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 understand the moderating effect of cohabitation with parents on the association between peer dating behavior and sexual behaviors among secondary vocational school students, so as to provide a scientific basis for preventing sexual behaviors among secondary vocational school students. Methods: From March to April 2021, an electronic questionnaire survey was conducted among 3 180 students from 6 vocational schools in Shanghai (urban, suburban, exurban) and Shaanxi (Shangluo, Ankang, Baoji) using cluster sampling. Spearman correlation analysis was used to investigate the relationship of cohabitation with parents, peer dating behavior and sexual behaviors among secondary vocational school students. Binary Logistic regression analysis was performed to investigate the role of cohabitation with parents on peer dating behavior and sexual behaviors among secondary vocational students. Results: There was a significant negative between cohabitation with parents and sexual ( r =-0.04); and there was a positive correlation between peer dating behavior and sexual behaviors ( r =0.24), as well as cohabitation with parents and peer dating behavior ( r =0.04)( P <0.05). Multivariable Logistic regression analysis showed an association between peer dating behavior and the occurrence of sexual behaviors ( OR=2.79-12.95, P <0.05). Cohabitation with parents played a moderating role in the association between peer dating behavior and sexual behaviors, and a signification interaction was found between cohabitation with parents and reporting that a small part or about half of their peers had dating behavior ( OR =0.48, P <0.05). Conclusions The more peers dating behavior are associated with a higher risk of sexual behaviors among secondary vocational school students, and cohabitation with parents can partly reduce this risk. School and family sexuality education for secondary vocational students should be strengthened to improve their interpersonal skills and decision-making, and ability to resist peer pressure, so as to reduce their risk of sexual behaviors.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Objective To investigate the effects of ganoderma lucidum polysaccharide peptide(GLPP)on renal function and oxidative damage in diabetic nephropathy mice and its mechanism.Methods C57 mice were randomly divided into normal group,model group,positive control group(10.0 mg·kg-1 losartan),experimental-L group(50 mg·kg-1 GLPP),experimental-M group(100 mg·kg-1 GLPP),experimental-H group(200 mg·kg-1 GLPP)and GLPP+Losartan group(200 mg·kg-1 GLPP+10.0 mg·kg-1 losartan).Each group had 10 mice.After 8 weeks of continuous gastric administration,blood glucose was detected;renal function and lipid metabolism related indexes were detected by biochemical analyzer;serum inflammatory factor expression was detected by enzyme linked immunosorbent assay(ELISA);protein expression was detected by Western blot;oxidative damage related indexes were detected by kit method;apoptosis was detected by Tunel method.Results The fasting blood glucose of mice in normal group,model group,positive control group,experimental-H group and GLPP+Losartan group were(4.69±0.16),(20.31±2.04),(10.22±0.98),(10.26±0.96)and(7.76±0.43)mmol·L-1;serum creatinine(Scr)level were(25.48±2.33),(68.34±4.78),(32.93±3.25),(36.37±2.36)and(28.30±1.19)μmol·L-1,respectively;malonaldehyde(MDA)content were(2.05±0.22),(6.71±0.57),(2.69±0.27),(3.21±0.32)and(2.19±0.11)nmol·L-1,respectively;Tunel positive cell rates were(3.39±0.27)％,(26.75±1.24)％,(6.81±0.71)％,(8.05±0.80)％,(5.33±0.33)％,respectively;nuclear factor kappa B(NF-κB p65)protein expression levels were 0.31±0.05,1.07±0.08,0.40±0.03,0.47±0.04 and 0.35±0.03,respectively.The model group compared with the normal group for the above indicators,the experimental-H group compared with the model group for the above indicators,and the GLPP+Losartan group was compared with the experimental-H group for the above indicators,all differences were statistically significant in statistics(all P＜0.05).Conclusion Ganoderma lucidum polysaccharide peptide may regulate Toll-like receptor 4/NF-κB pathway to inhibit apoptosis and inflammatory response,and improve oxidative damage in diabetic nephropathy mice.

ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.

An integrated evaluation model based on the combination of traditional trait identification and modern chemical analysis was used for the identification of key indexes of grade classification and the establishment of grade quality standard of Lycium barbarum fruits (LBF) from Ningxia genuine producing area. Under the guidance of herbal examination and market survey, particle size, skin color and imperfection were taken as the external trait indicators; focusing on the comprehensiveness and validity of the quality evaluation, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), zeaxanthin dipalmitate (ZD) and volatile ether extractives were used as important parameters involving in quality evaluation in addition to the pharmacopoeia test items and determination indicators such as moisture, ash, water-soluble extractives, Lycium barbarum polysaccharide (LBP) and betaine. The quality differences between different traits and grades of LBF as well as the relevance between character and quality were further investigated by mathematical and statistical means such as t-test analysis, correlation analysis and principal component analysis (PCA). Furthermore, the activities of extractives from different grades of LBF were compared at cellular level. The results showed that LBF were mainly preferred for large and red ones; except for betaine, the other index components were relatively high (moisture and ash were low) in the first-grade and samples with bright-red skin color. Besides, there was a significant correlation between skin color and moisture, water-soluble extractives, LBP, AA-2βG and ZD contents. Considering the results of the study and the market situation, the present study took the grain size and skin color of LBF as the core of evaluation, and at the same time introduced the multi-indicator components including LBP, AA-2βG, ZD, water-soluble extractives and moisture to further guide the grading, and established the quality standard of LBF grades combining the trait indexes and the chemical composition. More importantly, in vitro cell models both preliminarily confirmed that the first-grade LBF played a more significant "nourishing liver and kidney" effect, which provided an experimental basis for the quality control of LBF and promoted the realization of high quality and good price.

ObjectiveTo investigate the clinical features of patients with acute-on-chronic liver failure （ACLF） and bacterial infection and early warning indicators associated with multidrug-resistant infections. MethodsA retrospective analysis was performed for 130 patients with ACLF and bacterial infection who attended The Second Affiliated Hospital of Air Force Medical University from January 1， 2010 to December 31， 2021， and according to the drug susceptibility results， the patients were divided into multidrug-resistant （MDR） bacterial infection group with 80 patients and non-MDR bacterial infection group with 50 patients. General information and laboratory examination results were compared between the two groups to screen for the early warning indicators associated with MDR bacterial infection. The Student’s t-test was used for comparison of normally distributed continuous data with homogeneity of variance between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data or continuous data with heterogeneity of variance between two groups； the chi-square test or the Fisher’s exact test was used for comparison of categorical data between two groups. The binary logistic regression analysis and the receiver operating characteristic （ROC） curve were used to assess the predictive value of early warning indicators. ResultsAmong the 130 patients with ACLF and bacterial infection， sputum （27.7%） was the most common specimen for detection， followed by blood （24.6%）， urine （18.5%）， and ascites （17.7%）. Bacterial infections were dominated by Gram-negative bacteria （58.5%）. Of all bacteria， Escherichia coli （18.5%）， Klebsiella pneumoniae （14.6%）， and Enterococcus faecium （13.8%） were the most common pathogens. Gram-positive bacteria had a high resistance rate to the antibacterial drugs such as erythromycin （72.2%）， penicillin （57.4%）， ampicillin （55.6%）， and ciprofloxacin （53.7%）， while Gram-negative bacteria had a high resistance rate to the antibacterial drugs such as ampicillin （73.3%）， cefazolin （50.0%）， and cefepime （47.4%）. The patients with ACLF and bacterial infection had a relatively high rate of MDR bacterial infection （61.5%）. Comparison of clinical data between the two groups showed that compared with the patients with non-MDR bacterial infection， the patients with MDR bacterial infection had significantly higher levels of alanine aminotransferase （Z=2.089， P=0.037）， aspartate aminotransferase （Z=2.063， P=0.039）， white blood cell count （Z=2.207， P=0.027）， and monocyte count （Z=4.413， P<0.001）. The binary logistic regression analysis showed that monocyte count was an independent risk factor for MDR bacterial infection （odds ratio=7.120， 95% confidence interval ［CI］： 2.478‍ ‍—‍ ‍20.456，P<0.001） and had an area under the ROC curve of 0.686 （95%CI： 0.597‍ ‍—‍ ‍0.776） in predicting ACLF with MDR bacterial infection（P<0.001）， with the optimal cut-off value of 0.50×10⁹/L， a sensitivity of 0.725， and a specificity of 0.400. ConclusionACLF combined with bacterial infections is mainly caused by Gram-negative bacteria， with the common pathogens of Escherichia coli and Klebsiella pneumoniae and a relatively high MDR rate in clinical practice. An increase in monocyte count can be used as an early warning indicator to distinguish MDR bacterial infection from non-MDR bacterial infection.