In this study, we constructed a GLP-2R-HEK293 cell line and established a method for the determination of the in vitro biological activity of teduglutide based on HTRF, after optimizing experimental conditions and methodological verification. We also carried out relative potency detection of teduglutide pharmaceutical products using this method. The result showed that there was a quantitative-efficient relationship between the teduglutide activity and cAMP contents in GLP-2R-HEK293 cells, which conformed to four-parameter model. Method verification results of five concentrations of teduglutide (64%, 80%, 100%, 125% and 156%) met the requirements of the General Rules of Chinese Pharmacopoeia, 2020 edition, Volume IV (9401). We then analyzed the relative potency of three batches of teduglutide drug substances and three batches of drug products. The linearity, regression and parallelism of the obtained curves all fit the system suitability requirements. The relative potency of six batches of teduglutide was from 83% to 105%. In summary, the biological activity detection method established in this study was accurate, precise, simple and time-saving, which can be used for quality control of teduglutide pharmaceutical products.

This study constructed a LHCGR-CRE-luc-HEK293 transgenic cell line according to the activation of the cAMP signaling pathway after recombinant human chorionic gonadotropin binding to the receptor. The biological activity of recombinant human chorionic gonadotropin was assayed using a luciferase assay system. The relative potency of the samples was calculated using four-parameter model. And the method conditions were optimized to validate the specificity, relative accuracy, precision and linearity of the method. The results showed that there was a quantitative potency relationship of human chorinonic gonadotropin (hCG) in the method and it was in accordance with the four-parameter curve. After optimization, the conditions were determined as hCG dilution concentration of 2.5 μg·mL^-1, dilution ratio of 1∶4, cell number of 10 000-15 000 cells/well, and induction time of 6 h. The method had good specificity, relative accuracy with relative bias ranging from -8.9% to 3.4%, linear regression equation correlation coefficient of 0.996, intermediate precision geometric coefficient of variation ranging from 3.3% to 15.0%, and linearity range of 50% to 200%. This study successfully established and validated a reporter gene method to detect hCG biological activity, which can be used for hCG biological activity assay and quality control.

Objective:To investigate the expression of soluble lectin-like oxidized low den-sity lipoprotein receptor-1(sLOX-1)and C1q/tumor necrosis factor related protein 3(CTRP3)in the serum of patients with lower extremity arteriosclerosis obliterans(ASO)and their relationship with restenosis(ISR)after stent intervention.Methods:From June 2019 to June 2022,106 ASO pa-tients who underwent stent intervention in our hospital were regarded as the study subjects.One year after surgery,they were separated into a non ISR group(n=64)and an ISR group(n=42)based on their onset of ISR.The serum levels of sLOX-1 and CTRP3 were compared between the two groups;multivariate Logistic regression was applied to analyze the influencing factors of ISR after stent intervention in ASO patients;ROC curve was applied to analyze the predictive value of se-rum sLOX-1 and CTRP3 levels for ISR after stent intervention in ASO patients.Results:The se-rum sLOX-1 level in the ISR group was obviously higher than that in the non ISR group(P＜0.05),and the CTRP31 level was obviously lower than that in the non ISR group(P＜0.05).The complete occlusion,and sLOX-1 were independent risk factors for ISR after stent intervention in ASO pa-tients,while CTRP3 was a protective factor for ISR after stent intervention in ASO patients(P＜0.05).The AUC of the combination of serum sLOX-1 and CTRP3 in predicting ISR after stent in-tervention in ASO patients was 0.944,with a sensitivity of 97.62％and a specificity of 79.69％,which was superior to the individual predictions of sLOX-1 and CTRP3(Zcombineddetaction-sLOX-1=2.732,Zcombineddetection-CTRP3=2.143,P=0.006,0.032).Conclusion:After ASO stent intervention,the serum level of sLOX-1 was obviously increased in ISR patients,while the level of CTRP3 was obviously reduced,and the combination of the two fatcors has high predictive value for the occurrence of ISR in ASO patients after stent intervention.

Objective To investigate the effects of α7 nicotinic acetylcholine receptor(nAChR)agonist on β3-adrenoceptor agonist-induced impairment of white fat homeostasis and beige adipose formation and heat production in obese mice.Methods Forty obese C57BL/6J mice were randomized into high-fat feeding group,β3-adrenoceptor agonist-treated model group,α7 nAChR agonist group,and α7 nAChR inhibitor group(n=10),with another 10 mice with normal feeding as the blank control group.White adipose tissue from the epididymis of the mice were sampled for HE staining of the adipocytes.The expression levels of TNF-α,IL-1β,IL-10 and TGF-β in the white adipose tissue were determined by ELISA,and the mRNA levels of iNOS,Arg1,UCP-1,PRDM-16 and PGC-1α were detected using RT-qPCR.Western blotting was performed to detect the expression levels of NF-κB P65,p-JAK2,p-STAT3 in the white adipose tissue.Results Compared with those in the blank control group,the mice with high-fat feeding showed significantly increased body weight,more fat vacuoles in the white adipose tissue,increased volume of lipid droplets in the adipocytes,upregulated iNOS mRNA expression and protein expression of TNF-α and IL-1β,and lowered expression of Arg-1 mRNA and IL-10 and TGF-β proteins(P<0.01).Treatment with α7 nAChR significantly reduced mRNA levels of PRDM-16,PGC-1α and UCP-1,lowered TNF-α and IL-1β expressions,increased IL-10 and TGF-β expressions,and reduced M1/M2 macrophage ratio in the white adipose tissues(P<0.05 or 0.01).Conclusion Activation of α7 nAchR improves white adipose tissue homeostasis impairment induced by β3 agonist,promotes transformation of M1 to M2 macrophages,reduces inflammatory response in white adipose tissue,and promote beige adipogenesis and thermogenesis in obese mice.

In this study, the CHO_INSR_1284 transgenic cell line was employed as the target cell, utilizing homogeneous time-resolved fluorescence technology to establish a method for detecting the biological activity of insulin degludec. Key parameters were optimized, and validation was conducted in accordance with general principles 9401 and 1431 of the fourth section of the 2020 edition of the Chinese Pharmacopoeia. Results indicated a good dose-response relationship for insulin degludec in this method, aligning with a four-parameter curve. Following optimization, the cell seeding density was set at 3.5×10⁵ cell·mL^-1, the initial concentration of insulin degludec at 57.18 μg·mL^-1, with a four-fold dilution, a stimulation period of 45 minutes, and an incubation duration of 4 hours. This method demonstrated strong specificity, with the geometric variation coefficient (GCV%) for the five potency levels ranging from 4.1% to 10.6%. The linear regression equation from the linear fitting was y = 1.015x - 0.027 7, and R² = 0.999 6. The results confirmed the method's good intermediate precision and linearity. The regression term was highly significant (P < 0.01), neither the deviation from parallel terms nor the model mismatch terms were significant (P ≥ 0.05), complying with general rule 1431 of the fourth section of the 2020 edition of the Chinese Pharmacopoeia. This study established a method for detecting the biological activity of insulin degludec using homogeneous time-resolved fluorescence technology, suitable for evaluating the biological activity and quality control of insulin degludec products.

The Dionex CaboPac^TM PA10 BioLC^TM Analyical 2 mm × 250 mm column was used with a protective column (Dionex CaboPac^TM PA10 BioLC^TM Guard 2 mm × 50 mm). 100 mmol·L^-1 sodium hydroxide solution was used as eluent; the flow rate was 0.25 mL·min^-1. Sample tray temperature: 35 ℃. The pulse amperometric detector was adopted, and the waveform was Gold CWE, Ag-AgCl RE, Carbo, Quad. The samples were cultured with 8 concentrations of glycogen substrates (0.31, 1.25, 2.5, 5, 10, 20, 30, and 40 mg·mL^-1). D-Glucose concentrations were measured at 5 different time points (T0, T1, T2, T3 and T4). The glucose concentration from T1 to T4 minus the glucose concentration at T0. The reaction rate was calculated at different glycogen substrate concentrations. These reaction rates are plotted against substrate concentrations using Michaelis-Menten equation. The kinetic parameters were expressed as V_max (nmol·mg^-1·min^-1) and K_m (mg·mL^-1). The RSD of glucose standard curve R² (n = 6, linear range: 1.25-500 μmol·L^-1) was 0.1% and the RSD (n = 6) of the slope of the standard curve was 2.2%. The mean limit of quantitation was 0.14 μmol·L^-1, and the mean limit of detection was 0.05 μmol·L^-1. The RSD of K_m and V_max were 4.4% and 4.6% respectively in three separate experiments. The durability of the method was good. The method was developed for the on-line automatic determination of the hydrolysis kinetics of acid α-glucosidase (GAA) for injection by ion chromatography. The method has good precision, repeatability and durability, and can be used for the determination of glycogen hydrolysis kinetics of GAA for injection, and could reference value for the enzyme kinetics evaluation of recombinant enzyme replacement therapy.

According to the requirements of the regulatory authorities, degree of modification (DP) should be included in the characterisation of the PEGylated protein drug substance, and is one of the critical quality attributes for quality control. In this study, based on the fundamental assumption that the refractive index (RI) signal and the ultraviolet (UV) signal of PEGylated protein are equal to the sum of the corresponding signal produced by the polyethylene glycol (PEG) and protein parts of the conjugates in their uncoupled state, we developed a method to determine the DP of PEGylated recombinant human growth hormone (inpegsomatropin). In this method, 20 μL of 1 mg·mL^-1 human growth hormone (hGH) standard, 2 mg·mL^-1 PEG reference substance and 1 mg·mL^-1 drug substance solution were each injected to size exclusion chromatographic (SEC) column for separation, detected with ultraviolet and refractive index (UV-RI) detectors in series. Finally, the DP was calculated as the formula derived from the fundamental assumption. The developed SEC-UV-RI method showed good specificity, repeatability (RSD = 0.63%, n = 9) and accuracy, with a recovery of 100.0%, compared with the result obtained from the simultaneously established classical acid hydrolysis method, demonstrating pretty handleability and accessibility, and is appropriate for quality control test of DP for this drug substance.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in tertiary hospitals in major regions of China in 2022.Methods Clinical isolates from 58 hospitals in China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2022 Clinical &Laboratory Standards Institute(CLSI)breakpoints.Results A total of 318 013 clinical isolates were collected from January 1,2022 to December 31,2022,of which 29.5％were gram-positive and 70.5％were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species(excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi)was 28.3％,76.7％and 77.9％,respectively.Overall,94.0％of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 90.8％of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis showed significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 94.2％in the isolates from children and 95.7％in the isolates from adults.The resistance rate to carbapenems was lower than 13.1％in most Enterobacterales species except for Klebsiella,21.7％-23.1％of which were resistant to carbapenems.Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.1％to 13.3％.The prevalence of meropenem-resistant strains decreased from 23.5％in 2019 to 18.0％in 2022 in Pseudomonas aeruginosa,and decreased from 79.0％in 2019 to 72.5％in 2022 in Acinetobacter baumannii.Conclusions The resistance of clinical isolates to the commonly used antimicrobial agents is still increasing in tertiary hospitals.However,the prevalence of important carbapenem-resistant organisms such as carbapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a downward trend in recent years.This finding suggests that the strategy of combining antimicrobial resistance surveillance with multidisciplinary concerted action works well in curbing the spread of resistant bacteria.

Objective To investigate the distribution and antimicrobial resistance profiles of the common pathogens isolated from urine from 2015 to 2021 in the CHINET Antimicrobial Resistance Surveillance Program.Methods The bacterial strains were isolated from urine and identified routinely in 51 hospitals across China in the CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021.Antimicrobial susceptibility was determined by Kirby-Bauer method,automatic microbiological analysis system and E-test according to the unified protocol.Results A total of 261 893 nonduplicate strains were isolated from urine specimen from 2015 to 2021,of which gram-positive bacteria accounted for 23.8％(62 219/261 893),and gram-negative bacteria 76.2％(199 674/261 893).The most common species were E.coli(46.7％),E.faecium(10.4％),K.pneumoniae(9.8％),E.faecalis(8.7％),P.mirabilis(3.5％),P.aeruginosa(3.4％),SS.agalactiae(2.6％),and E.cloacae(2.1％).The strains were more frequently isolated from inpatients versus outpatients and emergency patients,from females versus males,and from adults versus children.The prevalence of ESBLs-producing strains in E.coli,K.pneumoniae and P.mirabilis was 53.2％,52.8％and 37.0％,respectively.The prevalence of carbapenem-resistant strains in E.coli,K.pneumoniae,P.aeruginosa and A.baumannii was 1.7％,18.5％,16.4％,and 40.3％,respectively.Lower than 10％of the E.faecalis isolates were resistant to ampicillin,nitrofurantoin,linezolid,vancomycin,teicoplanin and fosfomycin.More than 90％of the E.faecium isolates were ressitant to ampicillin,levofloxacin and erythromycin.The percentage of strains resistant to vancomycin,linezolid or teicoplanin was＜2％.The E.coli,K.pneumoniae,P.aeruginosa and A.baumannii strains isolated from ICU inpatients showed significantly higher resistance rates than the corresponding strains isolated from outpatients and non-ICU inpatients.Conclusions E.coli,Enterococcus and K.pneumoniae are the most common pathogens in urinary tract infection.The bacterial species and antimicrobial resistance of urinary isolates vary with different populations.More attention should be paid to antimicrobial resistance surveillance and reduce the irrational use of antimicrobial agents.

Objective To understand the distribution and changing resistance profiles of clinical isolates of Enterococcus in hospitals across China from 2015 to 2021.Methods Antimicrobial susceptibility testing was conducted for the clinical isolates of Enterococcus according to the unified protocol of CHINET program by automated systems,Kirby-Bauer method,or E-test strip.The results were interpreted according to the Clinical & Laboratory Standards Institute(CLSI)breakpoints in 2021.WHONET 5.6 software was used for statistical analysis.Results A total of 124 565 strains of Enterococcus were isolated during the 7-year period,mainly including Enterococcus faecalis(50.7％)and Enterococcus faecalis(41.5％).The strains were mainly isolated from urinary tract specimens(46.9％±2.6％),and primarily from the patients in the department of internal medicine,surgery and ICU.E.faecium and E.faecalis strains showed low level resistance rate to vancomycin,teicoplanin and linezolid(≤3.6％).The prevalence of vancomycin-resistant E.faecalis and E.faecium was 0.1％and 1.3％,respectively.The prevalence of linezolid-resistant E.faecalis increased from 0.7％in 2015 to 3.4％in 2021,while the prevalence of linezolid-resistant E.faecium was 0.3％.Conclusions The clinical isolates of Enterococcus were still highly susceptible to vancomycin,teicoplanin,and linezolid,evidenced by a low resistance rate.However,the prevalence of linezolid-resistant E.faecalis was increasing during the 7-year period.It is necessary to strengthen antimicrobial resistance surveillance to effectively identify the emergence of antibiotic-resistant bacteria and curb the spread of resistant pathogens.