2018, and discusses improvements in global public health capabilities and new tools for effective detection, control, and prevention of HAV outbreaks.

The mechanisms of DIRAS family members in role of tumor suppressor.

DIRAS family is a group of GTPases belonging to the RAS superfamily and shares homology with the pro-oncogenic Ras GTPases. Currently, accumulating evidence show that DIRAS family members could be identified as putative tumor suppressors in various cancers. The either lost or reduced expression of DIRAS proteins play an important role in cancer development, including cell growth, migration, apoptosis, autophagic cell death, and tumor dormancy. This review focuses on the latest research regarding the roles and mechanisms of the DIRAS family members in regulating Ras function, cancer development, assessing potential challenges, and providing insights into the possibility of targeting them for therapeutic use.

Evidence for calpains in cancer metastasis.

Metastatic dissemination represents the final stage of tumor progression as well as the principal cause of cancer-associated deaths. Calpains are a conserved family of calcium-dependent cysteine proteinases with ubiquitous or tissue-specific expression. Accumulating evidence indicates a central role for calpains in tumor migration and invasion via participating in several key processes, including focal adhesion dynamics, cytoskeletal remodeling, epithelial-to-mesenchymal transition, and apoptosis. Activated after the increased intracellular calcium concentration ( [Ca2+]i ) induced by membrane channels and extracellular or intracellular stimuli, calpains induce the limited cleavage or functional modulation of various substrates that serve as metastatic mediators. This review covers established literature to summarize the mechanisms and underlying signaling pathways of calpains in cancer metastasis, making calpains attractive targets for aggressive tumor therapies.

Serum chitinase activity prognosticates metastasis of colorectal cancer.

BACKGROUND: This study aimed to evaluate the value of chitinase activity in prognosticating the occurrence of metastasis in and prognosis of patients with colorectal cancer (CRC). METHODS: The chitinase activity in four different groups, namely 335 CRC patients without distant metastasis at their first visit (Group 1), 51 patients with CRC having synchronous liver metastasis (Group 2), 100 healthy age-matched controls (Group 3) and 40 patients with liver cancer (Group 4), were assayed using an enzyme-linked immunosorbent assay. The Cox proportional hazards ratio model and Kaplan-Meier curve were used to identify the association between chitinase activity and the clinical outcome of CRC patients without metastasis in the training set and testing set at their first visit. An in vitro Transwell experiment was performed to evaluate the migration of colon cancer cells. RESULTS: Patients with high chitinase activity had a significantly higher metastasis risk than those with low chitinase activity in the training and testing sets during follow-up, both at stage I/II and stage III. Further, multivariate analysis revealed that chitinase activity was an independent risk factor prognosticating liver metastases (P = 0.001). The combination of chitinase activity and lymph node metastasis status increased the accuracy of the prognosis of liver metastases after radical resection (P = 0.454E-011). In addition, chitinase promoted CRC cell migration in vitro. CONCLUSIONS: Chitinase activity can prognosticate the occurrence of metastasis in patients with CRC. Moreover, the combination of chitinase activity and N stage increased the power of prognosticating the occurrence of metastasis. Inhibiting chitinase activity may serve as a new strategy to treat metastases of CRC.

N6-methyladenosine modifications: interactions with novel RNA-binding proteins and roles in signal transduction.

RNA epigenetics has received a great deal of attention in recent years, and the reversible N6-methyladenosine (m6A) modification on messenger RNAs (mRNAs) has emerged as a widespread phenomenon. The vital roles of m6A in diverse biological processes are dependent on many RNA-binding proteins (RBPs) with 'reader' or 'nonreader' functions. Moreover, m6A effector proteins affect cellular processes, such as stem cell differentiation, tumor development and the immune response by controlling signal transduction. This review provides an overview of the interactions of m6A with various RBPs, including the 'reader' proteins (excluding the YT521-B homology (YTH) domain proteins and the heterogeneous nuclear ribonucleoproteins (hnRNPs)), and the functional 'nonreader' proteins, and this review focuses on their specific RNA-binding domains and their associations with other m6A effectors. Furthermore, we summarize key m6A-marked targets in distinct signaling pathways, leading to a better understanding of the cellular m6A machinery.

RNA helicase DDX5 participates in oxLDL-induced macrophage scavenger receptor 1 expression by suppressing mRNA degradation.

The DEAD box protein DDX5, an ATP-dependent RNA helicase, plays an important role in transcriptional regulation and is associated with solid tumors and leukemia. However, its role in oxLDL-induced lipid uptake in macrophages remains unclear. In this study, we detected the expression of DDX5 mRNA and protein in oxidized low-density lipoprotein (oxLDL)-treated human primary macrophages that were induced from monocytes isolated from human peripheral blood with or without several chemical inhibitors using quantitative real-time PCR (qRT-PCR) or Western blotting. We found that oxLDL induced DDX5 expression to be independent of both the MAPK and NF-κB pathways. We also found that DDX5 promoted macrophage lipid uptake by evaluating the fluorescence intensity of engulfed dil-oxLDL. Various scavenger receptors that participate in lipid uptake were detected in siR-DDX5 transfected macrophages using qRT-PCR and Western blotting. Macrophage scavenger receptor A (MSR1) was found to be involved the upregulation of DDX5-mediated lipid uptake. Through the use of a dual luciferase reporter assay system and incubation with cycloheximide (CHX) MG132 and actidione (ActD), we found that DDX5 promoted MSR1 protein expression by stabilizing MSR1 mRNA. Moreover, the mechanism involved in DDX5 regulation of MSR1 mRNA was also explored using mass spectrum analysis; Immunoprecipitations (IPs) and RNA- Immunoprecipitations (R-IPs) revealed that mettl3 was involved in DDX5-mediated MSR1 mRNA stabilization. In addition, we also demonstrated that DDX5 inhibited mettl3 to catalyze m6a methylation in MSR1 mRNA, which contributed to the maintenance of MSR1 mRNA stability. In conclusion, ox-LDL promotes DDX5 expression in macrophages, which interacts with mettl3 to stabilize MSR1 mRNA by decreasing the m6a modification of MSR1 mRNA, ultimately promoting lipid uptake in macrophages.

The phenotype of vascular smooth muscle cells co-cultured with endothelial cells is modulated by PDGFR-ß/IQGAP1 signaling in LPS-induced intravascular injury.

Background Sepsis, a leading cause of death in intensive care units, is generally associated with vascular dysfunction. However, its pathophysiological process has not been fully clarified, lacking in-depth knowledge of its pathophysiological process may hinder the improvement of diagnosis and therapy for sepsis. Hence, as the key parts of the vascular wall, the interaction between endothelial cells (ECs) and smooth muscle cells (SMCs) under septic situation need to be further studied. Methods ECs and SMCs were co-cultured using Transwell plates. Lipopolysaccharide (LPS) was used to induce sepsis. A scratch-wound assay was used to assess cell migration, and western blotting was used to assess the level of redifferentiation of SMCs as well as the expression of PDGFR-ß and IQGAP1. Results Co-culture with ECs reduced the redifferentiation of SMCs induced by LPS (10 µg/ml), which was characterized by increased migration ability and decreased expression of contractile proteins (e.g., SM22 and α-SMA). The production of TNF-α could decrease the level of PDGFR-ß in SMCs. Treatment of SMCs with the PDGFR-ß inhibitor imatinib (5 µM) was able to counteract LPS-induced SMC redifferentiation and reduce IQGAP1 protein expression, especially when SMCs were co-cultured with ECs. Conclusion The phenotype of vascular SMCs co-cultured with ECs was modulated by IQGAP1 through the PDGFR-ß pathway, which may lead to vascular remodeling and homeostasis in LPS-induced intravascular injury. This pathway could be a novel target for the treatment of vascular damage.

Structure-Property Relationships in Graphene-Based Strain and Pressure Sensors for Potential Artificial Intelligence Applications.

Wearable electronic sensing devices are deemed to be a crucial technology of smart personal electronics. Strain and pressure sensors, one of the most popular research directions in recent years, are the key components of smart and flexible electronics. Graphene, as an advanced nanomaterial, exerts pre-eminent characteristics including high electrical conductivity, excellent mechanical properties, and flexibility. The above advantages of graphene provide great potential for applications in mechatronics, robotics, automation, human-machine interaction, etc.: graphene with diverse structures and leverages, strain and pressure sensors with new functionalities. Herein, the recent progress in graphene-based strain and pressure sensors is presented. The sensing materials are classified into four structures including 0D fullerene, 1D fiber, 2D film, and 3D porous structures. Different structures of graphene-based strain and pressure sensors provide various properties and multifunctions in crucial parameters such as sensitivity, linearity, and hysteresis. The recent and potential applications for graphene-based sensors are also discussed, especially in the field of human motion detection. Finally, the perspectives of graphene-based strain and pressure sensors used in human motion detection combined with artificial intelligence are surveyed. Challenges such as the biocompatibility, integration, and additivity of the sensors are discussed as well.

Single-Step In Situ Acetylcholinesterase-Mediated Alginate Hydrogelation for Enzyme Encapsulation in CE.

A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with "egg-box" structure is triggered inside a capillary with releasing of Ca2+ by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis.