AILDE Computer-Aided Discovery of Novel Ibuprofen-Coumarin Antitumor Lead Compounds Targeting Cyclooxygenase-2.

Starting from three ibuprofen-coumarin hit compounds, we designed 18 derivative compounds targeting cyclooxygenase-2 (COX-2) by introducing different substituents onto them by using the computational auto in silico ligand directing evolution (AILDE) method. After synthesizing and testing the activity, we found that 6 representative compounds have micromolar enzyme inhibitory activity against COX-2. Additionally, 16 compounds have shown certain inhibitory activity in cervical cancer cells. Among these compounds, 6c (IC50 = 0.606 µM, HeLa) and 7g (IC50 = 0.783 µM, HeLa) have exhibited excellent activity, which is approximately 10 times better than the commercial drug gefitinib. According to molecular simulation results, the halogen atoms of 6c and 7g on the coumarin ring can form halogen bonds with COX-2, which significantly improves their activity compared to their hit compounds 6a and 7a. However, the key interactions were lost in binding with COX-1. The calculation results revealed that the two compounds are selective COX-2 inhibitors, with potential selectivity indexes of 6-fold and 5-fold, respectively. The cell-based activity of compounds 6c and 7g toward HEK293 cells demonstrates that our compounds possess an acceptable safety toward normal cells. The results indicate that 6c and 7g can serve as potential lead compounds for further lucubrate.

A transcriptome-wide analysis provides novel insights into how Metabacillus indicus promotes coral larvae metamorphosis and settlement.

BACKGROUND: Coral reefs experience frequent and severe disturbances that can overwhelm their natural resilience. In such cases, ecological restoration is essential for coral reef recovery. Sexual reproduction has been reported to present the simplest and most cost-effective means for coral reef restoration. However, larval settlement and post-settlement survival represent bottlenecks for coral recruitment in sexual reproduction. While bacteria play a significant role in triggering coral metamorphosis and settlement in many coral species, the underlying molecular mechanisms remain largely unknown. In this study, we employed a transcriptome-level analysis to elucidate the intricate interactions between bacteria and coral larvae that are crucial for the settlement process. RESULTS: High Metabacillus indicus strain cB07 inoculation densities resulted in the successful induction of metamorphosis and settlement of coral Pocillopora damicoris larvae. Compared with controls, inoculated coral larvae exhibited a pronounced increase in the abundance of strain cB07 during metamorphosis and settlement, followed by a significant decrease in total lipid contents during the settled stage. The differentially expressed genes (DEGs) during metamorphosis were significantly enriched in amino acid, protein, fatty acid, and glucose related metabolic pathways. In settled coral larvae induced by strain cB07, there was a significant enrichment of DEGs with essential roles in the establishment of a symbiotic relationship between coral larvae and their symbiotic partners. The photosynthetic efficiency of strain cB07 induced primary polyp holobionts was improved compared to those of the negative controls. In addition, coral primary polyps induced by strain cB07 showed significant improvements in energy storage and survival. CONCLUSIONS: Our findings revealed that strain cB07 can promote coral larval settlement and enhance post-settlement survival and fitness. Manipulating coral sexual reproduction with strain cB07 can overcome the current recruitment bottleneck. This innovative approach holds promise for future coral reef restoration efforts.

Exploring the Biosynthetic Potential of Tistrella Species for Producing Didemnin Antitumor Agents.

Didemnins are a class of cyclic depsipeptides derived from sea tunicates that exhibit potent anticancer, antiviral, and immunosuppressive properties. Although certain Tistrella species can produce didemnins, their complete biosynthetic potential remains largely unexplored. In this study, we utilize feature-based molecular networking to analyze the metabolomics of Tistrella mobilis and Tistrella bauzanensis, focusing on the production of didemnin natural products. In addition to didemnin B, we identify nordidemnin B and [hysp2]didemnin B, as well as several minor didemnin analogs. Heterologous expression of the didemnin biosynthetic gene cluster in a Streptomyces host results in the production of only didemnin B and nordidemnin B in limited quantities. Isotope-labeling studies reveal that the substrate promiscuity of the adenylation domains during biosynthesis leads to the accumulation of nordidemnin B and [hysp2]didemnin B. Additionally, precursor-directed biosynthesis is applied to generate eight novel didemnin derivatives by supplementing the culture with structurally related amino acids. Furthermore, we increased the titers of nordidemnin B and [hysp2]didemnin B by supplementing the fermentation medium with l-valine and l-isoleucine, respectively. Finally, both compounds undergo side-chain oxidation to enhance their biological activity, with their anticancer properties found to be as potent as plitidepsin.

A global microbiome analysis reveals the ecological feature of Tistrella and its production of the bioactive didemnins in the marine ecosystem.

Marine microorganisms like Tistrella are essential for producing bioactive compounds, including didemnins with antitumor and antiviral properties. However, our understanding of Tistrella's ecological features and didemnin production in natural environments is limited. In this study, we used genomics and metagenomics to show that Tistrella is widely distributed across natural habitats, especially in marine environments from the surface to 5000 m deep, with distinct non-random distribution patterns revealed by co-occurrence analysis. Importantly, transcriptional profiling of didemnin biosynthetic gene clusters indicates active in situ production of this compound within marine ecosystems. These findings enhance our understanding of Tistrella's ecology and secondary metabolite production in natural environments. Further research is needed to explore the ecological dynamics and functional impacts of Tistrella in these ecosystems.

Audiovisual Integration Decreases Inhibition of Return in Children With ADHD.

OBJECTIVES: Previous studies have widely demonstrated that inhibition of return (IOR) with audiovisual targets decreases due to audiovisual integration (AVI). It is currently unclear, however, whether the impaired AVI in children with ADHD has effects on IOR. The present study used the cue-target paradigm to explore differences between the IOR of audiovisual targets and the IOR of visual targets in ADHD and typically developing (TD) children. METHOD: A total of 81 native Chinese speakers aged 6 to 13 years were recruited, including 38 children with ADHD and 43 age- and sex-matched TD children. RESULTS: The results showed that there was a smaller magnitude of IOR with audiovisual targets as compared with visual targets in the two groups. Importantly, the reduction of IOR in audiovisual conditions was significantly smaller in children with ADHD than in children with TD. Race model analyses further confirmed that differences in IOR between ADHD and TD are due to deficits of audiovisual integration in ADHD. CONCLUSION: The results indicated that children with ADHD have impaired audiovisual integration, which has a minimal impact on IOR.

Electrophysiological evidence of nonspatial inhibition of return affecting audiovisual integration.

OBJECTIVE: The present study endeavored to investigate the potential neural underpinnings of disparities in audiovisual integration (AVI) between valid and invalid targets, modulated by nonspatial inhibition of return (IOR). Concurrently, we sought to delineate the distinct roles subserved by Chinese character primes and color block primes throughout this process. METHOD: We employed a prime-neutral cue-target paradigm, wherein 25 college students participated in the experiment. Behavioral measures encompassed the reaction time, IOR effect, multisensory response enhancement, and race model analysis. Besides, we examined the N200, N400, and P300 components elicited by the target stimulus presentation in a time-locked fashion to investigate the neural underpinnings of AVI disparities in the context of valid and invalid targets. RESULTS: Behavioral analyses unveiled a significant attenuation of AVI for valid targets, with this effect being particularly pronounced in trials involving Chinese character primes. Intriguingly, event-related potential (ERP) data evinced AVI within the N400 and P300 components. Moreover, the novelty of this study resides in identifying the P300 component as the principal neural correlate underpinning the attenuation of AVI arising from nonspatial IOR-a finding that was not replicated when employing color block primes. CONCLUSIONS: This research furnishes novel ERP evidence that elucidates the mechanisms through which nonspatial IOR modulates AVI. This contributes significantly to a broader understanding of the cognitive processes underpinning multisensory perception and attentional dynamics. These insights not only corroborate the late attention theory and the coactivation model but also lend credence to the context-updating hypothesis. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Upcycling the Spent Graphite Anode Into the Prelithiation Catalyst: A Separator Strategy Toward Anode-Free Cell Prototyping.

The substantial manufacturing of lithium-ion batteries (LIBs) requires sustainable, circular, and decarbonized recycling strategies. While efforts are concentrated on extracting valuable metals from cathodes using intricate chemical process, the direct, efficient cathode regeneration remains a technological challenge. More urgently, the battery supply chain also requires the value-added exploitation of retired anodes. Here, a "closed-loop" approach is proposed to upcycle spent graphite into the prelithiation catalyst, namely the fewer-layer graphene flakes (FGF), upon the exquisite tuning of interlayer spacing and defect concentration. Since the catalytic FGF mitigates the delithiation energy barrier from calcinated Li5FeO4 nanocrystalline, the composite layer of which cast on the polyolefin substrate thus enables a customized prelithiation capability (98% Li+ utilization) for the retired LiFePO4 recovery. Furthermore, the hydrophobic polymeric modification guarantees the moisture tolerance of Li5FeO4 agents, aligning with commercial battery manufacturing standards. The separator strategy well regulates the interfacial chemistry in the anode-free pouch cell (LiFePO4||Cu), the prototype of which balances the robust cyclability, energy density up to 386.6 Wh kg-1 as well as the extreme power output of 1159.8 W kg-1. This study not only fulfills the sustainable supply chain with graphite upcycling, but also establishes a generic, viable protocol for the anode-free cell prototyping.

Ginsenoside Re inhibits non-small cell lung cancer progression by suppressing macrophage M2 polarization induced by AMPKα1/STING positive feedback loop.

Tumor-associated macrophages (TAMs) in non-small cell lung cancer (NSCLC) promote tumor cell metastasis by interacting with cancer cells. Ginsenoside Re is capable of modulating the host immune system and exerts anticancer effects through multiple pathways. Both AMPK and STING are involved in the regulation of MΦ polarization, thereby affecting tumor progression. However, whether there is a regulatory relationship between them and its effect on MΦ polarization and tumor progression is unclear. The aim of this study was to provide mechanistic evidence that ginsenoside Re modulates MΦ phenotype through inhibition of the AMPKα1/STING positive feedback loop and thus exerts an antimetastatic effect in NSCLC immunotherapy. Cell culture models and conditioned media (CM) systems were constructed, and the treated MΦ were analyzed by database analysis, RT-PCR, Western blotting, flow cytometry, and immunofluorescence to determine the regulatory relationship between AMPK and STING and the effects of ginsenoside Re on MΦ polarization and tumor cells migration. The effects of ginsenoside Re (10, 20 mg/kg/day) on TAMs phenotype as well as tumor progression in mice were assessed by HE staining, immunohistochemical staining, and Western blotting. In this study, AMPKα1/STING positive feedback loop in NSCLC TAMs induced M2 type polarization, which in turn promoted NSCLC cell migration. In addition, ginsenoside Re was discovered to inhibit M2-like MΦ polarization, thereby inhibiting NSCLC cell migration. Mechanistically, Re was able to inhibit the formation of the AMPKα1/STING positive feedback loop, thereby inhibiting its induction of M2-like MΦ and consequently inhibiting the epithelial-mesenchymal transition (EMT) process of NSCLC cells. Furthermore, in mouse models, Re was found to suppress LLC tumor growth and colonization by inhibiting M2-type polarization of TAMs. Our finding indicates that ginsenoside Re can effectively modulate MΦ polarization and thus play an important role in antimetastatic immunotherapy of NSCLC.

Cantharidin overcomes IL-2Rα signaling-mediated vorinostat resistance in cutaneous T-cell lymphoma through reactive oxygen species.

BACKGROUND: Vorinostat (SAHA) is a histone deacetylase inhibitor that has shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, only a subset of patients with CTCL (30-35%) respond to SAHA and the response is not always sustainable. Thus, understanding the mechanisms underlying evasive resistance in this cancer is an unmet medical need to improve the efficacy of current therapies. PURPOSE: This study aims to identify factors contributing to resistance against SAHA in CTCL and ways to mitigate it. METHODS AND RESULTS: In this study, we demonstrated that attenuated reactive oxygen species (ROS) induces the expression of interleukin (IL)-2Rα, one of the IL-2 receptors, which drives resistance to SAHA in CTCL. We also determined that cantharidin could overcome SAHA resistance to CTCL by blocking IL-2Rα-related signaling via ROS-dependent manner. Mechanistically, accelerated translation of IL-2Rα contributes to excessive IL-2Rα protein formation as a result of reduced ROS levels in SAHA-resistant CTCL. At the same time, amplified IL-2R signals are evidenced by strengthened interaction of IL-2Rß with IL-2Rγ and Janus kinase/signal transducer and activator of transcription molecules, and by increased expression of protein kinase B (AKT)/mTOR and mitogen-activated protein kinase signaling. Moreover, cantharidin, an active constituent of Mylabris used in traditional Chinese medicine, markedly increased ROS levels, and thereby restrained IL-2Rα translation, resulting in suppression of downstream pathways in SAHA-resistant cells. Cantharidin is also found to synergize with SAHA and triggers SAHA-resistant cell death via IL-2R signaling both in vitro and in vivo. CONCLUSION: Our study uncovers a novel molecular mechanism of acquired SAHA resistance and also suggests that using cantharidin is a potential approach to overcome CTCL therapy resistance. Our findings underlie the therapeutic potential of cantharidin in treating CTCL.

Identification of 3-ketocapnine reductase activity within the human microbiota.

The microbial synthesis of sulfonolipids within the human body is likely involved in maintaining human health or causing diseases. However, the enzymes responsible for their biosynthesis remain largely unknown. In this study, we identified and verified the role of 3-ketocapnine reductase, the third-step enzyme, in the four-step conversion of l-phosphoserine into sulfobacin B both in vivo and in vitro. This finding builds upon our previous research into sulfonolipid biosynthesis, which focused on the vaginal bacterium Chryseobacterium gleum DSM 16776 and the gut bacterium Alistipes finegoldii DSM 17242. Through comprehensive gene mapping, we demonstrate the widespread presence of potential sulfonolipid biosynthetic genes across diverse bacterial species inhabiting various regions of the human body. These findings shed light on the prevalence of sulfonolipid-like metabolites within the human microbiota, suggesting a potential role for these lipid molecules in influencing the intricate biointeractions within the complex microbial ecosystem of the human body.

Alleviating Coral Thermal Stress via Inoculation with Quorum Quenching Bacteria.

In the background of global warming, coral bleaching induced by elevated seawater temperature is the primary cause of coral reef degradation. Coral microbiome engineering using the beneficial microorganisms for corals (BMCs) has become a hot spot in the field of coral reef conservation and restoration. Investigating the potential of alleviating thermal stress by quorum quenching (QQ) bacteria may provide more tools for coral microbial engineering remediation. In this study, QQ bacteria strain Pseudoalteromonas piscicida SCSIO 43740 was screened among 75 coral-derived bacterial strains, and its quorum sensing inhibitor (QSI) compound was isolated and identified as 2,4-di-tert-butylphenol (2,4-DTBP). Then, the thermal stress alleviating potential of QQ bacteria on coral Pocillopora damicornis was tested by a 30-day controlled experiment with three different treatments: control group (Con: 29 °C), high temperature group (HT: 31 °C), and the group of high temperature with QQ bacteria inoculation (HTQQ: 31 °C + QQ bacteria). The results showed that QQ bacteria SCSIO 43740 inoculation can significantly mitigate the loss of symbiotic algae and impairment of photosynthesis efficiency of coral P. damicornis under thermal stress. Significant difference in superoxide dismutase (SOD) and catalase (CAT) enzyme activities between HT and HTQQ was not observed. In addition, QQ bacteria inoculation suppressed the coral microbial community beta-dispersion and improved the stability of microbial co-occurrence network under thermal stress. It was suggested that QQ bacteria inoculation can alleviate coral thermal stress via reshaping microbial interaction and maintain community stability of coral microbiome. This study provided new evidence for the probiotic function of QQ bacteria in corals, which shedding light on the development of new microbiological tools for coral reef conservation.

Tactile temporal predictions: The influence of conditional probability.

Predicting the timing of incoming information allows brain to optimize information processing in dynamic environments. However, the effects of temporal predictions on tactile perception are not well established. In this study, two experiments were conducted to determine how temporal predictions interact with conditional probabilities in tactile perceptual processing. In Experiment 1, we explored the range of the interval between preceding ready cues and imperative targets in which temporal prediction effects can be observed. This prediction effect was observed for intervals of 500 and 1,000 ms. In Experiment 2, we investigated the benefits of temporal predictions on tactile perception while manipulating the conditional probability (setting the stimulus onset earlier or later than the predicted moment in short and long intervals). Our results revealed that this effect became stronger as the probability of the stimulus at the predicted time point increased under short-interval conditions. Together, our results show that the difficulty of transferring processing resources increases in temporally dynamic environments, suggesting a greater subjective cost associated with maladaptive responses to temporally uncertain events.

Physics Constrained High-Precision Data-Driven Modeling for Multi-Path Ultrasonic Flow Meter in Natural Gas Measurement.

Ultrasonic flow meters are crucial measuring instruments in natural gas transportation pipeline scenarios. The collected flow velocity data, along with the operational conditions data, are vital for the analysis of the metering performance of ultrasonic flow meters and analysis of the flow process. In practical applications, high requirements are placed on the modeling accuracy of ultrasonic flow meters. In response, this paper proposes an ultrasonic flow meter modeling method based on a combination of data learning and industrial physics knowledge. This paper builds ultrasonic flow meter flow velocity prediction models under different working conditions, combining pipeline flow field velocity distribution knowledge for data preprocessing and loss function design. By making full use of the characteristics of the physics and data learning, the prediction results are close to the real acoustic path flow velocity distribution; thus, the model has high accuracy and interpretability. Experiments are conducted to prove that the prediction error of the proposed method can be controlled within 1%, which can meet the needs of ultrasonic flow meter modeling and subsequent performance analysis in actual production.

Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates.

Antimicrobial resistance remains a significant global threat, driving up mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we report the discovery of new aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptide antibiotics through a synergistic approach combining biosynthetic rule-based omics mining and heterologous expression. We first bioinformatically identify 1172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing peptides formation from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully establish the connection between three identified BGCs and the biosynthesis of five peptide antibiotics via biosynthetic rule-guided metabolic analysis. Notably, we discover a class V lanthipeptide, massatide A, which displays excellent activity against gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration of 0.25 µg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a relatively low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing peptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

Sema4D Knockout Attenuates Choroidal Neovascularization by Inhibiting M2 Macrophage Polarization Via Regulation of the RhoA/ROCK Pathway.

Purpose: The aim of this study was to elucidate the role of Sema4D in the pathogenesis of senescence-associated choroidal neovascularization (CNV) and to explore its underlying mechanisms. Methods: In this study, we utilized a model of laser-induced CNV in both young (3 months old) and old (18 months old) mice, including those with or without Sema4D knockout. The expression and localization of Sema4D in CNV were assessed using PCR, Western blot, and immunostaining. Subsequently, the morphological and imaging examinations were used to evaluate the size of CNV and vascular leakage. Finally, the expression of M2 markers, senescence-related markers, and molecules involved in the RhoA/ROCK pathway was detected. Results: We found that Sema4D was predominantly expressed in macrophages within CNV lesions, and both the mRNA and protein levels of Sema4D progressively increased following laser photocoagulation, a trend more pronounced in old mice. Moreover, Sema4D knockout markedly inhibited M2 polarization in senescent macrophages and reduced the size and leakage of CNV, particularly in aged mice. Mechanistically, aging was found to upregulate RhoA/ROCK signaling, and knockout of Sema4D effectively suppressed the activation of this pathway, with more significant effects observed in aged mice. Conclusions: Our findings revealed that the deletion of Sema4D markedly inhibited M2 macrophage polarization through the suppression of the RhoA/ROCK pathway, ultimately leading to the attenuation of senescence-associated CNV. These data indicate that targeting Sema4D could offer a promising approach for gene editing therapy in patients with neovascular age-related macular degeneration.

An in-situ polymerization strategy for gel polymer electrolyte Si||Ni-rich lithium-ion batteries.

Coupling the Si-based anodes with nickel-rich LiNixMnyCo1-x-yO2 cathodes (x ≥ 0.8) in the energy-dense cell prototype suffers from the mechanical instability of the Li-Si alloys, cathode collapse upon the high-voltage cycling, as well as the severe leakage current at elevated temperatures. More seriously, the cathode-to-anode cross-talk effect of transitional metal aggravates the depletion of the active Li reservoir. To reconcile the cation utilization degree, stress dissipation, and extreme temperature tolerance of the Si-based anode||NMC prototype, we propose a gel polymer electrolyte to reinforce the mechanical integrity of Si anode and chelate with the transitional cations towards the stabilized interfacial property. As coupling the conformal gel polymer electrolyte encapsulation with the spatial arranged Si anode and NMC811 cathode, the 2.7 Ah pouch-format cell could achieve the high energy density of 325.9 Wh kg-1 (based on the whole pouch cell), 88.7% capacity retention for 2000 cycles, self-extinguish property as well as a wide temperature tolerance. Therefore, this proposed polymerization strategy provides a leap toward the secured Li batteries.

Fully dense generative adversarial network for removing artifacts caused by microwave dielectric effect in thermoacoustic imaging.

Microwave-induced thermoacoustic (TA) imaging (MTAI) combines pulsed microwave excitation and ultrasound detection to provide high contrast and spatial resolution images through dielectric contrast, which holds great promise for clinical applications. However, artifacts caused by microwave dielectric effect will seriously affect the accuracy of MTAI images that will hinder the clinical translation of MTAI. In this work, we propose a deep learning-based method fully dense generative adversarial network (FD-GAN) for removing artifacts caused by microwave dielectric effect in MTAI. FD-GAN adds the fully dense block to the generative adversarial network (GAN) based on the mutual confrontation between generator and discriminator, which enables it to learn both local and global features related to the removal of artifacts and generate high-quality images. The practical feasibility was tested in simulated, experimental data. The results demonstrate that FD-GAN can effectively remove the artifacts caused by the microwave dielectric effect, and shows superiority in denoising, background suppression, and improvement of image distortion. Our approach is expected to significantly improve the accuracy and quality of MTAI images, thereby enhancing the diagnostic accuracy of this innovative imaging technique.

The human brain deals with violating general color or depth knowledge in different time courses.

Utilizing the high temporal resolution of event-related potentials (ERPs), we compared the time course of processing incongruent color versus 3D-depth information. Participants were asked to judge whether the food color (color condition) or 3D structure (3D-depth condition) was congruent or incongruent with their previous knowledge and experience. The behavioral results showed that the reaction times in the congruent 3D-depth condition were slower than those in the congruent color condition. The reaction times in the incongruent 3D-depth condition were slower than those in the incongruent color condition. The ERP results showed that incongruent color stimuli induced a larger N270, larger P300, and smaller N400 components in the fronto-central region than the congruent color stimuli. Incongruent 3D-depth stimuli induced a smaller N1 in the occipital region, larger P300 and smaller N400 in the parietal-occipital region than congruent 3D-depth stimuli. The time-frequency analysis found that incongruent color stimuli induced a larger theta band (360-580 ms) activation in the fronto-central region than congruent color stimuli. Incongruent 3D-depth stimuli induced larger alpha and beta bands (240-350 ms) activation in the parietal region than congruent 3D-depth stimuli. Our results suggest that the human brain deals with violating general color or depth knowledge in different time courses. We speculate that the depth perception conflict was dominated by solving the problem with visual processing, whereas the color perception conflict was dominated by solving the problem with semantic violation.

Apolipoprotein O modulates cholesterol metabolism via NRF2/CYB5R3 independent of LDL receptor.

Apolipoprotein O (APOO) plays a critical intracellular role in regulating lipid metabolism. Here, we investigated the roles of APOO in metabolism and atherogenesis in mice. Hepatic APOO expression was increased in response to hyperlipidemia but was inhibited after simvastatin treatment. Using a novel APOO global knockout (Apoo-/-) model, it was found that APOO depletion aggravated diet-induced obesity and elevated plasma cholesterol levels. Upon crossing with low-density lipoprotein receptor (LDLR) and apolipoprotein E (APOE) knockout hyperlipidemic mouse models, Apoo-/- Apoe-/- and Apoo-/- Ldlr-/- mice exhibited elevated plasma cholesterol levels, with more severe atherosclerotic lesions than littermate controls. This indicated the effects of APOO on cholesterol metabolism independent of LDLR and APOE. Moreover, APOO deficiency reduced cholesterol excretion through bile and feces while decreasing phospholipid unsaturation by inhibiting NRF2 and CYB5R3. Restoration of CYB5R3 expression in vivo by adeno-associated virus (AAV) injection reversed the reduced degree of phospholipid unsaturation while decreasing blood cholesterol levels. This represents the first in vivo experimental validation of the role of APOO in plasma cholesterol metabolism independent of LDLR and elucidates a previously unrecognized cholesterol metabolism pathway involving NRF2/CYB5R3. APOO may be a metabolic regulator of total-body cholesterol homeostasis and a target for atherosclerosis management. Apolipoprotein O (APOO) regulates plasma cholesterol levels and atherosclerosis through a pathway involving CYB5R3 that regulates biliary and fecal cholesterol excretion, independently of the LDL receptor. In addition, down-regulation of APOO may lead to impaired mitochondrial function, which in turn aggravates diet-induced obesity and fat accumulation.