Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

Aim To establish a reliable in vitro evaluation sys-tem to assess human liver UGTs activities and the inhibitory effect of drugs on human liver UGTs and validate its accuracy and stability.Methods Six probe substrate of the six major subtypes of human liver microsomal UGT enzymes were select-ed.The activity of human liver microsomal UGT enzymes was determined under optimized incubation conditions using a metab-olite generation method.The concentrations of metabolites were determined using a validated liquid chromatography-tandem mass spectrometry(LC-MS/MS)method.Known inhibitors were used to verify the established incubation system.Results The estab-lished LC-MS/MS analysis method complied with the require-ments of bioanalysis.In the incubation system of this experi-ment,known UGT inhibitors exhibited significant inhibitory effects.Conclusions This study successfully establishes an ac-curate and reliable in vitro evaluation system,which can be used to assess the in vitro inhibition of drugs on UGT enzymes,provi-ding important guidance for drug development and rational clini-cal medication.

This study was aimed at investigating the effect of lymphocyte activation gene-3(LAG3)on liver B lymphocyte subsets and their functions in WT and LAG3-KO mice infected with Echinococcus multilocularis(E.multilocularis).In a mouse model of E.multilocularis infection,the expression and localization of CD19 and α-SMA in liver were detected by immu nohistochemistry.CD80,CD86 and MHC-Ⅱ molecules expressed on B cells and their subsets in mice liver were detected by flow cytometry.After 12 weeks of infection,the area and percentage of CD19 in LAG3-KO group was slightly higher than that in WT group,but the difference was not statistically(t=-1.241、-1.237,P＞0.05).The area and percentage of a-SMA in LAG3-KO group was higher than that in WT group(t=-3.224、-3.227,P＜0.05).The proportion of CD80 and MHC-Ⅱ molecules expressed on liver B cells in LAG3-KO group was up-regulated(t=-2.379,-3.321,P＜0.05).The percentage of liver B2 cells in LAG3-KO group was higher than that in WT group(t=-2.695,P＜0.05).The expression of CD80 on Blb cells in LAG3-KO group was significantly up-regulated(t=-5.315,P＜0.001).The proportion of CD80 of B2 cells in LAG3-KO group was lower than that in WT group(t=2.806,P＜0.05).The expression of MHC-Ⅱ molecule in B2 cells in LAG3-KO group was up-regulated(t=-4.227,P＜0.01).It is suggested that LAG3 molecules affected the B cell subsets and func-tion of mouse liver in the middle stage of E.multilocularis infection,especially B2 lymphocytes.LAG3 molecule exerted an in-hibitory effect on the activation of B cells and the expression of MHC-class Ⅱ molecules,suggesting that it may be involved in B cell exhaustion caused by E.multilocularis.

Oligoasthenozoospermia(OAS)is one of the most common types of male infertility,which,however,still lacks ef-fective treatment.An increasing number of studies have shown the potential therapeutic value of omega-3 polyunsaturated fatty acid(ω-3 PUFA)in the treatment of OAS.This article presents an overview of the studies on the effects of ω-3 PUFA on fatty acid composition and metabolism,inflammatory response,and oxidative stress in OAS,hoping to provide some new ideas for the treatment of the dis-ease.

Objective:To investigate the effects of Xiongcan Yishen Formula(XYF)on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury(OSI)induced by H2O2.Methods:An oxidative stress injury model was established in mouse TM3 Leydig cells using H2O2 induction.The modeled TM3 cells were randomly divided into OSI group,XYF group,the ferroptosis inhibitor Ferrostatin-1(F-1)group,and F-1+XYF group,which were respectively intervened with blank serum,20%drug-containing serum,2μmol/L F-1,and2μmol/L F-1+20%drug-containing serum.A control group(normal TM3 cells+blank serum)was also set up.The morphology of cells in each group was observed,and the levels of testosterone,superoxide dismutase(SOD),reactive oxygen spe-cies(ROS),malondialdehyde(MDA),ferritin heavy chain 1(FTH1),solute carrier family 7 member 11(SLC7A11),glutathione(GSH),glutathione peroxidase 4(GPX4),fatty acid CoA ligase 4(FACL4),total iron ions,and ferrous ions were detected.Re-sults:Compared with the model group,the control group showed significantly decreased expression of ROS,MDA,FACL4,total iron,and ferrous ions(P<0.05),and significantly increased levels of testosterone,SOD,GSH,FTH1,SLC7A11,and GPX4(P<0.05).The male silkworm kidney-tonifying formula group significantly promoted testosterone secretion by TM3 cells and upregulated the expression of FTH1,SLC7A11,GPX4,GSH,and SOD in TM3 cells(P<0.05),while significantly downregulating ROS,MDA,FACL4,total iron ions,and ferrous ions(P<0.05).Conclusion:Following H2O2 exposure,oxidative stress can induce ferroptosis in mouse TM3 Leydig cells.XYF can antagonize OSI and ferroptosis in TM3 cells by activating the SLC7A11/GSH/GPX4 axis,which may underlie the mechanism of XYF in the treatment of male late-onset hypogonadism.

Objective To dynamically observe the changes in hypoxia-inducible factor 1α(HIF-1α)and Bcl-2/adenovirus E1B19kDa-interacting protein 3(BNIP3)in children with traumatic brain injury(TBI)and evaluate their clinical value in predicting the severity and prognosis of pediatric TBI.Methods A prospective study included 47 children with moderate to severe TBI from January 2021 to July 2023,categorized into moderate(scores 9-12)and severe(scores 3-8)subgroups based on the Glasgow Coma Scale.A control group consisted of 30 children diagnosed and treated for inguinal hernia during the same period,with no underlying diseases.The levels of HIF-1α,BNIP3,autophagy-related protein Beclin-1,and S100B were compared among groups.The predictive value of HIF-1α,BNIP3,Beclin-1,and S100B for the severity and prognosis of TBI was assessed using receiver operating characteristic(ROC)curves.Results Serum levels of HIF-1α,BNIP3,Beclin-1,and S100B in the TBI group were higher than those in the control group(P＜0.05).Among the TBI patients,the severe subgroup had higher levels of HIF-1α,BNIP3,Beclin-1,and S100B than the moderate subgroup(P＜0.05).Correlation analysis showed that the serum levels of HIF-lα,BNIP3,Beclin-1,and S100B were negatively correlated with the Glasgow Coma Scale scores(P＜0.05).After 7 days of treatment,serum levels of HIF-1α,BNIP3,Beclin-1,and S100B in both non-surgical and surgical TBI patients decreased compared to before treatment(P＜0.05).ROC curve analysis indicated that the areas under the curve for predicting severe TBI based on serum levels of HIF-1α,BN1P3,Beclin-1,and S100B were 0.782,0.835,0.872,and 0.880,respectively(P＜0.05),and for predicting poor prognosis of TBI were 0.749,0.775,0.814,and 0.751,respectively(P＜0.05).Conclusions Serum levels of HIF-1α,BNIP3,and Beclin-1 are significantly elevated in children with TBI,and their measurement can aid in the clinical assessment of the severity and prognosis of pediatric TBI.IChinese Journal of Contemporary Pediatrics,2024,26(4):378-384]

Objective To observe the clinical efficacy of joint needling method combined with ultrasound in the treatment of qi stagnation and blood stasis type of patellofemoral pain syndrome(PFPS).Methods Eighty-six patients with qi stagnation and blood stasis type of PFPS were randomly divided into observation group and control group,with 43 cases in each group.The control group was given western medicine conventional treatment combined with functional exercise,and the observation group was given joint needling method combined with ultrasound treatment on the basis of the control group.Both groups were treated for 2 consecutive weeks.After 2 weeks of treatment,the clinical efficacy of the two groups was evaluated,and the changes in the Visual Analogue Scale(VAS)scores of knee pain and the Kujala scale scores of the two groups were observed before and after treatment.The changes in active range of motion(AROM)of the affected knee joint were compared before and after treatment between the two groups.Results(1)After treatment,the VAS scores of the two groups of patients were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the level of VAS scores,and the difference was statistically significant(P＜0.05).(2)After treatment,the Kujala scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the level of Kujala scores,and the difference was statistically significant(P＜0.05).(3)After treatment,the AROM of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the level of AROM,and the difference was statistically significant(P＜0.05).(4)The total effective rate was 95.35%(41/43)in the observation group and 81.40%(35/43)in the control group.The efficacy of the observation group was superior to that of the control group,and the difference was statistically significant(P＜0.05).Conclusion The joint needling method combined with ultrasound can significantly relieve the pain symptoms of patients with PFPS and promote the recovery of knee joint function,and the clinical efficacy is remarkable.

ObjectiveTo identify and analyze the possible influencing factors of obesity among public primary school students in Minhang District， Shanghai. MethodsBasic data， collected through questionnaire stars， was imported with merged physical examination data into Excel to form a database. Data were collected by questionnaire and analyzed by SPSS 22.00. Independent sample t-test was used for the data with normal distribution. Nonparametric test was used for the data with non-normal distribution. χ² test was used for the quantitative data. Logistic regression was used for univariate and multivariate analysis. ResultsAccording to the national physical health standards for students （revised in 2014） ， the prevalence rate of obesity among primary school students in Minhang district was 29.63% . The prevalence of overweight and obesity was 32.1% in boys and 26.9% in girls. The trend of overweight and obesity in different grades was similar among different genders .A single factor analysis showed that， being the only child （χ²=9.300， P<0.01） ， father obesity（χ²=106.417， P<0.001） or mother obesity（χ²=118.534， P<0.001） ， mother's overweight during pregnancy （χ²=159.731， P<0.001） ， caregiver's role as grandparents （χ²=38.736， P<0.001） ， low education level （χ²=111.309， P<0.001） had effects on overweight and obesity of children while the differences had statistical significance. Pupil's bad eating habit （χ²=18.905，P<0.001） and preference （χ²=22.901， P<0.001） ， insufficient sleep time （χ²=61.685， P<0.001） ， long-time using of electronic products （χ²=37.182， P<0.001） and low-time of exercise （χ²=15.328， P<0.001） also affected the obesity of primary school students. Among the five factors of family， diet， psychology， static state and dynamic state， the influencing factors of obesity in primary school students from grade one to grade four were being the only child， obese father and/or mother， overweight mother during pregnancy， caregivers without higher education. In terms of diet， it was mainly based on eating habits before bedtime， as well as partial and picky eating habits. In terms of psychology， the main psychological source was the pressure of learning in school. There was a clear relationship between the pace of learning and not keeping up. Static analysis revealed a correlation with lack of sleep. After assigning values based on eye hygiene recommendations for the use of electronic products， it was found that there was no direct correlation between obesity in primary school students and the duration of electronic product use. Dynamic analysis showed that not taking part in school physical exercise was related to obesity. ConclusionThe obesity of primary school students is influenced by family， diet， psychology， static and dynamic factors. The intervention of obesity in primary school needs the joint efforts of school， society， family， self-cognition and so on.

A major impedance to neuronal regeneration after peripheral nerve injury(PNI)is the activation of various programmed cell death mechanisms in the dorsal root ganglion.Ferroptosis is a form of pro-grammed cell death distinguished by imbalance in iron and thiol metabolism,leading to lethal lipid peroxidation.However,the molecular mechanisms of ferroptosis in the context of PNI and nerve regeneration remain unclear.Ferroportin(Fpn),the only known mammalian nonheme iron export protein,plays a pivotal part in inhibiting ferroptosis by maintaining intracellular iron homeostasis.Here,we explored in vitro and in vivo the involvement of Fpn in neuronal ferroptosis.We first delineated that reactive oxygen species at the injury site induces neuronal ferroptosis by increasing intracellular iron via accelerated UBA52-driven ubiquitination and degradation of Fpn,and stimulation of lipid peroxidation.Early administration of the potent arterial vasodilator,hydralazine(HYD),decreases the ubiquitination of Fpn after PNI by binding to UBA52,leading to suppression of neuronal cell death and significant ac-celeration of axon regeneration and motor function recovery.HYD targeting of ferroptosis is a promising strategy for clinical management of PNI.

Objective To understand the respiratory protection competency of staff in hospitals.Methods Staff from six hospitals of different levels and characteristics in Beijing were selected,including doctors,nurses,medical technicians,and servicers,to conduct knowledge assessment on respiratory protection competency.According to exposure risks of respiratory infectious diseases,based on actual cases and daily work scenarios,content of respira-tory protection competency assessment was designed from three aspects:identification of respiratory infectious di-seases,transmission routes and corresponding protection requirements,as well as correct selection and use of masks.The assessment included 6,6,and 8 knowledge points respectively,with 20 knowledge points in total,all of which were choice questions.For multiple-choice questions,full marks,partial marks,and no mark were given respective-ly if all options were correct,partial options were correct and without incorrect options,and partial options were correct but with incorrect options.Difficulty and discrimination analyses on question of each knowledge point was conducted based on classical test theory.Results The respiratory protection competency knowledge assessment for 326 staff members at different risk levels in 6 hospitals showed that concerning the 20 knowledge points,more than 60％participants got full marks for 6 points,while the proportion of full marks for other questions was relatively low.Less than 10％participants got full marks for the following 5 knowledge points:types of airborne diseases,types of droplet-borne diseases,conventional measures for the prevention and control of healthcare-associated infec-tion with respiratory infectious diseases,indications for wearing respirators,and indications for wearing medical protective masks.Among the 20 knowledge questions,5,1,and 14 questions were relatively easy,medium,and difficult,respectively;6,1,4,and 9 questions were with discrimination levels of ≥0.4,0.30-0.39,0.20-0.29,and ≤0.19,respectively.Conclusion There is still much room for hospital staff to improve their respiratory protection competency,especially in the recognition of diseases with different transmission routes and the indications for wearing different types of masks.