Objective To develop a gas chromatography-tandem mass spectrometry(GC-MS/MS)method for the simultaneous determination of ephedrine and pseudoephedrine in urine.Methods Urine samples containing ephedrine and pseudoephedrine components were extracted with ethyl acetate,centrifuged to collect the supernatant and evaporated to dryness under a nitrogen stream and then derivatized with heptafluorobutyric anhydride 60 μL at 70 ℃ for 30 min,and re-evaporated under nitrogen,and then solubilized with 50 μL of methanol,and then analyzed by GC-MS/MS.Results The method demonstraed excellent linearity for ephedrine(0.05～10 μg/mL,r=0.999 8)and pseudoephedrine(0.02～5 μg/mL,r=0.999 5).Extraction recoveries ranged from 89.4％～95.8％(ephedrine)and 90.3％～93.8％(pseudoephedrine).Limits of detection and quantification of ephedrine and pseudoephedrine were 0.005 μg/mL and 0.01 μg/mL,the intra-day precision and accuracy were less than 5.87％and 9.56％,respectively,and the inter-day precision and accuracy were less than 7.54％and 9.27％,respectively.The stability of ephedrine and pseudoephedrine in urine in 15 d was good under the conditions of room temperature and-20 ℃.Conclusion The GC-MS/MS analytical method for the analysis of ephedrine and pseudoephedrine components in urine established in this study is accurate,stable and sensitive,which can provide data technical support for the forensic toxicological analysis of amphetamine-type drugs or new psychoactive substances in the cathinone group.

Objective:To analyze the effect of nucleotide sequence variants in the 5′ untranslated region (5′UTR) of Helicobacter pylori (Hp) cagA on mRNA secondary structure, as well as its regulatory role in cytotoxin-associated gene A (CagA) protein expression and bacterial virulence. Methods:The upstream nucleotide sequence of cagA was amplified by polymerase chain reaction (PCR) from 37 Hp strains, and the PCR products were sequenced. MEGA 5.0 software and RNAfold prediction software were used to analyze the nucleotide sequence variants of cagA 5′UTR and the changes in mRNA secondary structure of this region, respectively. Western blot was used to detect the expression level of CagA protein in Hp strains, and the regulatory effect of cagA 5′UTR variants on the difference in CagA protein expression was analyzed. An Hp-infected AGS cell model was established to evaluate bacterial adhesion rate; quantitative PCR (qPCR) was used to analyze the mRNA transcription levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α); enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of IL-8 and TNF-α proteins. Results:Nucleotide sequence alignment of cagA 5′UTR from 37 Hp strains showed that sequence differences were mainly concentrated in the -53motif, -10motif, + 34motif, and + 86motif regions. mRNA secondary structure prediction analysis revealed three types based on the StemB stem-loop structure: type Ⅰ (no StemB stem-loop), type ⅡA (StemB stem-loop with 3-4 base partial pairing), and type ⅡB (StemB stem-loop with 5 base full pairing). Western blot analysis showed that the CagA protein expression level was the highest in type Ⅰ Hp strains (1.72±0.29) and the lowest in type ⅡB strains (0.81±0.26), with a statistically significant difference between the two types ( P=0.030). The adhesion rate of type Ⅰ Hp strains to AGS cells was (52.90±11.17)%, which was higher than that of type Ⅱ strains [(21.27±6.16)%]. qPCR results showed that the mRNA transcription levels of IL-8 and TNF-α in AGS cells induced by type Ⅰ Hp strains were higher than those induced by type Ⅱ strains (140.23±24.47 vs 76.16±8.76, P=0.069; 55.20±9.04 vs 21.26±6.16, P=0.036). ELISA analysis further indicated that the secretion levels of IL-8 and TNF-α proteins in AGS cells induced by type Ⅰ Hp strains were also higher than those induced by type Ⅱ strains [(344.66±62.62)pg/ml vs (302.13±66.27)pg/ml, P=0.665; (131.04±4.94)pg/ml vs (79.17±11.32)pg/ml, P=0.014]. Conclusions:The cagA 5′UTR region of Hp strains exhibits significant nucleotide sequence variants. Hp strains with no StemB stem-loop (type Ⅰ) in the mRNA secondary structure show significantly increased CagA protein expression and higher bacterial pathogenic potential.

The epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation is a rare subtype of mutations in non-small cell lung cancer (NSCLC). Patients with advanced NSCLC carrying the EGFR ex20ins mutation tend to have poor responses to traditional EGFR tyrosine kinase inhibitors (TKIs), chemotherapy, and immunotherapy, leading to a poor clinical prognosis. Significant progress has been made in the development of new drugs targeting the EGFR ex20ins mutation. The research on new drugs targeting EGFR ex20ins mutations has made significant progress. The main ones include new EGFR-TKIs (such as sunvozertinib, mobocertinib, and furmetinib, etc.), bispecific antibodies (such as amivantamab, JMT101, and GB263T, etc.), and emerging drugs such as AUY922. These agents have demonstrated promising efficacy in clinical trials, improving the objective response rate and progression-free survival of patients, and are expected to improve overall survival. An in-depth analysis of the mechanism of action and clinical trial progress of these novel targeted drugs for EGFR ex20ins-mutated NSCLC can offer new therapeutic strategies for patients with EGFR ex20ins-mutated NSCLC.

Objective To investigate the transgenerational effects of paternal PM2.5 exposure on offspring growth and development,and to preliminarily elucidate the role of sperm DNA methylation modifications in mediating these effects.Methods Eight-week-old male C57BL/6 mice were randomly divided into filtered air(FA),unfiltered air(UA),and concentrated PM2.5(CAP)groups,with 10 animals in each group.The exposure was conducted from November 2019 to April 2020,and then,these male mice were mated with unexposed females to generate F1 offspring,which were bred successively to produce F2 and F3 generations.All the offspring were living in PM2.5-free environment.The birth body weight,birth number,and sex ratio of the offspring were recorded,body weight growth was monitored,and organ coefficients of the heart,liver,lung,and brain were calculated.Whole-genome methylation sequencing was performed on the sperm DNA of the CAP group,FA group,and their F1 generation offspring to screen for differentially methylated regions,and the genes and pathways associated with these regions were analyzed.Results When compared with the F1～F3 offspring of the FA group,the CAP group had significantly reduced birth body weight in the F1 generation(P<0.05),no statistical differences were observed in the birth body weight in the F2 and F3 generations(P>0.05),or either in the sex ratio and birth number among the F1,F2 and F3 generations.Compared with the FA group offspring,the F1～F3 offspring of CAP group exhibited delayed body weight gain,especially in the males(P<0.05),the CAP-F1 male generation had obviously elevated liver organ coefficient(P<0.01),but no statistical changes were observed in the heart,lung,or brain coefficients among the F1～F3 generations.Between the FA group and the CAP group,37 997 differentially methylated regions were detected,with a reduction of approximately 50%in the number of differentially methylated regions in the F1 generation.Differentially methylated genes in F0 and F1 sperm were potentially related to developmental processes,including imprinting genes(Gnas,Igf2)and metabolic genes(Ppard,Rps6kb1).Conclusion Paternal exposure to PM2.5 leads to reduced birth weight and intergenerational growth retardation in offspring.Its impact on phenotypic effects is gradually weakened during intergenerational transmission.Changes in the methylation of development-related genes in sperm may be one of the mechanisms mediating this intergenerational effect.

Objective This study aimed to investigate the effects of sleep interruption(SI)cycles on emotional behavior in ICR mice,and to establish a mouse model of comorbid anxiety and depression induced by SI.Methods Seventy-two male ICR mice(4～5 weeks old)were divided randomly into a blank group and a model group.Mice in the model group were subjected to SI stress modeling for 1,2,and 3 weeks,respectively.After modeling,emotional behaviors were evaluated using open-field,elevated plus maze,light-dark box,marble-burying,and forced-swimming tests.Serum corticosterone levels were detected by enzyme-linked immunosorbent assay.Results Mice in the model group buried significantly more marbles after 1 week of SI stress,compared with the blank group(P＜0.05).After 2 weeks of stress,mice in the model group also showed a significant decrease in the number of crossings in the light-dark box(P＜0.05)and a significant increase in the number of marbles buried(P＜0.01)compared with the control group.After 3 weeks of stress,mice in the model group showed a significant increase in the number of marbles buried(P＜0.05),a significant decrease in the number of crossings in the light-dark box(P＜0.05),and a significant increase in immobility time in the forced-swim test(P＜0.01).Conclusions ICR mice exhibited significant anxiety-related behaviors after 2 weeks of SI modeling and significant anxiety-and depressive-related behavioral changes after 3 weeks.Three weeks of SI stress can be used to establish a model of comorbid anxiety and depression.

Objective To develop a gas chromatography-tandem mass spectrometry(GC-MS/MS)method for the simultaneous determination of ephedrine and pseudoephedrine in urine.Methods Urine samples containing ephedrine and pseudoephedrine components were extracted with ethyl acetate,centrifuged to collect the supernatant and evaporated to dryness under a nitrogen stream and then derivatized with heptafluorobutyric anhydride 60 μL at 70 ℃ for 30 min,and re-evaporated under nitrogen,and then solubilized with 50 μL of methanol,and then analyzed by GC-MS/MS.Results The method demonstraed excellent linearity for ephedrine(0.05～10 μg/mL,r=0.999 8)and pseudoephedrine(0.02～5 μg/mL,r=0.999 5).Extraction recoveries ranged from 89.4％～95.8％(ephedrine)and 90.3％～93.8％(pseudoephedrine).Limits of detection and quantification of ephedrine and pseudoephedrine were 0.005 μg/mL and 0.01 μg/mL,the intra-day precision and accuracy were less than 5.87％and 9.56％,respectively,and the inter-day precision and accuracy were less than 7.54％and 9.27％,respectively.The stability of ephedrine and pseudoephedrine in urine in 15 d was good under the conditions of room temperature and-20 ℃.Conclusion The GC-MS/MS analytical method for the analysis of ephedrine and pseudoephedrine components in urine established in this study is accurate,stable and sensitive,which can provide data technical support for the forensic toxicological analysis of amphetamine-type drugs or new psychoactive substances in the cathinone group.

Objective This study aimed to investigate the effects of sleep interruption(SI)cycles on emotional behavior in ICR mice,and to establish a mouse model of comorbid anxiety and depression induced by SI.Methods Seventy-two male ICR mice(4～5 weeks old)were divided randomly into a blank group and a model group.Mice in the model group were subjected to SI stress modeling for 1,2,and 3 weeks,respectively.After modeling,emotional behaviors were evaluated using open-field,elevated plus maze,light-dark box,marble-burying,and forced-swimming tests.Serum corticosterone levels were detected by enzyme-linked immunosorbent assay.Results Mice in the model group buried significantly more marbles after 1 week of SI stress,compared with the blank group(P＜0.05).After 2 weeks of stress,mice in the model group also showed a significant decrease in the number of crossings in the light-dark box(P＜0.05)and a significant increase in the number of marbles buried(P＜0.01)compared with the control group.After 3 weeks of stress,mice in the model group showed a significant increase in the number of marbles buried(P＜0.05),a significant decrease in the number of crossings in the light-dark box(P＜0.05),and a significant increase in immobility time in the forced-swim test(P＜0.01).Conclusions ICR mice exhibited significant anxiety-related behaviors after 2 weeks of SI modeling and significant anxiety-and depressive-related behavioral changes after 3 weeks.Three weeks of SI stress can be used to establish a model of comorbid anxiety and depression.

Objective:To construct bacterial cytoplasmic membrane nanovesicles(BMV)with overexpressing programmed death 1(PD-1),denoted as BMV-PD-1 and evaluate the targeting efficacy of BMV-PD-1 towards transplanted lung tumor tissues in mice.Methods:The fusion plasmid ClyA-PD-1-EGFP fused by PD-1 and Cytolysin A(ClyA)was transferred into Escherichia coli BL21-Codonplus through plasmid transformation.Laser confocal microscopy,SDS-PAGE,and WB were used to detect the expression of the fusion protein ClyA-PD-1-EGFP.Bacterial membranes were extracted and processed with an extruder to generate BMV-PD-1.TEM and NTA were utilized to assess the morphology,size distribution,and zeta potential of BMV-PD-1,while WB was used to verify the presence of PD-1 protein.Laser confocal imaging was conducted to monitor the uptake of BMV-PD-1 by Lewis lung cancer cells.A C57BL/6J mouse subcutaneous transplant tumor model of LLC lung cancer cells was constructed,and the tumor targeting of BMV-PD-1 was evaluated by small animal imaging system.Results:Laser confocal microscopy images demonstrated that the plasmid ClyA-PD-1-EGFP was transferred into BL21-Codonplus and successfully expressed into protein.SDS-PAGE results suggested that ClyA-PD-1-EGFP was overexpressed in BL21-Codonplus.WB analysis indicated that PD-1 was expressed in bacteria and highly expressed in BMV-PD-1(P<0.001).NTA and TEM analyses revealed that BMV-PD-1 were spherical vesicles with a diameter of(145±14)nm and a negative surface charge.Laser confocal imaging showed that the high expression of PD-1 significantly increased the uptake of BMV-PD-1 by lung cancer cells(P<0.01).In vivo imaging of small animals further confirmed that the high expression of PD-1 can effectively improve cancer targeting of BMV-PD-1(P<0.01).Conclusion:In this study,bacterial plasma membrane nanovesicles BMV-PD-1 with high PD-1 expression are successfully constructed,and it is found that PD-1 overexpression markedly improve the mouse lung cancer xenograft tissue targeting specificity of BMV-PD-1,laying the groundwork for further development of BMV-PD-1 as a carrier for targeted drug delivery systems in tumors.

Objective:To analyze the effect of nucleotide sequence variants in the 5′ untranslated region (5′UTR) of Helicobacter pylori (Hp) cagA on mRNA secondary structure, as well as its regulatory role in cytotoxin-associated gene A (CagA) protein expression and bacterial virulence. Methods:The upstream nucleotide sequence of cagA was amplified by polymerase chain reaction (PCR) from 37 Hp strains, and the PCR products were sequenced. MEGA 5.0 software and RNAfold prediction software were used to analyze the nucleotide sequence variants of cagA 5′UTR and the changes in mRNA secondary structure of this region, respectively. Western blot was used to detect the expression level of CagA protein in Hp strains, and the regulatory effect of cagA 5′UTR variants on the difference in CagA protein expression was analyzed. An Hp-infected AGS cell model was established to evaluate bacterial adhesion rate; quantitative PCR (qPCR) was used to analyze the mRNA transcription levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α); enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of IL-8 and TNF-α proteins. Results:Nucleotide sequence alignment of cagA 5′UTR from 37 Hp strains showed that sequence differences were mainly concentrated in the -53motif, -10motif, + 34motif, and + 86motif regions. mRNA secondary structure prediction analysis revealed three types based on the StemB stem-loop structure: type Ⅰ (no StemB stem-loop), type ⅡA (StemB stem-loop with 3-4 base partial pairing), and type ⅡB (StemB stem-loop with 5 base full pairing). Western blot analysis showed that the CagA protein expression level was the highest in type Ⅰ Hp strains (1.72±0.29) and the lowest in type ⅡB strains (0.81±0.26), with a statistically significant difference between the two types ( P=0.030). The adhesion rate of type Ⅰ Hp strains to AGS cells was (52.90±11.17)%, which was higher than that of type Ⅱ strains [(21.27±6.16)%]. qPCR results showed that the mRNA transcription levels of IL-8 and TNF-α in AGS cells induced by type Ⅰ Hp strains were higher than those induced by type Ⅱ strains (140.23±24.47 vs 76.16±8.76, P=0.069; 55.20±9.04 vs 21.26±6.16, P=0.036). ELISA analysis further indicated that the secretion levels of IL-8 and TNF-α proteins in AGS cells induced by type Ⅰ Hp strains were also higher than those induced by type Ⅱ strains [(344.66±62.62)pg/ml vs (302.13±66.27)pg/ml, P=0.665; (131.04±4.94)pg/ml vs (79.17±11.32)pg/ml, P=0.014]. Conclusions:The cagA 5′UTR region of Hp strains exhibits significant nucleotide sequence variants. Hp strains with no StemB stem-loop (type Ⅰ) in the mRNA secondary structure show significantly increased CagA protein expression and higher bacterial pathogenic potential.

ObjectiveTo explore the syndrome elements and evolving patterns of patients with hepatitis B virus-related acute on chronic liver failure （HBV-ACLF） at different stages. MethodsClinical information of 1,058 hospitalized HBV-ACLF patients， including 618 in the early stage， 355 in the middle stage， and 85 in the late stage， were collected from 18 clinical centers across 12 regions nationwide from January 1， 2012 to February 28， 2015. The “Hepatitis B-related Chronic and Acute Liver Failure Chinese Medicine Clinical Questionnaire” were designed to investigate the basic information of the patients， like the four diagnostic information （including symptoms， tongue， pulse） of traditional Chinese medicine （TCM）， and to count the frequency of the appearance of the four diagnostic information. Factor analysis and cluster analysis were employed to determine and statistically analyze the syndrome elements and patterns of HBV-ACLF patients at different stages. ResultsThere were 76 four diagnostic information from 1058 HBV-ACLF patients， and 53 four diagnostic information with a frequency of occurrence ≥ 5% were used as factor analysis entries， including 36 symptom information， 12 tongue information， and 5 pulse information. Four types of TCM patterns were identified in HBV-ACLF， which were liver-gallbladder damp-heat pattern， qi deficiency and blood stasis pattern， liver-kidney yin deficiency pattern， and spleen-kidney yang-deficiency pattern. In the early stage， heat （39.4%， 359/912） and dampness （27.5%， 251/912） were most common， and the pattern of the disease was dominated by liver-gallbladder damp-heat pattern （74.6%， 461/618）； in the middle stage， dampness （30.2%， 187/619） and blood stasis （20.7%， 128/619） were most common， and the patterns of the disease were dominated by liver-gallbladder damp-heat pattern （53.2%， 189/355）， and qi deficiency and blood stasis pattern （27.6%， 98/355）； and in the late stage， the pattern of the disease was dominated by qi deficiency （26.3%， 40/152） and yin deficiency （20.4%， 31/152）， and the patterns were dominated by qi deficiency and blood stasis pattern （36.5%， 31/85）， and liver-gallbladder damp-heat pattern （25.9%， 22/85）. ConclusionThere are significant differences in the distribution of syndrome elements and patterns at different stages of HBV-ACLF， presenting an overall trend of evolving patterns as "from excess to deficiency， transforming from excess to deficiency"， which is damp-heat → blood stasis → qi-blood yin-yang deficiency.