Effective annotation of in vivo drug metabolites using liquid chromatography-mass spectrometry (LC-MS) remains a formidable challenge. Herein, a metabolic reaction-based molecular networking (MRMN) strategy is introduced, which enables the "one-pot" discovery of prototype drugs and their metabolites. MRMN constructs networks by matching metabolic reactions and evaluating MS² spectral similarity, incorporating innovations and improvements in feature degradation of MS² spectra, exclusion of endogenous interference, and recognition of redundant nodes. A minimum 75% correlation between structural similarity and MS² similarity of neighboring metabolites was ensured, mitigating false negatives due to spectral feature degradation. At least 79% of nodes, 49% of edges, and 97% of subnetworks were reduced by an exclusion strategy of endogenous ions compared to the Global Natural Products Social Molecular Networking (GNPS) platform. Furthermore, an approach of redundant ions identification was refined, achieving a 10%-40% recognition rate across different samples. The effectiveness of MRMN was validated through a single compound, plant extract, and mixtures of multiple plant extracts. Notably, MRMN is freely accessible online at https://yaolab.network, broadening its applications.

Objective To investigate the effect and mechanism of circular RNA_0009910(circ_0009910)on the proliferation and apoptosis of non-small cell lung cancer cells.Methods A549 cells were randomly divided into groups and transfected as follows:si-NC group,si-circ_0009910 group,miR-NC group,miR-34a-5p group,si-circ_0009910+anti-miR-NC group,and si-circ_0009910+anti-miR-34a-5p group.After successful transfec-tion,cell proliferation was measured using the CCK-8 assay.Dual-luciferase reporter assays were used to vali-date the target gene of circ_0009910.Apoptosis of A549 cells was analyzed by flow cytometry.Protein expres-sion levels of B-cell lymphoma-2(Bcl-2),transmembrane receptor protein 1(Notch1),cell proliferation-asso-ciated nuclear antigen(Ki-67),Bcl-2-associated X protein(Bax),and cysteine aspartic acid protease-3(Caspase-3)were detected by Western blot.Cells from each group were subcutaneously injected into the abdo-mens of male nude mice to establish xenograft models.Tumor volume,tumor weight,and protein expression in tumor tissues were measured.Results Compared with the si-NC group,the si-circ_0009910 group showed significantly reduced A549 cell proliferation,increased apoptosis,decreased expression of Ki-67,Notch1,and Bcl-2,and increased expression of Bax and Caspase-3(P＜0.05).Compared with the miR-NC group,the miR-34a-5p group exhibited significantly reduced luciferase activity after transfection with wild-type circ_0009910(P＜0.05).The si-circ_0009910 group showed upregulated miR-34a-5p expression compared to the si-NC group.The miR-34a-5p group demonstrated weaker proliferation,higher apoptosis,downregulated Bcl-2,Notch1,and Ki-67,and upregulated Bax and Caspase-3 compared to the miR-NC group(P＜0.05).Compared with the si-circ_0009910+anti-miR-NC group,the si-circ_0009910+anti-miR-34a-5p group exhibited en-hanced proliferation,reduced apoptosis,upregulated Bcl-2,Notch1,and Ki-67,and downregulated Bax and Caspase-3(P＜0.05).In vivo,the si-circ_0009910 and miR-34a-5p groups showed smaller tumor volumes and weights,downregulated Notch1,Ki-67,and Bcl-2,and upregulated Bax and Caspase-3 in tumor tissues com-pared to their respective control groups(P＜0.05).Conclusion Low expression of circ_0009910 inhibits A549 cell proliferation and promotes apoptosis,potentially via the miR-34a-5p/Notch1 signaling pathway.

ObjectiveTo explore the association between short-term exposure to atmospheric fine particulate matter （PM_2.5） and ozone （O₃） and systemic inflammatory indicators in patients with pneumonia， and to identify the susceptible populations. MethodsFrom September 2018 to April 2020， data of 1 480 patients admitted for pneumonia was collected from a tertiary hospital in Taiyuan City. Generalized additive models （GAMs） were used to explore the associations between PM_2.5 and O₃ exposure and inflammatory indicators of patients with pneumonia； and to explore the susceptibility factors and susceptible populations to PM_2.5 and O₃ exposures through stratified analyses. ResultsThe short-term exposure to PM_2.5 was associated with changes in peripheral blood C-reation protein （CRP）， erythrocyte sedimentation （ESR）， easinophil （EOS）， neutrophil （NEU） and neutrophil-lymphocyte ratio （NLR） in patients with pneumonia， and there were different degrees of hysteresis effects， with the effect values reaching a maximum at lag03， lag03， lag0， lag03， lag03， respectively， which were 4.13% （95%CI： 0.43%‒7.84%）， 3.10% （95%CI： 0.24%‒5.97%）， 5.27% （95%CI： 3.12%‒7.42%）， 1.85% （95%CI： 0.36%‒3.34%）， and 2.53% （95%CI： 0.53%‒4.74%） for every 10 μg·m^-3 of PM_2.5. The changes in O₃ concentration were associated with the elevation of peripheral blood PCT and ESR in patients with pneumonia， and their effect values all reached the maximum at lag01 d， every 1 μg·m^-3 of O₃ elevation increased by 0.38% （95%CI： 0.04%‒0.73%） and 0.47% （95%CI： 0.19%‒0.76%）， respectively. Stratified analyses showed that the associations of PM_2.5 with peripheral blood CRP， ESR， NEU， and NLR in pneumonia patients were more significant in males， the elderly， and those with onset in the cold season； the associations of O₃ with peripheral blood PCT and ESR in pneumonia patients were more significant in the elderly and those with onset in the warm season， and the peripheral blood CRP and PCT in female patients with pneumonia were more susceptible to the changes of O₃. ConclusionShort-term exposure to atmospheric PM_2.5 and O₃ are positively associated with changes in inflammatory indicators in patients with pneumonia， and the effects of PM_2.5 on patients with pneumonia are more extensive than those of O₃， with a longer lag effect. In addition， elderly patients with pneumonia are more sensitive to air pollution， male patients with pneumonia are more sensitive to PM_2.5， and female patients with pneumonia are more sensitive to O₃. Cold and warm seasons can exacerbate the effects of PM_2.5 and O₃ on inflammatory indicators in patients with pneumonia， respectively， and the patients must be protected well.

Background: Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing’s syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Methods: Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected.A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing–Bablok regression and Bland–Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Results: Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r = 0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r = 0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LCMS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8–1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Conclusions Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Objective To investigate the expression of CKS1B in endometrial carcinoma(EC)and its relationship with clinicopathological features and prognosis.Methods The expression profile data and clinical data of CKS1B from the TCGA and GTEx databases were downloaded to investigate the expression of CKS1B in EC and its relationship with clinicopathological features.The expression of CKS1B at the protein level was verified using the UALCAN database.The relationship between CKS1B expression and clinicopathological parameters was analyzed by Logistic regression.The r program perform enrichment analysis on CKS1B co-expressed genes in the TCGA database.Finally,CKS1B mRNA expression was discovered in the cell lines Ishikawa and HEC-1-A by quantitative real-time PCR(qRT-PCR).CKS1B protein expression was detected in EC tissues and adjacent tissues by Western Bolt(WB).Results CKS1B mRNA and protein were remarkably higher in EC tissues than in normal endometrium,and the differences were statistically significant(P<0.05).The level of CKS1B mRNA expression was strongly correlated with FIGO stage(F=42.994),histological grade(F=70.350),histological type(F=87.341)and age(F=40.097)(all P<0.05).The results of the Kaplan-Meier method showed that patients with high CKS1B mRNA expression had a lower overall survival rate(Log-rank x2=1.175,P<0.01).Multifactorial COX analysis showed that FIGO stage(HR=3.065,95%CI:1.906～4.926)and CKS1B expression(HR=1.856,95%CI:1.154～2.985)were independent risk factors affecting the prognosis of patients with EC(P<0.05).GO analysis showed that CKS1B was mainly involved in nuclear division and chromosome separation.KEGG analysis showed it was mainly enriched in the cell cycle,spliceosome and DNA replication.Further verification showed that CKS1B mRNA was highly expressed in Ishikawa and HEC-1-A cell lines(F=44.560,P<0.001),CKS1B protein was highly expressed in EC tissues(t=14.900,P<0.001).Conclusion CKS1B is upregulated in EC and is linked to clinicopathological variables in the patients.It may play a role in the development of EC by regulating the cell cycle,and it is expected to be a new marker for the diagnosis and prognosis of EC.

Objective To investigate the mechanism by which lectin-like oxidized low density lipoprotein receptor-1(LOX-1)regulates hyperglycemic-induced myocardial fibroblast(CFs)fibrosis through the glycogen synthase kinase-3β(GSK-3β)/signal transducer and activator of transcription 3(STAT3)pathway.Methods CFs were isolated,cultured and identified.LOX-1 RNAi lentiviral vector was constructed and infected CFs.The experimental groups were as follows:Normal control(NC)group,High glucose(HG)group,LV-LOX-1,LV-Con group,Hypertonic(HPG)group.After LV-LOX-1 and LV-Con were infected with CFs,adding 25 mmol/L glucose to culture CFs for 24 h,they were denoted as HG+LV-LOX-1 group and HG+LV-Con group.Cells in HG+LV-LOX-1 group and HG+LV-Con group were treated with 10 μ mol/L SB216763 and 10 μ mol/L STATTIC for 24 h,respectively,and then they were recorded as HG+LV-LOX-1+SB216763 group,HG+LV-Con+SB216763 group,HG+LV-LOX-1+STATTIC group and HG+LV-Con+STATTIC group.CCK-8 was used to detect the activity of CFs,and the expression levels of mRAN and protein of LOX-1,collagen type I(COL-I),thioredoxin 5(TXNDC5),GSK-3β,STAT3,p-GSK-3β and p-STAT3 were detected by qRT-PCR and Western blot.Results CFs infected with LOX-1 RNAi lentiviral vector were obtained,which showed green under fluorescence microscopy.Compared with HG and HG+LV-Con groups,the mRNA expressions of LOX-1,COL-I and TXNDC5 were decreased in HG+LV-LOX-1 group(P<0.05).Compared with HG+LV-LOX-1 group,mRNA expressions of COL-I and TXNDC5 were decreased in HG+LV-LOX-1+SB216763 and HG+LV-LOX-1+STATTIC groups(P<0.05).Compared with HG and HG+LV-Con groups,p-GSK-3β protein expression was increased in HG+LV-LOX-1 group(P<0.05),while LOX-1,p-STAT3,COL-I,TXNDC5 protein expression was decreased in HG+LV-LOX-1 group(P<0.05).Compared with HG+LV-LOX-1 group,p-GSK-3β protein expression was increased in HG+LV-LOX-1+SB216763 group(P<0.05),while the protein expressions of p-STAT3,COL-I and TXNDC5 were decreased in HG+LV-LOX-1+SB216763 and HG+LV-LOX-1+STATTIC groups(P<0.05).Conclusion LOX-1,GSK-3β,STAT3,TXNDC5,and COL-I are involved in high glucose induced CFs fibrosis.LOX-1 promotes the expression of TXNDC5 and COL-I through GSK-3β/STAT3 pathway,and inhibition of LOX-1 can inhibit high glucose induced CFs fibrosis.

Objective:To investigate the effect of curcumin-chitosan microspheres on the inflammatory response in ulcerative colitis (UC) mice via the Toll like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) pathway.Methods:BALB/c mice were induced to establish a UC model by drinking 5% dextran sulfate sodium (DSS) aqueous solution. According to the random number table method, the mice were randomly divided into model group, empty chitosan microsphere group, curcumin group, curcumin-chitosan microsphere group and curcumin-chitosan microsphere+lipopolysaccharide group, with 12 mice in each group. 0.5% carboxymethyl cellulose solution, 19 mg/ml empty chitosan microsphere solution, 12 mg/ml curcumin solution, 19 mg/ml curcumin-chitosan microsphere solution by gavage, 19 mg/ml curcumin-chitosan microsphere solution by gavage+3 mg/ml lipopolysaccharide solution by intraperitoneal injection were administered at an administration volume of 5 ml/kg. Another 12 BALB/c mice drank purified water were taken as the control group. The disease activity index (DAI) score and colon length of mice in each group were determined. The permeability of the intestinal mucosal barrier of mice in each group was evaluated by detecting the level of fluorescein isothiocyanate (FITC)-labelled dextran. Hematoxylin-eosin (HE) staining was used to detect the pathological morphology of the colon tissue in each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory factors [interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β, IL-18] in serum and colon tissue of mice in each group. The expression levels of TLR4/MyD88/NF-κB pathway related proteins in colon tissues of mice in each group were detected by Western blotting.Results:The DAI score [(0.42±0.49) points] of the curcumin-chitosan microsphere group was lower than that of the model, curcumin, empty chitosan microsphere, and curcumin-chitosan microsphere+lipopolysaccharide groups [(3.50±0.26), (2.33±0.41), (2.83±0.35), and (3.33±0.25) points], and the length of colon [(6.17±0.38) cm] was greater than that of the model, curcumin, empty chitosan microsphere, and curcumin-chitosan microsphere+lipopolysaccharide groups [(3.89±0.23), (5.13±0.34), (4.78±0.31), and (4.01±0.27) cm], and the differences were statistically significant (all P<0.05). The FITC-labelled dextran level in the curcumin-chitosan microsphere group [(2.51±0.70) μg/ml] was lower than that in the model, curcumin, empty chitosan microsphere, and curcumin-chitosan microsphere+lipopolysaccharide groups [(10.13±0.71), (5.47±0.63), (4.52±0.60), (9.63±0.58) μg/ml], and the differences were statistically significant (all P<0.05). The villus height [(160.47±11.34) μm] of the curcumin-chitosan microsphere group was higher than that of the model, curcumin, empty chitosan microsphere, and curcumin-chitosan microsphere+ lipopolysaccharide groups [(102.13±7.65), (134.26±9.42), (124.85±8.36), and (106.94±7.81) μm], the mucosal thickness [(253.89±18.38) μm] was higher than that of the model, curcumin, empty chitosan microsphere, and curcumin-chitosan microsphere+lipopolysaccharide groups [(171.86±12.51), (218.34±13.12), (204.75±14.36), and (175.23±12.74) μm], and the differences were statistically significant (all P<0.05). The serum levels of IL-6, TNF-α, IL-1β, and IL-18 in the curcumin-chitosan microspheres group were (25.58±5.43), (49.37±7.82), (69.85±15.74), and (43.01±7.69) ng/L, respectively, which were lower than those of the model group [(130.69±10.24), (172.14±11.76), (214.54±25.72), and (165.85±18.74) ng/L], the curcumin group [(56.87±6.82), (98.76±10.65), (125.43±18.67), and (94.64±16.85) ng/L], the empty chitosan microspheres group [(68.24±8.67), (113.53±12.13), (149.76±20.35), and (117.93±15.63) ng/L], the curcumin-chitosan microspheres+lipopolysaccharide group [(121.75±9.89), (163.85±10.86), (203.12±24.86), and (154.12±18.54) ng/L], and the differences were statistically significant (all P<0.05). The levels of IL-6, TNF-α, IL-1β, and IL-18 in colon tissue of mice in the curcumin-chitosan microsphere group were (16.94±4.02), (31.12±4.25), (39.13±6.73), and (41.23±7.50) ng/g respectively, which were lower than those of the model group [(92.63±7.21), (114.32±8.24), (142.37±13.41), and (165.85±18.74) ng/g], the curcumin group [(46.86±6.25), (67.95±6.84), (75.20±8.64), and (84.93±11.84) ng/g], and the empty chitosan microsphere group [(57.12±6.73), (79.31±7.76), (89.76±10.52), and (98.74±13.62) ng/g], the curcumin-chitosan microspheres+lipopolysaccharide group [(86.53±7.46), (105.43±7.93), (133.81±12.95), and (154.90±18.39) ng/g], and the differences were statistically significant (all P < 0.05). The relative expression levels of TLR4 and MyD88 proteins and phosphorylated NF-κB p65 (p-NF-κB p65)/NF-κB p65 (0.32±0.07, 0.38±0.09, and 0.09±0.03) in the colon tissue of curcumin-chitosan microspheres group were lower than those of the model group (1.34±0.13, 1.40±0.16, and 0.89±0.09), the curcumin group (0.73±0.09, 0.79±0.11, and 0.39±0.05), the empty chitosan microsphere group (0.82±0.11, 0.89±0.13, and 0.47±0.08), the curcumin-chitosan microsphere+ lipopolysaccharide group (1.31±0.12, 1.36±0.14, and 0.87±0.08), and the differences were statistically significant (all P<0.05). Conclusions:Curcumin-chitosan microspheres can alleviate inflammation, reduce colon tissue damage, and restore intestinal mucosal barrier function in UC mice, which may be achieved by inhibiting TLR4/MyD88/NF-κB signaling activation.

Humans ; Consensus ; Pancreatic Neoplasms/therapy* ; Neuroendocrine Tumors/therapy*

OBJECTIVE To clarify the core content of traditional Chinese medicine （TCM） policy in the provinces of China， so as to provide reference for optimizing the structure of the policy system of traditional Chinese medicine in China and assisting the inheritance and innovation of TCM industry in various regions. METHODS The websites of directly affiliated organs in 31 provinces， excluding Hong Kong， Macao and Taiwan， were retrieved to collect the TCM policies released from 2000 to 2021. The importance of keywords in the TCM policies of each province was measured based on term frequency-inverse documentation frequency （TF-IDF） keyword extraction method， and the similarities and differences were analyzed among TCM policies. RESULTS & CONCLUSIONS A total of 99 documents related to TCM policies of various provinces were obtained in this study， most of which were released after 2016. The theme of national TCM policy covered four aspects： building TCM talent team， perfecting TCM service system， strengthening TCM resource management and promoting TCM industry innovation. The TF-IDF values of “medical institutions”“traditional Chinese medicine”“medical treatment” were higher than other keywords in each province， indicating that the provinces paid more attention to the construction of TCM service system and the management of TCM resources than other aspects. Anhui and Jiangsu， Beijing and Henan， Hubei and Jilin， Hubei and Tianjin， and Hubei and Yunnan had the more degree of similarity in TCM policies， which all contained 16 of the same keywords. Therefore， the above regions should be encouraged to strengthen exchanges and cooperation and realize mutual promotion and joint development. Among all the keywords whose importance ratio was greater than 0.2，“ Tibetan medicine” was unique to Qinghai and Tibet，“ disease type” was unique to Guangdong， and the TF-IDF value of “supervision and management” in Beijing was higher， indicating that the emphasis of TCM policy formulation in different provinces was various. Meanwhile， the top 10 keywords of TF- IDF value in all provinces did not have words related to financial input， and the TF-IDF values of “informatization” in most provinces did not rank at the top. It is suggested to increase financial input or encourage social financing， and add “Internet+new business” in the field of TCM.

Objective:To construct an entrustable professional activities (EPAs) assessment framework for undergraduate medical students suited to the national conditions in China.Methods:The Delphi method was used to construct an EPAs assessment framework for medical undergraduates. Twenty-one clinical experts across China were invited to participate in two rounds of Delphi consultation.Results:All the 21 experts completed the two rounds of Delphi consultation. The effective questionnaire response rates of the two rounds of consultation were 100.0% (21/21). For the first-round expert consultation, the W values of importance and feasibility scores of EPAs were 0.182 and 0.173 (both P<0.05), respectively. For the second-round expert consultation, the W values of importance and feasibility scores of EPAs were 0.167 and 0.152 (both P<0.05), respectively. According to the second-round Delphi consultation, the importance and feasibility scores of all 14 EPAs indicators were >3.5 points, with the coefficients of variation <0.25 points. We preliminarily established 12 EPAs indicators and 42 key assessment points and determined the expected entrustment levels of each EPA at different stages for medical undergraduates. Conclusion:This study preliminarily constructed an EPAs assessment framework for medical undergraduates, which provides a new evaluation method for the cultivation of medical undergraduates in China.