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.

Biomolecular condensates are formed through phase separation of biomacromolecules such as proteins and RNAs. These condensates exhibit liquid-like properties that can futher transition into more stable material states. They form complex internal structures via multivalent weak interactions, enabling precise spatiotemporal regulations. However, the use of inconsistent and non-standardized terminology has become increasingly problematic, hindering academic exchange and the dissemination of scientific knowledge. Therefore, it is necessary to discuss the terminology related to biomolecular condensates in order to clarify concepts, promote interdisciplinary cooperation, enhance research efficiency, and support the healthy development of this field.

ObjectiveTraditional Chinese medicine (TCM) constitutes a valuable cultural heritage and an important source of antitumor compounds. Poria (Poria cocos (Schw.) Wolf), the dried sclerotium of a polyporaceae fungus, was first documented in Shennong’s Classic of Materia Medica and has been used therapeutically and dietarily in China for millennia. Traditionally recognized for its diuretic, spleen-tonifying, and sedative properties, modern pharmacological studies confirm that Poria exhibits antioxidant, anti-inflammatory, antibacterial, and antitumor activities. Pachymic acid (PA; a triterpenoid with the chemical structure 3β-acetyloxy-16α-hydroxy-lanosta-8,24(31)-dien-21-oic acid), isolated from Poria, is a principal bioactive constituent. Emerging evidence indicates PA exerts antitumor effects through multiple mechanisms, though these remain incompletely characterized. Neuroblastoma (NB), a highly malignant pediatric extracranial solid tumor accounting for 15% of childhood cancer deaths, urgently requires safer therapeutics due to the limitations of current treatments. Although PA shows multi-mechanistic antitumor potential, its efficacy against NB remains uncharacterized. This study systematically investigated the potential molecular targets and mechanisms underlying the anti-NB effects of PA by integrating network pharmacology-based target prediction with experimental validation of multi-target interactions through molecular docking, dynamic simulations, and in vitro assays, aimed to establish a novel perspective on PA’s antitumor activity and explore its potential clinical implications for NB treatment by integrating computational predictions with biological assays. MethodsThis study employed network pharmacology to identify potential targets of PA in NB, followed by validation using molecular docking, molecular dynamics (MD) simulations, MM/PBSA free energy analysis, RT-qPCR and Western blot experiments. Network pharmacology analysis included target screening via TCMSP, GeneCards, DisGeNET, SwissTargetPrediction, SuperPred, and PharmMapper. Subsequently, potential targets were predicted by intersecting the results from these databases via Venn analysis. Following target prediction, topological analysis was performed to identify key targets using Cytoscape software. Molecular docking was conducted using AutoDock Vina, with the binding pocket defined based on crystal structures. MD simulations were performed for 100 ns using GROMACS, and RMSD, RMSF, SASA, and hydrogen bonding dynamics were analyzed. MM/PBSA calculations were carried out to estimate the binding free energy of each protein-ligand complex. In vitro validation included RT-qPCR and Western blot, with GAPDH used as an internal control. ResultsThe CCK-8 assay demonstrated a concentration-dependent inhibitory effect of PA on NB cell viability. GO analysis suggested that the anti-NB activity of PA might involve cellular response to chemical stress, vesicle lumen, and protein tyrosine kinase activity. KEGG pathway enrichment analysis suggested that the anti-NB activity of PA might involve the PI3K/AKT, MAPK, and Ras signaling pathways. Molecular docking and MD simulations revealed stable binding interactions between PA and the core target proteins AKT1, EGFR, SRC, and HSP90AA1. RT-qPCR and Western blot analyses further confirmed that PA treatment significantly decreased the mRNA and protein expression of AKT1, EGFR, and SRC while increasing the HSP90AA1 mRNA and protein levels. ConclusionIt was suggested that PA may exert its anti-NB effects by inhibiting AKT1, EGFR, and SRC expression, potentially modulating the PI3K/AKT signaling pathway. These findings provide crucial evidence supporting PA’s development as a therapeutic candidate for NB.

Aim To investigate the effect of benzyl iso-thiocyanate (BITC) on the proliferation of mouse U14 cervical cancer cells and to explore the mechanism of cytotoxicity based on transcriptomic data analysis. Methods The effect of BITC on U14 cell activity was detected by MTT, nuclear morphological changes were observed by Hochest 33258 and fluorescent inverted microscope, cell cycle and apoptosis were determined by flow cytometry, and the transcriptome database of U14 cells before and after BITC (20 μmol · L

Aim To discover the potential active compounds and possible mechanisms in rheumatoid arthritis (RA) treatment with Zhi-Huang-Zhi-Tong powder (ZHZTP) by using network pharmacology and in vitro study. Methods The active ingredient targets and disease targets of Zhihuang Zhitong Powder were searched and screened by database; they intersected to get a common target; and the "drug-component-target" relationship network diagram was constructed for GO and KEGG enrichment analysis of the overlapping genes; then the core components were docked with the core targets. Finally, based on the inflammation model of HUVECs in vitro, the efficacy and mechanism of Zhihuang Zhitong powder were verified by MTT method, plate scratch test and Western blot. Results Active compounds involved in RA treatment were screened in the present study, and the top two were ursolic acid and emodin, all playing crucial roles in RA treatment with ZHZTP. Additionally, the key target was AKTA, TNF and IL-6. GO and KEGG enrichment analysis revealed that ZHZTP regulated BP, MF and CC, and also focused on regulating AKTA, TNF and IL-6 signaling pathway. Molecular docking showed that interactions between key active compounds and key targets were stable. In vitro ZHZTP significantly inhibited cell viability and migration of TNF-a-stimulated HUVECs, and the involved mechanism may be associated with PI3K/AKT/m-TOR signaling. Conclusions The present study reveals that the potential active compounds of ZHZTP are ursolic acid and emodin, and moreover, the involved mechanisms of ZHZTP for RA treatment are associated with PI3 K/AKT/m-TOR signaling.

Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ

Objective To investigate the therapeutic effect and mechanism of Jianwei Xiaozhang Tablets on rats with precancerous lesions of gastric cancer(PLGC).Methods Forty male SD rats were randomly divided into the normal group,the model group,the folic acid group and the Jianwei Xiaozhang Tablets group,with 10 rats in each group.In addition to the normal group,the other three groups of rats were prepared by gavage with Ranitidine Aqueous Solution combined with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)solution drinking method for the preparation of PLGC model.After successful modeling,drugs were administered accordingly for 7 weeks.The changes in body mass of rats during modeling and drug administration were recorded,the gross view of the stomach was observed and scored pathologically,the coefficients of spleen and liver were determined,the pathological changes in gastric tissue were observed by hematoxylin-eosin(HE)staining,enzyme-linked immunosorbent assay(ELISA)was used to measure serum gastrin(GAS),motilin(MTL)and glucagon(GC),Alisin Blue-Periodic Acid Schiff's(AB-PAS)staining was used to observe the thickness of the mucosal layer of gastric tissues,the expressions of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),and endothelial-type nitric oxide synthase(eNOS)proteins in gastric tissues were detected by protein immunoblotting(Western Blot),and the expression of vascular endothelial growth factor A(VEGFA)protein in gastric tissues was detected by immunofluorescence staining.Results Compared with the normal group,the body mass of rats in the model group grew slowly during the experimental period,gastric macroscopic pathological scores were significantly increased(P＜0.01),splenic coefficient and hepatic coefficient were significantly decreased(P＜0.01),the gastric tissues showed cuprocyte hyperplasia and intestinal chemotaxis,gastric tissues'inflammation scores were significantly increased(P＜0.01),the serum GAS content was significantly increased(P＜0.01),and the MTL,GC contents were significantly reduced(P＜0.05),and the thickness of the mucous membrane layer of gastric tissue was significantly reduced(P＜0.05),the protein expression levels of PI3K,p-PI3K,Akt,p-Akt and eNOS were reduced(P＜0.01),and the protein expression level of VEGFA was reduced(P＜0.01);compared with the model group,the above indexes of the Jianwei Xiaozhang Tablets group and the folic acid group were all significantly improved(P＜0.05 or P＜0.01),among which,the Jianwei Xiaozhang Tablets group had a better improvement effect in the proliferation of cup cells and intestinal chemotaxis in gastric tissues,the content of serum GAS,and the thickness of the mucous layer in gastric tissues.Conclusion The mechanism of the improvement of PLGC in rats by Jianwei Xiaozhang Tablets may be related to the activation of the PI3K-Akt-eNOS pathway,which in turn promotes the angiogenesis and repair of gastric damaged tissues.

Objective To investigate the protective effect and mechanism of acellular nerve allografts(ANA)combined with electroacupuncture on spinal ganglia in rats with sciatic nerve injury(SNI).Methods Totally 50 male adult SD rats were randomly selected for this experiment.Ten rats were prepared for the ANA.Forty male SD rats were randomly divided into normal group,model group,ANA group and combinational group,with 10 rats in each group.The SNI model was established by cutting off the nerves 10 mm at the 5 mm on the inferior border of piriformis after separating the right sciatic nerves.The rats in the ANA group were bridged with ANA to the two broken ends of injured nerves.The rats in the combinational group were treated with electroacupuncture 2 days after ANA bridging,Huantiao(GB30)and Yanglingquan(GB34)were performed as the acupuncture points,each electroacupuncture lasted 15 minutes and 7 days as a course of treatment,4 courses in all.Sciatic nerve conduction velocity was measured by electrophysiology to evaluate the regeneration of damaged axons.Morphology of spinal ganglia was observed by Nissl staining.The expression of nerve growth factor(NGF)and brain-derived neurotrophic factor(BDNF)were detected by Western blotting and immunofluorescent staining.Results Compared with the normal group,the sciatic nerve conduction velocity in model group decreased significantly(P<0.01),Nissl bodies in neurons of spinal ganglia were swollen and dissolved,with incomplete structure and the number decreased dramatically(P<0.01),while the level of NGF and BDNF also decreased significantly(P<0.01).Compared with the model group,the sciatic nerve conduction velocity in ANA and combinational groups strongly increased(P<0.01),the damage of Nissl bodies in neurons of spinal ganglia reduced and the number obviously increased(P<0.01),the level of NGF and BDNF increased considerably(P<0.01).Compared with the ANA group,the sciatic nerve conduction velocity in combinational group increased significantly(P<0.01),the morphology of Nissl bodies in neurons of spinal ganglia were more regular and the number increased(P<0.01),moreover,the level of NGF also increased significantly(P<0.01).Conclusion ANA combined with electroacupuncture can enhance the sciatic nerve conduction velocity,improve the morphology of neurons in spinal ganglia and play a protective effect on spinal ganglia.The mechanism can be related to the higher expression of NGF and BDNF proteins,especially the expression of NGF protein.

Objective To explore the eradication rate of human papillomavirus(HPV)and gestational outcome of patients with high-grade squamous intraepithelial disease of the cervix(HSIL)after loop electrosurgical excision procedure(LEEP)by transvaginal dissection of the vesicorectal form the cervix.Methods A total of 53 patients treated with LEEP by transvaginal dissection of the vesicorectal form the cervix in Obstetrics and Gynecology Hospital,Fudan University from Jan to Dec,2019 were investigated.Clinical information of cervical cytological examination,HPV test and cervical biopsy under colposcopy were followed up for 6,12 and 24 months post-LEEP were collected.HPV infection in these 53 patients were compared before and after LEEP surgery.The rate of successful fertility of the cohort,the HPV conversion rate of patients with hysterectomy and LEEP done were compared.The association between the pathological type and positive surgical margin and the association between HPV infection type and positive surgical margin were analyzed.Results HPV infection rate of was 94.3%(50/53)and the proportion of HPV16 and/or 18 infection was 75.5%(40/53).Mono-HPV infection rate(69.8%,37/53)was significantly higher than mixed HPV infection rate(22.7%,13/53).Thirty-eight patients(71.7%)were found with positive surgical margin in previous LEEP operation.Fifteen patients had recurrence(28.3%)and 40 patients(75.5%)successfully delivered baby after surgery.Postoperative pathology was mainly HSIL,accounting for 66%(30/53),and 28.3%patients(15/53)had no pathological change.Forty cases had satisfying fertility-conservative operation outcome with negative surgical margin,and 38 patients eradicated HPV infection after LEEP,which took up 95%of patients with satisfying fertility-conservative operation.There was no significant difference of positive resection margin rate in between groups of HPV16/18 infection and other types.Five cases had successful delivery(12.5%,5/40)with 1 case of vaginal delivery and 4 cases of cesarean section.Among these 5 cases,3 cases undertook preventive cervical cerclage,with 1 case of vaginal delivery and 2 cases of cesarean sections.Conclusion HPV eradication rate and surgical outcome could be significantly improved by LEEP with transvaginal dissection of the vesicorectal from the cervix,which satisfied the fertility preservation of females at reproductive age.

Objective To evaluate the clinical application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)in analyzing the homology of Acinetobacter baumannii(AB).Methods After excluding repetitive strains from multiple specimens of the same patient or environment,a total of 46 AB strains isolated from patients'sputum and environmental specimens of neurological intensive care unit(ICU)in a tertiary first-class general hospital from May 2020 to February 2021 were collected.Strains were detected by VITEK-MS mass spectrometer.Cluster analysis was performed by SARAMIS Premium software,and verified by multilocus sequence typing(MLST).Results Cluster analysis and comparison of MALDI-TOF MS and MLST found that among the 46 AB strains,39 were the type MS-a of MALDI-TOF MS,of which 22 strains were the clus-ter MT-A of MLST,including ST208(n=3),ST540(n=3),ST195(n=8),ST369(n=5),ST136(n=1),ST436(n=1)and ST1893(n=1);16 strains were MT-B,including type ST381(n=4),type ST469(n=11),and type ST938(n=1);one strain was cluster MT-C(ST1821);one strain of type MS-b was ST381;two strains of type MS-c were ST369;one strain of type MS-d was ST195;two strains of type MS-e were ST540 and ST369,respectively;one strain of type MS-f was STN1.Conclusion As a homology analysis method,MALDI-TOF MS still has certain limitations such as low consistency with MLST results,low resolution and specificity,thus cannot replace MLST technology.