CLMP is a tumor suppressor that determines all-trans retinoic acid response in colorectal cancer.

CAR-like membrane protein (CLMP) is a tight junction-associated protein whose mutation is associated with congenital short bowel syndrome (CSBS), but its functions in colorectal cancer (CRC) remain unknown. Here, we demonstrate that CLMP is rarely mutated but significantly decreased in CRC patients, and its deficiency accelerates CRC tumorigenesis, growth, and resistance to all-trans retinoic acid (ATRA). Mechanistically, CLMP recruits ß-catenin to cell membrane, independent of cadherin proteins. CLMP-mediated ß-catenin translocation inactivates Wnt(Wingless and INT-1)/ß-catenin signaling, thereby suppressing CRC tumorigenesis and growth in ApcMin/+, azoxymethane/dextran sodium sulfate (AOM/DSS), and orthotopic CRC mouse models. As a direct target of Wnt/ß-catenin, cytochrome P450 hydroxylase A1 (CYP26A1)-an enzyme that degrades ATRA to a less bioactive retinoid-is upregulated by CLMP deficiency, resulting in ATRA-resistant CRC that can be reversed by administering CYP26A1 inhibitor. Collectively, our data identify the anti-CRC role of CLMP and suggest that CYP26A1 inhibitor enable to boost ATRA's therapeutic efficiency.

Effects of food emulsifiers on high fat-diet-induced obesity, intestinal inflammation, changes in bile acid profile, and liver dysfunction.

Obesity has become one of the most prevalent health concerns of our time. A long-term high-fat diet is closely related to obesity. Food emulsifiers are incorporated into high-fat foods to enhance the texture and stability. Whether food emulsifiers exacerbate obesity and metabolic disorders induced by a high-fat diet remains unclear. This study aimed to investigate the effects of polysorbate-80 (P80) and polyglycerol polyricinoleate (PGPR) on lipid metabolism, bile acid profile, and gut microbiota in normal and high-fat-diet-induced obesity in mice. The results of this study showed that P80 and PGPR had little effect on body weight but significantly increased epididymal-fat weight, total energy intake, and blood lipid levels. P80 and PGPR stimulated colon inflammation and improved the expression of inflammatory factors in the colon and liver significantly. P80 and PGPR changed the bile acid profile. However, P80 and PGPR did not aggravate inflammation, obesity and alter bile acid profile by altering the composition of the gut microbiota. The results of this study provide an experimental reference for the rational use of food additives and the adjustment of dietary structure, which are important and have application value.

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from "glucose repression" are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Micro-histology combined with cytology improves the diagnostic accuracy of endometrial lesions.

BACKGROUND: In the study, we aimed to evaluate the ability of micro-histology combined with cytology to improve the quality of slides and diagnose endometrial lesions. METHODS: Endometrial specimens were collected from Li Brushes. Every specimen was prepared for micro-histological and cytological slides, using cell block (CB) and liquid-based cytology (LBC) technologies. Semi-quantitative scoring system was used to evaluate the qualities of slides. CB slides were assessed by 5-category scoring system. Diagnostic accuracy was calculated in LBC, CB, and LBC + CB groups based on the histological gold standard. Endometrial atypical hyperplasia, and endometrial cancer were considered positive, whereas others were considered negative. RESULTS: A total of 167 patients were enrolled. CB slides were inferior to LBC slides only in cellularity (p < 0.001), but superior in the other six parameters (all p < 0.001). The satisfaction rate of micro-histology accounted for 92.3%. The accuracy index in the CB group was higher than in the LBC group in terms of sensitivity (85.5% vs. 82.7%) and specificity (98.9% vs. 95.7%). The sensitivity and specificity in the LBC + CB group were increased to 94.2% and 99.0%, respectively. CONCLUSIONS: The quality of micro-histological slides was higher than that of cytological slides. By combining micro-histology with cytology, higher accuracy was achieved for endometrial lesions diagnosis.

Targeting intratumor heterogeneity suppresses colorectal cancer chemoresistance and metastasis.

Intratumor heterogeneity (ITH) is a barrier to effective therapy. However, it is largely unknown how ITH is established at the onset of tumor progression, such as in colorectal cancer (CRC). Here, we integrate single-cell RNA-seq and functional validation to show that asymmetric division of CRC stem-like cells (CCSC) is critical for early ITH establishment. We find that CCSC-derived xenografts contain seven cell subtypes, including CCSCs, that dynamically change during CRC xenograft progression. Furthermore, three of the subtypes are generated by asymmetric division of CCSCs. They are functionally distinct and appear at the early stage of xenografts. In particular, we identify a chemoresistant and an invasive subtype, and investigate the regulators that control their generation. Finally, we show that targeting the regulators influences cell subtype composition and CRC progression. Our findings demonstrate that asymmetric division of CCSCs contributes to the early establishment of ITH. Targeting asymmetric division may alter ITH and benefit CRC therapy.

Clinically Applicable Pathological Diagnosis System for Cell Clumps in Endometrial Cancer Screening via Deep Convolutional Neural Networks.

OBJECTIVES: The soaring demand for endometrial cancer screening has exposed a huge shortage of cytopathologists worldwide. To address this problem, our study set out to establish an artificial intelligence system that automatically recognizes and diagnoses pathological images of endometrial cell clumps (ECCs). METHODS: We used Li Brush to acquire endometrial cells from patients. Liquid-based cytology technology was used to provide slides. The slides were scanned and divided into malignant and benign groups. We proposed two (a U-net segmentation and a DenseNet classification) networks to identify images. Another four classification networks were used for comparison tests. RESULTS: A total of 113 (42 malignant and 71 benign) endometrial samples were collected, and a dataset containing 15,913 images was constructed. A total of 39,000 ECCs patches were obtained by the segmentation network. Then, 26,880 and 11,520 patches were used for training and testing, respectively. On the premise that the training set reached 100%, the testing set gained 93.5% accuracy, 92.2% specificity, and 92.0% sensitivity. The remaining 600 malignant patches were used for verification. CONCLUSIONS: An artificial intelligence system was successfully built to classify malignant and benign ECCs.

Intestinal Microflora Changes in Patients with Mild Alzheimer's Disease in a Chinese Cohort.

BACKGROUND: Understanding the relationship between Alzheimer's disease (AD) and intestinal flora is still a major scientific topic that continues to advance. OBJECTIVE: To determine characterized changes in the intestinal microbe community of patients with mild AD. METHODS: Comparison of the 16S ribosomal RNA (rRNA) high-throughput sequencing data was obtained from the Illumina MiSeq platform of fecal microorganisms of the patients and healthy controls (HC) which were selected from cohabiting caregivers of AD patients to exclude environmental and dietary factors. RESULTS: We found that the abundance of several bacteria taxa in AD patients was different from that in HC at the genus level, such as Anaerostipes, Mitsuokella, Prevotella, Bosea, Fusobacterium, Anaerotruncus, Clostridium, and Coprobacillus. Interestingly, the abundance of Akkermansia, an emerging probiotic, increased significantly in the AD group compared with that in the HC group. Meanwhile, the quantity of traditional probiotic Bifidobacteria of the AD group also rose. CONCLUSION: These alterations in fecal microbiome of the AD group indicate that patients with mild AD have unique gut microbial characteristics. These specific AD-associated intestinal microbes could serve as novel potential targets for early intervention of AD.

Chromatin Remodeling of Colorectal Cancer Liver Metastasis is Mediated by an HGF-PU.1-DPP4 Axis.

Colorectal cancer (CRC) metastasizes mainly to the liver, which accounts for the majority of CRC-related deaths. Here it is shown that metastatic cells undergo specific chromatin remodeling in the liver. Hepatic growth factor (HGF) induces phosphorylation of PU.1, a pioneer factor, which in turn binds and opens chromatin regions of downstream effector genes. PU.1 increases histone acetylation at the DPP4 locus. Precise epigenetic silencing by CRISPR/dCas9KRAB or CRISPR/dCas9HDAC revealed that individual PU.1-remodeled regulatory elements collectively modulate DPP4 expression and liver metastasis growth. Genetic silencing or pharmacological inhibition of each factor along this chromatin remodeling axis strongly suppressed liver metastasis. Therefore, microenvironment-induced epimutation is an important mechanism for metastatic tumor cells to grow in their new niche. This study presents a potential strategy to target chromatin remodeling in metastatic cancer and the promise of repurposing drugs to treat metastasis.

Creatine promotes cancer metastasis through activation of Smad2/3.

As one of the most popular nutrient supplements, creatine has been highly used to increase muscle mass and improve exercise performance. Here, we report an adverse effect of creatine using orthotopic mouse models, showing that creatine promotes colorectal and breast cancer metastasis and shortens mouse survival. We show that glycine amidinotransferase (GATM), the rate-limiting enzyme for creatine synthesis, is upregulated in liver metastases. Dietary uptake, or GATM-mediated de novo synthesis of creatine, enhances cancer metastasis and shortens mouse survival by upregulation of Snail and Slug expression via monopolar spindle 1 (MPS1)-activated Smad2 and Smad3 phosphorylation. GATM knockdown or MPS1 inhibition suppresses cancer metastasis and benefits mouse survival by downregulating Snail and Slug. Our findings call for using caution when considering dietary creatine to improve muscle mass or treat diseases and suggest that targeting GATM or MPS1 prevents cancer metastasis, especially metastasis of transforming growth factor beta receptor mutant colorectal cancers.

CD146 is a Novel ANGPTL2 Receptor that Promotes Obesity by Manipulating Lipid Metabolism and Energy Expenditure.

Obesity and its related complications pose an increasing threat to human health; however, targetable obesity-related membrane receptors are not yet elucidated. Here, the membrane receptor CD146 is demonstrated to play an essential role in obesity. In particular, CD146 acts as a new adipose receptor for angiopoietin-like protein 2 (ANGPTL2), which is thought to act on endothelial cells to activate adipose inflammation. ANGPTL2 binds to CD146 to activate cAMP response element-binding protein (CREB), which then upregulates CD146 during adipogenesis and adipose inflammation. CD146 is present in preadipocytes and mature adipocytes, where it is mediated by its ligands ANGPTL2 and galectin-1. In preadipocytes, CD146 ablation suppresses adipogenesis, whereas the loss of CD146 in mature adipocytes suppresses lipid accumulation and enhances energy expenditure. Moreover, anti-CD146 antibodies inhibit obesity by disrupting the interactions between CD146 and its ligands. Together, these findings demonstrate that ANGPTL2 directly affects adipocytes via CD146 to promote obesity, suggesting that CD146 can be a potential target for treating obesity.

Clinical Evaluation of Li Brush Endometrial Samplers for Diagnosing Endometrial Lesions in Women With Intrauterine Devices.

Background: For women with intrauterine devices (IUDs), it is difficult to sample the endometrium when abnormal uterine bleeding occurs or when regular screening of endometrial cancer is proposed. The purpose of this study is to evaluate the validity of endometrial sampling using Li Brush in IUD users. Methods: This study was a prospective cohort study and conducted in two parts. Part I was to assess the impact of Li Brush on the position of IUDs. Transvaginal ultrasound was used to locate IUDs before and after sampling. Part II was to explore the diagnostic accuracy of Li Brush in detecting endometrial lesions. IUD users with irregular uterine bleeding were recruited in the IUD group and IUD non-users who arranged for dilatation and curettage (D&C) were recruited in the control group. The endometrium was sampled by Li Brush for cells and by D&C for tissues in both groups. The satisfactoriness of sampling and validity of Li Brush were evaluated. Results: Seventeen cases in part I confirmed no significant difference in the position of IUDs before and after sampling (p = 0.20). 112 IUD users and 139 IUD non-users were recruited in part II. Li Brush achieved 94.64 and 92.09% satisfactory sampling rates in the IUD group and control group, respectively, without statistically significant difference between the two groups (p = 0.42). The Sensitivity and specificity of Li Brush for detection of endometrial lesions in IUD group were 95.35 and 87.76% respectively. Conclusions: Li Brush used for endometrial biopsy did not affect the position of IUDs and had high yield of satisfactory samples and good validity for endometrial diagnoses. It was feasible to screen endometrial lesions by Li Brush for women with IUDs.

A Clinical Comparative Study of Two Different Endometrial Cell Samplers for Evaluation of Endometrial Lesions by Cytopathological Diagnosis.

PURPOSE: Cytopathology detecting for endometrial cancer is becoming accepted, and Tao Brush is the most widely used sampler for endometrial cells. This study aims to compare the effectiveness between Li brushes and Tao brushes for the diagnosis of endometrial lesions and to evaluate the diagnostic accuracy of endometrial cytology compared with histology. METHODS: There were 109 patients needing dilation and curettage (D&C) and 21 patients needing hysterectomies included from November 2017 to April 2018. Every patient was sampled by Tao brush and Li brush before D&C or hysterectomy performed. The cytological results were compared based on the gold standard histological results of D&C or hysterectomy. RESULTS: The sensitivity of Li brush cytology for detecting endometrial cancer and atypical hyperplasia was estimated at 83.33%, specificity at 100%, positive predictive value (PPV) at 100%, and negative predictive value (NPV) at 98.02%, respectively. While for the Tao brush, it was 91.67% of sensitivity, 96.04% of specificity, 73.33% of PPV, and 98.98% of NPV, respectively. The kappa value was 0.767, which indicated a substantial agreement. Cytology by both two brushes had a lower insufficient sample rate (2.75% of Tao brush, 4.59% of Li brush) than did D&C (11.93%). DISCUSSION: Endometrial cytology is a reliable approach for evaluating endometrium with a lower insufficient sample rate. Cytology sampled by both Li brushes and Tao brushes has a high accuracy with histological diagnosis in detecting endometrial cancer and atypical hyperplasia. Combining social and economic benefits, the Li brush may be a better endometrial cell collector.

Comparison of Cervical Cytopathological Diagnosis Using Innovative Qi Brush and Traditional Cervex-Brush® Combi.

Objectives: To compare the effectiveness between Qi brush and Cervex-Brush® Combi for the diagnosis of cervical lesions. Methods: After we registered a random-control clinical trial on the Chinese Clinical Trial Registry (No. XJTU1AF2017LSK-25), cervical cell samples were successively collected with both Qi brush and Cervex-Brush® Combi before undergoing colposcope. Colposcopy with biopsy was performed later. Histological diagnosis was regarded as the gold standard in this study. The following indices of the two brushes were compared: sampling degree of satisfaction and presence rate of metaplastic cells, together with sensitivity (Se), specificity (Sp), false positives (FP), false negatives (FN), positive predictive value (PPV), and negative predictive value (NPV). The kappa value was used to measure the inter-rater agreement of the Qi brush and Cervex-Brush® Combi in diagnosing cervical lesions. Results: In total, 74 patients were enrolled in this study. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Qi brush were 57.14, 86.84, 76.19, and 73.33%, respectively. For the Cervex-Brush® Combi, they were 26.92, 88.89, 63.63, and 62.75%, respectively. In addition, the Qi brush had a higher satisfied sampling rate (89.19%) than the Cervex-Brush® Combi (83.78%), and the P-value was 0.336 using Chi-square test. The kappa value was 0.444, which indicated a medium agreement between these two brushes, and the sensitivity of the Qi brush was higher than that of the Cervex-Brush® Combi, with significant statistical difference (P = 0.039<0.05). Conclusions: The Qi brush was more effective than the Cervex-Brush® Combi for sampling and also had a slightly higher accuracy in diagnosing in cytology. In terms of social and economic benefits, the Qi brush may be a better cervical cytology collector.

Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.

Accumulated unfolded proteins in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR) to increase ER protein folding capacity. ER proteostasis and UPR signaling need to be regulated in a precise and timely manner. Here, we identify phosphorylation of protein disulfide isomerase (PDI), one of the most abundant and critical folding catalysts in the ER, as an early event during ER stress. The secretory pathway kinase Fam20C phosphorylates Ser357 of PDI and responds rapidly to various ER stressors. Phosphorylation of Ser357 induces an open conformation of PDI and turns it from a "foldase" into a "holdase", which is critical for preventing protein misfolding in the ER. Phosphorylated PDI also binds to the lumenal domain of IRE1α, a major UPR signal transducer, and attenuates excessive IRE1α activity. Importantly, PDI-S359A knock-in mice display enhanced IRE1α activation and liver damage under acute ER stress. We conclude that the Fam20C-PDI axis constitutes a post-translational response to maintain ER proteostasis and plays a vital role in protecting against ER stress-induced cell death.

Development of a highly efficient and specific L-theanine synthase.

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Validation of Molecular Typing for Endometrial Screening Test That Predicts Benign and Malignant Lesions.

The aim of this study is to examine the immunocytochemical expression of p53, Ki-67, and CA125 in endometrial brush samples for endometrial cancer. Forty-four patients were recruited with liquid-based cytology preparations during a 5-month period. Both the histological and cytological samples were assessed by histology based on hematoxylin and eosin (H&E), and the expression of p53, CA125, and Ki-67 in endometrial cells was examined by immunocytochemistry. The percentage and intensity of endometrial cells were scored on a scale of 0-3. The final score was calculated by the addition of all partial scores, and then Probit model was used to predict the possibility for malignant lesions. The mean immunoreactivity score of the three immunocytochemical biomarkers (p53, CA125, and Ki-67) in the positive group (including atypical hyperplastic cells and malignant cells) was significantly higher than in the negative group (benign cells and non-atypical hyperplastic cells). The possibility value of the positive group was also significantly higher than the negative group (P < 0.05). The cutoff value of the possibility value was 0.754, the sensitivity and specificity of which were 86.4 and 95.5%. The assessment of p53, CA125, and Ki-67 combined with the prediction model is valuable for the detection of endometrial cancer and atypical hyperplasia in endometrial cytology.

Targeting the functional interplay between endoplasmic reticulum oxidoreductin-1α and protein disulfide isomerase suppresses the progression of cervical cancer.

BACKGROUND: Endoplasmic reticulum (ER) oxidoreductin-1α (Ero1α) and protein disulfide isomerase (PDI) constitute the pivotal pathway of oxidative protein folding, and are highly expressed in many cancers. However, whether targeting the functional interplay between Ero1α and PDI could be a new approach for cancer therapy remains unknown. METHODS: We performed wound healing assays, transwell migration and invasion assays and xenograft assays to assess cell migration, invasion and tumorigenesis; gel filtration chromatography, oxygen consumption assay and in cells folding assays were used to detect Ero1α-PDI interaction and Ero1α oxidase activity. FINDINGS: Here, we report that elevated expression of Ero1α is correlated with poor prognosis in human cervical cancer. Knockout of ERO1A decreases the growth, migration and tumorigenesis of cervical cancer cells, through downregulation of the H2O2-correlated epithelial-mesenchymal transition. We identify that the conserved valine (Val) 101 of Ero1α is critical for Ero1α-PDI complex formation and Ero1α oxidase activity. Val101 of Ero1α is specifically involved in the recognition of PDI catalytic domain. Mutation of Val101 results in a reduced ER, retarded oxidative protein folding and decreased H2O2 levels in the ER of cervical cancer cells and further impairs cell migration, invasion, and tumor growth. INTERPRETATION: Our study identifies the critical residue of Ero1α for recognizing PDI, which underlines the molecular mechanism of oxidative protein folding for tumorigenesis and provides a proof-of-concept for cancer therapy by targeting Ero1α-PDI interaction. FUND: This work was supported by National Key R&D Program of China, National Natural Science Foundation of China, and Youth Innovation Promotion Association, CAS.

Promiscuous enzymatic activity-aided multiple-pathway network design for metabolic flux rearrangement in hydroxytyrosol biosynthesis.

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.

Biomineralization Synthesis of the Cobalt Nanozyme in SP94-Ferritin Nanocages for Prognostic Diagnosis of Hepatocellular Carcinoma.

Nanomaterials with intrinsic enzyme-like activities (nanozymes) have emerged as promising agents for cancer theranostics strategies. However, size-controllable synthesis of nanozymes and their targeting modifications are still challenging. Here, we report a monodispersed ferritin-based cobalt nanozyme (HccFn(Co3O4)) that specifically targets and visualizes clinical hepatocellular carcinoma (HCC) tissues. The cobalt nanozyme is biomimetically synthesized within the protein shell of the HCC-targeted ferritin (HccFn) nanocage, which is enabled by the display of HCC cell-specific peptide SP94 on the surface of ferritin through a genetic engineering approach. The intrinsic peroxidase-like activity of HccFn(Co3O4) nanozymes catalyzes the substrates to make color reaction to visualize HCC tumor tissues. We examined 424 clinical HCC specimens and verified that HccFn(Co3O4) nanozymes distinguish HCC tissues from normal liver tissues with a sensitivity of 63.5% and specificity of 79.1%, which is comparable with that of the clinically used HCC-specific marker α fetoprotein. Moreover, the pathological analysis indicates that the HccFn(Co3O4) nanozyme staining result is a potential predictor of prognosis in HCC patients. Staining intensity is positively correlated to tumor differentiation degree ( P = 0.0246) and tumor invasion ( P < 0.0001) and negatively correlated with overall survival ( P = 0.0084) of HCC patients. Together, our study demonstrates that ferritin is an excellent platform for size-controllable synthesis and targeting modifications of nanozymes, and the HccFn(Co3O4) nanozyme is a promising reagent for prognostic diagnosis of HCC.