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: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

Background: Maturity-onset diabetes of the young (MODY) due to variants of hepatocyte nuclear factor 1-beta (HNF1β) (MODY5) has not been well studied in the Chinese population. This study aimed to estimate its prevalence and evaluate the application of a clinical screening method (Faguer score) in Chinese early-onset diabetes (EOD) patients. Methods: Among 679 EOD patients clinically diagnosed with type 2 diabetes mellitus (age at diagnosis ≤40 years), the exons of HNF1β were sequenced. Functional impact of rare variants was evaluated using a dual-luciferase reporter system. Faguer scores ≥8 prompted multiplex ligation-dependent probe amplification (MLPA) for large deletions. Pathogenicity of HNF1β variants was assessed following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Two rare HNF1β missense mutations (E105K and G454R) were identified by sequencing in five patients, showing functional impact in vitro. Another patient was found to have a whole-gene deletion by MLPA in 22 patients with the Faguer score above 8. Following ACMG guidelines, six patients carrying pathogenic or likely pathogenic variant were diagnosed with MODY5. The estimated prevalence of MODY5 in Chinese EOD patients was approximately 0.9% or higher. Conclusion MODY5 is not uncommon in China. The Faguer score is helpful in deciding whether to perform MLPA analysis on patients with negative sequencing results.

ObjectiveTo establish a rat model of osteoarthritis and study the anti-inflammatory effects and mechanisms of fraxetin. MethodsEighteen 8-week-old male SPF-grade SD rats were randomly divided into three groups: Rats in the blank group received a right articular cavity injection of 50 μL of normal saline for 1 week; the model and intervention groups were injected with monosodium iodoacetate (MIA) into the right joint cavity to induce osteoarthritis, while the intervention group subsequently received fraxetin (5 mg·kg^-1·d^-1) for 1 week. Four weeks after drug intervention, abdominal aortic blood was collected. The animals were then euthanized, and knee joint cartilage were collected. The cartilage samples were stained with hematoxylin-eosin, safranin O-fast green, and toluidine blue for histopathological examination and scoring using the Mankin and OARSI scoring systems. The trabecular bone volume/total volume (Tb.BV/TV), trabecular bone surface density/total volume (Tb.BS/TV), and trabecular number (Tb.N) of each group were compared and analyzed using a micro-CT scanning system. The expression levels of various inflammatory factors [tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], and cartilage oligomeric matrix protein (COMP) were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of mitogen-activated protein kinase p38 (p38 MAPK), phosphorylation-p38 MAPK (p-p38 MAPK), c-Jun N-terminal kinase (JNK), and phosphorylation-JNK (p-JNK) were measured by western blotting. ResultsThe staining of cartilage sections of rat knee joints showed that the articular surface defects in the model group were severe, while the cartilage destruction in the intervention group was relatively reduced. Micro-CT results showed that Tb.BV/TV, Tb.BS/TV and Tb.N in the intervention group were significantly higher than those in the model group (P < 0.05); the Mankin score in the model group was significantly higher than that in the blank group (P < 0.05), the Mankin score in the intervention group was significantly lower than that in the model group (P < 0.05); while the OARSI score in the intervention group was significantly lower than that in the model group (P < 0.05). The results of the enzyme-linked immunosorbent assay showed that the serum levels of TNF-α, IL-1β, IL-6, and COMP in the model group were significantly higher than those in the blank group (all P < 0.05), while those in the intervention group were significantly lower than in the model group (P < 0.05). Western blot results showed that the expression levels of p-p38 MAPK and p-JNK in the knee cartilage tissue were significantly lower in the intervention group than in the model group (both P < 0.05), and significantly higher in the model group than in the blank group (both P < 0.05). ConclusionFraxetin may play a therapeutic role in a monosodium iodoacetate-induced rat model of osteoarthritis through the p38 MAPK pathway.

ObjectiveTo establish a rat model of osteoarthritis and study the anti-inflammatory effects and mechanisms of fraxetin. MethodsEighteen 8-week-old male SPF-grade SD rats were randomly divided into three groups: Rats in the blank group received a right articular cavity injection of 50 μL of normal saline for 1 week; the model and intervention groups were injected with monosodium iodoacetate (MIA) into the right joint cavity to induce osteoarthritis, while the intervention group subsequently received fraxetin (5 mg·kg^-1·d^-1) for 1 week. Four weeks after drug intervention, abdominal aortic blood was collected. The animals were then euthanized, and knee joint cartilage were collected. The cartilage samples were stained with hematoxylin-eosin, safranin O-fast green, and toluidine blue for histopathological examination and scoring using the Mankin and OARSI scoring systems. The trabecular bone volume/total volume (Tb.BV/TV), trabecular bone surface density/total volume (Tb.BS/TV), and trabecular number (Tb.N) of each group were compared and analyzed using a micro-CT scanning system. The expression levels of various inflammatory factors [tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], and cartilage oligomeric matrix protein (COMP) were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of mitogen-activated protein kinase p38 (p38 MAPK), phosphorylation-p38 MAPK (p-p38 MAPK), c-Jun N-terminal kinase (JNK), and phosphorylation-JNK (p-JNK) were measured by western blotting. ResultsThe staining of cartilage sections of rat knee joints showed that the articular surface defects in the model group were severe, while the cartilage destruction in the intervention group was relatively reduced. Micro-CT results showed that Tb.BV/TV, Tb.BS/TV and Tb.N in the intervention group were significantly higher than those in the model group (P < 0.05); the Mankin score in the model group was significantly higher than that in the blank group (P < 0.05), the Mankin score in the intervention group was significantly lower than that in the model group (P < 0.05); while the OARSI score in the intervention group was significantly lower than that in the model group (P < 0.05). The results of the enzyme-linked immunosorbent assay showed that the serum levels of TNF-α, IL-1β, IL-6, and COMP in the model group were significantly higher than those in the blank group (all P < 0.05), while those in the intervention group were significantly lower than in the model group (P < 0.05). Western blot results showed that the expression levels of p-p38 MAPK and p-JNK in the knee cartilage tissue were significantly lower in the intervention group than in the model group (both P < 0.05), and significantly higher in the model group than in the blank group (both P < 0.05). ConclusionFraxetin may play a therapeutic role in a monosodium iodoacetate-induced rat model of osteoarthritis through the p38 MAPK pathway.

Objective To investigate the mechanism of naringenin resisting lower extremity deep venous thrombosis(LEDVT)in rats.Methods Sixty rats were randomly divided into 6 groups,i.e.,sham-operation group,model group,naringenin low-,medium-,and high-dose groups,and naringenin high-dose + STING agonist 2.5 hexamethylene cacodylate(DMXAA)group,with 10 rats in each group.The coagulation indexes[D-dimer,thrombin time(TT),activated partial thromboplastin time(APTT),prothrombin time(PT)],inflammation indexes[interleukin 1β(IL-1β),interleukin 6(IL-6),tumor necrosis factor α(TNF-α)]and oxidative stress indexes[malondialdehyde(MDA),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)];Hematoxylin-eosin(HE)staining to detect thrombus formation in venous tissues;wet and dry mass of thrombus were detected;ultrastructure of venous thrombus was detected by transmission electron microscope(TEM);protein expressions of cyclic GMP-AMP synthase(cGAS)and stimulator of interferon genes(STING)in venous thrombus tissue were detected by Western Blot.Results(1)Compared with the sham-operation group,rats in the model group showed an increase in D-dimer levels,IL-1β,IL-6,TNF-α levels,MDA content,thrombus wet and dry mass,and a decrease in TT,APTT,PT,SOD activity,and GSH-Px activity(all P＜0.05);and compared with the model group,rats in naringen's low-,medium-,and high-dose groups showed a decrease in D-dimer levels,IL-1β,IL-6,TNF-α levels,MDA content,thrombus wet and dry mass,TT,APTT and PT,SOD activity and GSH-Px activity were increased(P＜0.05)in a dose-dependent manner compared with the model group;compared with the naringenin high-dose group,rats in the naringenin high-dose + DMXAA group,D-dimer levels,IL-1β,IL-6,TNF-α levels,MDA content,thrombus wet and dry mass were elevated,TT,APTT and PT,SOD activity and GSH-Px activity were decreased(P＜0.05).(2)Compared with the sham-operation group,the expression levels of cGAS and STING proteins in the venous thrombus tissues of rats in the model group were elevated(P＜0.05);compared with the model group,the expression levels of cGAS and STING proteins in the venous thrombus tissues of rats in the naringeno low-,medium-and high-dose groups were significantly reduced(P＜0.05);cGAS and STING protein expression levels in the naringenin high-dose + DMXAA group were significantly higher than those in the naringenin high-dose group(P＜0.05).Conclusion Naringenin can inhibit the activation of cGAS/STING signalling pathway,thereby inhibiting the inflammatory response and resisting oxidative stress,and thus alleviating the LEDVT.

ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.

italic>Platycodon grandiflorum (Jacq.) A. DC is one of the most commonly used bulk medicinal herbs. It has important value in the fields of medicine, food and cosmetics, and its market demand is increasing year by year, and it has a good development prospect. In this study, based on 403 distribution records and 8 environmental variables, we used Maxent model to predict the potential distribution of P. grandiflorum under climate change. The results showed that the model simulation effect was the best when the Maxent parameter was FC (feature combination) = LQPH (L: linear features; Q: quadratic features; P: product features; H: hinge features) and RM (regularization multiplier) = 2.1, AUC (area under curve) = 0.901, and the model prediction showed that P. grandiflorum was widely distributed in 28 provinces of China under the current climate conditions, with a suitable area of 2 337 419.98 km². Under the influence of climate change in the future, the area of suitable habitat of P. grandiflorum showed a decreasing trend, and the distribution center as a whole showed a trend of northward and eastward shift. The distribution area of P. grandiflorum in the three main producing areas is affected by climate change, and the overall trend is that the future suitable distribution area of Chifeng in Inner Mongolia increases, while the future suitable distribution area of Zibo in Shandong and Taihe, Bozhou in Anhui decreases or even disappears under the SSP5-8.5 scenario (shared socioeconomic pathways). It is suggested to maintain and protect the ecological environment and germplasm resources of the reserved area suitable for the growth of P. grandiflorum, and increase the cultivation research in the expansion area of P. grandiflorum, so as to lay a foundation for the subsequent expansion of P. grandiflorum planting and to promote the protection and sustainable development of P. grandiflorum resources.