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.

Inhibiting the death receptor 3(DR3)signaling pathway in group 3 innate lymphoid cells(ILC3s)pre-sents a promising approach for promoting mucosal repair in individuals with ulcerative colitis(UC).Paeoniflorin,a prominent component of Paeonia lactiflora Pall.,has demonstrated the ability to restore barrier function in UC mice,but the precise mechanism remains unclear.In this study,we aimed to delve into whether paeoniflorin may promote intestinal mucosal repair in chronic colitis by inhibiting DR3 signaling in ILC3s.C57BL/6 mice were subjected to random allocation into 7 distinct groups,namely the control group,the 2％dextran sodium sulfate(DSS)group,the paeoniflorin groups(25,50,and 100 mg/kg),the anti-tumor necrosis factor-like ligand 1A(anti-TL1A)antibody group,and the IgG group.We detected the expression of DR3 signaling pathway proteins and the proportion of ILC3s in the mouse colon using Western blot and flow cytometry,respectively.Meanwhile,DR3-overexpressing MNK-3 cells and 2％ DSS-induced Rag1-/-mice were used for verification.The results showed that paeoniflorin alleviated DSS-induced chronic colitis and repaired the intestinal mucosal barrier.Simultaneously,paeoniflorin inhibited the DR3 signaling pathway in ILC3s and regulated the content of cytokines(interleukin-17A,granulocyte-macrophage colony stimulating factor,and interleukin-22).Alternatively,paeoniflorin directly inhibited the DR3 signaling pathway in ILC3s to repair mucosal damage indepen-dently of the adaptive immune system.We additionally confirmed that paeoniflorin-conditioned me-dium(CM)restored the expression of tight junctions in Caco-2 cells via coculture.In conclusion,paeoniflorin ameliorates chronic colitis by enhancing the intestinal barrier in an ILC3-dependent manner,and its mechanism is associated with the inhibition of the DR3 signaling pathway.

The disorder of group 3 innate lymphoid cells(ILC3)subgroup,such as the predominance of NCR-ILC3 but the deple-tion of NCR+ILC3,is unfavorable to damaged intestinal barrier repair,which leads to the prolongations and obstinacy of ulcerative colitis(UC).Our previous studies had shown that luteolin promoted NCRILC3 differentitating into NCR+ILC3 to improving the de-pletion of NCR+ILC3 in UC mice,while the mechanism is unclear.This article aimed to explore the underlying mechanism of luteolin enhancing the proportion NCR+ILC3.UC mice model was established with 2％DSS and Notch signaling was blocked,then luteolin was used to intervene.The results showed that the effect of luteolin on ameliorating disease symptoms in UC mice,including inhibit-ing the weight loss,reducing the pathological damage of colon mucosa,etc.,was diminished with blocking Notch signaling pathway.In addition,luteolin increased the proportion of NCR+ILC3,NCR+MNK3 and IL-22+ILC3,decreased intestinal permeability,pro-moted mucin secretion,and promoted ZO-1 and Occludin expression,the above effect of luteolin was neutralized by Notch inhibitor LY-411575.Luteolin activated the abnormally blocked Notch signaling pathway in UC mice.And molecular docking predicted the af-finity of luteolin for RBPJ to be-7.5 kcal·mol-1 in mouse,respectively;the affinity of luteolin for Notchl and RBPJ was respectively scored to be-6.4 kcal·mol-1 and-7.7 kcal·mol-1 homo sapiens.These results proved that luteolin is positive for enhancing the propor-tion of NCR+ILC3 via Notch signaling,and it provides a basis for targeting NCR+ILC3 for restoring intestinal barrier function to alle-viating ulcerative colitis.

Aim To explore the effect and mechanism of the artesunate(ART)on hepatocellular carcinoma(HCC).Methods The cell lines MHCC-97H and HCC-LM3 were used to be detected.MTT and clone formation were used to determine the cell proliferation;Wound healing was used to detect the cell migration;Transwell was used to test the cell invasion.Flow-cy-tometry was used to detect cell apoptosis and cell cy-cle.RNA-seq and qRT-PCR was used to detect the genes expression.Results The proliferation,migra-tion and invasion of treated cells were obviously inhibi-ted(P＜0.01).Moreover,the apoptosis rate in-creased significantly,so did the proportion of G2/M cells.Transcriptomic analysis identified GADD45A as a potential target of ART through RNA-sequencing da-ta,and suggested that ART might induce apoptosis and cell cycle arrest through regulating the expression of GADD45A.In addition,the results of mechanism studies and signaling analysis suggested that GADD45A had interaction with its upstream gene NACC1(nucle-us accumbens associated 1).Moreover,after ART treatment,the expressions of GADD45A and NACC1 were changed significantly.Conclusion ART may be a potential drug to resist HCC by affecting the expres-sion of GADD45A and its upstream gene NACC1,which provides a new drug,a new direction and a new method for the clinical treatment of HCC.

Objective To investigate the implementation of surveillance,prevention and control measures for healthcare-associated infection(HAI)in maternal and child healthcare(MCH)institutions,and provide policy evi-dence for optimizing HAI prevention and control in MCH institutions.Methods Stratified sampling was conducted among the MCH institutions at provincial,municipal and county levels in 8 provinces/autonomous regions.A uni-fied questionnaire was designed and the online survey was conducted through"Questionnaire Star".Results The data from 123 MCH institutions were included in the analysis.90.24％of the MCH institutions carried out compre-hensive surveillance on HAI.The ratios of MCH institutions which implemented targeted surveillance on HAI in neonatal intensive care unit(NICU),surgical site infection,multidrug-resistant organisms(MDROs)and HAI in intensive care units(non-NICU excluded)were 89.66％,85.96％,80.77％,and 74.19％,respectively.51.22％MCH institutions adopted information surveillance system on HAI cases.94.31％MCH institutions carried out surveillance on hand hygiene compliance.Over 90％MCH institutions carried out surveillance on environment hy-giene in high-risk departments.71.54％MCH institutions conducted centralized cleaning,disinfection,sterilization and supply for reusable medical instruments in the central sterile supply department(CSSD).Over 90％MCH insti-tutions established three-level pre-examination triage systems.86.18％set up transitional wards.MCH institutions generally adopted a management model with established effective communication,full appointment visits,and sepa-rate visits for special medical groups,such as registered pregnant women,high-risk newborns,healthcare groups,and long-term rehabilitation patients.However,the ratio of institutions conducting on-line follow-up visits was less than 50％.Conclusion MCH institutions have generally carried out comprehensive and targeted surveillance on HAI.Information surveillance need to be facilitated.Hand hygiene and environmental hygiene surveillance has been popularized to a certain extent at all levels of MCH institutions.The cleaning,disinfection,sterilization,and supply processes of reusable medical devices in a few MCH institutions are not standardized.Special medical populations get effective management.On-line healthcare is to be further promoted.

Based on the long bud stage phenotype of a new Lonicera japonica Flos variety "Huajin 6", using "Huajin 6" and "Da Mao Hua" as materials, probing the mechanism of its phenotype formation. Detection of endogenous Jasmonic acid hormones (JAs) content; the genes related to jasmonic acid (JA) synthesis were identified by transcriptome analysis of Lonicera japonica; flower buds and flowers of "Huajin 6" and "Da Mao Hua" were collected at different periods, and the qRT-PCR (quantitative real-time PCR) technique was used to analyze the trend of the expression of synthesis-related enzyme genes in Lonicera japonica Flos during the bud stage. The study found that the content of JAs in "Huajin 6" Lonicera japonica Flos was significantly lower than that in "Da Mao Hua"; applying exogenous methyl-jasmonate (MeJA) to "Huajin 6" can restore its flowering phenotype, making it close to wild type Lonicera japonica Flos; there are significant differences in the expression of two allene oxide synthase genes (AOS), three lipoxygenase genes (LOX), and two allene oxide cyclase genes (AOC) in the flowers and buds of "Huajin 6" and "Da Mao Hua" at different periods. It is hypothesized that the low expression of JA synthesis-related enzyme genes in " Huajin 6" leads to the blockage of JA synthesis, which causes the formation of the long bud phenotype. This study laid a certain foundation for the genetic breeding of Lonicera japonica, provided a new idea for the improvement of Lonicera japonica varieties, and provided a reference for the study of JAs in plant flower organs.

Trihelix transcription factors play important roles in plant light responses, growth and development, and stress responses. However, Trihelix has not yet been reported in Eucommia ulmoides. In this study, bioinformatics methods were used to comprehensively identify and analyze the expression patterns of the Trihelix gene family in E. ulmoides, aiming to provide a basis for further functional studies of EuGTs genes. A total of 9 Trihelix gene family members were identified in E. ulmoides, encoding proteins with 339 to 883 amino acids, with isoelectric points ranging from 5.13 to 9.39 and relative molecular weights between 36 992.06 and 97 871.61. Subcellular localization results showed that only EuGT-2 was localized in chloroplasts, while the others were located in the nucleus. The Trihelix gene family was categorized into six subfamilies: GT-1, GT-2, SH4, SIP1, GTγ, and GTδ. EuGTs were distributed among three subfamilies: SH4, GT-1, and GT-2, containing 1, 6, and 2 Trihelix proteins, respectively, with 2 to 17 exons. The promoters of EuGTs contained various cis-acting elements related to hormones, stress, photoperiod, and growth and development. Collinearity analysis revealed 5 collinear gene pairs between E. ulmoides and Arabidopsis thaliana, and 14 collinear gene pairs between E. ulmoides and Populus. Expression pattern analysis showed that EuGTs exhibited tissue-specific expression: EuGT-1, EuGT-2 had the highest expression levels in leaves, EuGT-4, EuGT-6, EuGT-9 had the highest transcriptional levels in marginal peel, and EuGT-5、EuGT-8 were predominantly expressed in the xylem. As leaves developed, EuGTs showed a trend of asynchronous changes. No significant differences in EuGTs expression were observed between male and female flowers, with high expression levels mainly during the induction stage of flowering. The qRT-PCR analysis indicated that most EuGTs genes were most highly expressed in the leaves of E. ulmoides, while EuGT-5 was highly expressed in the stems. Under 200 mmol·L~(-1) NaCl treatment, most EuGTs genes exhibited an initial increase followed by a decrease in expression, significantly responding to salt stress. This study provides important genetic resources for further exploration of EuGTs gene functions and germplasm innovation in E. ulmoides.
Plant Proteins/metabolism* ; Gene Expression Regulation, Plant ; Eucommiaceae/chemistry* ; Phylogeny ; Multigene Family/genetics* ; Gene Expression Profiling ; Transcription Factors/metabolism* ; Genome, Plant/genetics*

This study explored the growth-promoting effect and mechanism of the endophytic bacterium Kocuria rosea on Rehmannia glutinosa, aiming to provide a scientific basis for the development of green bacterial fertilizer. R. glutinosa 'Jinjiu' was treated with K. rosea, and the shoot parameters including leaf length, leaf width, plant width, and stem diameter were measured every 15 days. After 120 days, the shoots and roots were harvested. The root indicators(root number, root length, root diameter, root fresh weight, root dry weight, root volume, and root vitality) and secondary metabolites(catalpol, rehmannioside A, rehmannioside D, verbascoside, and leonuride) were determined. The R. glutinosa growth-promoting mechanism of K. rosea was discussed from the effect of K. rosea on the nutrient element content in R. glutinosa and rhizosphere soil and the genome information of this plant. After application of K. rosea, the maximum increases in leaf length, leaf width, plant width, and stem diameter were 35.67%(60 d), 25.39%(45 d), 40.17%(60 d), and 113.85%(45 d), respectively. The root number, root length, root diameter, root volume, root fresh weight, root dry weight, and root viability increased by 41.71%, 45.10%, 48.61%, 94.34%, 101.55%, 147.61%, and 42.08%, respectively. In addition, the content of rehmannioside A and verbascoside in the root of R. glutinosa increased by 76.67% and 69.54%, respectively. K. rosea promoted the transformation of nitrogen(N), phosphorus(P), and potassium(K) in the rhizosphere soil into the available state. Compared with that in the control, the content of available N(54.60 mg·kg~(-1)), available P(1.83 μmol·g~(-1)), and available K(83.75 mg·kg~(-1)) in the treatment with K. rosea increased by 138.78%, 44.89%, and 14.34%, respectively. The content of N, P, and K in the treatment group increased by 293.22%, 202.63%, and 23.80% in the roots and by 23.60%, 107.23%, and 134.53% in the leaves of R. glutinosa, respectively. K. rosea carried the genes related to colonization(rbsB, efp, bcsA, and gmhC), N, P, and K metabolism(narG, narH, narI, nasA, nasB, GDH2, pyk, aceB, ackA, CS, ppa, ppk, ppk2, pstS, pstA, pstB, and pstC), and indole-3-acetic acid and zeatin synthesis(iaaH and miaA). Further studies showed that K. rosea could colonize the roots of R. glutinosa and secrete indole-3-acetic acid(3.85 μg·mL~(-1)) and zeatin(0.10 μg·mL~(-1)). In summary, K. rosea promotes the growth of R.ehmannia glutinosa by enhancing the nutrient uptake, which provides a theoretical basis for the development of plant growth-promoting microbial products.
Rehmannia/metabolism* ; Endophytes/metabolism* ; Plant Roots/growth & development* ; Micrococcaceae/genetics* ; Data Mining ; Plant Leaves/metabolism* ; Genomics ; Rhizosphere

In this study, ultra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS~E) was used to analyze the plasma components of Danzhi Xiaoyao Formula after oral administration. Forty-nine plasma components were found in the serum of rats by comparing the compound extract, drug-containing serum, and blank serum. Components, such as 6-hydroxycoumarin, poricoic acid F, deoxoglabrolide, 30-norhederagenin, kanzonol R, 3',6'-di-O-galloylpaeoniflorin, 16α-hydroxytrametenolic acid, 16-deoxyporicoic acid B, 3-O-acetyl-16α-hydroxytrametenolic acid, and 16α,25-dihydroxydehydroeburiconic acid, were first found in rat serum. Behavioral tests, including the tail suspension test, novel object recognition test, and novelty-suppressed feeding test, were conducted for behavioral analysis. It was confirmed that this formula had therapeutic effects on perimenopausal depression. Furthermore, in combination with the network pharmacology method, 53 core targets including MAPK1, HRAS, AKT1, EGFR, and ESR1 were screened, and these targets participated in 165 signaling pathways, including PI3K-AKT, AMPK, VEGFA, MAPK, and HIF-1. In summary, the potential effects of Danzhi Xiaoyao Formula in treating perimenopausal depression are associated with mechanisms in accelerating inflammation repair, improving neuroplasticity, affecting neurotransmitters, regulating estrogen levels, and promoting new blood vessel formation.
Animals ; Rats ; Chromatography, High Pressure Liquid ; Depression/drug therapy* ; Network Pharmacology ; Perimenopause ; Phosphatidylinositol 3-Kinases ; Drugs, Chinese Herbal/pharmacology* ; Molecular Docking Simulation

AIM: To investigate the effect of the intravitreal injection of vascular endothelial growth factor-A165(VEGF-A165)on the scleral remodeling of guinea pigs with form-deprivation myopia(FDM).METHODS: A total of 120 tricolor guinea pigs, aged three weeks, were randomly divided into 6 groups, with 20 in each group. The blank group did not undergo any intervention. In the FDM group, only the FDM model was established. In the phosphate buffer saline(PBS)group, 2.5 μL of PBS was injected into the vitreous cavity before establishing the FDM model. In the 1ng group, 5ng group, and 10ng group, VEGF-A165 was injected into the vitreous cavity at concentrations of 1, 5 and 10ng, respectively, before the establishment of the FDM model. The FDM model was established by covering the right eyes of guinea pigs with translucent balloons for 14d. The diopter and axial length of the right eyes were measured before and after covering. After 14d, the content of dopamine(DA)in retina was measured by high performance liquid chromatography. Additionally, the mRNA and protein expression levels of matrix metalloproteinase-2(MMP-2), tissue inhibitor of matrix metalloproteinase-2(TIMP-2), transforming growth factor(TGF)-β1, TGF-β2 and α-smooth muscle actin(α-SMA)in sclera were detected by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.RESULTS: Before covering, there were no significant differences in the diopter and axial length of the right eyes of guinea pigs in all groups(P>0.05). After 14d of modeling, when compared with the blank group, FDM group showed an increase in the degree of myopia in the right eye, a prolongation of the axial length, a decrease in the content of DA in the retina, and an increase in the expression of MMP-2, TGF-β2 and α-SMA in the sclera. Conversely, the expression of TIMP-2 and TGF-β1 were decreased(P<0.01). However, in comparison to the FDM group, the degree of myopia in the 1ng, 5ng, and 10ng groups of guinea pigs decreased, the growth trend of axial length slowed, the content of DA in the retina increased, and the expression of MMP-2, TGF-β2 and α-SMA in the sclera decreased. Furthermore, the expression of TIMP-2 and TGF-β1 in the sclera increased(P<0.01). As the concentration of intravitreal injection of VEGF-A165 increased, the degree of myopia in the right eye of guinea pigs gradually increased, and the axial length gradually prolonged. The content of DA in the retina gradually decreased, the expression of MMP-2, TGF-β2, and α-SMA in the sclera gradually increased, while the expression of TIMP-2 and TGF-β1 decreased gradually.CONCLUSION: Intravitreal injection of VEGF-A165 can increase the content of DA in the retina of FDM guinea pigs, affect the expression of MMP-2, TIMP-2, TGF-β1, TGF-β2 and α-SMA in the sclera, and inhibit scleral remodeling of guinea pigs. Notably, the VEGF-A165 at the concentration of 1ng showed the most significant efficacy.