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.

The auxin/indole-3-acetic acid (Aux/IAA) gene family is an important regulator for plant growth hormone signaling, involved in plant growth, development, as well as response to environmental stresses. In the present study, we identified SmIAA7 which is potentially associated with Salvia miltiorrhiza leaf development through comparatively analyzed the transcriptome data from different pinnate leaves. SmIAA7 was successfully isolated from S. miltiorrhiza using the specific primers. Then subsequent bioinformatic analysis, prokaryotic expression and purification, subcellular localization, and induction expression analysis under auxin and abiotic stress were performed. The full-length of SmIAA7 contained an ORF of 684 bp encoding a protein of 227 amino acid with a molecular weight of 25.3 kD. Conserved domain analysis showed that SmIAA7 contains the conserved Aux_IAA domain (pfam02309). Sequence analysis and phylogenetic tree analysis results indicated SmIAA7 was phylogenetically close to IAA7 and IAA14 from other plants, suggesting SmIAA7 involved in plant growth and development as well as response to environmental stresses. The prokaryotic expression vector pET28a-SmIAA7 was constructed and SmIAA7 recombinant protein was successfully expressed in E. coli Rosetta (DE3) strain. Subcellular localization experiment demonstrated that SmIAA7 localized in the nucleus of plant cells. Real-time fluorescence quantitative PCR results showed that the expression level of SmIAA7 was upregulated in response to auxin. Drought, low temperature, and salt stress significantly increased the transcript level of SmIAA7 gene. This study lays a foundation for further elucidating the role of SmIAA7 in leaf development, signal transduction, and stress defense in S. miltiorrhiza.

Objective:To evaluate the consistency between architectural distortion（AD）observed on coronal images in three-dimensional ultrasonography（3D-US）and full-field digital mammography（FFDM），and to analyze the influencing factors of the presence of AD on 3D-US coronal images.Methods:A retrospective study was conducted on 96 female patients（97 lesions）with AD identified via FFDM that examined at Affiliated Hangzhou First People's Hospital，School of Medicine，Westlake University from January 2019 to December 2021，all of which with corresponding 3D-US results. Kappa analysis was used to assess the consistency between AD on 3D-US and FFDM.The differences of the lesion characteristics on FFDM（glandular density，presence of calcifications，and degree of distortion），3D-US features（number of lesions，presence of mass or non-mass lesions，and skipping sign in coronal images），clinical pathological characteristics（age，location，and pathology）between groups with AD and without AD in 3D-US were compared. Multivariate Logistic regression was used to identify the influencing factors.Results:Among the FFDM-identified AD lesions，62（63.92%，62/97）lesions showed AD on 3D-US coronal images，including 30 malignant lesions（48.39%，30/62）；35（36.08%，35/97）lesions without AD on 3D-US，in which 4（11.43%，4/35）were malignant. The consistency of distortion degree between FFDM and 3D-US was moderate（Kappa=0.455， P<0.001），with no statistically significant difference（ χ2=2.882， P=0.143）. The probability of malignancy was higher in typical AD compared to atypical AD. The comparisons of age，location，number of lesions，presence of skipping sign in 3D-US，breast density，and presence of calcification in the lesion showed no statistically significant differences between the groups with and without AD（all P >0.05）.The pathological results of malignancy，presence of lesions in 3D-US（mass and non-mass lesions），and the degree of distortion in FFDM showed statistically significant differences（all P < 0.05）. The multivariate Logistic regression analysis indicated that there were no independent risk factors for the appearance of AD in 3D-US（all P > 0.05）. Conclusions:In breast FFDM AD lesions，the incidence of 3D-US coronal plane AD was 63.92%，and the consistency between the degree of distortion in FFDM and 3D-US was moderate. The probability of malignancy is higher with typical AD. Pathological malignancy，presence of lesions in 3D-US（both mass and non-mass lesions），and typical AD in FFDM are more likely to correspond with AD in the coronal.

OBJECTIVE: To explore the potential causal relationship between intervertebral disc degeneration and certain autoimmune diseases. METHODS: Genome-wide association study (GWAS) data of 5 autoimmune diseases were obtained from large-scale GWAS databases. Data on internal vertebral disc degeneration (IVDD) were derived from the FinnGen consortium, which included 294, 770 controls and 41, 669 cases. A two-sample bidirectional Mendelian randomization (MR) analysis was performed to investigate the potential causal relationship between the 5 autoimmune diseases and IVDD. Multiple analytical methods were adopted, including MR methods such as inverse variance weighting(IVW), MR-Egger, weighted median, weighted mode, and simple mode. Cochran's Q test, leave-one-out analysis, and MR-Egger intercept test were conducted to assess heterogeneity, robustness, and pleiotropy. For the robustness of the results, MR-PRESSO was used to detect outliers, and MR analysis was re-conducted after removing the outliers. RESULTS: The MR analysis results showed that there might be a bidirectional causal relationship between ankylosing spondylitis(AS) and IVDD:AS on IVDD, OR=1.038, 95%CI (1.024, 1.053), P=0.000;and IVDD on AS, OR=2.117, 95%CI(1.065, 4.207), P=0.032. There might be a positive correlation between IVDD and rheumatoid arthritis(RA) as well as systemic lupus erythematosus(SLE):IVDD on RA, OR=1.184, 95%CI(1.071, 1.309), P=0.001;and IVDD on SLE, OR=1.678, 95%CI(1.187, 2.372), P=0.003. There was no significant correlation between ulcerative colitis(UC), autoimmune thyroiditis(ATD) and IVDD. After removing outliers by MR-PRESSO and re-conducting MR analysis, the results did not change qualitatively. Sensitivity analysis indicated that the results were robust to potential sources of bias. CONCLUSION AS and IVDD may be risk factors for each other, and IVDD may be a potential risk factor for RA and SLE. These findings provide a basis for guiding the prevention and combined diagnosis and treatment of IVDD, AS, RA, and SLE, while the specific underlying mechanisms still require further experimental basic research.
Humans ; Intervertebral Disc Degeneration/etiology* ; Mendelian Randomization Analysis ; Autoimmune Diseases/complications* ; Genome-Wide Association Study ; Spondylitis, Ankylosing/genetics* ; Arthritis, Rheumatoid/genetics*

OBJECTIVES: To assess the preliminary efficacy and safety of a dose-intensified C5VD regimen (cisplatin, 5-fluorouracil, vincristine, and doxorubicin) in children with locally advanced hepatoblastoma. METHODS: This prospective study enrolled 24 children with newly diagnosed, locally advanced hepatoblastoma who received the dose-intensified C5VD regimen at Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, and Shanghai Children's Hospital between January 2020 and December 2023. Clinical characteristics, treatment outcomes, and chemotherapy-related toxicities were analyzed. RESULTS: Of the 24 patients, 13 were male and 11 were female, with a median age at diagnosis of 18.7 months (range: 3.5-79.4 months). All patients achieved complete macroscopic resection of hepatic lesions without liver transplantation. Serum alpha-fetoprotein levels decreased significantly after two chemotherapy cycles. During a median follow-up of 38.4 months (range: 15.8-50.7 months), all patients maintained continuous complete remission, with 3-year event-free survival and overall survival rates of 100%. Across 144 chemotherapy cycles, the incidence rates of grade 3-4 neutropenia, thrombocytopenia, and infections were 97%, 77%, and 71%, respectively; no treatment-related deaths occurred. Notably, 5 patients (21%) developed Brock grade ≥3 hearing loss, of whom 1 required a hearing aid. CONCLUSIONS The dose-intensified C5VD regimen demonstrates significant efficacy with an overall favorable safety profile in the treatment of newly diagnosed, locally advanced pediatric hepatoblastoma. Grade 3-4 myelosuppression and infection are the predominant toxicities. However, high‑dose cisplatin-induced ototoxicity remains a concern, highlighting the need for improved otoprotective strategies.
Humans ; Hepatoblastoma/pathology* ; Male ; Female ; Infant ; Liver Neoplasms/pathology* ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Child, Preschool ; Prospective Studies ; Doxorubicin/adverse effects* ; Child ; Cisplatin/adverse effects* ; Vincristine/adverse effects* ; Fluorouracil/adverse effects*

Objective： The aim of this study is to explore the predictive value of biparametric magnetic resonance imaging(bpMRI)for pelvic lymph node metastasis in prostate cancer patients with PSA≤20 μg/L and establish a nomogram. Methods： The imaging data and clinical data of 363 patients undergoing radical prostatectomy and pelvic lymph node dissection in the First Affiliated Hospital of Nanjing Medical University from July 2018 to December 2023 were retrospectively analyzed. Univariate analysis and multivariate logistic regression were used to screen independent risk factors for pelvic lymph node metastasis in prostate cancer, and a nomogram of the clinical prediction model was established. Calibration curves were drawn to evaluate the accuracy of the model. Results： Multivariate logistic regression analysis showed extrocapusular extension (OR=8.08,95%CI=2.62－24.97, P<0.01), enlargement of pelvic lymph nodes (OR=4.45,95%CI=1.16－17.11,P=0.030), and biopsy ISUP grade(OR=1.97,95%CI=1.12－3.46, P=0.018)were independent risk factors for pelvic lymph node metastasis. The C-index of the prediction model was 0.834, which indicated that the model had a good prediction ability. The actual value of the model calibration curve and the prediction probability of the model fitted well, indicating that the model had a good accuracy. Further analysis of DCA curve showed that the model had good clinical application value when the risk threshold ranged from 0.05 to 0.70.Conclusion： For prostate cancer patients with PSA≤20 μg/L, bpMRI has a good predictive value for the pelvic lymph node metastasis of prostate cancer with extrocapusular extension, enlargement of pelvic lymph nodes and ISUP grade≥4.
Humans ; Male ; Prostatic Neoplasms/diagnostic imaging* ; Lymphatic Metastasis ; Retrospective Studies ; Nomograms ; Prostate-Specific Antigen/blood* ; Lymph Nodes/pathology* ; Pelvis ; Predictive Value of Tests ; Prostatectomy ; Lymph Node Excision ; Risk Factors ; Magnetic Resonance Imaging ; Logistic Models ; Middle Aged ; Aged

Next generation sequencing (NGS) technology is playing an increasingly important role in the diagnosis of genetic diseases. Whole exome sequencing (WES), which targets the coding regions of the genome, has been widely used in the diagnosis of genetic diseases for its low cost and high efficiency. However, compared to conventional methods, the Next Generation Sequencing (NGS) process is intricate, and there is variability in the expertise of data analysts and variant interpreters, which may lead to inconsistencies in the outcomes. To ensure the quality of testing and enhance the diagnostic rate of diseases, this consensus has provided recommendations regarding the laboratory setup, operational procedures, data analysis, result interpretation, and quality control for WES, with an aim to standardize its application in the detection of genetic disorders.

OBJECTIVES: To investigate the therapeutic effect of Ziwuliuzhu acupuncture in a rat model of insomnia and its regulatory effect on the glutamic acid (Glu)/γ-aminobutyric acid (GABA)-glutamine (Gln) metabolic loop. METHODS: Forty male SD rats were randomly assigned to control group, model group, Najia group and Nazi group (n=10). In the latter 3 groups, rat models of insomnia were established by intraperitoneal injections of p-chlorophenylalanine and verified using a sodium pentobarbital-induced sleep test. After modeling, the rats in Najia and Nazi groups received acupuncture for 7 days at specifically chosen sets of acupoints based on the Ziwuliuzhu rationale in traditional Chinese medicine. Pathological changes in the hypothalamic tissue of the rats were examined with HE staining, and the levels of Glu and GABA in the hypothalamus were determined with high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Immunohistochemistry was used to detect the expressions of GABAA receptors (GABAARs) in the hypothalamus, and the expression levels of glutamate decarboxylase (GAD65/67) and glutamine synthetase (GS) were determined with Western blotting. RESULTS: Compared with the model group, the rats in Najia and Nazi groups exhibited decreased Glu levels and GABAA receptor expression and increased GABA levels with a decreased Glu/GABA ratio in the hypothalamus. Ziwuliuzhu acupuncture significantly increased the protein expressions of GAD65 and GAD67 and lowered the expression of GS in the hypothalamus in the rat models of insomnia. CONCLUSIONS Ziwuliuzhu acupuncture produces sedative and hypnotic effects in rat models of insomnia possibly by regulating Glu and GABA-Gln metabolism to restore the excitatory/inhibitory balance between Glu and GABA.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; gamma-Aminobutyric Acid/metabolism* ; Sleep Initiation and Maintenance Disorders/therapy* ; Glutamine/metabolism* ; Glutamic Acid/metabolism* ; Acupuncture Therapy ; Hypothalamus/metabolism* ; Receptors, GABA-A/metabolism* ; Acupuncture Points

Psoriasis is a chronic inflammatory skin disease, and its pathogenesis is largely modulated by abnormal angiogenesis. Previous research has indicated that AlkB homolog 5 (ALKBH5), an important demethylase affecting N⁶-methyladenosine (m⁶A) modification, plays a role in regulating angiogenesis in cardiovascular and eye diseases. Our present study found that ALKBH5 was upregulated and co-localized with cluster of differentiation 31 (CD31) in the skin of IMQ group compared with control group. ALKBH5-deficient mice decreased IMQ-induced psoriatic dermatitis and exhibited histological improvements, including decreased epidermal thickness, hyperkeratosis, numbers of dermal capillary vessels and inflammatory cell infiltration. ALKBH5-KO mice alleviated angiogenesis in psoriatic lesions by downregulating the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. Additionally, the expression of ALKBH5 was significantly upregulated in IL-17A-induced human umbilical vein endothelial cells (HUVECs), which further promoted the expression of angiogenesis-related cytokines and endothelial cell proliferation. Cell proliferation and angiogenesis were suppressed in ALKBH5 knockdown group, whereas ALKBH5 overexpression promoted these processes. The regulation of angiogenesis in HUVECs by ALKBH5 was facilitated through the AKT-mTOR pathway. Collectively, ALKBH5 plays a pivotal role in psoriatic dermatitis and angiogenesis, which may offer a new potential targets for treating psoriasis.
Animals ; Psoriasis/chemically induced* ; Mice ; Humans ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; AlkB Homolog 5, RNA Demethylase/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; Mice, Knockout ; Disease Models, Animal ; Signal Transduction ; Male ; Skin/blood supply* ; Mice, Inbred C57BL ; Angiogenesis

Objective To investigate the therapeutic effect of Ziwuliuzhu acupuncture in a rat model of insomnia and its regulatory effect on the glutamic acid(Glu)/γ-aminobutyric acid(GABA)-glutamine(Gln)metabolic loop.Methods Forty male SD rats were randomly assigned to control group,model group,Najia group and Nazi group(n=10).In the latter 3 groups,rat models of insomnia were established by intraperitoneal injections of p-chlorophenylalanine and verified using a sodium pentobarbital-induced sleep test.After modeling,the rats in Najia and Nazi groups received acupuncture for 7 days at specifically chosen sets of acupoints based on the Ziwuliuzhu rationale in traditional Chinese medicine.Pathological changes in the hypothalamic tissue of the rats were examined with HE staining,and the levels of Glu and GABA in the hypothalamus were determined with high-performance liquid chromatography(HPLC)-mass spectrometry(MS)/MS.Immunohistochemistry was used to detect the expressions of GABAA receptors(GABAARs)in the hypothalamus,and the expression levels of glutamate decarboxylase(GAD65/67)and glutamine synthetase(GS)were determined with Western blotting.Results Compared with the model group,the rats in Najia and Nazi groups exhibited decreased Glu levels and GABAA receptor expression and increased GABA levels with a decreased Glu/GABA ratio in the hypothalamus.Ziwuliuzhu acupuncture significantly increased the protein expressions of GAD65 and GAD67 and lowered the expression of GS in the hypothalamus in the rat models of insomnia.Conclusion Ziwuliuzhu acupuncture produces sedative and hypnotic effects in rat models of insomnia possibly by regulating Glu and GABA-Gln metabolism to restore the excitatory/inhibitory balance between Glu and GABA.