TATA-Box Binding Protein O-GlcNAcylation at T114 Regulates Formation of the B-TFIID Complex and Is Critical for Metabolic Gene Regulation.

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Here, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9-edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

Structural characterization of N-acyl-homoserine lactones from bacterial quorum sensing using LC-MS/MS analyses after Paternò-Büchi derivatization in solution.

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs.

Research on the Sensing Characteristics of an Integrated Grid-like Sensor Based on a Triboelectric Nanogenerator.

With the development of the integration and miniaturization of sensing devices, the concept of self-sensing devices has been proposed. A motion state is self-sensed via the structure or integration of an actuator in the construction of a sensing unit. This device is then used to capture the perception and measurement of states such as position, displacement, and speed. A triboelectric nanogenerator converts mechanical energy into electrical energy through the coupling effect of contact generation and electrostatic induction, which represents one of the reliable ways through which to realize integrated sensing. In this world, the power generation technology of the TENG is applied to a sensing device. The sensing characteristics of a grid-like TENG are designed and analyzed in freestanding triboelectric mode. Firstly, a relation model of displacement, velocity, voltage, and charge is established. The charge-transfer increment and current amounts are linearly related to the velocity. The open-circuit voltage has a positive relationship with the displacement. The maximum open-circuit voltage and the maximum charge transfer are fixed values, and they are only related to the inherent parameters of a triboelectric nanogenerator. Next, the sensor model is constructed using COMSOL Multiphysics 6.0. The simulation results show that the relationships between output voltage and charge transfer, as well as those between the increments of charge transfer, velocity, and displacement, are consistent with the results derived from the formula. Finally, a performance test of the designed sensor is carried out, and the results are consistent with the theoretical deduction and simulation. After analysis and processing of the output electrical signal by the host computer, it can feedback the frequency and speed value of the measured object. In addition, the output signal is stable, and there is no large fluctuation or attenuation during the 521-s vibration test. Because the working unit of the sensor is thin filmed, it is small in size, easy to integrate, and has no external power supply; moreover, it can be integrated into a device to realize the self-sensing of a motion state.

High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry.

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.

CAFS: An Attention-Based Co-Segmentation Semi-Supervised Method for Nasopharyngeal Carcinoma Segmentation.

Accurate segmentation of nasopharyngeal carcinoma is essential to its treatment effect. However, there are several challenges in existing deep learning-based segmentation methods. First, the acquisition of labeled data are challenging. Second, the nasopharyngeal carcinoma is similar to the surrounding tissues. Third, the shape of nasopharyngeal carcinoma is complex. These challenges make the segmentation of nasopharyngeal carcinoma difficult. This paper proposes a novel semi-supervised method named CAFS for automatic segmentation of nasopharyngeal carcinoma. CAFS addresses the above challenges through three mechanisms: the teacher-student cooperative segmentation mechanism, the attention mechanism, and the feedback mechanism. CAFS can use only a small amount of labeled nasopharyngeal carcinoma data to segment the cancer region accurately. The average DSC value of CAFS is 0.8723 on the nasopharyngeal carcinoma segmentation task. Moreover, CAFS has outperformed the state-of-the-art nasopharyngeal carcinoma segmentation methods in the comparison experiment. Among the compared state-of-the-art methods, CAFS achieved the highest values of DSC, Jaccard, and precision. In particular, the DSC value of CAFS is 7.42% higher than the highest DSC value in the state-of-the-art methods.

Lightweight Compound Scaling Network for Nasopharyngeal Carcinoma Segmentation from MR Images.

Nasopharyngeal carcinoma (NPC) is a category of tumours with a high incidence in head-and-neck. To treat nasopharyngeal cancer, doctors invariably need to perform focal segmentation. However, manual segmentation is time consuming and laborious for doctors and the existing automatic segmentation methods require large computing resources, which makes some small and medium-sized hospitals unaffordable. To enable small and medium-sized hospitals with limited computational resources to run the model smoothly and improve the accuracy of structure, we propose a new LW-UNet network. The network utilises lightweight modules to form the Compound Scaling Encoder and combines the benefits of UNet to make the model both lightweight and accurate. Our model achieves a high accuracy with a Dice coefficient value of 0.813 with 3.55 M parameters and 7.51 G of FLOPs within 0.1 s (testing time in GPU), which is the best result compared with four other state-of-the-art models.

DCNet: Densely Connected Deep Convolutional Encoder-Decoder Network for Nasopharyngeal Carcinoma Segmentation.

Nasopharyngeal Carcinoma segmentation in magnetic resonance imagery (MRI) is vital to radiotherapy. Exact dose delivery hinges on an accurate delineation of the gross tumor volume (GTV). However, the large-scale variation in tumor volume is intractable, and the performance of current models is mostly unsatisfactory with indistinguishable and blurred boundaries of segmentation results of tiny tumor volume. To address the problem, we propose a densely connected deep convolutional network consisting of an encoder network and a corresponding decoder network, which extracts high-level semantic features from different levels and uses low-level spatial features concurrently to obtain fine-grained segmented masks. Skip-connection architecture is involved and modified to propagate spatial information to the decoder network. Preliminary experiments are conducted on 30 patients. Experimental results show our model outperforms all baseline models, with improvements of 4.17%. An ablation study is performed, and the effectiveness of the novel loss function is validated.

Native Hydrophobic Interaction Chromatography Hyphenated to Mass Spectrometry for Characterization of Monoclonal Antibody Minor Variants.

Conventionally, hydrophobic interaction chromatography (HIC) uses mobile phases with high salt concentration that are not compatible with mass spectrometry (MS). Here we describe development of an HIC method coupled with MS detection (HIC-MS) utilizing an aqueous mobile phase with a low concentration of a volatile salt for characterizing recombinant monoclonal antibody (mAb) post-translational modifications (PTMs). The ability of HIC to separate the oxidation and free thiol variants of the mAbs enables their isolation and rapid characterization of these attributes under native conditions, an important step toward understanding the role they play.

Quantitative Determination of Protein-Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry.

High throughput protein-ligand interaction screening assays employing mass spectrometric detection are widely used in early stage drug discovery. Mass spectrometry-based screening approaches employ a target protein added to a pool of small-molecule compounds, and binding is assessed by measuring ligands denatured from the complexes. Direct analysis of protein-ligand interactions using native mass spectrometry has been demonstrated but is not widely used due to the detection limit on protein size, the requirement of volatile buffers, and the necessity for specialized instrumentation to preserve weak interactions under native conditions. Here we present a robust, quantitative, and automated online size-exclusion chromatography-native mass spectrometry (SEC-nMS) platform for measuring affinities of noncovalent protein-small-molecule interactions on an Orbitrap mass spectrometer. Indoleamine 2,3-dioxygenase 1, a catabolic enzyme, and inhibitory ligands were employed as a demonstration of the method. Efficient separation and elution enabled preservation of protein-ligand complexes and increased throughput. The high sensitivity and intra charge state resolution at high m/ z offered by the Exactive Plus EMR Orbitrap allowed for protein ligand affinity quantitation and resolved individual compounds close in mass. Vc50 values determined via collision-induced dissociation experiments enabled the evaluation of complex stability in the gas phase and were found to be independent of the extent of complex formation. For the first time, Vc50 determinations were achieved on an inline SEC-nMS platform. Systematic comparison of our method with optimized chip-based nanoelectrospray infusion served as a reference for ligand screening and affinity quantitation and further revealed the advantages of SEC-MS.

Interface modeling of magnetorheological elastomers subjected to variable working strain.

A magnetorheological elastomer (MRE) is a type of particle-matrix composite material, whose properties depend on the strain to which it is subjected in different applications. This paper proposes an interface model in which the magnetorheological characteristics of an MRE are described in terms of the effect of variable strain on the strength of interfacial bonding between the particles and the matrix. The model can describe the whole process of interface change from a strong interface to a strong-weak mixed interface and then to a weak interface under variable strain. The results indicate that the combined effects of the magnetic flux density, particle content, and strain amplitude are responsible for the magnetorheological performance of the MRE. The maximum value of the shear modulus under large strain is decreased by 0.75 × 105 Pa compared to the value under small strain. This model opens new opportunities for the development of high-performance MREs and MRE-based devices under variable strain conditions.

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

AANL (Agrocybe aegerita lectin 2) is a new facile tool to probe for O-GlcNAcylation.

O-linked N-acetylglucosamine (O-GlcNAcylation) is an important post-translational modification on serine or threonine of proteins, mainly observed in nucleus or cytoplasm. O-GlcNAcylation regulates many cell processes, including transcription, cell cycle, neural development and nascent polypeptide chains stabilization. However, the facile identification of O-GlcNAc is a major bottleneck in O-GlcNAcylation research. Herein, we report that a lectin, Agrocybe aegerita GlcNAc-specific lectin (AANL), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and surface plasmon resonance (SPR) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 µM, which is consistent with the result tested through isothiocyanate (ITC) assay reported before (Jiang S, Chen Y, Wang M, Yin Y, Pan Y, Gu B, Yu G, Li Y, Wong BH, Liang Y, et al. 2012. A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. Biochem J. 443:369-378.). Confocal imaging shows that AANL co-localizes extensively with NUP62, a heavily O-GlcNAcylated and abundant nuclear pore glycoprotein. Furthermore, O-GlcNAc-modified peptides could be effectively enriched in the late flow-through peak from simple samples by using affinity columns Sepharose 4B-AANL or POROS-AANL. Therefore, using AANL affinity column, we identified 28 high-confidence O-linked HexNAc-modified peptides mapped on 17 proteins involving diverse cellular progresses, including transcription, hydrolysis progress, urea cycle, alcohol metabolism and cell cycle. And most importantly, major proteins and sites were not annotated in the dbOGAP database. These results suggest that the AANL lectin is a new useful tool for enrichment and identification of O-GlcNAcylated proteins and peptides.

Structural and Functional Characterization of a Hole-Hole Homodimer Variant in a "Knob-Into-Hole" Bispecific Antibody.

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of CH3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

Characterization of Chain Pairing Variants of Bispecific IgG Expressed in a Single Host Cell by High-Resolution Native and Denaturing Mass Spectrometry.

Bispecific antibodies, including bispecific IgG, show some promise in clinical trials as a means to extend the therapeutic potential of antibodies. Bispecific IgG can be made by separate expression and purification of each parent half antibody followed by in vitro reconstitution. Generating bispecific IgG by coexpression of two different light and heavy chains in a single host cell is potentially more efficient because it obviates the need for two separate cell lines and purification processes. However, this workflow may produce unwanted mispaired IgG species in addition to the desired bispecific IgG. Development and identification of designs that facilitate cognate light chain pairing may benefit from more refined methods to identify and quantify low levels of mispaired IgG. Using an anti-IL-4/IL-13 bispecific IgG, a mass spectrometric characterization method was developed using native or denaturing conditions by direct infusion into an Exactive Plus Extended Mass Range Orbitrap instrument. The high mass resolving power of the instrument allows unambiguous identification and accurate quantification of all light and heavy chain pairing variants in a mixture of bispecific IgG assembled in vivo upon coexpression down to 1% impurity. Preferential pairing of the anti-IL-13 light chain to its cognate heavy chain was observed, which may be leveraged to guide the design of a single-cell solution for streamlined production of bispecific IgG. Additionally, the utility of native mass spectrometry in deconvoluting complex antibody mixtures and in antigen-binding experiments to understand the contribution of doubly light chain mispaired bispecific IgG was demonstrated.

Systematic analysis of protein phosphorylation networks from phosphoproteomic data.

In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes.

Total Synthesis of Cyclotripeptidic Natural Products Anacine, Aurantiomide C, Polonimides A and C, and Verrucine F.

The total synthesis of cyclotripeptidic natural products possessing a central piperazino[2,1-b]quinazolin-3,6-dione core is described through an original strategy involving the pivotal cyclocondensation of an electrophilic homoserine lactone intermediate. The alkylidene group was spontaneously installed by autoxidation during the cyclocondensation process, while the propionamide side chain was introduced through the nickel-catalyzed aminocarbonylation of a bromoethyl intermediate. This last reaction is unprecedented on such highly functionalized intermediates. Finally, we explored structural modifications and interconversions of the natural products. Overall, this work led to anacine, aurantiomide C, polonimides A and C, and verrucine F.

In situ annealing achieves an ultrafast synthesis of high coercive strontium ferrite foams and beyond.

Strontium ferrite nanostructures have attracted intensive interest recently due to the increasing demand for cost-effective features and good chemical corrosion resistance of magnetic materials, yet the ultrafast synthesis of strontium ferrite with desired coercivity is still experiencing a severe challenge. Herein, porous strontium ferrite foams with a coercivity up to 23.35 kOe were prepared by ultrafast in situ annealing for 1 min based on an auto-combustion strategy. The high coercivity of strontium ferrite benefits from the increasing magnetocrystalline anisotropy caused by the ion substitution and the appropriate grain size close to the critical single-domain size of strontium ferrite. In addition, this ultrafast synthesis can be extended to prepare a series of porous spinel, lanthanide-based perovskites, and their high-entropy counterpart foams. We also demonstrate that this strategy is feasible for preparing biphasic composite oxide foams. Furthermore, this work provides important guidance for the design of porous permanent magnet materials and the efficient preparation of porous oxide foam materials.

Tissue inhibitor of metalloproteinases 1 is involved in ROS-mediated inflammation via regulating matrix metalloproteinase 1 expression in the sea cucumber Apostichopus japonicus.

Tissue inhibitors of metalloproteinases (TIMPs) serve as matrix metalloproteinase (MMP) inhibitors in the pathogenesis of inflammatory diseases in vertebrates. We cloned and characterised the TIMP1 gene from Apostichopus japonicus using RACE approaches (designated as AjTIMP1). For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjTIMP1 mRNAs in coelomocytes was detected at 24 h (23.44-fold) and remained at high levels (4.01-fold) until 72 h. Similarly, AjTIMP1 expression was upregulated in primary coelomocytes exposed to 10 µg mL-1 LPS. AjTIMP1 was expressed in all tissues, and the highest expression was observed in the body wall. Functional investigation revealed an imbalance in the ratio of AjMMP1/AjTIMP1 in the skin ulceration syndrome (SUS) diseased group; it was sharply up-regulated to 3.97:1 compared with the healthy group. Furthermore, when AjTIMP1 was knocked down using small interfering RNA (siRNA-KD) to 0.4-fold, AjMMP1 and AjMMP19 were upregulated to 1.99- and 1.85-fold, respectively. AjTIMP1 siRNA-KD can promote ROS production by 26.2%, whereas AjMMP1 siRNA-KD can eliminate the increase in ROS. In inflamed tissues, collagen I and III levels were decreased by 33.1% and 33.6%, respectively, in the AjTIMP1 siRNA group at 24 h AjTIMP1 was involved in the inflammatory response by mediating ROS formation and collagen degradation.

Dihuang-Yinzi Alleviates Cognition Deficits via Targeting Energy-Related Metabolism in an Alzheimer Mouse Model as Demonstrated by Integration of Metabolomics and Network Pharmacology.

Energy metabolism disturbance and the consequent reactive oxygen species (ROS) overproduction play a key and pathogenic role in the onset and progression of Alzheimer's disease (AD). Dihuang-Yinzi (DHYZ) is a traditional Chinese herbal prescription clinically applied to treat AD and other neurodegenerative diseases for a long time. However, the systematical metabolic mechanism of DHYZ against AD remains largely unclear. Here we aimed to explore the mechanism of DHYZ in the treatment of AD comprehensively in an in vivo metabolic context by performing metabolomics analysis coupled with network pharmacology study and experimental validation. The network pharmacology was applied to dig out the potential target of DHYZ against AD. The metabolomics analysis based on UPLC-HRMS was carried out to profile the urine of 2× Tg-AD mice treated with DHYZ. By integrating network pharmacology and metabolomics, we found DHYZ could ameliorate 4 key energy-related metabolic pathways, including glycerophospholipid metabolism, nicotinate/nicotinamide metabolism, glycolysis, and tricarboxylic acid cycle. Besides, we identified 5 potential anti-AD targets of DHYZ, including DAO, HIF1A, PARP1, ALDH3B2, and ACHE, and 14 key differential metabolites involved in the 4 key energy-related metabolic pathways. Furthermore, DHYZ depressed the mitochondrial dysfunction and the resultant ROS overproduction through ameliorating glycerophospholipid metabolism disturbance. Thereby DHYZ increased nicotinamide adenine dinucleotide (NAD+) content and promoted glycolysis and tricarboxylic acid (TCA) cycle, and consequently improved oxidative phosphorylation and energy metabolism. In the present study, we provided a novel, comprehensive and systematic insight into investigating the therapeutic efficacy of DHYZ against AD via ameliorating energy-related metabolism.