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.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Humans ; PPAR gamma/metabolism* ; Multiple Sclerosis ; Transcription Factors/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha

Objective To analyze the stress changes of thoracic vertebra(T)11-sacrum(S)titanium rods in patients with ankylosing spondylitis after vertebral column decancellation(VCD)osteotomy,and provide reference for the selection and improvement of titanium rods before surgery.Methods The original data of the continuous scanning tomographic images of patients with ankylosing spondylitis after VCD osteotomy were imported into Mimics 21.0 in DICOM format,and T11-S vertebrae,screws and titanium rods were respectively reconstructed.They were imported into 3-Matic to establish a preliminary geometric modeling,and then processed with noise removal,paving,smoothing,etc.The improved model was imported into Hypermesh 10 software for grid division,and the material was imported into ANSYS 19.2 to display the finite element model after attribute assignment,Set the boundary and load conditions,and measure the stress value at the connection between the screw and the titanium rod.Results Under neutral position,forward bending,lateral bending,and axial rotation conditions,the titanium rod had the highest stress at the upper vertebrae(T11)and the lowest stress at the top vertebrae(L3);Under the backward extension condition,the titanium rod has the highest stress at the lower end vertebra(L5).Conclusion In the upper and lower vertebrae,it is possible to consider increasing the diameter of the titanium rod,enhancing its hardness,or changing it to a double rod.

Objective To investigate the effects of total flavones from Oxytropis falcata Bunge on hepatic fibrosis(HF)induced by carbon tetrachloride and liver transforming growth factor(TGF-β)/Smad signaling pathway.Methods Forty-eight male rats were randomly divided into normal group(intraperitoneal injection of peanut oil,intragastric administration of 0.9％NaCl),model group(intraperitoneal injection of 40％CC14 peanut oil solution induced HF model,intragastric administration of 0.9％NaCl),positive control group(modeling,intragastric administration of 0.2 mg·kg-1 of colchicine),experimental-L,-M,-H groups(modeling,intragastric administration of 100,200 and 400 mg·kg-1 of total flavonoid extract of Oxytropis falcata Bunge),8 individuals in each group,for 4 consecutive weeks.The histopathological changes were observed by hematoxylin-eosin and Masson staining.Serum liver function and liver fibrosis were measured;erum inflammatory factors were detected;fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to determine gene expression in liver.Results The pathological injury of liver tissue in the model group was serious,and a large number of inflammatory factors and collagen fibers were accumulated,while the rest of the treatment groups had different degrees of remission.In normal group,model group,positive control group,experimental-L,-M,-H groups,glutamic-pyruvic transaminase levels were(49.28±12.44),(5 885.42±948.37),(4 454.60±489.27),(4 650.47±843.53),(3 761.75±887.30)and(3 544.90±1 066.75)μg·L-1;glutamic-oxaloacetic transaminase levels were(186.90±46.89),(5 936.23±793.81),(3 971.37±780.28),(4 360.30±863.35),(3 943.10±439.47)and(3 971.38±631.08)μg·L-1;hyaluronic acid levels were(45.08±17.16),(104.32±36.06),(66.83±20.09),(70.30±21.07),(60.00±9.68)and(59.02±10.73)μg·L-1;laminin levels were(23.13±3.89),(60.85±13.66),(35.67±9.92),(39.98±9.39),(36.55±12.21)and(34.68±24.83)μg·L-1;type Ⅲ procollagen level were(24.98±5.34),(82.58±30.14),(40.70±16.14),(51.08±23.21),(43.60±12.48)and(44.20±11.66)p±g·L-1;interleukin(IL)-1β levels were(37.63±1.24),(46.10±3.23),(39.22±2.36),(41.33±0.93),(40.25±2.04)and(39.18±2.23)pg·mL-1;tumor necrosis factor-α levels were(314.58±20.56),(383.71±16.97),(349.00±7.93),(348.88±25.11),(325.75±27.84)and(335.07±21.33)pg·mL-1;TGF-β1 mRNA expression of relative quantity respectively were 1.00±0.00,60.99±15.70,9.61±1.59,7.37±1.09,6.41±0.64,6.87±1.09;Smad7 mRNA relative expression were 1.00±0.00,0.34±0.05,0.21±0.03,0.35±0.02,0.38±0.02,0.42±0.03.The above indexes in the model group were compared with the normal group,and the above indexes in the experimental-M,-H groups were compared with the model group,and the differences were statistically significant(P＜0.05,P＜0.01,P＜0.001).Conclusion Total flavonoids of Oxytropis falcata Bunge have protective effects on CC14-induced liver fibrosis in rats,and the mechanism may be related to the regulation of TGF-β/Smad pathway.

Objective To analyze the risk factors and survival status of Epstein-Barr virus(EBV)infection in pa-tients with aplastic anemia(AA)after haploid allogeneic hematopoietic stem cell transplantation(Haplo-HSCT).Methods Clinical data of 78 AA patients who underwent Haplo-HSCT in the hematology department of a hospital from January 1,2019 to October 31,2022 were analyzed retrospectively.The occurrence and onset time of EBV viremia,EBV-related diseases(EBV diseases),and post-transplant lymphoproliferative disorders(PTLD)were ob-served,risk factors and survival status were analyzed.Results Among the 78 patients,38 were males and 40 were females,with a median age of 33(9-56)years old;53 patients experienced EBV reactivation,with a total inci-dence of 67.9％,and the median time for EBV reactivation was 33(13,416)days after transplantation.Among pa-tients with EBV reactivation,49 cases(62.8％)were simple EBV viremia,2 cases(2.6％)were possible EBV di-seases,and 2 cases(2.6％)were already confirmed EBV diseases(PTLD).Univariate analysis showed that age 1＜40 years old at the time of transplantation,umbilical cord blood infusion,occurrence of acute graft-versus-host disease(aGVHD)after transplantation,and concurrent cytomegalovirus(CMV)infection were independent risk fac-tors for EBV reactivation in AA patients after Haplo-HSCT.Multivariate analysis showed that concurrent CMV in-fection was an independent risk factor for EBV reactivation in A A patients after Haplo-HSCT(P=0.048).Ritu-ximab intervention before stem cell reinfusion was a factor affecting the duration of EBV reactivation(P＜0.05).The mortality of EBV viremia,EBV diseases,and PTLD alone were 8.2％,50.0％,and 100％,respectively.The 2-year overall survival rate of patients with and without EBV reactivation were 85.3％,and 90.7％,respectively,difference was not statistically significant(P=0.897).However,patients treated with rituximab had 2-year lower survival rate than those who did not use it,with a statistically significant difference(P=0.046).Conclusion EBV reactivation is one of the serious complications in AA patients after Haplo-HSCT,which affects the prognosis and survival of patients.

Objective Polymorphic microsatellite markers developed for Apodemus peninsulae can enrich its genetic data and lay a foundation for genetic quality control and gene mapping.Methods Microsatellite loci were screened based on the genome sequence of Apodemus peninsulae,and microsatellite primers were identified.The genetic diversity of the population was analyzed by multiplex PCR.Results Thirty microsatellite markers were successfully developed and evaluated using 60 samples of Apodemus peninsulae.A total of 152 alleles were detected,with an average of 5.067 alleles per locus.The average observed heterozygosity was 0.592.The average Shannon index was 1.265.The average polymorphism information content was 0.598.Conclusions Based on the microsatellite loci developed in this study,the genetic diversity of Apodemus peninsulae can be effectively analyzed,laying a foundation for establishing genetic quality standards and detection method.

Objective:To investigate the clinicopathologic characteristics, gene mutation profile and prognostic influencing factors of diffuse large B-cell lymphoma (DLBCL) complicated with follicular lymphoma (FL) (DLBCL/FL).Methods:The clinicopathological data of 50 DLBCL/FL patients admitted to Rui Jin Hospital Affiliated of Shanghai Jiao Tong University School of Medicine from February 2018 to November 2021 were retrospectively analyzed. Targeted sequencing was performed to assess the mutation profile of 55 lymphoma-related genes. The clinicopathological characteristics were summarized to evaluate the short-term therapeutic efficacy of all patients. Kaplan-Meier method was used to analyze the overall survival (OS) and progression-free survival (PFS) of patients. Cox regression risk models were used to assess the factors affecting the OS and PFS.Results:Among 50 DLBCL/FL patients, 23 cases (46%) were male, 22 cases (44%) had an international prognosis index (IPI) score ≥ 2 points, 16 cases (32%) were double-expression lymphoma (DEL) and 4 cases (8%) were double-hit lymphoma (DHL). The complete response (CR) and overall response rates were 68% (34/50) and 78% (39/50), respectively after the first-line therapy. The median follow-up time was 23.3 months (5.1-50.9 months). The 2-year OS rate was 82.1% and 2-year PFS rate was 67.1%; and the median OS and PFS were not reached. Targeted sequencing results showed that the mutation frequencies of KMT2D, MYD88, TP53, BTG2, DTX1, EZH2, CD70, CREBBP, DUSP2, HIST1H1C, HIST1H1E and PRDM1 genes in this cohort were more than 15%. Multivariate Cox regression analysis showed that male ( HR = 4.264, 95% CI 1.144-15.896, P = 0.031) and IPI score ≥ 2 points ( HR = 6.800, 95% CI 1.771-37.741, P = 0.007) were independent risk factors of PFS in newly diagnosed DLBCL/FL patients, and TP53 mutation ( HR = 4.992, 95% CI 1.027-24.258, P = 0.046) was an risk influencing factor of OS. Conclusions:The proportion of male and female DLBCL/FL patients is similar, with a small proportion of DHL. Mutations of KMT2D, MYD88 and TP53 genes are commonly found in DLBCL/FL patients. Generally, DLBCL/FL patients can have a high overall response and good prognosis. Male and IPI score ≥ 2 points are the independent risk factors of PFS, and TP53 mutation is an independent risk factor of OS in DLBCL/FL patients.