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.

The early response of plant auxin gene family Aux/IAA (auxin/indole-3-acetic acid) and its interaction with auxin response factor (ARF) are important pattern to regulate plant growth and development. This work identified 28 StoIAA and 24 StoARF members based on the whole genome data of the medicinal plant Senna tora L., which were classified into 10 and 8 subfamilies, respectively. Phylogenetic tree and collinearity analysis showed that S. tora has close evolutionary relationship with the IAA and ARF homologous genes of Glycine max, Medicago truncatula, and the segment duplication events dominate the expansion of StoIAA and StoARF. Gene structure analysis showed that the vast majority of StoIAA and StoARF contain characteristic conserved domain. Transcriptome data showed that StoIAAs and StoARFs were expressed in leaves, roots and seeds, some members had tissue specific expression. The StoIAA and StoARF promoter region most contain functional elements related to stress response, growth and development, hormone induction and secondary metabolism. In addition, gene expression analysis showed that many StoIAAs and StoARFs can quickly respond to drought and salt stress and exhibited same expression patterns under both stress condition. The yeast two-hybrid experiment confirmed that StoARF8 and StoARF10 exhibit varying degrees of interaction with multiple StoIAA proteins, respectively. The above results provide a basis for further biological functional analysis of the Aux/IAA and ARF gene family of S. tora.

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 study the application of CE-Chirp in the evaluation of hearing impairment in forensic medicine by testing the auditory brainstem response(ABR)in adults using CE-Chirp to ana-lyze the relationship between the V-wave response threshold of CE-Chirp ABR test and the pure tone hearing threshold.Methods Subjects(aged 20-77 with a total of 100 ears)who underwent CE-Chirp ABR test in Changzhou De'an Hospital from January 2018 to June 2019 were selected to obtain the V-wave response threshold,and pure tone air conduction hearing threshold tests were conducted at 0.5,1.0,2.0 and 4.0 kHz,respectively,to obtain pure tone listening threshold.The differences and statistical differences between the average pure tone hearing threshold and V-wave response threshold were compared in different hearing levels and different age groups.The correlation,differences and statistical differences between the two tests at each frequency were analyzed for all subjects.The lin-ear regression equation for estimating pure tone hearing threshold for all subjects CE-Chirp ABR V-wave response threshold was established,and the feasibility of the equation was tested.Results There was no statistical significance in the CE-Chirp ABR response threshold and pure tone hearing threshold dif-ference between different hearing level groups and different age groups(P>0.05).There was a good correlation between adult CE-Chirp ABR V-wave response threshold and pure tone hearing threshold with statistical significance(P<0.05),and linear regression analysis showed a significant linear correla-tion between the two(P<0.05).Conclusion The use of CE-Chirp ABR V-wave response threshold can be used to evaluate subjects'pure tone hearing threshold under certain conditions,and can be used as an audiological test method for forensic hearing impairment assessment.

Objective To establish a rapid screening method for 34 emerging contaminants in surface water by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS).Methods The pretreatment conditions of solid phase extraction(SPE)were op-timized by orthogonal experimental design and the surface water samples were concentrated and ex-tracted by Oasis? HLB and Oasis? MCX SPE columns in series.The extracts were separated by Kine-tex? EVO C18 column,with gradient elution of 0.1%formic acid aqueous solution and 0.1%formic acid methanol solution.Q-TOF-MS'fullscan'and'targeted MS/MS'modes were used to detect 34 emerging contaminants and to establish a database with 34 emerging contaminants precursor ion,prod-uct ion and retention times.Results The 34 emerging contaminants exhibited good linearity in the con-centration range respectively and the correlation coefficients(r)were higher than 0.97.The limit of de-tection was 0.2-10 ng/L and the recoveries were 81.2%-119.2%.The intra-day precision was 0.78%-18.70%.The method was applied to analyze multiple surface water samples and 6 emerging contaminants were detected,with a concentration range of 1.93-157.71 ng/L.Conclusion The method is simple and rapid for screening various emerging contaminants at the trace level in surface water.

Thrombosis is a key factor that increases the mortality rate of COVID-19 patients and causes long COVID sequelae. Guanxinning Tablet (GXNT), which is composed of Salvia miltiorrhiza and Ligusticum Chuanxiong, has significant antithrombotic activity, but the similarities and differences between its anti-conventional thrombus and microthrombus induced by COVID-19 remain unclear. In this paper, the main active components, potential targets and mechanisms of GXNT in the treatment of thrombus and microthrombus caused by COVID-19 were preliminarily revealed by using anti-platelet experiments in vitro, network pharmacology analysis, molecular docking technology and molecular biology experiments. The results of platelet aggregation and adhesion experiments in vitro showed that GXNT had significant anti-platelet aggregation and adhesion activities in a dose-dependent manner. Using network pharmacology analysis, it was revealed that salvianolic acid B, tanshinone IIA, caffeic acid and ligustrazine in GXNT could resist thrombus and microthrombus caused by COVID-19 through key targets as the high mobility group box 1 protein (HMGB1), tumor necrosis factor (TNF), interleukin 6 (IL6) and AKT serine/threonine kinase 1 (AKT1). HMGB1 signaling pathway is one of its key common mechanisms. Western blot also indicated that GXNT significantly inhibited the expression of HMGB1 protein in platelets. In summary, this paper explores the similarities and differences between the mechanism of GXNT against conventional thrombus and microthrombus caused by COVID-19 and provides drug reference and theoretical basis for clinical prevention and treatment of long COVID sequelae. The animal experiment has been approved by the Experimental Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (No. TCM-LAEC2023187g1549).

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.

In this study, we established a novel bioassay to determine the activity of polyethylene glycolated recombinant human growth hormone (PEG-rhGH) using Nb2-11 cells. We performed experimental condition optimization and methodological verification, and then detected the relative potency of PEG-rhGH products using this method. We demonstrated that the bioactivity of PEG-rhGH in promoting Nb2-11 cell proliferation displays a dose-response relationship, which conformed to the four-parameter model. Using PEG-rhGH reference as a control, we analyzed the relative potency of six batches of PEG-rhGH products, as well as linearity, regression and parallelism of the obtained curves. The relative potency of six batches of PEG-rhGH products was 95% to 105%. These results implied that the new bioassay established may be employed in quality control of PEG-rhGH products.

Objective To analyze the expression changes of related mRNA and microRNA(miRNA)during spinal cord regeneration after tail amputation of Gekko japonicus, and to explore the biological effects of differentially expressed mRNA and miRNA during spinal cord regeneration. Methods Fifty Gekko japonicus, the tail amputation model of Gekko japonicus was constructed, divided into normal group, 15 days tail amputation group, and 25 days tail amputation group, 5 in each group, repeat the experiment 3 times, 5 spare. Samples of each group were collected, RNA of each group was extracted and high-throughput sequencing. Bioinformatics analysis identifies differentially expressed mRNA and miRNA between groups, Gene Ontology(GO) enrichment analysis of differentially expressed mRNA functional annotations, and construction of miRNA and mRNA gene regulatory networks related to spinal cord regeneration. Results The differential expression of mRNA and miRNA in the normal and newborn spinal cords of Gekko japonicus was analyzed by sequencing. The 15 days and 25 days tail amputation groups identified 538 and 510 differential mRNA expressions and 446, 127 differential miRNA expressions, respectively. GO analysis found that the differentially expressed mRNA aggregated in biological processes related to cell proliferation and neurodevelopment. In the spinal cord regeneration-related miRNA and its target gene regulatory network, 21 mRNA expression was down-regulated in the 15 days tail amputation group, which was regulated negatively by 41 up-regulated miRNAs; 12 mRNA expression was up-regulated and was regulated by 29 down-regulated miRNAs. In the 25 days tail amputation group, 8 mRNA expression was down-regulated and regulated negatively by 10 up-regulated miRNAs; 20 mRNA expression was up-regulated and regulated by 32 down-regulated miRNAs. Conclusion Through the analysis of the differential expression of miRNA and mRNA in the regenerated spinal cord of Gekko japonicus, the expression changes of mRNA and miRNA in spinal cord regeneration were initially revealed, which provided experimental data for elucidating the molecular mechanism of spinal cord regeneration.