Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, which increases the incidence of colorectal cancer (CRC). In the pathophysiology of IBD, ubiquitination/deubiquitination plays a critical regulatory function. Josephin domain containing 2 (JOSD2), a deubiquitinating enzyme, controls cell proliferation and carcinogenesis. However, its role in IBD remains unknown. Colitis mice model developed by dextran sodium sulfate (DSS) or colon tissues from individuals with ulcerative colitis and Crohn's disease showed a significant upregulation of JOSD2 expression in the macrophages. JOSD2 deficiency exacerbated the phenotypes of DSS-induced colitis by enhancing colon inflammation. DSS-challenged mice with myeloid-specific JOSD2 deletion developed severe colitis after bone marrow transplantation. Mechanistically, JOSD2 binds to the C-terminal of inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and preferentially cleaves K63-linked polyubiquitin chains at the K134 site, suppressing IMPDH2 activity and preventing activation of nuclear factor kappa B (NF-κB) and inflammation in macrophages. It was also shown that JOSD2 knockout significantly exacerbated increased azoxymethane (AOM)/DSS-induced CRC, and AAV6-mediated JOSD2 overexpression in macrophages prevented the development of colitis in mice. These outcomes reveal a novel role for JOSD2 in colitis through deubiquitinating IMPDH2, suggesting that targeting JOSD2 is a potential strategy for treating IBD.

OBJECTIVES: To verify whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiotoxicity by reducing inflammation via regulating the AMPK/NLRP3 pathway. METHODS: C57/bl6 mice and H9c2 cells treated with DOX to mimic cardiotoxicity were randomly divided into Sham (or control) group, DOX group, DOX+Hes group, DOX+Hes+compound C (CC, an AMPK inhibitor) group. Cardiac function and myocardial pathologies of the mice were evaluated, and the changes in H9c2 cell morphology and viability were assessed. Lactate dehydrogenase (LDH) activity in mouse myocardial tissues and H9c2 cells was measured using ELISA, and H9c2 cell apoptosis was detected with TUNEL staining. In both H9c2 cells and the myocardial tissues of the mice, cellular expression levels of TNF-α, IL-6 and IL-1β mRNAs and cleaved caspase-3, Bcl2, Bax, IL-1β, IL-18, p-AMPK, AMPK, p-mTOR, mTOR, NLRP3, ASC and caspase-1 proteins were detected using RT-PCR and Western blotting. RESULTS: DOX treatment caused cell swelling, decreased cell viability and increased LDH activity in H9c2 cells, resulting also in significantly increased cell apoptosis and cleaved caspase-3 expression and decreased Bcl2/Bax ratio. The DOX-treated mice showed obvious myocardial fiber swelling and inflammatory infiltration, decreased cardiac function and significantly increased myocardial LDH activity. In H9c2 cells, DOX treatment significantly increased the mRNA expressions of TNF-α, IL-6 and IL-1β and protein expressions of IL-1β and IL-18, lowered the expressions of p-AMPK and p-mTOR, and increased the expressions of NLRP3, ASC and caspase-1. Hes treatment obviously reduced these toxic effects of DOX in H9c2 cells, but its protective effects were blocked by application of compound C. CONCLUSIONS Hes reduces DOX-induced cardiotoxicity by inhibiting inflammation via regulating the AMPK/NLRP3 pathway.
Animals ; Doxorubicin/toxicity* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Mice, Inbred C57BL ; Mice ; Signal Transduction/drug effects* ; Cardiotoxicity ; AMP-Activated Protein Kinases/metabolism* ; Apoptosis/drug effects* ; Cell Line ; Myocytes, Cardiac/drug effects* ; Rats

In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

ObjectiveBased on non-targeted metabolomics， to analyze the regulation of endogenous differential metabolites in serum of type 2 diabetes mellitus（T2DM） rats by Fuling Yunhua granules， and to clarify the metabolic pathways through which this granules exerted its effect on improving T2DM. MethodSeventy SD rats， half male and half female， were randomly divided into the control group， model group， and high， medium， low dose groups of Fuling Yunhua granules（20.70， 10.35， 5.18 g·kg^-1 in raw drug amount） and the positive drug group（pioglitazone hydrochloride tablets， 8.1 mg·kg^-1）. Except for the control group， other groups were fed with high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin（STZ） to establish a T2DM rat model. After successful modeling， the treatment groups were administered the corresponding drugs by gavage， and the control group and model group were treated with an equal volume of saline by gavage， once/d， for 28 d. Fasting blood glucose（FBG） and glycosylated hemoglobin A1c（GHbA1c） levels were measured in all groups of rats during the administration period， and hematoxylin-eosin（HE） staining was used to observe the pathomorphological changes in the pancreatic tissues of rats at the end of the administration period. The endogenous metabolite levels in rat serum were detected by ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-LTQ-Orbitrap MS）， and the data were processed using principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）. Differential metabolites were identified by the Human Metabolome Database（HMDB） and the Kyoto Encyclopedia of Genes and Genomes（KEGG）， and screened for differential metabolites with variable importance in the projection（VIP） value>1， P<0.05， and fold change（FC）<0.6 or FC>1. And the metabolic pathway enrichment analysis of the screened differential metabolites was performed by MetaboAnalyst 5.0， then the screened differential metabolites were diagnosed and evaluated by the receiver operating characteristic（ROC） curves. ResultCompared with the control group， the FBG level of rats in the model group increased significantly（P<0.01）， the GHbA1c content tended to increase， but the difference was not statistically significant， and the pancreatic tissue of rats was obviously damaged， the number of pancreatic islets decreased， and the pancreatic β-cells were obviously reduced， atrophied and enlarged. Compared with the model group， the FBG levels of rats in the high dose group of Fuling Yunhua granules and the positive drug group were significantly reduced after 2 weeks of administration（P<0.05， P<0.01）， the GHbA1c content of rats in the high dose group of Fuling Yunhua granules was significantly reduced（P<0.05）， and the pancreatic tissue lesions of rats in the different dose groups of Fuling Yunhua granules were reduced. The results of non-targeted metabolomics showed that 46 differential metabolites were significantly changed in the model group compared with the blank group. Pathway enrichment analysis found that T2DM mainly affected biological processes including biosynthesis of primary bile acid， D-amino acid metabolism， steroid hormone biosynthesis， and glycerophospholipid metabolism in rats. Compared with the model group， the levels of 8 differential metabolites in the high dose group of Fuling Yunhua granules were significantly adjusted， and the pathway enrichment analysis found that D-amino acid metabolism， retinol metabolism， glycine， serine and threonine metabolism， tryptophan metabolism and other metabolic pathways were mainly involved. ROC curves further analysis revealed that the four characteristic differential markers of 11-cis-retinol， D-piperidinic acid， D-serine， and p-cresol sulfate had high diagnostic value for the treatment of T2DM with Fuling Yunhua granules. ConclusionFuling Yunhua granules can improve the symptoms of T2DM rats by regulating the amino acid metabolic and retinol metabolic pathways through the modulation of endogenous differential metabolites.

Objective:To study the expression and clinical significance of alpha-1 antitrypsin (A1AT) in the serum of patients with antiphospholipid syndrome (APS).Methods:The study recruited 131 patients with APS, 48 patients with other autoimmune diseases (8 patients with rheumatoid arthritis, 8 with osteoarthritis, and 32 with systemic erythematous sores), and 49 healthy people were recruited. The patients were admitted to Peking University People's Hospital during January 2019 to June 2022. A1AT expression in the serum of patients with APS and its clinical significance were investigated. Blood samples were collected and the concentration of A1AT in the samples was determined by enzyme-linked immunosorbent assay (ELISA). The correlation between A1AT and clinical and laboratory parameters of APS patients was analyzed. Statistical analysis and graphing were performed using GraphPadPrism 10.1.2. The categorical variables were subjected to the χ2 test, and the continuous variables were subjected to the normal distribution test. If the sample were normally distributed, the independent sample t-test (with homogeneity of variance) or the Welch's t-test (with heterogeneity of variance) was used for comparison between the 2 groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups; Otherwise, and the variables were described as M( Q1, Q3), the Mann Whitney U-test was used for comparison between 2 groups, and the Kruskal-Wallis U-test was used for comparison among multiple groups. The Kruskal-Wallis H test was used for multiple comparisons. If the samples were normally distributed, Spearman correlation analysis was used to determine the correlation, otherwise, Pearson correlation analysis was used. Results:The serum A1AT concentrations were significantly higher in APS patients than in patients with other autoimmune diseases [2 048.0(670.6, 2 904.0) μg/ml vs. 1 099.0(0, 1 855.0) μg/ml, U=1 990, P<0.001] and healthy people [2 048.0(670.6, 2 904.0) μg/ml vs. 739.5 (0, 1 232.0) μg/ml, U=1 485, P<0.001]. No statistically significant difference was observed between patients with other autoimmune diseases and healthy people [1 099.0(0, 1 855.0) μg/ml vs. 739.5 (0, 1 232.0) μg/ml, U=924, P=0.060]. Mean serum A1AT concentrations were also higher in patients with both a history of adverse pregnancy and thrombosis than in those with morbid pregnancy only [(3 212 ±1 744)μg/ml vs. (1 965 ±1 500) μg/ml, t=2.27, P=0.026] and thrombosis only [(3 212 ±1 744)μg/ml vs. (1 963 ±1745)μg/ml, t=2.01, P=0.048]. Mean serum A1AT concentrations were higher in patients with both arterial and venous thrombosis than in those with only venous thrombosis [(3 390 ±2 286) μg/ml vs. (2 148 ±1 648) μg/ml, t=3.04, P=0.004]. The mean A1AT concentration was higher in patients with recurrent thrombosis than in patients with single thrombosis [(2 709 ±1 941) μg/ml vs. (1 805 ±1 627) μg/ml, t=2.10, P=0.040]. Using the 95% upper limit of A1AT concentration in healthy controls (1 066 μg/ml) as a cut-off value, the risk of recurrent thrombosis was higher in A1AT-positive than negative TAPS patients [51.0%(25/49) vs. 26.1%(6/23), χ2=3.97, P=0.046]. In terms of laboratory indicators, there was a significant positive correlation between serum A1AT concentration and ESR level ( r=0.28, P=0.045), a significant negative correlation with C4 level ( r=-0.24, P=0.025). There was a significant positive correlation with fibrinogen (FIB) concentration( r=0.25, P=0.027). A1AT was an effective diagnostic marker of APS [AUC(95% CI)=0.769(0.699, 0.847), P<0.001], with a sensitivity of 71.8%, a specificity of 73.7%, and Youden index of 0.452. Conclusion:A1AT was is significantly elevated in the serum of patients with APS and may be associated with the severity of thrombotic event.

Objective:Taking the main international collaborative innovation network management modes for clinical research as a reference, to promote the construction of a collaborative innovation network for clinical research in China.Methods:Using literature research, case study, comparative analysis, and interview methods, this study systematically studied the collaborative innovation network management modes of clinical research in the United States, the United Kingdom, Japan, South Korea, and China from the perspective of integrating and sharing clinical research resources.Results:Based on the comparative results and drawing on foreign experience, suggestions were proposed to optimize the management mode of the clinical research collaborative innovation network in China and promote the construction of clinical research collaborative innovation network.Conclusions:It is recommended to establish national medical research institutions, increase investment in clinical research funds, build a platform for integrating volunteer resources based on clinical research collaborative innovation networks, and establish a clinical sample resource service platform to further supplement and improve the structure and layout of China’s clinical research collaborative innovation network.

In the central nervous system,CX3CL1 is a transmembrane chemokine expressed on neurons,and its specific receptor CX3CR1 is located on microglia.The combination of CX3CL1 and CX3CR1 is regarded as the bond of interaction between neurons and microglia.Bidirectional communication between microglia and neurons is essential for maintaining brain homeostasis and overcoming neuroinflammation,which is thought to play an important role in neurode-generative and cerebrovascular diseases.This article reviews the mechanisms of CX3CL1/CX3CR1 signaling pathway in the proliferation,metabolism,activation,polarization and regulation of synaptic plasticity of microglia,and uses this sig-naling pathway as a target to regulate the role of microglia in the disease process,which may provide a new target for the treatment of inflammation-related diseases.

OBJECTIVE To establish a quantitative analysis of multi-components by single marker （QAMS） method for simultaneous determination of 10 ganoderic acids in Ganoderma lucidum. METHODS Using ganoderic acid A as internal reference， high-performance liquid chromatography （HPLC） method was adopted to calculate relative correction factors of the other 9 components， such as ganoderic acid B， ganoderic acid C2， ganoderic acid D， ganoderic acid F， ganoderic acid H， ganoderenic acid A， ganoderenic acid B， ganoderenic acid C， ganoderenic acid D； the contents of above ganoderic acids were calculated with relative correction factors， and compared with the results of external standard method. RESULTS The linear relationship of ganoderic acid A， ganoderic acid B， ganoderic acid C2， ganoderic acid D， ganoderic acid F， ganoderic acid H， ganoderenic acid A， ganoderenic acid B， ganoderenic acid C and ganoderenic acid D were 0.032-3.996， 0.040-4.971， 0.037-4.568， 0.028-3.558， 0.033-4.177， 0.044-5.440， 0.032-3.944， 0.040-4.994， 0.045-5.593 and 0.035-4.342 mg/mL （all R 2≥0.999 2）， respectively. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 2%. Their average recovery rates were 99.43%， 100.25%， 98.50%， 99.88%， 100.59%， 99.64%， 98.50%， 99.40%， 99.64% and 99.76%， respectively （RSD＜2%， n＝6）. Relative correction factors of ganoderic acid B， ganoderic acid C2， ganoderic acid D， ganoderic acid F， ganoderic acid H， ganoderenic acid A， ganoderenic acid B， ganoderenic acid C and ganoderenic acid D were 1.788 5， 1.288 2， 1.126 4， 1.698 5， 0.885 4， 5.468 1， 4.210 9， 5.780 8， 4.290 3， respectively. Relative errors between the content obtained by QAMS method and external standard method for G. lucidum from different origins were within ±12%. CONCLUSIONS It is feasible that the contents of 10 ganoderic acids are determined simultaneously by QAMS method， using ganoderic acid A as internal reference. This method shows good precision and reproducibility and can be used for the quality control of G. lucidum.

Objective To explore the predictive value of contrast-enhanced echocardiography com-bined with serum levels of CD137 and insulin-like growth factor binding protein 6(IGFBP-6)for cardiovascular adverse events(MACE)in elderly patients with stable coronary heart disease(CHD)after percutaneous coronary intervention(PCI).Methods A total of 108 elderly patients with stable CHD(CHD group)who visited Department of Cardiology of Changsha First Hospital from March 2020 to March 2022 were recruited in this study.They were grouped into a non-MACE group(81 cases)and a MACE group(27 cases)according to whether MACE occurred after PCI.Another 100 healthy individuals who taking physical examination during the same period served as control group.Their serum CD137 and IGFBP-6 levels were detected,and the contrast agent filling speed(β value)and maximum number of microbubbles(A value)were calculated based on the results of contrast-enhanced echocardiography.Their general clinical data were col-lected.ROC curve analysis and multivariate logistic regression analysis were used to analyze the data.Results The serum levels of CD137 and IGFBP-6 were significantly higher,while the β value and A value were obviously lower in the CHD group than the control group(P＜0.01).And the serum levels were notably higher,and the β value and A value were remarkably lower in the MACE group than the non-MACE group(P＜0.01).The AUC of cardiac ultrasound parameters βvalue and A value combined with serum CD137 and IGFBP-6 to predict MACE after PCI in CHD patients was 0.930,which was significantly higher than the AUC value of every single indicator(P＜0.01).β value,A value,CD137 and IGFBP-6 levels were all risk factors for the occurrence of MACE in CHD patients after PCI(P＜0.01).Conclusion Contrast-enhanced echocardiography,serum CD137 and IGFBP-6 levels have certain predictive value for MACE in elderly CHD patients after PCI,and combined detection has higher predictive value.

Plant metabolites are important for plant development and human health. Plants of celery (Apiumgraveolens L.) with different-colored petioles have been formed in the course of long-term evolution. However, the composition, content distribution, and mechanisms of accumulation of metabolites in different-colored petioles remain elusive. Using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), 1159 metabolites, including 100 lipids, 72 organic acids and derivatives, 83 phenylpropanoids and polyketides, and several alkaloids and terpenoids, were quantified in four celery cultivars, each with a different petiole color. There were significant differences in the types and contents of metabolites in celery with different-colored petioles, with the most striking difference between green celery and purple celery, followed by white celery and green celery. Annotated analysis of metabolic pathways showed that the metabolites of the different-colored petioles were significantly enriched in biosynthetic pathways such as anthocyanin, flavonoid, and chlorophyll pathways, suggesting that these metabolic pathways may play a key role in determining petiole color in celery. The content of chlorophyll in green celery was significantly higher than that in other celery cultivars, yellow celery was rich in carotenoids, and the content of anthocyanin in purple celery was significantly higher than that in the other celery cultivars. The color of the celery petioles was significantly correlated with the content of related metabolites. Among the four celery cultivars, the metabolites of the anthocyanin biosynthesis pathway were enriched in purple celery. The results of quantitative real-time polymerase chain reaction (qRT-PCR) suggested that the differential expression of the chalcone synthase (CHS) gene in the anthocyanin biosynthesis pathway might affect the biosynthesis of anthocyanin in celery. In addition, HPLC analysis revealed that cyanidin is the main pigment in purple celery. This study explored the differences in the types and contents of metabolites in celery cultivars with different-colored petioles and identified key substances for color formation. The results provide a theoretical basis and technical support for genetic improvement of celery petiole color.
Anthocyanins ; Apium/metabolism* ; Chlorophyll/metabolism* ; Color ; Gene Expression Regulation, Plant ; Humans ; Metabolomics ; Plant Proteins/genetics* ; Tandem Mass Spectrometry