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 investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

AIM To establish a quantitative analysis of multi-components by single-marker(QAMS)method for the simultaneous content determination of gastrodin,parishin E,syringin,parishin B,parishin C,ferulic acid,parishin A,buddleoside,harpagoside and cinnamic acid in Tianma Toufengling Capsules.METHODS The analysis was performed on a 30℃thermostatic GL Science InertsilTM ODS-3 column(150 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1%phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 220,280 nm.Syringin was used as an internal standard to calculate the relative correction factors of the other nine constituents,after which the content determination was made.RESULTS Ten constituents showed good linear relationships within their own ranges(r≥0.999 7),whose average recoveries were 98.53%-102.22%with the RSDs of 1.26%-2.68%.The result obtained by QAMS approximated those obtained by external standard method.CONCLUSION This accurate and specific method can be used for the quality control of Tianma Toufengling Capsules.

Objective To investigate disease characteristics and hospitalization costs of children with Mycoplasma Pneumoniae pneumonia(MPP)admitted to Shanghai municipal medical hospitals from 2019 to 2023.Methods Depending on the Shanghai Municipal Hospital Pediatric Alliance,we retrospectively investigated community acquired MPP pediatric patients hospitalized in 22 municipal hospitals with pediatric qualifications(including 4 children's hospitals)in Shanghai from Jan 2019 to Dec 2023.We collected the patients'diagnosis codes,gender,age,length of hospital stay,hospitalization costs,and whether they progressed to severe Mycoplasma pneumoniae pneumonia(SMPP).Results From 2019 to 2023,a total of 29 045 hospitalized children with MPP were treated,with 6 035 cases(20.8%)identified as SMPP in the 22 hospitals.Trend analysis revealed a rising trend with years in the proportion of SMPP patients(χ2trend=365.498,P<0.001).Among the 4 children's hospitals,there were 18 710 cases with MPP,including 4 078 cases(21.8%)of SMPP.The proportion of SMPP patients also showed an increasing trend with years(χ2trend=14.548,P<0.001),and the proportion in 2023(23.0%)was higher than that in previous years with statistical significance.There were statistical differences in the seasonal distribution of MPP cases between different years,with higher proportions in summer and autumn overall.The age distribution of hospitalized MPP children varied among different years,with school-age children accounting for the majority(56.8%)in 2023.There was no difference in the distribution of severe cases between different genders,but there were differences in the proportion of severe cases among different age groups in different years,with a gradual increase in severe cases among children aged 1 to 3 years(χ2trend=191.567,P<0.001).The average length of hospital stay for MPP during the epidemic was higher than that during non-epidemic periods,and there were statistically significant differences in the average length of hospital stay between different years(P<0.001).The individual hospitalization costs during the epidemic were higher than in other years,and there were statistically significant differences in individual hospitalization costs between different years(P<0.001).The total hospitalization costs were still higher in 2019 and 2023.The individual hospitalization costs for SMPP were higher than for non-SMPP cases.Conclusion MPP outbreaks occurred in Shanghai in 2019 and 2023,with the higher proportions in summer and autumn overall.Compared to previous years,the number of hospitalized MPP children in Shanghai was higher in 2023,with a higher proportion of SMPP cases,especially among children under 3 years old.The individual per capita hospitalization expenses for SMPP cases were higher than for non-SMPP cases.

Background: and Purpose Orthostatic hypotension (OH) is common in patients with Parkinson’s disease (PD). Early recognition OH is required with sensitive assessments. The purpose of this study was to determine whether blood pressure (BP) changes during exercise can predict the occurrence of OH in PD. Methods: This prospective cohort study included 80 consecutive patients with PD. All patients agreed to participate in a baseline evaluation and cardiopulmonary exercise test (CPET).According to the initial active standing test (AST), those without OH (PD-nonOH) at baseline had their AST results followed up for 6 months. The main outcome was defined as whether patients without OH at baseline would develop OH after 6 months. Logistic regression analysis was applied to identify the relevant variables. A nomogram was constructed based on clinical features and identified variables. The concordance index (C-index) and area under the receiver operating characteristic curve (AUC) were used to evaluate the accuracy and predictive ability of the nomogram, respectively. Results: CPET results indicated that peak load, peak heart rate, heart rate recovery at 1 min, and systolic BP change (ΔSBP) were lower in those with OH than in the PD-nonOH group (p<0.05) at baseline. Logistic regression analysis indicated that peak load and ΔSBP during CPET had significant effects on OH (p<0.05). Age, sex, peak load, and ΔSBP were used to construct the nomogram model (C-index=0.761). The prediction model had an AUC of 0.782 (95% confidence interval=0.649–0.889) and a specificity and sensitivity of 70.0% and 81.8%, respectively. Conclusions This study has identified predictive factors for OH development in patients with PD. CPET could be used as a complementary examination to identify patients at a high risk of OH.

Currently,the research or publications related to the clinical comprehensive evaluation of Chinese patent medicine are increasing,which attracts the broad attention of all circles. According to the completed clinical evaluation report on Chinese patent medicine,there are still practical problems and technical difficulties such as unclear responsibility of the evaluation organization,unclear evaluation subject,miscellaneous evaluation objects,and incomplete and nonstandard evaluation process. In terms of evaluation standards and specifications,there are different types of specifications or guidelines with different emphases issued by different academic groups or relevant institutions. The professional guideline is required to guide the standardized and efficient clinical comprehensive evaluation of Chinese patent medicine and further improve the authority and quality of evaluation. In combination with the characteristics of Chinese patent medicine and the latest research achievement at home and abroad,the detailed specifications were formulated from six aspects including design,theme selection,content and index,outcome,application and appraisal,and quality control. The guideline was developed based on the guideline development requirements of China Assoication of Chinese medicine. After several rounds of expert consensus and public consultation,the current version of the guideline has been developed.
Medicine, Chinese Traditional ; Nonprescription Drugs ; Consensus ; China ; Reference Standards ; Drugs, Chinese Herbal

Hepatotoxicity induced by herbal medicines such as Gynura japonica, which contains large amount of pyrrolizidine alkaloids (PAs) such as senecionine (SEN), is among the most serious problems of herbal drug-induced liver injury, yet there is no effective treatment in clinic. We have previously reported that ritonavir (the well-known CYP3A4 inhibitor) protected rats against Gynura japonica-induced liver injury in rats, which was closely related to the inhibition of the metabolic activation of PAs. A large number of lignans have been identified in Schisandrae Chinensis Fructis and are reported to attenuate drug-induced liver injuries by modulating the drug metabolism enzymes. Therefore, the present study investigated the protective effect and potential mechanism of schisandrol A (SoA, a representative lignan identified in Schisandrae Chinensis Fructis) against SEN-induced hepatotoxicity in mice. All experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM210604002). Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Shanghai University of Traditional Chinese Medicine. Liver injury was induced by a single gavage of SEN (150 μmol·kg^-1); mice in the protection group were gavaged with SoA (116 μmol·kg^-1) 7 days before SEN treatment. The results show that SoA dramatically alleviated SEN-induced liver injury in mice. Mice in the protection group showed decreased serum activities for alanine aminotransferase and aspartate aminotransferase; in addition, the hepatic necrosis and sinusoidal hemorrhage in SEN-treated mice were markedly attenuated in the protection group. The serum contents of SEN metabolites in mice were decreased. In vitro studies were performed by using human liver microsomes and proved that SoA inhibits CYP3A4 to decrease the metabolism of SEN. These studies indicate that SoA attenuated SEN-induced liver injury in mice, which was closely related to the inhibition of the metabolic activation of SEN. These results provide a better understanding of the relationship between CYP3A4 and PA-induced toxicity. This work also will be helpful in developing effective treatments for SEN-induced liver injury based on inhibition of its metabolic activation, and in making reasonable evaluations of the safety of herbal medicines containing PAs such as G. japonica.

AIM: To analyze the stability of different concentrations of fluorescein sodium solution on the detection of tear break-up time(TBUT).METHODS:A retrospective study. A total of 150 cases(150 eyes)who came to our dry eye clinic with good cooperation from August 2019 to September 2021 were selected for the study, and the subjects were randomly divided into five groups, which were fluorescein sodium(FLS, 0.5%), FLS(1.0%), FLS(1.5%), FLS(2.0%)and fluorescein sodium parallel(FLSP), with 30 patients in each group(all the right eyes were the subject eyes). Each group was dripped with the corresponding fluorescein sodium. The FLSP group was the fluorescent test strip detection group. The slit lamp image scores of different concentration groups were compared, the survival time of sodium fluorescein at the instant, 2, 5, 10, 15 and 30min points was observed in each group, and the mean value of TBUT in each group was recorded.RESULTS: The image score of FLS(0.5%)group was significantly higher than that of the other four groups(t=7.746, 21.483, 116.190, 38.730, all P<0.01). The image score of FLS(1.0%)group was significantly higher than that of FLS(1.5%)and FLS(2.0%)group(t=10.742, 15.492, all P<0.01). The survival time of fluorescein in FLS(0.5%)group was significantly shorter than that of the other four groups(t=8.226, 7.458, 9.159, 12.347, all P<0.01). The survival time of fluorescein in FLS(1.5%)group was significantly longer than that of FLS(1.0%)and FLS(2.0%)group(t=15.428, 13.274, all P<0.05). TBUT in FLS(0.5%)group was significantly higher than that of the other four groups at 2min(t=22.767, 22.345, 15.494, 17.213, all P<0.01), and was significantly lower than that of the other four groups at 10min(t=23.266, 25.353, 10.183, 22.025, all P<0.01). The mean first TBUT of FLS(1.5%)group was significantly shorter than that of the other four groups(t=25.236, 21.374, 19.658, 72.341, all P<0.01), and the mean first TBUT of FLSP group was significantly longer than that of the other four groups(t=22.487, 30.267, 60.247, 40.857, all P<0.01). There was no significant correlation between TBUT and ocular surface disease index(OSDI)and tear river height(rs=-0.072, 0.219, P=0.689, 0.112). TBUT was positively correlated with tear secretion(rs=0.674, P<0.01).CONCLUSION: FLS(0.5%)had higher image quality but it was only suitable for observing staining within 5min, and the FLSP group was more suitable for clinical observation of corneal fluorescence staining for a longer period; FLS(1.5%)was the most stable and reliable concentration and dose for the detection of TBUT.

Numerous in vitro studies have shown that most pyrrolizidine alkaloids (PAs) are hepatotoxic after being metabolically activated by cytochrome P450 (CYP) 3A4. However, the key role of CYP3A4 has not been confirmed in vivo. Therefore, the CYP3A4 chemical inhibitor ritonavir was employed in this work and the effect of ritonavir on Gynura japonica-induced liver injury in rats was investigated. All experiments were approved by the Animal Research Committee of Shanghai University of Traditional Chinese Medicine. Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Shanghai University of Traditional Chinese Medicine. Acute liver injury was induced by a single gavage of Gynura japonica extracts (GJE, 8 g·kg^-1); rats in the protection group were gavaged with ritonavir (RIT, 30 mg·kg^-1) 1 h before GJE treatment. The results show that RIT could significantly attenuate GJE-induced liver injury in rats. Rats in the protection group showed decreased serum activities for alanine aminotransferase and aspartate aminotransferase, as well as lower total bile acids. In addition, the infiltration of inflammatory cells, sinusoidal hemorrhage, and hepatic necrosis in GJE-treated rats were markedly attenuated in the protection group. The content of pyrrole-protein adducts (PPAs), a recommended biomarker for PA-induced hepatotoxicity in clinics, was determined at 10 min to 24 h after GJE treatment. The content of 13 bile acids was also quantified. RIT treatment reduced the content of PPAs in serum dramatically and restored the impaired bile acid homeostasis caused by GJE. These studies indicate that RIT attenuated Gynura japonica-induced liver injury in rats, which was closely related to the inhibition of the metabolic activation of PAs and the regulation of bile acid metabolism. These results provide a better understanding of the relationship between CYP3A4 and PA-induced toxicity. This work will also be helpful in developing effective treatments for PA-induced liver injury and making a reasonable evaluation of the safety of drugs containing PAs in clinic.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwise. The incidence rate of HCC is high and is easy to metastasis and recurrence, which seriously affects human health. Traditional chemical drugs have some challenges such as toxicity, side effects, and multidrug resistance, thus it is urgent to find new drugs and effective targets. Here we synthesized a novel chemical, protonic bis-phenanthroline (H-BP), and the antitumor effect was investigated in the study. The results showed that H-BP could selectively inhibit the proliferation of tumor cells and cause HCC apoptosis. And also, in HCC tumor-bearing mice, H-BP could effectively prevent the growth of tumor mass, even completely eliminate the tumor at medium dose (5 mg·kg^-1) and high dose (10 mg·kg^-1), and meanwhile H-BP has no significant effect on the body weight of mice. The experimental protocol was approved by the Animal Ethics Committee of Southwest University, and the experimental operation was strictly carried out in accordance with the ethical principles of animal use and care. Mechanism studies showed that H-BP induced HCC apoptosis was related to down-regulation the expression of pleomorphic adenoma gene like-2 (PLAGL2), a oncogene transcription factor, resulting in the down-regulation of PLAGL2 downstream proteins hypoxia inducible factor and β-catenin. This study not only introduces the dimerization method to form novel compounds that will provide a new approach for drug design, but also suggests that PLAGL2 may be an effective target in tumor therapy.