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.

Objective:To explore the clinical application and effect of FilmArray ? meningitis/encephalitis (ME) panel in identifying pathogens of central nervous system (CNS) infections in children. Methods:Molecular biology study.Cerebrospinal fluid (CSF) samples were prospectively obtained through lumbar puncture from children with suspected CNS infections admitted to the Department of Infectious Diseases at Beijing Children′s Hospital, Capital Medical University from May to November 2019.These samples were subjected to both routine clinical pathogen testing and FilmArray ME panel testing.Polymerase chain reaction was used to validate all samples.Independent sample t-test or Mann-Whitney U-test was used for comparative analysis of the results and influence factors obtained by the two detection methods. Results:A total of 113 cases of suspected CNS infections were enrolled.Routine clinical testing yielded 17 cases, with a positive rate of 15.0%, including 4 positive CSF cultures, with a positivity rate of 3.5%.FilmArray ME panel detected 23 positive cases, with a positive rate of 20.4%.FilmArray ME panel detected bacteria in 7 cases, viruses in 13 cases, fungi in 1 case, and both viruses and bacteria in 2 cases.Among the common pathogens detected, FilmArray ME panel obtained the results on average 2.7 days in advance.Conclusions:Compared with CSF culture, FilmArray ME panel has the advantages of shorter detection period, higher positive detection rate, and higher virus detection rate.

Objective:To investigate the association of urinary cadmium level with body mass index (BMI) and body circumferences among the older adults over 65 years old in 9 longevity areas of China.Methods:Subjects were older adults over 65 years old from the Healthy Aging and Biomarkers Cohort Study (HABCS) between 2017 and 2018 conducted in 9 longevity areas in China. A total of 1 968 older adults were included in this study. Information including socio-demographic characteristics, lifestyles, diet intake, and health status was collected by using questionnaires and physical examinations. Urine samples were collected to detect urinary cadmium and creatinine levels. Body circumferences included waist circumference, hip circumference and calf circumference. Subjects were divided into three groups (low:<0.77 μg/g·creatinine, middle:0.77-1.69 μg/g·creatinine, high:≥1.69 μg/g·creatinine) by tertiles of creatinine-adjusted urinary cadmium concentration. Multiple linear regression models were used to analyze the association of creatinine-adjusted urinary cadmium level with BMI and body circumferences. The dose-response relationship of creatinine-adjusted urinary cadmium concentration with BMI and body circumferences was analyzed by using restrictive cubic splines fitting multiple linear regression model.Results:The mean age of subjects was (83.34±11.14) years old. The median (Q1, Q3) concentration of creatinine-adjusted urinary cadmium was 1.13 (0.63, 2.09) μg/g·creatinine, and the BMI was (22.70±3.82) kg/m 2. The mean values of waist circumference, hip circumference, and calf circumference were (85.42±10.68) cm, (92.67±8.90) cm, and (31.08±4.76) cm, respectively. After controlling confounding factors, the results of the multiple linear regression model showed that for each increment of 1 μg/g·creatinine in creatinine-adjusted urinary cadmium, the change of BMI, waist circumference, hip circumference, and calf circumference in the high-level group was -0.28 (-0.37, -0.19) kg/m 2, -0.74 (-0.96, -0.52) cm, -0.78 (-0.96, -0.61) cm, and -0.20 (-0.30, -0.11) cm, respectively. The restrictive cubic splines curve showed a negative nonlinear association of creatinine-adjusted urinary cadmium with BMI ( Pnonlinear<0.001) and negative linear associations of creatinine-adjusted urinary cadmium with waist circumference ( Plinear<0.001), hip circumference ( Plinear<0.001), and calf circumference ( Plinear<0.001). Conclusion:Urinary cadmium level is significantly associated with decreased BMI, waist circumference, hip circumference and calf circumference among older adults over 65 years old in 9 longevity areas of China.

Objective:To investigate the association of urinary cadmium level with body mass index (BMI) and body circumferences among the older adults over 65 years old in 9 longevity areas of China.Methods:Subjects were older adults over 65 years old from the Healthy Aging and Biomarkers Cohort Study (HABCS) between 2017 and 2018 conducted in 9 longevity areas in China. A total of 1 968 older adults were included in this study. Information including socio-demographic characteristics, lifestyles, diet intake, and health status was collected by using questionnaires and physical examinations. Urine samples were collected to detect urinary cadmium and creatinine levels. Body circumferences included waist circumference, hip circumference and calf circumference. Subjects were divided into three groups (low:<0.77 μg/g·creatinine, middle:0.77-1.69 μg/g·creatinine, high:≥1.69 μg/g·creatinine) by tertiles of creatinine-adjusted urinary cadmium concentration. Multiple linear regression models were used to analyze the association of creatinine-adjusted urinary cadmium level with BMI and body circumferences. The dose-response relationship of creatinine-adjusted urinary cadmium concentration with BMI and body circumferences was analyzed by using restrictive cubic splines fitting multiple linear regression model.Results:The mean age of subjects was (83.34±11.14) years old. The median (Q1, Q3) concentration of creatinine-adjusted urinary cadmium was 1.13 (0.63, 2.09) μg/g·creatinine, and the BMI was (22.70±3.82) kg/m 2. The mean values of waist circumference, hip circumference, and calf circumference were (85.42±10.68) cm, (92.67±8.90) cm, and (31.08±4.76) cm, respectively. After controlling confounding factors, the results of the multiple linear regression model showed that for each increment of 1 μg/g·creatinine in creatinine-adjusted urinary cadmium, the change of BMI, waist circumference, hip circumference, and calf circumference in the high-level group was -0.28 (-0.37, -0.19) kg/m 2, -0.74 (-0.96, -0.52) cm, -0.78 (-0.96, -0.61) cm, and -0.20 (-0.30, -0.11) cm, respectively. The restrictive cubic splines curve showed a negative nonlinear association of creatinine-adjusted urinary cadmium with BMI ( Pnonlinear<0.001) and negative linear associations of creatinine-adjusted urinary cadmium with waist circumference ( Plinear<0.001), hip circumference ( Plinear<0.001), and calf circumference ( Plinear<0.001). Conclusion:Urinary cadmium level is significantly associated with decreased BMI, waist circumference, hip circumference and calf circumference among older adults over 65 years old in 9 longevity areas of China.

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.

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.

AIM To study the chemical constituents form the leaves of Castanopsis orthacantha Fance and their α-glucosidase inhibitory activities.METHODS The methanol extract form the leaves of C.orthacanth was isolated and purified by various column chromatography methods,such as MCI gel CHP 20P,Sephadex LH-20,Diaion HP20SS,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The α-glucosidase inhibitory activities were determined by PNPG method.RESULTS Eighteen compounds were isolated and identified as protocatechuic acid(1),gallic acid(2),3-O-α-L-arabininopyranosyl-4-hydroxybenzoic acid(3),3-O-galloyl shikimic acid(4),methyl 4-epi-shikimate-3-O-gallate(5),5-O-galloyl shikimic acid(6),5-O-caffeoylshikimic acid(7),6-O-galloyl-glucoside(8),1,6-di-O-galloyl-β-D-pyranogluloside(9),1,3-di-O-galloyl-α-D-glucoside(10),2,3-di-O-galloyl-D-glucoside(11),β-O-methylgluco-2,3-digalloyl esters(12),(3R,1'S)-[1'-(6"-O-galloyl-β-D-gluco-pyranosyl)oxyethyl]-3-hydroxy-dihydrofuran-2(3H)-one(13),4-O-D-(6'-O-galloyl)glucopyranyl-(E)-p-coumaryl acid(14),chestanin(15),1-desgalloyl eugeniin(16),picrorhiza acid(17),11-methyl chebulate(18).The IC50 values of compounds 2 and 16 were(0.12±0.059),(0.00089±0.00025)mmol/L,respectively.CONCLUSION All compounds are isolated from the leaves of C.orthacantha for the first time.Compounds 2 and 16 have strong α-glucosidase inhibitory activities.

Objective:To study the therapeutic effect of cardiac rehabilitation in patients with acute myocardial infarction(AMI)after percutaneous coronary intervention(PCI)and its effect on inflammatory factors.Methods:A total of 94 AMI patients after PCI admitted in Hospital of China University of Geosciences(Wuhan)between June 2020 and August 2021 were selected and randomly divided into control group(n=47,routine therapy)and study group(n=47,individualized cardiac rehabilitation therapy based on routine therapy).Cardiopulmonary exercise indexes,blood lipid indexes,inflamma-tory factors and cardiac function indexes were compared between two groups before and 3 months after treatment.Results:Compared with control group after treatment,participants in study group had significant higher anaerobic threshold[(15.46±3.07)ml·min-1·kg-1 vs.(19.37±3.27)ml·min 1·kg-1],maximal oxygen uptake[(21.26±4.05)ml·min-1·kg-1 vs.(25.31±4.10)ml·min-1·kg-1],left ventricular ejection fraction(LVEF)[(52.20±5.75)％vs.(57.91±5.17)％],6min walking distance(6MWD)[(430.58±43.87)m vs.(481.52±44.57)m]and levels of high density lipoprotein cholesterol(HDL-C)[(1.30±0.32)mmol/L vs.(1.49±0.35)mmol/L]and interleukin(IL)-10[(5.45±0.55)pg/ml vs.(6.19±0.59)pg/ml](P＜0.01 all),and significant lower levels of triglyceride(TG)[(1.88±0.65)mmol/L vs.(1.54±0.63)mmol/L],total cholesterol(TC)[(3.49±0.54)mmol/L vs.(3.13±0.47)mmol/L],low density lipoprotein cholesterol(LDL-C)[(2.12±0.59)mmol/L vs.(1.57±0.52)mmol/L],tumor necrosis factor-α(TNF-α)[(128.34±14.5)pg/ml vs.(99.28±8.51)pg/ml],leptin(LEP)[(623.49±61.27)pg/ml vs.(483.57±47.61)pg/ml]and N terminal pro B-type natriuretic peptide(NT-proBNP)[(396.59±18.12)pg/ml vs.(302.96±17.56)pg/ml](P＜0.05 or＜0.01).Conclusion:Cardiac rehabilitation therapy can significantly im-prove the blood lipid levels,cardiac function and exercise endurance,and effectively reduce the levels of inflammatory fac-tors in AMI patients after PCI,which is an important component of secondary prevention in these patients.

Objective To investigate the correlation between the level of N-terminal pro-Brain natriuretic peptide(NT-proBNP)and cardiorespiratory fitness following acute exposure to high altitude.Methods Forty-six subjects were recruited from the Second Affiliated Hospital of Army Medical University in June 2022,including 19 males and 27 females.After completing cardiopulmonary exercise test(CPET),serological detection of myocardial cell-related markers,and multiple metabolites at a plain altitude(300 meters above sea level),all subjects flew to a high-altitude location(3900 meters above sea level).Biomarker testing and CPET were repeated on the second and third days after arrival at high altitude.Changes in serum biomarker and key CPET indicators before and after rapid ascent to high altitude were compared,and the correlation between serum levels of various myocardial cell-related markers and metabolites and high altitude cardiorespiratory fitness was analyzed.Results Compared with the plain altitude,there was a significant decrease in maximal oxygen uptake after rapid ascent to high altitude[(25.41±6.20)ml/(kg.min)vs.(30.17±5.01)ml/(kg.min),P<0.001].Serum levels of NT-proBNP,Epinephrine(E),plasma renin activity(PRA),angiotensin Ⅱ(Ang Ⅱ),angiotensin-converting enzyme 2(ACE2)and leptin(LEP)significantly increased,with all differences being statistically significant(P<0.05)after acute high altitude exposure.In contrast,no statistically significant differences were observed for creatine kinase MB(CK-MB),cardiac troponin I(cTnI),myoglobin(Myo)and norepinephrine(NE)(P>0.05).Correlation analysis showed a significant negative correlation between NT-proBNP at plain altitude(r=-0.768,P<0.001)and at high altitude(r=-0.791,P<0.001)with maximal oxygen uptake at high altitude.Multivariate linear regression analysis indicated that maximal oxygen uptake at plain altitude(t=2.069,P=0.045),NT-proBNP at plain altitude(t=-2.436,P=0.020)and at high altitude(t=-3.578,P=0.001)were independent influencing factors of cardiorespiratory fitness at high altitude.Conclusion Cardiorespiratory fitness significantly decreases after rapid ascent to high altitude,and the baseline NT-proBNP level at plain altitude is closely related to cardiorespiratory fitness at high altitude,making it a potential predictor indicator for high altitude cardiorespiratory fitness.

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.