OBJECTIVEThe aim of this study was to establish a standard protocol for detection of EGFR mutations in cytologic specimens.

METHODS287 cytologic samples were collected from the patients who were suspected of having lung cancer at six hospitals in Beijing. A detection protocol for EGFR mutations was designed. Two comparative experiments were carried out for the coincidence in EGFR mutation rates between direct sequencing (Seq) and amplification refractory mutation system (ARMS) methods, and between 40 matched cytologic samples with formaldehyde-fixed paraffin embedded (FFPE) cytologic blocks and cytospin slides.

RESULTSTumor cells were found in 236 out of 287 cases (82.2%, 236/287) . Among them, there were 31 cases (13.1%, 31/236) of low tumor cell content samples and 205 cases (86.9%, 205/236) of high tumor cell content samples. 180 cases in the high tumor cell content samples (87.8%, 180/205) were diagnosed to be consistent with NSCLC. 25 out of 194 cases were ruled out or indefinite to be diagnosed as NSCLC by immunohistochemistry. By direct sequencing, the mutation rate of EGFR was 27.8% (50/180) in NSCLC samples and 28.2% (50/177) in adenocarcinoma samples (high tumor content samples) . By ARMS, the mutation rate of EGFR was 45.6% (82/180) in NSCLC samples and 46.3% (82/177) in adenocarcinoma samples (high tumor content samples). The EGFR mutation rate in low tumor content samples was 38.7% (12/31) , there was no significant difference in EGFR mutation rates between the groups of low tumor cell content samples and high tumor cell content samples (P = 0.12). The concordance rate of EGFR mutation rates was 100% between scraping tumor cells from slides samples and from FFEP blocks in the 40 matched samples. Forty-eight out of 180 definitive NSCLC patients received Gefitinib therapy. The FPS was 12 months in the gefitinib-treated ARMS⁺ group and 2 months in the ARMS⁻ group (P < 0.001), and the OS was 19 months in the gefitinib-treated ARMS⁺ group and 7 months in the ARMS⁻ group (P = 0.003), but no significant differences were found in the efficacy (PFS and OS) of Gefitinib between Seq⁺ and Seq⁻ groups (P = 0.227, P = 0.510, respectively), and Seq⁺/ARMS⁺ and Seq⁻/ARMS⁺ groups (P = 0.354, P = 0.334, respectively).

CONCLUSIONSThe detection protocol for EGFR mutations in cytological specimens introduced in this study is tested to be reliable and feasible. Pathological evaluation and immunohistochemistry are important in the detection procedure of EGFR mutations in cytologic specimens. High sensitivity methods should be selected for detection of EGFR mutations in cytologic samples.

Adenocarcinoma ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Humans ; Lung Neoplasms ; diagnosis ; epidemiology ; metabolism ; Mutation ; Mutation Rate ; Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor ; genetics ; metabolism

Objective: Network pharmacology combines drug and disease targets with biological information networks based on the integrity and systematicness of the interactions between drugs and disease targets. This study aims to explore the molecular basis of Hanshi Zufei formula for treatment of COVID-19 based on network pharmacology and molecular docking techniques. Methods: Using TCMSP, the chemical constituents and molecular targets of Atractylodis Rhizoma, Citri Reticulatae Pericarpium, Magnoliae Officinalis Cortex, Pogostemonis Herba, Tsaoko Fructus, Ephedrae Herba, Notopterygii Rhizoma et Radix, Zingiberis Rhizoma Recens, and Arecae Semen were investigated. The predicted targets of novel coronavirus were screened using the NCBI and GeneCards databases. To further screen the drug-disease core targets network, the corresponding target proteins were queried using multiple databases (Biogrid, DIP, and HPRD), a protein interaction network graph was constructed, and the network topology was analyzed. The molecular docking studies were also performed between the network's top 15 compounds and the coronavirus (SARS-CoV-2) 3CL hydrolytic enzyme and angiotensin conversion enzyme II (ACE2). Results: The herb-active ingredient-target network contained nine drugs, 86 compounds, and 49 drug-disease targets. Gene ontology (GO) enrichment analysis resulted in 1566 GO items (P < 0.05), among which 1438 were biological process items, 35 were cell composition items, and 93 were molecular function items. Fourteen signal pathways were obtained by enrichment screening of the KEGG pathway database (P < 0.05). The molecular docking results showed that the affinity of the core active compounds with the SARS-CoV-2 3CL hydrolase was better than for the other compounds. Conclusion: Several core compounds can regulate multiple signaling pathways by binding with 3CL hydrolase and ACE2, which might contribute to the treatment of COVID-19.

OBJECTIVE：To establish the fingerprint of ethanol extract and acetone extract from Anemarrhena asphodeloides and its different processed products ，and to investigate the spectrum-effect relationship between the fingerprint and the antioxidant activity. METHODS ：HPLC method and HPLC-ELSD method were adopted. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of acetonitrile- 0.2％ acetic acid at the flow rate of 1.0 mL/min. The column temperature was 30 ℃，and the detection wavelength was set at 258 nm. The sample size was 10 μL. The determination was performed on XDB-C 18 columnwith mobile phase consisted of acetonitrile-0.1％ acetic acid （gradient elution ）at the flow rate of 0.9 mL/min. The column temperature was 30 ℃ . The temperature of atomizer was 40 ℃ and the flow rare of N 2 was 1.6 mL/min. The sample size was 10 μL. Using mangiferin and timosaponin B Ⅱ as reference ，Fingerprint Similarity Eva- com luation System of TCM Chromatogram （2004A edition ）was adopted to draw the fingerprint of ethanol extract and acetoneextract from 20 batches of A. asphodeloides and its different processed products to confirm common peaks. Using scave nging rate of 1，1-diphenyl-2-trinitrophenylhydrazine（DPPH）radical as index，antioxidant activities of ethanol extract and acetone extract from 20 batches of A. asphodeloides and its processed products were investigated. Using scavenging rate of DPPH radical as dependent variable ，common peak area as independent variable ，PLSR was used to analyze the spectrum-effect relationship of ethanol extract and acetone extract from A. asphodeloides with antioxidantion activity. RESULTS ：Eight peaks （M1-M8）were identified in the fingerprints of ethanol extracts from 20 batches of processed A. asphodeloides . Mangiferin （chromatogram peak M 7）was identified with similarity of 0.389-1.000；seven comon peaks （S1-S7）and timosaponin B Ⅱ（peak S 5）were identified in the fingerprint of acetone extract ，and the similarity was 0.044-0.999. DPPH radical scavenging rate of ethanol extract from 20 batches of A. asphodeloides and its processed products was 21.23％- 81.39％，and A. asphodeloides was significantly lower than salt-processed A. asphodeloides with salt wine-processed A. asphodeloides （P＜0.001）；and that of acetone extract was 49.73％-83.78％，and A. asphodeloides was significantly higher than stir-baked A. asphodeloides with salt ，wine or fire （P＜0.001）. The standardized regression coefficients of peaks M 2-M7 in the spectrum of ethanol extract from A. asphodeloides were all greater than 0，which was positively correlated with antioxidant activity. Only the variable importance projection （VIP）value of peak M 7 was greater than 1，which had an important contribution. The standardized regression coefficients of peaks S 4-S7 in the acetone extract spectrum of A. asphodeloides were greater than 0，and were positively correlated with antioxidant activity. The order of VIP values was peak S 5＞S6＞S4，and the VIP values were all greater than 1. CONCLUSIONS：The fingerprint of the different processed products A. asphodeloides and its antioxidant activity spectral effect relationship were successfully established ；mangiferin（peak M 7）may be the main antioxidant substance of ethanol extract from A. asphodeloides . Timosaponin B Ⅱ（peak S 5），peak S 6 and peak S 4 may be the main antioxidant substance in acetone extract from A. asphodeloides .

Background The safety of drinking water is closely related to people's health. In recent years, relevant studies have identified some health related problems with drinking water in Inner Mongolia Autonomous Region. The complex and diverse natural environment embraced by the vast jurisdiction of the region may lead to uneven drinking water quality across the region. Objective To evaluate eight chemicals including arsenic, cadmium, chromium (hexavalent), lead, mercury, fluoride, trichloromethane, and carbon tetrachloride in urban drinking water in Inner Mongolia Autonomous Region in 2021, and to provide reference for optimizing urban water supply system and ideas for further developing strategies to promote population health. Methods A total of 1228 monitoring sites were set up in urban areas of Inner Mongolia, and water samples were collected once in dry season (May) and once in wet season (August−September). Eight chemicals of interest in drinking water were detected according to the Standard examination methods for drinking water, and assessed for health risks using the health risk assessment model recommended by the United States Environmental Protection Agency (USEPA) and following the Technical guide for environmental health risk assessment of chemical exposure. Mann-Whitney U test was used to compare the concentrations of eight chemicals in urban drinking water by water seasons and water sample types. Results In 2021, a total of 2381 samples of urban drinking water were tested in the Inner Mongolia Autonomous Region, including 1195 samples in wet season and 1186 samples in dry season; 389 samples of finished water and 1992 samples of tap water. The positive rates of arsenic and fluoride were 26.25% and 96.77%, respectively. The positive rates of cadmium, chromium (hexavalent), lead, mercury, trichloromethane, and carbon tetrachloride were 6.22%, 16.63%, 6.09%, 16.67%, 18.98%, and 8.36%, respectively. The exceeding standard rate of fluoride was 4.87%. Trichloromethane and carbon tetrachloride were qualified in all samples. There were statistical differences in the concentrations of arsenic, cadmium, chromium (hexavalent), lead, and carbon tetrachloride in urban drinking water between water seasons (Z=−3.847, P<0.05; Z=2.464, P=0.014; Z=−3.129, P=0.002; Z=4.341, P<0.05; Z=4.342, P<0.05). Only fluoride concentration was found statistically different among different water sample types (Z=−2.287, P=0.022). The non-carcinogenic risks of ingestion and dermal exposure to each chemical in drinking water by water seasons and water sample types were all less than 1, but the P₉₅ total non-carcinogenic risks of oral exposure were greater than 1. The P₉₅ carcinogenic risks of oral exposure to some chemicals in drinking water by water seasons and water sample types were>10⁻⁴, which suggested carcinogenic risks, while the carcinogenic risks of dermal explore to chemicals were all less than 10⁻⁶. Conclusion In 2021, urban drinking water in Inner Mongolia Autonomous Region is generally safe, but arsenic, cadmium, chromium (hexavalent), lead, mercury, and fluoride still exceed the national limits, posing certain health risks.