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.

Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism* ; Autophagy/drug effects* ; Apoptosis/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Cell Line ; bcl-X Protein/metabolism* ; Osteoblasts/metabolism* ; Rats ; Osteoporosis/physiopathology* ; Male ; Rats, Sprague-Dawley ; Osteogenesis/drug effects*

The polymerase 1 and transcript release factor (PTRF)-cytoplasmic phospholipase A2 (cPLA2) phospholipid remodeling pathway facilitates tumor proliferation in glioma. Nevertheless, blockade of this pathway leads to the excessive activation of oncogenic receptors on the plasma membrane and subsequent drug resistance. Here, CD26/dipeptidyl peptidase 4 (DPP4) was identified through screening of CRISPR/Cas9 libraries. Suppressing PTRF-cPLA2 signaling resulted in the activation of the epidermal growth factor receptor (EGFR) pathway through phosphatidylcholine and lysophosphatidylcholine remodeling, which ultimately increased DPP4 transcription. In turn, DPP4 interacted with EGFR and prevented its ubiquitination. Linagliptin, a DPP4 inhibitor, facilitated the degradation of EGFR by blocking its interaction with DPP4. When combined with the cPLA2 inhibitor AACOCF3, it exhibited synergistic effects and led to a decrease in energy metabolism in glioblastoma cells. Subsequent in vivo investigations provided further evidence of a synergistic impact of linagliptin by augmenting the sensitivity of AACOCF3 and strengthening the efficacy of temozolomide. DPP4 serves as a novel target and establishes a constructive feedback loop with EGFR. Linagliptin is a potent inhibitor that promotes EGFR degradation by blocking the DPP4-EGFR interaction. This study presents innovative approaches for treating glioma by combining linagliptin with AACOCF3 and temozolomide.

Objective To analyze the characteristics of methadone-related poisoning cases and provide a reference for forensic identification.Methods A total of 71 cases of methadone-related poisoning re-ported from 1998 to 2023 in China and 26 cases of methadone-related deaths reported from 2013 to 2018 in Italy were retrieved from databases including PubMed,Wanfang and CNKI.The general infor-mation,forensic pathological and toxicological characteristics were analyzed.Results Among the 71 methadone-related poisoning cases in China,there were 54 cases(76.06%)of poisoning without death and 17 cases(23.94%)of death from poisoning.There were 54 male cases(76.06%),and 51 cases(71.83%)aged 19 to 39 years old.There were 35 cases(49.30%)of poisoning caused by methadone alone,and 32 cases(45.07%)were poisoning caused by methadone combined with other substances or drugs including heroin and benzodiazepines.Most of the poisoned showed coma,respiratory depres-sion and miosis.Signs of asphyxia were often found by autopsy.The mass concentration of methadone detected in the blood of 6 deceased ranged from 0.112 to 3.000 mg/L.Among the 26 methadone-related deaths in Italy,22 cases were male(84.62%).There were 6 cases(23.08%)caused by methadone alone,and 20 cases(76.92%)died from methadone combined with other substances or drugs.The mass concentration of methadone in blood ranged from 0.181 to 4.059 mg/L.Conclusion The propor-tions of poisoning cases caused by methadone alone and methadone combined with other substances or drugs are comparable in China.The majority of deceased caused by methadone poisoning shows typi-cal triad of coma,respiratory depression and miosis,which helps forensic experts determine the cause of death related to methadone.Additionally,it is necessary to increase the routine testing of the con-centration of methadone and its combined substances or drugs in deceased,and collect data for the in-terpretation of the results of related cases.

This article reports a first case of combined infection with severe fever with thrombocytopenia syndrome(SFTS)and scrub typhus in Dalian City.The patient was admitted to the hospital due to recurrent fever for 7 days and loss of consciousness for 1 day.Pathogen metagenomic sequencing(mNGS),SFTSV quantitative PCR,and enzyme-linked immunosorbent assay(ELISA)IgM tests were performed,showing positive results for Orientia tsutsugamushi and SFTSV nucleic acids.Based on clinical manifestations and epidemiological history,the patient was diagnosed with combined infections.

The abuse of novel phenylcyclohexylpyridine drugs poses a significant threat to societal safety. The novel psychoactive substance 2-methyl-deschloroketamine (2-MDCK), belonging to the phenylcyclohexylpyridine class, has recently surfaced as a new compound. However, there is a lack of understanding regarding its metabolic pathways and the identification of suitable biomarkers. In this study, a human liver microsomal model was established, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technology was applied to investigate the in vitro metabolism and products of 2-MDCK. The results indicate that 2-MDCK undergoes various metabolic reactions, including dehydrogenation, deamination, hydroxylation, and demethylation, leading to the formation of eight metabolites. By conducting actual testing on hair samples from 2-MDCK abusers, the existence of six metabolites was confirmed. Comparing the metabolic products in the liver microsomal in vitro model, M3.1 and M4.1 were identified as biomarkers for 2-MDCK consumption. Five samples of abusers of 2-methyl-dechloroketamine were provided by the Anti-Drug Brigade of the Hangzhou Public Security Bureau. Negative hair samples were provided by laboratory volunteers, and all samples were obtained with the informed consent of the volunteers. These findings provide a scientific basis for the detection and identification of 2-MDCK and its metabolites, as well as crucial support for the study of the metabolic mechanisms of similar novel psychoactive substances in the phenylcyclohexylpyridine class. This research holds significant importance in addressing the issue of abuse of new psychoactive substances.

Aim To investigate the role of metabolites of eicosapentaenoic acid (EPA) in promoting the transdifferentiation of pancreatic α cells to β cells. Methods Male C57BL/6J mice were injected intraperitoneally with 60 mg/kg streptozocin (STZ) for five consecutive days to establish a type 1 diabetes (T1DM) mouse model. After two weeks, they were randomly divided into model groups and 97% EPA diet intervention group, 75% fish oil (50% EPA +25% DHA) diet intervention group, and random blood glucose was detected every week; after the model expired, the regeneration of pancreatic β cells in mouse pancreas was observed by immunofluorescence staining. The islets of mice (obtained by crossing GCG

Objective:To explore the prognostic value of MUC13 expression in gastric cancer(GC)patients and its impact on the biological behavior of GC cells.Methods:Comprehensive anal-ysis of the expression pattern of MUC genes in GC tissues based on the TCGA database to screen for differentially expressed genes.Spearman correlation analysis determined the correlation of ex-pression between MUC genes in GC tissues.Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway(KEGG)enrichment analysis were used to explore the potential biological functions of MUC genes.Univariate COX regression analysis was performed to explore the relationship between all differentially expressed MUC genes and the prog-nosis of GC patients to screen out MUC genes that were significantly related to the prognosis of GC.Clinical GC tissue samples were used to further verify the expression of MUC13 through im-munofluorescence,and its relationship with the clinicopathological characteristics and prognosis of GC was analyzed.siRNA was used to silence the expression of MUC13 in GC cells,and the effect of MUC13 on cell proliferation,migration and invasion was analyzed through CCK-8,colony forma-tion and Transwell experiments.Results:Among all MUC members,the expression levels of MUC1,MUC2,MUC3A,MUC4,MCU5B,MUC12,and MUC13 were significantly upregulated in GC tissues(P＜0.05).There are certain interactions between these MUC genes,and they are mainly en-riched in pathways related to digestive system processes,epithelial structure maintenance,apical plasma membrane,saliva secretion,etc.Importantly,upregulation of MUC13 in GC tissues indicates poor patient prognosis(Log-rank P＜0.05).In addition,MUC13 expression was significantly correlat-ed with the age(P＜0.001)of GC patients and tumor size(P=0.035).Further cell function experiments showed that after silencing MUC13,the proliferation ability of GC cells was significantly reduced(P＜0.05),while their migration and invasion abilities were not significantly affected(P＞0.05).Con-clusions:Highly expressed MUC13 is closely related to the poor prognosis of gastric cancer,par-ticipates in the regulation of tumor progression and is a potential therapeutic target and prognostic marker for gastric cancer.

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannan-binding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP. Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays. Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05). Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.