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.

OBJECTIVE To study the interventional effect and mechanism of 1，8-cineole on pancreatic β cell ferroptosis induced by type 2 diabetes. METHODS In vitro ferroptosis model was established in pancreatic β cells of mice by using high glucose. The effects of low-dose and high-dose 1，8-cineole （0.25， 0.5 μmol/L） on the level of Fe2+ in pancreatic β cells were investigated. The effects of 1，8-cineole （0.5 μmol/L） combined with ferroptosis inducer Erastin （20 μmol/L） and ferroptosis inhibitor Ferrostatin-1 （20 μmol/L） on the protein expressions of glutathione peroxidase-4 （GPX4） and cyclooxygenase-2 （COX2） were also detected. The type 2 diabetes model mice were established by feeding high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin. The effects of low-dose and high-dose 1，8-cineole （50， 200 mg/kg） on the pathological morphology of pancreatic tissue， the content of iron as well as the protein expressions of GPX4 and COX2 were investigated. RESULTS The results of the cell experiment showed that compared with the model group， pretreatment with 1，8-cineole significantly reduced intracellular Fe2+ levels and upregulated GPX4 protein expression， while downregulated COX2 protein expression in pancreatic β cells （P＜0.05）. After combining with Ferrostatin-1， the expression trends of the above two proteins were the same， while there was no statistically significant difference after combining with Erastin. The results of animal experiments showed that compared with the model group， after intervention with 1，8-cineole， the structure of the pancreatic islets in mice recovered intact and their morphology improved； the iron content of pancreatic tissue and protein expression of COX2 were decreased significantly （P＜0.05）， while protein expression of GPX4 was increased significantly （P＜0.05）. CONCLUSIONS 1，8-cineole could ameliorate pancreatic β cell injury induced by diabetes， the mechanism of which may be related to reducing intracellular iron deposition and regulating ferroptosis-related proteins.

Objective To analyze the clinical efficacy of the Qianyang Fengsui Dan(combined with flying needle therapy)in the treatment of kidney-yang deficiency type of insomnia.Methods A retrospective study was conducted to select 82 patients with insomnia admitted to the Department of Traditional Chinese Medicine of Dezhou Hospital of Traditional Chinese Medicine from November 2020 to November 2021,and they were divided into an observation group and a control group according to whether or not they were treated with Qianyang Fengsui Dan combined with flying needle therapy,with 41 cases in each group.The control group was treated with Estazolam,while the observation group was treated with Qianyang Fengsui Dan combined with flying needle therapy on the basis of the treatment of the control group,and the course of treatment was 1 month.The changes of Pittsburgh Sleep Quality Index(PSQI)scores and Epworth Sleepiness Scale(ESS)scores,as well as polysomnographic parameters were observed before and after treatment in the two groups.The changes of γ-aminobutyric acid(GABA),glutamate(GA),substance P(SP),and neuropeptide Y(NPY)levels were compared before and after treatment between the two groups.And followed up for 1 year to compare the incidence of relapce of the two groups of patients.Results(1)The total effective rate was 95.12%(39/41)in the observation group and 63.41%(26/41)in the control group,and the efficacy of the observation group was superior to that of the control group,and the difference was statistically significant(P＜0.05).(2)After treatment,PSQI scores and ESS scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving PSQI scores and ESS scores,and the differences were statistically significant(P＜0.05).(3)After treatment,sleep efficiency,awakening time,sleep latency,REM,and total sleep time were significantly improved in the two groups(P＜0.05),and the observation group was significantly superior to the control group in improving sleep efficiency,awakening time,sleep latency,REM,and total sleep time,and the differences were statistically significant(P＜0.05).(4)After treatment,the serum GABA,GA,SP,and NPY levels of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the serum GABA,GA,SP,and NPY levels,and the differences were all statistically significant(P＜0.05).(5)After treatment,follow-up for 1 year,the recurrence rate of the observation group was 0,and there were 7 cases of recurrence in the control group,and the recurrence rate of the control group was 17.07%(7/41),and the recurrence rate of the observation group was lower than that of the control group,and the difference was statistically significant(P＜0.05).Conclusion The combination of flying needle therapy and Qianyang Fengsui Dan can effectively relieve insomnia and fatigue in patients with insomnia,reduce daytime drowsiness,regulate the release of blood monoamine neurotransmitters,and reduce the relapse rate,and its efficacy is superior to that of simple western medicine treatment.

BACKGROUND:Numerous scholars have previously researched certain greater tuberosity fractures and the procedures used to treat them.Few researchers,however,have studied the comminuted split fracture of the greater tuberosity of the humerus(Liu-Gang type IV)with rotator cuff tear in great detail. OBJECTIVE:To compare the clinical therapeutic effect of open repair position modified calcaneal plate combined with suture anchors and proximal humeral internal locking system(PHILOS)plate in the treatment of comminuted fracture of split-type greater tuberosity of humerus combined with rotator cuff tears(Liu-Gang type IV). METHODS:Case data of 30 patients with comminuted fracture of split-type greater tuberosity of humerus combined with rotator cuff tears(Liu-Gang type IV)from May 2012 to May 2022 were retrospectively analyzed.They were divided into the modified calcaneal plate combined with suture anchor group(group A)and the PHILOS with#2 Johnson group(group B),with 15 cases in each group.Intraoperative blood loss,surgical time,and incision length of all patients were recorded.Pain visual analog scale score,Constant-Murley score,as well as shoulder joint abduction,forward flexion,external rotation,and dorsal expansion activities during the last follow-up(>1 year)were evaluated. RESULTS AND CONCLUSION:(1)The surgical incision length and operation time were shorter,and blood loss was less in group A than those in group B(P<0.05).(2)No significant difference in visual analog scale score and Constant-Murley score was detected between the two groups(P>0.05).(3)During the last follow-up,forward flexion in group A was better than that in group B(P<0.05).No significant difference in abduction,external rotation,and dorsal expansion was determined between group A and group B(P>0.05).(4)In terms of complications,there was 1 case of shoulder joint pain and discomfort in group A(7%),2 cases of subacromial impingement syndrome,2 cases of upward movement of nodules,and 2 cases of shoulder joint pain(40%)in group B.There were significant differences in complication rates between the two groups(P=0.031).(5)In summary,the modified calcaneal plate combined with suture anchors in the treatment of comminuted fracture of split-type greater tuberosity of humerus combined with rotator cuff tears(Liu-Gang type IV)could better restore the forward flexion function of the shoulder joint and has a small incision,less blood loss,shorter operation time and fewer complications.

Objective To explore the clinical efficacy of double beam double tunnel enhanced reconstruction technique in the treatment of knee anterior cruciate ligament(ACL)training injuries.Methods Twenty-nine cases of ACL injury of knee joint from January 2021 to December 2021 were retrospectively analyzed.All the cases were underwent ligament reconstruc-tion surgery.Cases were grouped by surgical technique:there were 14 patients in conventional reconstruction group,including 13 males and 1 female,aged from 22 to 31 years old with an average of(27.07±7.28)years old,autogenous hamstring tendon was used for ligament reconstruction.There were 15 patients in the enhanced reconstruction group,including 13 males and 2 females,aged from 25 to 34 years old with an average of(29.06±4.23)years old,double tunnel ligament reconstruction,the autogenous hamstring muscle was used as the anteromedial bundle,and the posterolateral bundle was replaced by a high-strength line.The difference between knee tibial anterior distance,Lysholm score,International Knee Literature Committee(IKDC)subjective score,Tegner motor level score and visual analog scale(VAS)at 6th and 12th months after the surgery,limb symmetry index(LSI)were recorded at the last follow-up and surgery-related adverse effects during follow-up.Results All patients were followed up,ranged from 13 to 15 months with an average of(13.7±0.8)months.There were no serious adverse reactions related to surgery during the period.There was no statistical difference between the preoperative general data and the observation index of the two groups(P＞0.05).The difference in tibial anterior distance at 6 and 12 months in the enhanced re-construction group(1.45±0.62)mm and(1.74±0.78)mm which were lower those that in the conventional reconstruction group(2.42±0.60)mm and(2.51±0.63)mm(P＜0.05).There was no significant difference in postoperative Lysholm score,Tegner motor level score,IKDC score,VAS,and limb symmetry index at the last follow-up(P＞0.05).Conclusion The enhanced recon-struction technique can more effectively maintain the stability of the knee joint and has no significant effect on the postoperative knee joint function compared with the traditional ligament reconstruction technique.The short-term curative effect is satisfac-tory,and it is suitable for the group with high sports demand.

Objective To elucidate the molecular genetic etiology of patients with disorders of sex development(DSD)using whole exome sequencing(WES),thereby enhancing our understanding of the underlying mechanisms of sexual development abnormalities.Methods Retrospective analysis was conducted on clinical data of 60 DSD patients diagnosed in the First People's Hospital of Yunnan Province between March 2008 and August 2021,with an additional family study for one proband.Genomic DNA was extracted from patients for WES analysis.Single nucleotide polymorphism(SNP)and insertions/deletion(InDel)tests were identified using SAMtools software in conjunction with established SNP and InDel databases.Copy number variations(CNVs)at the exon level were detected using ExomeDepth,while the potential pathogenicity of mutations was predicted with PolyPhen-2,Mutation taster and PyMol software,with Sanger sequencing employed for confirmation.Results The study included 22 patients with 46,XX DSD and 38 with 46,XY DSD.Among the 46,XX DSD patients,the SRY gene was detected in 14 patients.In the remaining 8 patients and a proband's families,single nucleotide site variations(SNVs)of NR5A1,PROKR2 and ANOS1 genes were identified in 2 patients,and CNVs in CYP21A2 gene were found in 4 patients.The pathogenicity of CYP21A2 EX1 Dup has been previously reported,while the remaining 3 CNVs were of uncertain significance,and no DSD-related mutations were detected in 2 patients.In the WES analysis of 46,XY DSD patients,10 pathogenic or likely pathogenic SNVs across 5 genes(SRY,AR,SRD5A2,CYP17A1,and NR5A1)were identified in 14 patients.Additionally,5 likely pathogenic CNVs involving the CYP21A2,AKR1C2,CBX2,and NR5A1 genes were detected in 5 patients,comprising 3 deletions and 2 duplications.Novel SNVs in NR5A1(c.722G>T,c.48C>G)and ANOS1 c.564A>T were identified,with no prior reports in relevant databases.The pathogenicity of CYP21A2 EX1 Dup is documented in related databases,while the remaining CNVs have not been previously reported.Conclusion The utilization of WES technology has enhanced the diagnostic potential for DSD,broadened the spectrum of known DSD-related gene mutations,and deepened our comprehension of DSD pathogenesis,offering valuable support for genetic counseling.

Objective:To investigate the neuroprotective effect of hesperidin combined with enriched environment on ferroptosis in collagenase-induced intracerebral hemorrhage(ICH) mouse model as well as the ferroptosis mechanism.Methods:ICH model was established by injecting collagenase Ⅶ into caudate putamen nucleus. Ninty C57BL/6J mice were randomly divided into 6 groups according to a random number table: sham group, intracerebral hemorrhage group, enriched environment group, hesperidin group, enriched environment and hesperidin group (combination group), and combination group + Nrf2 specific inhibitor ML385 (inhibitor group), with 15 mice in each group. The mice in inhibitor group, intracerebral hemorrhage group, enriched environment group, hesperidin group and combination group were injected with 0.5 μL collagenase type Ⅶ solution (0.075 U/μL, dissolved with 0.9% NaCl solutin) for ICH modeling, and the mice in sham group were injected with 0.9% normal saline. The hesperidin group, combination group, and inhibitor group were given hesperidin solution (dissolved in 0.5% carboxymethyl cellulose sodium) by gavage within 6 hours after the modeling surgery. The sham group, intracerebral hemorrhage group, and enriched environment group were given equal volumes of 0.5% carboxymethyl cellulose sodium solution by gavage. The inhibitor group was intraperitoneally injected with Nrf2 specific inhibitor ML385 (30 mg/kg, dissolved in 5% DMSO), while the other groups were intraperitoneally injected with an equal volume of 5% DMSO. Both gastric perfusion and intraperitoneal injection were completed within 6 hours after the end of modeling operation, once a day for 14 days. After the postoperative recovery of the mice, the enriched environment group, combination group, and inhibitor group were placed in enriched environment cages, while the sham group, intracerebral hemorrhage group, and hesperidin group were placed in regular cages. After all intervention were completed, all mice were evaluated using the modified neurological severity score (mNSS). Then the mice were subjected to brain water content detection, Prussian blue staining, ELISA detection of changes in malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS), glutathione peroxidase 4 (Gpx4), PCR and Western blot detection of nuclear factor E2 related factor 2 (Nrf2) and Gpx4 at the mRNA level and protein level. The GraphPad Prism 9 software was used for statistical analysis. ANOVA was used for multi-group comparison, and Tukey test was used for multiple comparisons.Results:(1) There was a statistically significant difference in mNSS scores among the 6 groups ( F=66.35, P<0.001). The mNSS score of the intracerebral hemorrhage group(8.00±1.46) was higher than that of the sham group(0.86±0.83)( P<0.05). The mNSS scores of the enriched environment group (6.47±1.13) and hesperidin group (6.13±1.25) were lower than that of the intracerebral hemorrhage group, but higher than that of the combination group (4.53±1.30)(all P<0.05). (2) There was a statistically significant difference in the percentage of brain water content among the 6 groups ( F=33.29, P<0.001). The percentage of brain water content in the intracerebral hemorrhage group was higher than that in the sham group.The percentage of brain water content in the enriched environment group and hesperidin group were lower than that in the intracerebral hemorrhage group, but higher than that in the combination group (all P<0.05). (3) The result of Prussian blue staining showed that iron deposition in the intracerebral hemorrhage group was higher than that in the sham group, while the iron depositions in the enriched environment group and hesperidin groups were lower than that in the intracerebral hemorrhage group, but higher than that in the combination group(all P<0.05). (4) There were statistically significant differences in the expression levels of Nrf2 and Gpx4 mRNA and protein among the 6 groups ( F=27.73, 31.24, 26.79, 13.79, all P<0.001). The mRNA and protein levels of Nrf2 and Gpx4 in the combination group were higher than those in the enriched environment group, hesperidin group, but higher than those in the inhibitor group(all P<0.05). (5) The results of ELISA showed that the levels of MDA, Gpx4, ROS, and SOD in the brain tissues of 6 groups were statistically significant ( F=111.20, 21.53, 29.45, 22.75, all P<0.001). Among them, the MDA and ROS in the combination group ((14.05±0.57) nmol/mL, (75.46±3.40) ng/mL) were lower than those in the enriched environment group ((18.17±2.51) nmol/mL, (97.23±3.43) ng/mL), hesperidin group ((17.24±0.68) nmol/mL, (90.02±9.46) ng/mL) and the inhibitor group ((17.08±0.64) nmol/mL, (101.07±3.38) ng/mL), while Gpx4 and SOD ((340.40±31.21) pg/mL, (62.55±2.81) ng/mL) were higher than those in the enriched environment group ((267.81±27.17) pg/mL, (50.47±8.38) ng/mL), hesperidin group ((271.55±34.36) pg/mL, (50.55±8.19) ng/mL) and the inhibitor group ((235.65±72.54)pg/mL, (52.67±3.56)ng/mL)(all P<0.05). Conclusion:Enriched environment and hesperidin can inhibit ferroptosis after ICH by activating the Nrf2/GPX4 pathway, exerting neuroprotective effects on ICH mouse models, and the combined treatment of the enriched environment and hesperidin has a more significant effect.

Objective:To establish a general method for the simultaneous determination of hydroxyphenyl esters and quaternary ammonium bacteriostatic agents in eye drops.Methods:The chromatographic analysis was per-formed on an Agilent C18 column(4.6 mm ×250 mm,5 μm)with 1％triethylamine solution(pH adjusted to 5.0 with phosphoric acid)as mobile phase A and methanol as mobile phase B.Gradient elution was performed at col-umn temperature of 40 ℃.The detection wavelength was 214 nm,the flow rate was 1 mL·min-1,and the injec-tion volume was 20 μL.Results:Methylparaben,ethylparaben,propylparaben,butylparaben,benzalkonium chlo-ride and benzalkonium bromide were 0.11-559.0,0.10-513.0,0.10-258.8,0.11-270.5,1.07-537.0 and 1.03-512.8 μg·mL-1,respectively.The linear range was good(r＞0.999).The average recoveries of meth-ylparaben,benzalkonium bromide and benzalkonium chloride were 104.7％(RSD=1.3％),102.6％(RSD=1.1％)and 100.9％(RSD=1.1％),respectively.The contents of bacteriostatic agent in 100 batches of eye drops from 36 varieties of 12 enterprises were determined,and the accurate results were obtained.Conclusion:This meth-od provides a reference for the content quality control and safety evaluation of bacteriostatic agents in eye drops.

Objective To compare the contents of alkaloid in Aconiti Kusnezoffii Radix with different degrees of processing and their effects on electrocardiogram and vagus nerve action potential in SD rats;To establish a toxicity evaluation method for Aconiti Kusnezoffii Radix and its processed products.Methods HPLC was used to determine the contents of six alkaloid components,including benzoyl neoaconitine,benzoyl aconitine,benzoyl subeaconitine,neoaconitine,subeaconitine and aconitine in Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix Cocta of soaking and boiled for 30 minutes,boiled for 4 h without soaking,soaking and boiled for 4 h,and soaking and boiled for 8 h.SD rats were subjected to sublingual drainage with Aconiti Kusnezoffii Radix and four types of processed products.The vagus nerve action potential and electrocardiogram of rats before and after administration were recorded,and the correlation analysis between alkaloid content and vagus nerve action potential and heart rate was conducted.Results The contents of alkaloid of the four processed products showed that the content of diester alkaloids was soaking and boiled for 30 min>boiled for 4 h without soaking>soaking and boiled for 4 h>soaking and boiled for 8 h;monoester alkaloid content:boiled for 4 h without soaking>soaking and boiled for 30 min>soaking and boiled for 4 h>soaking and boiled for 8 h.After the administration of each sample,compared with the basal discharge,the vagus nerve discharge of rats with different processed products was changed by sublingual administration,heart rate increased,the degree of arrhythmia increased,and it varied with the degree of processing;the results of correlation analysis showed that there was a significant correlation between the nerve discharge area and the contents of diester alkaloids and total alkaloids.Conclusion The action potential of the vagus nerve and electrocardiogram of rats after sublingual drainage administration can reflect the degree of numbness in the tongue,which can provide reference for the study of tongue sensation of Aconiti Kusnezoffii Radix.

Objective:To investigate the value of the combined ultrasound imaging radiomics model for predicting progression-free survival in endocrine therapy for prostate cancer.Methods:A total of 283 prostate cancer patients who received endocrine treatment at the Inner Mongolia Autonomous Region People′s Hospital and the First Hospital of Hohhot from July 2018 to January 2023 were retrospectively collected, of which 198 patients from the Inner Mongolia Autonomous Region People′s Hospital were randomly divided into the training set and the validation set according to the ratio of 7∶3, and 85 patients from the First Hospital of Hohhot served as an independent external test set. They were classified into a progression group and a non-progression group based on whether the patients progressed to desmoplasia-resistant prostate cancer 18 months after the start of endocrine treatment.Based on the two-dimensional ultrasound images, the imaging radiomics features were extracted and the imaging radiomics score (Rad-score) were constructed, the immunopathology and other clinical data were analysed, and three prediction models were constructed using logistic regression: the clinical model, the ultrasonography model, and the ultrasonography-clinical combined model, respectively. The predictive efficacy and clinical utility of the models were assessed by the ROC curves and clinical decision curves.Results:Five ultrasonographic features were included in the ultrasound model; the prostate-specific antigen nadir, the neutrophil-to-lymphocyte ratio before treatment, and the expression level of tumour proliferating cell nuclear antigen 67 (Ki-67) were incorporated into the clinical model; and the Rad score computed from the output of the ultrasound model for the screening features, together with the prostate-specific antigen nadir (PSA nadir), the neutrophil to lymphocyte ratio (NLR) before treatment, and the expression level of Ki-67 were used to construct the ultrasound-clinical joint model. The joint model achieved the highest predictive performance in both the training and validation sets of the three groups of models, with the area under the curve of 0.85 and 0.84, and the clinical decision curve showed good clinical benefit.Conclusions:The combined ultrasound-clinical model constructed in this study based on two-dimensional ultrasound images of prostate cancer before endocrine therapy can predict progression-free survival of endocrine therapy for prostate cancer more accurately.