Metabolite discovery through global annotation of untargeted metabolomics data.

Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.

Personalized application of antimicrobial drugs in pediatric patients with augmented renal clearance: a review of literature.

The effect of renal functional status on drug metabolism is a crucial consideration for clinicians when determining the appropriate dosage of medications to administer. In critically ill patients, there is often a significant increase in renal function, which leads to enhanced drug metabolism and potentially inadequate drug exposure. This phenomenon, known as augmented renal clearance (ARC), is commonly observed in pediatric critical care settings. The findings of the current study underscore the significant impact of ARC on the pharmacokinetics and pharmacodynamics of antimicrobial drugs in critically ill pediatric patients. Moreover, the study reveals a negative correlation between increased creatinine clearance and blood concentrations of antimicrobial drugs. The article provides a comprehensive review of ARC screening in pediatric patients, including its definition, risk factors, and clinical outcomes. Furthermore, it summarizes the dosages and dosing regimens of commonly used antibacterial and antiviral drugs for pediatric patients with ARC, and recommendations are made for dose and infusion considerations and the role of therapeutic drug monitoring. CONCLUSION:  ARC impacts antimicrobial drugs in pediatric patients. WHAT IS KNOWN: • ARC is inextricably linked to the failure of antimicrobial therapy, recurrence of infection, and subtherapeutic concentrations of drugs. WHAT IS NEW: • This study provides an updated overview of the influence of ARC on medication use and clinical outcomes in pediatric patients. • In this context, there are several recommendations for using antibiotics in pediatric patients with ARC: 1) increase the dose administered; 2) prolonged or continuous infusion administration; 3) use of TDM; and 4) use alternative drugs that do not undergo renal elimination.

Prediction of cytochrome P450-mediated bioactivation using machine learning models and in vitro validation.

Cytochrome P450 (P450)-mediated bioactivation, which can lead to the hepatotoxicity through the formation of reactive metabolites (RMs), has been regarded as the major problem of drug failures. Herein, we purposed to establish machine learning models to predict the bioactivation of P450. On the basis of the literature-derived bioactivation dataset, models for Benzene ring, Nitrogen heterocycle and Sulfur heterocycle were developed with machine learning methods, i.e., Random Forest, Random Subspace, SVM and Naïve Bayes. The models were assessed by metrics like "Precision", "Recall", "F-Measure", "AUC" (Area Under the Curve), etc. Random Forest algorithms illustrated the best predictability, with nice AUC values of 0.949, 0.973 and 0.958 for the test sets of Benzene ring, Nitrogen heterocycle and Sulfur heterocycle models, respectively. 2D descriptors like topological indices, 2D autocorrelations and Burden eigenvalues, etc. contributed most to the models. Furthermore, the models were applied to predict the occurrence of bioactivation of an external verification set. Drugs like selpercatinib, glafenine, encorafenib, etc. were predicted to undergo bioactivation into toxic RMs. In vitro, IC50 shift experiment was performed to assess the potential of bioactivation to validate the prediction. Encorafenib and tirbanibulin were observed of bioactivation potential with shifts of 3-6 folds or so. Overall, this study provided a reliable and robust strategy to predict the P450-mediated bioactivation, which will be helpful to the assessment of adverse drug reactions (ADRs) in clinic and the design of new candidates with lower toxicities.

Isolation of Lactobacillus acidophilus strain and its anti-obesity effect in a diet induced obese murine model.

Intestinal microbiota is a potential determinant of obesity, with probiotic bile salt hydrolase (BSH) as one of the key mechanisms in the anti-obesity effects. In this study, we present a Lactobacillus acidophilus GOLDGUT-LA100 (LA100) with high BSH activity, good gastric acid and bile salt tolerance, and a potential anti-obesity effect. LA100's anti-obesity effects were evaluated in a high-fat diet-induced, obese mouse model. LA100 administration alleviates high-fat diet-induced pathophysiological symptoms, such as body weight gain, high serum glucose and cholesterol level, hepatic lipid accumulation, and adipose inflammation. These results demonstrate concrete anti-obesity benefit in animal models and show promising applications in future clinical studies.

A Bifidobacterium animalis subsp. lactis strain that can suppress Helicobacter pylori: isolation, in vitro and in vivo validation.

The administration of probiotics is an effective approach for treatment of Helicobacter pylori, which is associated with human gastrointestinal diseases and cancers. To explore more effective probiotics for H. pylori infection elimination, bacteria from infant feces were screened in this study. We successfully isolated the Bifidobacterium animalis subsp. lactis strains and evaluated its efficacy to inhibit H. pylori growth in vitro and in vivo. The results showed that a B. animalis strain (named BB18) sustained a high survival rate after incubation in gastric juice. The rapid urease test suggested that B. animalis BB18 reduced pathogen loads in H. pylori-infected Mongolian gerbils. Alleviation of H. pylori infection-induced gastric mucosa damage and decreased levels inflammatory cytokines were observed after the B. animalis BB18 administration. These findings demonstrated that B. animalis BB18 can inhibit H. pylori infection both in vitro and in vivo, suggesting its potential application for the prevention and eradication therapy of H. pylori infection.

Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells.

Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7(-/-)) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.

Glucose feeds the TCA cycle via circulating lactate.

Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of 13C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, 13C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours.

Two-Step Enzymolysis of Antarctic Krill for Simultaneous Preparation of Value-Added Oil and Enzymolysate.

Antarctic krill is a crucial marine resource containing plenty of high-valued nutrients. However, krill oil as a single product has been developed by the current solvent extraction with high cost. From the perspective of comprehensive utilization of Antarctic krill, this study proposed a novel two-step enzymolysis-assisted extraction in attempt to produce value-added oil and enzymolysate simultaneously. After two-step chitinase/protease hydrolysis, the lipid yield increased from 2.09% to 4.18%, reaching 112% of Soxhlet extraction. The method greatly improved the yields of main components while reducing the impurity content without further refining. After optimization, the oil contained 246.05 mg/g of phospholipid, 80.96 mg/g of free eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and 0.82 mg/g of astaxanthin. The by-product enzymolysate was abundant in water-soluble proteins (34.35 mg/g), oligopeptides (13.92 mg/g), amino acids (34.24 mg/g), and carbohydrates (5.79 mg/g), which was a good source of functional nutrients. In addition, both oil and enzymolysate showed high antioxidant capacity. This novel method could simultaneously provide oil and enzymolysate amounting for 58.61% of dried krill.

Glycemic control and neonatal outcomes in women with gestational diabetes mellitus treated using glyburide, metformin, or insulin: a pairwise and network meta-analysis.

AIMS: We aimed to assess the comparative efficiency and safety of the use of glyburide, metformin, and insulin in gestational diabetes mellitus (GDM). METHODS: We searched for randomized controlled trials that compared glyburide, metformin, and insulin in GDM. Data regarding glycemic control and neonatal safety were collected and analyzed in pairwise and network meta-analyses. RESULTS: A total of 4533 individuals from 23 trials were included. Compared with glyburide, metformin reduced 2-h postprandial blood glucose (2HPG) to a greater extent (standard mean difference (SMD) 0.18; 95% credible interval (CI) 0.01, 0.34). There were significantly lower prevalence of neonatal hypoglycemia (risk difference (RD) - 0.07; 95%CI - 0.11, - 0.02) and preeclampsia (RD - 0.03; 95%CI - 0.06, 0) in the metformin group than in the insulin group. The metformin group had significantly lower birth weight (SMD - 0.17; 95%CI - 0.25, - 0.08) and maternal weight gain (SMD - 0.61; 95%CI - 0.86,- 0.35) compared with the insulin group. Network meta-analysis suggested that metformin had the highest probability of successfully controlling glycemia and preventing neonatal complications. CONCLUSIONS: The present meta-analysis suggests that metformin may be as effective as insulin for glycemic control and is the most promising drug for the prevention of neonatal and maternal complications.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis.

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs.

Curcumin-Loaded Blood-Stable Polymeric Micelles for Enhancing Therapeutic Effect on Erythroleukemia.

Curcumin has high potential in suppressing many types of cancer and overcoming multidrug resistance in a multifaceted manner by targeting diverse molecular targets. However, the rather low systemic bioavailability resulted from its poor solubility in water and fast metabolism/excretion in vivo has hampered its applications in cancer therapy. To increase the aqueous solubility of curcumin while retaining the stability in blood circulation, here we report curcumin-loaded copolymer micelles with excellent in vitro and in vivo stability and antitumor efficacy. The two copolymers used for comparison were methoxy-poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) and N-(tert-butoxycarbonyl)-l-phenylalanine end-capped mPEG-PCL (mPEG-PCL-Phe(Boc)). In vitro cytotoxicity evaluation against human pancreatic SW1990 cell line showed that the delivery of curcumin in mPEG-PCL-Phe(Boc) micelles to cancer cells was efficient and dosage-dependent. The pharmacokinetics in ICR mice indicated that intravenous (i.v.) administration of curcumin/mPEG-PCL-Phe(Boc) micelles could retain curcumin in plasma much better than curcumin/mPEG-PCL micelles. Biodistribution results in Sprague-Dawley rats also showed higher uptake and slower elimination of curcumin into liver, lung, kidney, and brain, and lower uptake into heart and spleen of mPEG-PCL-Phe(Boc) micelles, as compared with mPEG-PCL micelles. Further in vivo efficacy evaluation in multidrug-resistant human erythroleukemia K562/ADR xenograft model revealed that i.v. administration of curcumin-loaded mPEG-PCL-Phe(Boc) micelles significantly delayed tumor growth, which was attributed to the improved stability of curcumin in the bloodstream and increased systemic bioavailability. The mPEG-PCL-Phe(Boc) micellar system is promising in overcoming the key challenge of curcumin's to promote its applications in cancer therapy.

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate.

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Thermal-Driven Orderly Assembly of Ir-atomic Chains on α-MnO2 with Enhanced Performance for Acidic Oxygen Evolution.

The development of acid-stable oxygen evolution reaction electrocatalysts is essential for high-performance acidic water electrolysis. Herein, we report the results of one-dimensional (1D) nanorods (NRs) IrCeMnO@Ir containing ~20 wt . % Iridium (Ir) as an efficient anode electrocatalyst, synthesized via a one-step cation exchange strategy. Owing to the presence of 1D channels of the nanorod architecture and the unique electronic structure, the IrCeMnO@Ir exhibited 69 folds more mass activity than that of commercial IrO2 as well as over 400 h stability with only a 20 mV increase in overpotential. DFT calculations and control experiments demonstrated that CeO2 serves as an electron buffer to accelerate the kinetics of the rate-determined step for the significantly enhanced activity and suppress the over-oxidation of Ir species as well as their dissolution for impressively promoted stability under practical conditions. Our work opens up a feasible strategy to boost OER activity and stability simultaneously.

The Burgeoning Significance of Liquid-Liquid Phase Separation in the Pathogenesis and Therapeutics of Cancers.

Liquid-liquid phase separation (LLPS) is a physiological phenomenon that parallels the mixing of oil and water, giving rise to compartments with diverse physical properties. Biomolecular condensates, arising from LLPS, serve as critical regulators of gene expression and control, with a particular significance in the context of malignant tumors. Recent investigations have unveiled the intimate connection between LLPS and cancer, a nexus that profoundly impacts various facets of cancer progression, including DNA repair, transcriptional regulation, oncogene expression, and the formation of critical membraneless organelles within the cancer microenvironment. This review provides a comprehensive account of the evolution of LLPS from the molecular to the pathological level. We explore the mechanisms by through which biomolecular condensates govern diverse cellular physiological processes, encompassing gene expression, transcriptional control, signal transduction, and responses to environmental stressors. Furthermore, we concentrate on potential therapeutic targets and the development of small-molecule inhibitors associated with LLPS in prevalent clinical malignancies. Understanding the role of LLPS and its interplay within the tumor milieu holds promise for enhancing cancer treatment strategies, particularly in overcoming drug resistance challenges. These insights offer innovative perspectives and support for advancing cancer therapy.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

Accurate Density Prediction of Sesquiterpenoid HEDFs and the Multiproperty Computing Server SesquiterPre.

Accurate and rapid evaluation of density is crucial for evaluating the packing and combustion characteristics of high-energy-density fuels (HEDFs). This parameter is pivotal in the selection of high-performance HEDFs. Our study leveraged a polycyclic compound density data set and quantum chemical (QC) descriptors to establish a correlation with the target properties using the XGBoost algorithm. We utilized a recursive feature elimination method to simplify the model and developed a concise and interpretable density prediction model incorporating only six QC descriptors. The model demonstrated robust performance, achieving coefficients of determination (R 2) of 0.967 and 0.971 for internal and external test sets, respectively, and root-mean-square errors (RMSE) of 0.031 and 0.027 g/cm3, respectively. Compared to the other two mainstream methods, the marginal discrepancy between the predicted and actual molecular densities underscores the model's superior predictive ability and more usefulness for energy density calculation. Furthermore, we developed a web server (SesquiterPre, https://sespre.cmdrg.com/#/) that can simultaneously calculate the density, enthalpy of combustion, and energy density of sesquiterpenoid HEDFs, which greatly facilitates the use of researchers and is of great significance for accelerating the design and screening of novel sesquiterpenoid HEDFs.

Expression and purification of active receptor interacting protein 1 kinase using a baculovirus system.

Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.

Supporting nanoscale zero-valent iron onto shrimp shell-derived N-doped biochar to boost its reactivity and electron utilization for selenite sequestration.

Nanoscale zero-valent iron (nZVI) has been widely used in the reductive removal of contaminants from water, yet it still fights against the inherent passive cover and the raise of medium pH. In this study, nZVI was supported onto a nitrogen-doped biochar (NBC) that was prepared by pyrolyzing shrimp shell for efficiently sequestrating aqueous selenite (Se(IV)). The resultant composite (NBC-nZVI) revealed a higher reactivity and electron utilization efficiency (EUE) than the bare nZVI in Se(IV) sequestration because of the positive charge, the buffering effect and the good conductivity of NBC. The kinetic rate and EUE of NBC-nZVI were increased by 143.4% and 15.3% compared to the bare nZVI, respectively, at initial pH of 3.0. The high removal capacity of 605.4 mg g-1 for NBC-nZVI was obtained at Se(IV) concentration of 1000 mg L-1, initial pH of 3.0, NBC-nZVI dosage of 1.0 g L-1 and contact time of 12 h. Moreover, NBC-nZVI exhibited a strong tolerance to solution pHs and coexisting compounds (e.g., humic acid) and could reduce the Se(IV) concentration from 5.0 mg L-1 to below the limit of drinking water (50 µg L-1) in real-world samples. This work exemplified a utilization of shrimp shell-derived NBC to simultaneously enhance the reactivity and EUE of nZVI for reductively removing contaminants.

Multilayered NiMo/CoMn/Ni Cathodic Electrodes with Enhanced Activity and Stability toward Alkaline Water Electrolysis.

In this study, multilayered NiMo/CoMn/Ni cathodic electrodes were prepared by the multilayered electrodeposition method. The multilayered structure includes a nickel screen substrate, CoMn nanoparticles at the bottom, and cauliflower-like NiMo nanoparticles at the top. The multilayered electrodes have a lower overpotential, preferable stability, and better electrocatalytic performance than monolayer electrodes. In a three-electrode system, the overpotentials of the multilayered NiMo/CoMn/Ni cathodic electrodes at 10 and 500 mA/cm2 are only 28.7 and 259.1 mV, respectively. The overpotential rise rate of the electrodes after constant current tests at 200 and 500 mA/cm2 was 4.42 and 8.74 mV/h, respectively, and the overpotential rise rate after 1000 cycles of cyclic voltammetry of the electrodes was 1.9 mV/h, while the overpotential rise rate after the three stability tests of the nickel screen was 5.49, 11.42, and 5.1 mV/h. According to the Tafel extrapolation polarization curve, the Ecorr and Icorr of the electrodes were -0.3267 V and 1.954 × 10-5 A/cm2, respectively. The charge transfer rate of the electrodes is slightly slower than that of the monolayer electrodes, indicating that its corrosion resistance is more excellent. An electrolytic cell was designed for the overall water-splitting test, and the current density of the electrodes was 121.6 mA/cm2 at 1.8 V. In addition, the stability of the electrodes is excellent after intermittent testing for 50 h, which can greatly reduce power consumption and is more suitable for industrial overall water-splitting tests. In addition, the three-dimensional model was used to simulate the three-electrode system and alkaline water electrolytic cell system, and the simulation results are consistent with the experimental results. The hydrogen adsorption free energy (ΔGH) of the electrodes was -1.0191 eV, which was evaluated by density functional theory (DFT). The ΔGH is closer to zero than that of the monolayer electrodes, indicating that the surface has stronger adsorption of hydrogen atoms.