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: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

Background: s/Aims: Non-invasive models stratifying clinically significant portal hypertension (CSPH) are limited. Herein, we developed a new non-invasive model for predicting CSPH in patients with compensated cirrhosis and investigated whether carvedilol can prevent hepatic decompensation in patients with high-risk CSPH stratified using the new model. Methods: Non-invasive risk factors of CSPH were identified via systematic review and meta-analysis of studies involving patients with hepatic venous pressure gradient (HVPG). A new non-invasive model was validated for various performance aspects in three cohorts, i.e., a multicenter HVPG cohort, a follow-up cohort, and a carvediloltreating cohort. Results: In the meta-analysis with six studies (n=819), liver stiffness measurement and platelet count were identified as independent risk factors for CSPH and were used to develop the new “CSPH risk” model. In the HVPG cohort (n=151), the new model accurately predicted CSPH with cutoff values of 0 and –0.68 for ruling in and out CSPH, respectively. In the follow-up cohort (n=1,102), the cumulative incidences of decompensation events significantly differed using the cutoff values of <–0.68 (low-risk), –0.68 to 0 (medium-risk), and >0 (high-risk). In the carvediloltreated cohort, patients with high-risk CSPH treated with carvedilol (n=81) had lower rates of decompensation events than non-selective beta-blockers untreated patients with high-risk CSPH (n=613 before propensity score matching [PSM], n=162 after PSM). Conclusions Treatment with carvedilol significantly reduces the risk of hepatic decompensation in patients with high-risk CSPH stratified by the new model.

Aldehyde oxidase (AOX) is a molybdoenzyme that is primarily expressed in the liver and is involved in the metabolism of drugs and other xenobiotics. AOX-mediated metabolism can result in unexpected outcomes, such as the production of toxic metabolites and high metabolic clearance, which can lead to the clinical failure of novel therapeutic agents. Computational models can assist medicinal chemists in rapidly evaluating the AOX metabolic risk of compounds during the early phases of drug discovery and provide valuable clues for manipulating AOX-mediated metabolism liability. In this study, we developed a novel graph neural network called AOMP for predicting AOX-mediated metabolism. AOMP integrated the tasks of metabolic substrate/non-substrate classification and metabolic site prediction, while utilizing transfer learning from ¹³C nuclear magnetic resonance data to enhance its performance on both tasks. AOMP significantly outperformed the benchmark methods in both cross-validation and external testing. Using AOMP, we systematically assessed the AOX-mediated metabolism of common fragments in kinase inhibitors and successfully identified four new scaffolds with AOX metabolism liability, which were validated through in vitro experiments. Furthermore, for the convenience of the community, we established the first online service for AOX metabolism prediction based on AOMP, which is freely available at https://aomp.alphama.com.cn.

ObjectiveTo investigate the safety and efficacy of endoscopic ultrasound-guided tissue adhesive injection with the assistance of metal clips in the treatment of cirrhotic patients with gastric varices and gastric-renal shunt （GRS）. MethodsThe patients who attended Beijing Ditan Hospital， Capital Medical University， due to liver cirrhosis and gastric varices from February to June 2023 were enrolled， and all patients were confirmed to have GRS and received endoscopic ultrasound-guided tissue adhesive injection with the assistance of metal clips. The primary evaluation index was alleviation or disappearance of varicose veins after surgery， and the secondary evaluation indices were surgical completion and complications. ResultsA total of 11 patients were enrolled in this study， among whom there were 7 male patients and 4 female patients， with a median age of 55 years. Of all patients， 1 had Child class A liver function， 7 had Child class B liver function， and 3 had Child class C liver function. The maximum （median） diameter of the shunt was 8 mm， and the minimum （median） diameter of the shunt was 4 mm. The median blood flow velocity of the target vessel was 11 cm/s before treatment and 5 cm/s after occlusion with metal clips. The median amount of tissue adhesive injected was 2 mL， and the amount of lauromacrogol used was 1 mL. Disappearance of blood flow signals was observed in all patients after surgery （100%）， and the success rate of surgery was 100%. No patient experienced rebleeding after follow-up for 6 weeks. Gastroscopy at 1 month after surgery showed that gastric varices were eradicated or almost disappeared in 9 patients and were alleviated in 2 patients. ConclusionEndoscopic ultrasound-guided tissue adhesive injection with the assistance of metal clips is a feasible， safe， and effective treatment method for cirrhotic patients with gastric varices and GRS.

BACKGROUND:Deferoxamine mesylate is a potential anti-osteoporosis drug with iron chelation,vascular regeneration,and antioxidant effects.Recent studies have shown that the application of deferoxamine mesylate can be extended to the field of tissue regeneration engineering. OBJECTIVE:To investigate whether deferoxamine mesylate can promote the repair effect of iron overload osteoporotic rats after bone grafting for mandibular bone defects by simulating the state of iron accumulation in patients with postmenopausal osteoporosis with high iron intervention in osteoporotic rats. METHODS:An iron accumulation ovariectomized osteoporosis model was firstly constructed.The model group underwent bilateral ovariectomy,and the intraperitoneal injection of ferric ammonium citrate(90 mg/kg,twice a week,for 11 weeks)was started in the 2nd week,while the sham-operated group had some fat around the ovaries removed and was given an equal amount of saline for 11 weeks.After the successful modeling,the experimental rats were divided into sham-operated group(n=6),high iron ovariectomtized group(n=6)and high iron ovariectomized deferoxamine mesylate treatment group(deferoxamine mesylate group,n=6).Bone defects of 5 mm in diameter were established in the rat's bilateral mandibles and implanted with Bio-Oss bone powder.Intraperitoneal injection of deferoxamine mesylate(100 mg/kg,3 times a week)was started on postoperative day 4 in the deferoxamine mesylate group,and equal volume of saline was given in the sham-operated and high iron ovariectomized groups.The bone samples of the mandible,liver and blood were taken at 2 and 12 weeks after bone grafting for Prussian blue staining of the jaw and liver and ELISA detection of serum ferritin to detect iron levels in various body tissues;hematoxylin-eosin staining and Masson staining were performed to observe inflammatory cell infiltration and early osteogenesis in the bone defect area;tartrate resistant acid phosphatase staining was performed to observe osteoclast differentiation;ELISA was performed to detect serum calcitonin and type I collagen C-terminal peptide levels;and Micro-CT and hematoxylin-eosin staining were performed to observe osteogenesis in the middle and late stages. RESULTS AND CONCLUSION:The number of tibial trabeculae was reduced and the trabeculae were sparsely arranged in the high iron ovariectomized group.Iron levels in the liver,jaw bone and serum were significantly higher in the high iron ovariectomized group than the sham-operated group at 2 weeks after bone grafting,while the iron levels were significantly decreased after deferoxamine mesylate intervention(P＜0.05).In the early stage of bone defect repair,more inflammatory cell infiltration,less new bone matrix and less type I collagen fiber production were observed in the high iron ovariectomized group than in the sham-operated group(P＜0.05);after deferoxamine mesylate treatment,inflammatory cell infiltration was reduced,a small amount of new bone matrix was produced and collagen fibers increased significantly(P＜0.05).In the middle and late stages of bone defect repair,Micro-CT results showed a reduction in new bone production in the high iron ovariectomized group compared with the sham-operated group and increased new bone matrix after deferoxamine mesylate treatment(P＜0.05).Compared with the sham-operated group,the osteoclast number,serum calcitonin level,and serum type I collagen C-terminal peptide level were increased in the high-iron ovariectomized group,while the osteoclast number was decreased and bone metabolic indexes were improved after treatment with deferoxamine mesylate.To conclude,in ovariectomized rats with high iron intervention,elevated iron levels can be seen in multiple tissues,accompanied by reduced new bone production in the mandibular bone defect area.Deferoxamine mesylate can improve bone metabolism and inhibit osteoclast activity by removing iron deposits in tissues,improve bone formation in iron-accumulated osteoporotic rats,and promote bone healing in the mandibular bone defect area.

BACKGROUND:Interleukin-4 can promote the osteogenic effect of bone substitute materials,but its molecular mechanism is not yet clear.Further elucidating the mechanism of interleukin-4 promoting osteogenic effect can help find safe,economical,and effective methods for the regeneration treatment of alveolar bone defects in patients. OBJECTIVE:To explore the effect of interleukin-4 intervention on polarization transformation of macrophages and osteogenic differentiation of bone marrow mesenchymal stem cells and its possible mechanism. METHODS:RAW264.7 cells in the M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours.RAW264.7 cells in the interleukin-4+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours and then interleukin-4 was added for 24 hours.RAW264.7 cells in the interleukin-4+AG+M1 group were induced with interferon gamma + lipopolysaccharide for 24 hours,and then interleukin-4 and AG-490,a JAK/STAT pathway inhibitor,were added for 24 hours.After intervention,immunofluorescence staining was used to analyze the expression of inducible nitric oxide synthase and CD206,the phenotypic marker protein of macrophages.ELISA kit was used to detect the expression of interleukin-10 and tumor necrosis factor-α in the supernatant of cell culture.The gene expressions of nodular receptor protein-3(NLRP3),interleukin-1β,and caspase-1 were detected by RT-qPCR.The expression levels of tyrosine protein kinase 1(JAK1)/phosphorylated tyrosine protein kinase 1(p-JAK1),signal transduction and transcription activator 6(STAT6)/phosphorylated signal transduction and transcription activator 6(p-STAT6),NLRP3,pro-interleukin-1β and pro-caspase-1 were detected by western blot assay.Then,RAW264.7 cells in the above four groups were indirectly co-cultured with bone marrow mesenchymal stem cells by transwell for 24 hours,followed by alkaline phosphatase staining and alizarin red staining.The mRNA expressions of alkaline phosphatase,collagen type I,and osteocalcin were detected by RT-qPCR. RESULTS AND CONCLUSION:(1)Immunofluorescence and ELISA results showed that interleukin-4 intervention could promote the expression of CD206 and interleukin-10 in M2 macrophages,and inhibit the secretion of inducible nitric oxide synthase and tumor necrosis factor-α.(2)RT-qPCR results showed that interleukin-4 could suppress the expression of NLRP3,interleukin-1β,and caspase-1 mRNAs.(3)Western blot assay showed that interleukin-4 could promote the expression of JAK1/p-JAK1,STAT6/p-STAT6 and NLRP3 proteins.(4)The alkaline phosphatase staining and alizarin red staining of bone marrow mesenchymal stem cells co-cultured with the interleukin-4+M1 group were significantly enhanced,and the mRNA expressions of alkaline phosphatase,collagen type I,and osteocalcin were significantly increased.It is concluded that interleukin-4 may inhibit the activation of NLRP3 by up-regulating JAK1/STAT6 pathway,thus promoting the transformation of macrophages from M1 polarization to M2 polarization,and finally enhancing the osteogenic differentiation ability of bone marrow mesenchymal stem cells.

AIM To establish a quantitative analysis of multi-components by single-marker(QAMS)method for the simultaneous content determination of gastrodin,parishin E,syringin,parishin B,parishin C,ferulic acid,parishin A,buddleoside,harpagoside and cinnamic acid in Tianma Toufengling Capsules.METHODS The analysis was performed on a 30℃thermostatic GL Science InertsilTM ODS-3 column(150 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1%phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 220,280 nm.Syringin was used as an internal standard to calculate the relative correction factors of the other nine constituents,after which the content determination was made.RESULTS Ten constituents showed good linear relationships within their own ranges(r≥0.999 7),whose average recoveries were 98.53%-102.22%with the RSDs of 1.26%-2.68%.The result obtained by QAMS approximated those obtained by external standard method.CONCLUSION This accurate and specific method can be used for the quality control of Tianma Toufengling Capsules.

Background::Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods::The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. Results::Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions::The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.