Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

Patellofemoral pain syndrome(PFPS)is one of the main diseases leading to patellofemoral joint dysfunction,which seriously affects the quality of life of patients.This disease is classified as"knee impediment"and"tendon impediment"in TCM,and acupuncture treatment has a significant effect on this disease.This article combed the clinical studies on acupuncture treatment of PFPS in recent years around filiform needling,warm acupuncture and moxibustion,fire needle,needle knife,electro-acupuncture and other acupuncture treatments,and discussed the clinical effects and application scope of different treatments.Studies have shown that acupuncture therapy can effectively improve the symptoms and function of PFPS through a variety of mechanisms,such as regulating the function of the central nervous system,promoting the absorption of inflammatory factors and the clearance of joint effusion,and improving local blood circulation.These findings provide an important reference for the clinical treatment of PFPS.

Objective:This study aims to explore the application value of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors.Methods:We retrospectively analyzed the clinical data of 15 patients with complex adrenal tumors treated at the General Hospital of Southern Theater Command from May 2022 to June 2023. The cohort comprised 5 males and 10 females with an average age of (47.6±7.8) years and a body mass index (BMI) of 26.5 (23.8-27.9) kg/m 2. Among the patients, 3 had a BMI ≥28 kg/m 2, 2 had diabetes, 6 had hypertension, and 1 had coronary heart disease. Preoperative endocrine hormone examination revealed abnormal blood catecholamines in 5 cases and abnormal blood cortisol in 2 cases. Ultrasound and CT scans indicated that 9 tumors were located on the left side and 6 on the right, with 4 cases showing tumor compression on adjacent large blood vessels or organs. The average tumor diameter was (7.61±2.79) cm, with 10 cases having a diameter ≥ 6 cm. All patients underwent laparoscopic adrenalectomy assisted by robots through the transperitoneal approach. The surgeries were performed in a lateral position under general anesthesia. The "liftoff" modular method was utilized to separate the treatment of adrenal tumors into lateral, medial, dorsal, cephalic, and adrenal renal plane sides. Tumors were appropriately manipulated during the operations to achieve a "liftoff" shape. Different modular dissociation steps were adopted based on the size and location of the left and right adrenal tumors. The left adrenal gland was dissected in the order of medial and dorsal, adrenal renal plane side, and lateral and cephalic sides, while the right adrenal gland was dissected in the order of lateral and dorsal, adrenal renal plane side, and medial and cephalic sides. Postoperative related indicators and follow-up status of patients were recorded and analyzed. Results:All 15 surgeries were successfully completed without any conversions to open adrenalectomy, with an average operation time of 118 (102-130) minutes and an average intraoperative blood loss of 102 (69-163) ml. The postoperative drainage time was 4 (3-5) days, and the postoperative hospital stay was 6 (4-7) days. The postoperative pathological diagnoses included 5 cases of pheochromocytoma, 3 cases of macronodular adrenal hyperplasia, 6 cases of adrenocortical adenoma, and 1 case of myelolipoma. Follow-up for 6-12 months after surgery showed good recovery and no recurrence.Conclusions:The application of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors is safe and feasible. It efficiently aids in tumor removal and holds significant clinical application value.

Objective:To investigate the tissue methylation features of adenocarcinoma patients presenting as pulmonary nodules on CT scans.Methods:A retrospective analysis was conducted on 70 adenocarcinoma patients with pulmonary nodules diagnosed at the Shanghai General Hospital from June 1, 2022 to January 20, 2024. Participants were assigned to two groups using the random number table, with 40 in the discovery group and 30 in the validation group. In the discovery group, tissue samples were analyzed using reduced representation bisulfite sequencing (RRBS) technology to compare the average methylation levels between cancer tissues and paired adjacent non-cancerous tissues. Differentially methylated regions (DMRs) were screened for analysis of their distribution across various genomic functional elements, and hierarchical clustering was plotted. GO and KEGG pathway enrichment analyses were further conducted on the DMRs. Subsequently, candidate DMRs associated with lung adenocarcinoma were validated using TCGA lung adenocarcinoma cohort and targeted bisulfite sequencing technology in the validation group. The comparison of methylation levels between groups was conducted using t-tests or non-parametric tests, while rates and composition ratios were analyzed using chi-square tests or Fisher′s exact test.Results:In discovery cohort, the average methylation level in cancer tissues was lower compared to adjacent normal tissues [(42.369±4.627) vs (44.370±4.046), t=?2.059, P=0.043]. A total of 37 995 DMRs were identified, including 16 889 upregulated regions and 21 106 downregulated regions, predominantly locating in promoter regions (48.917%), introns (36.457%), and exons (10.812%). The DMR clustering heatmap revealed two distinct clusters corresponding to cancer tissues and adjacent non-cancerous tissues. GO analysis showed that DMRs associated genes were mainly located in the cell membrane and nuclear chromatin, and were primarily involved in RNA polymerase Ⅱ-related transcription and regulation. KEGG pathway enrichment analysis indicated that DMRs associated genes were mainly involved in neuroactive ligand-receptor interaction, cancer pathways, calcium signaling pathway, cAMP signaling pathway, and MAPK signaling pathway. Validation in the TCGA cohort confirmed 11 potential characteristic DMRs. In the validation group, TBS confirmed that the methylation levels of DMRs associated with MIR10B, DMRTA2, HOPX, TFAP2B and MARCH11 in cancer tissues were significantly higher than those in adjacent non-cancerous tissues [11.200(4.305, 27.088) vs 2.650(1.298, 4.645), Z=?4.539, P<0.05; 18.610(13.600, 33.025) vs 8.675(5.488, 13.085), Z=?4.554, P<0.05; 17.600(2.183, 76.015) vs 1.085(0.898, 1.835), Z=?5.131, P<0.05; 5.250(3.220, 7.693) vs 3.495(2.165, 4.383), Z=?2.861, P<0.05; 11.515(7.525, 21.033) vs 7.830(5.518, 11.488), Z=?2.440, P<0.05 ], and the differences were statistically significant. Conclusions:Lung adenocarcinoma tissue exhibits different methylation patterns compared with adjacent normal lung tissue. The identified DMRs are involved in the regulation of several key pathways. Results from the TCGA cohort and an independent validation group support the potential diagnostic value of DMRs such as MIR10B, DMRTA2, HOPX, TFAP2B, and MARCH11 in lung adenocarcinoma, though their clinical application requires further validation.

Objective·To verify the performance and explore the clinical application value of a multi-modal pulmonary nodule diagnosis model combined with metabolic fingerprints,protein biomarker CEA and Image-AI via random forest(MPI-RF).Methods·This study enrolled 289 patients with pulmonary nodules who were admitted to the Shanghai Chest Hospital,Shanghai Jiao Tong University School of Medicine and were detected by low-dose helical computed tomography(LDCT).The patients were divided into malignant nodule group(n=197)and benign nodule group(n=92)based on postoperative pathological results,and the basic information of the two groups was collected and compared.Electrochemiluminescence was used to detect the preoperative serum CEA levels of the patients in the two groups,matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)was used to detect the serum metabolic fingerprints,and the CT image artificial intelligence model Image-AI was used to calculate the image scores.CEA data,serum metabolic fingerprints data and image scores were integrated and input into MPI-RF to calculate the malignant probability score of each patient.The receiver operator characteristic curve(ROC curve)and area under the curve(AUC)were used to evaluate the performance of different models,and the DeLong test was used for comparative analysis,including the diagnostic performance of MPI-RF in different types(solid nodule,pure ground-glass nodule and part-solid nodule)and sizes(diameter<8 mm and diameter≥8 mm)of pulmonary nodules,the diagnostic performance comparison of MPI-RF with Mayo Clinic model,veterans administration(VA)model and Brock model,and the diagnostic performance comparison of MPI-RF with lung imaging reporting and data system(Lung-RADS)in benign and malignant nodules.Results·MPI-RF had good diagnostic performance in the differentiation of benign and malignant pulmonary nodules(AUC=0.887,95%CI 0.848?0.925,sensitivity 81.22%,specificity 83.70%).Among them,the AUC of MPI-RF for solid nodules was 0.877(95%CI 0.820?0.934),for part-solid nodules was 0.858(95%CI 0.771?0.946),and for pure ground-glass nodules was 0.978(95%CI 0.923?1.000).The AUC of MPI-RF was 0.840(95%CI 0.716?0.963)for nodules within 8 mm diameter and 0.891(95%CI 0.849?0.933)for nodules larger than 8 mm diameter.Compared with the existing models,the diagnostic performance of MPI-RF was better than that of Mayo Clinic model,VA model and Brock model(all P=0.000).Compared with Lung-RADS,MPI-RF had better diagnostic performance in the total samples and different types of nodules(all P=0.000).Conclusion·MPI-RF is a model for the differential diagnosis of benign and malignant pulmonary nodules with excellent performance,and has potential clinical application value.

Objective Based on construction and verification of the lumbar finite element model, the simulation calculation and injury prediction on dynamic response of normal lumbar model and L5 unilateral and bilateral spondylolysis models of the pilot were carried out, so as to explore the influence of persistent flight overload on normal and spondylolysis lumbar vertebrae of the pilot. Methods The precise finite element model of lumbavertebrae was established using reverse engineering software and computer-aided engineering (CAE) technology based on CT images. The validity of the lumbar vertebrae model was verified by static and dynamic in vitro experiments. The biomechanical simulation analysis on normal and spondylolysis lumbar vertebrae of the pilotunder persistent overload was carried out, and the spinal injury was predicted and analyzed by dynamic response index (DRI) injury evaluation and prediction method. Results The maximum isthmus stress of L5 vertebra in unilateral and bilateral spondylolysis models were 105. 29 MPa and 126. 32 MPa, respectively, which were significantly higher than those in normal model. The L4-5 and L5-S1 intervertebral discs of the spondylolysis model were more prone to premature degenerative changes than those of normal model. Combined with DRI spinal injury prediction method, the probability of spinal injury in normal lumbar vertebrae, lumbar vertebrae with L5 unilateral and bilateral spondylolysis were 0. 001 4% , 2. 26% and 3. 21% , respectively, and the probability of spinal injury was significantly increased after the occurrence of spondylolysis. Conclusions The spondylolysis increases the load of lumbar isthmus under flight overload. The results provide more accurate data support for the formulation of training programs and the development of protective devices to ensure flight safety

With the development of economy and medicine, chronic diseases have become a more and more prominent problem affecting children′s physical and mental development.Juvenile idiopathic arthritis(JIA)is a common rheumatic disease in children.As an important medium of immune response, cytokines play a role in systemic juvenile idiopathic arthritis(SJIA)and its complication such as macrophage activation syndrome(MAS), which has attracted more and more attention.Many studies have found that a variety of cytokines play an inflammatory or anti-inflammatory role in the occurrence and development of SJIA and MAS.Therefore, this paper summarizes the specific mechanism of action and clinical significance of related cytokines in SJIA and MAS, and reviews the progress of related drug therapy targeting cytokines.By exploring the pathogenesis of SJIA, we hope to provide theoretical basis for identifying new therapeutic targets.

Objective To simulate dynamic response of cervical spine of the pilot during typical maneuver flight movements using finite element method, as well as make analysis and prediction on damage failure of the pilot neck during flight by impact injury and fatigue injury model of biological tissues.Methods A geometrically accurate finite element model of the neck was constructed, and validity of the model was verified by relevant examples. Then, the acceleration curves of centrifugal trainer under different modes were loaded for numerical simulation, and impact injury and fatigue injury of tissues were predicted by using the universal cervical injury criterion and the fatigue damage model of biological tissues.Results The maximum stress of the vertebrae and intervertebral disc caused by overload impact was 66.53 MPa and 58.63 MPa respectively during typical maneuver flight. According to the Nij injury criteria, the maximum Nij was 0.096, which was lower than the injury tolerance threshold of 1, and would not cause direct acute injury to cervical tissues. Based on fatigue damage model of biological tissues, it was found that cancellous bone suffered fatigue failure under the condition of uninterrupted repeated loading for more than 40 000 times. Considering the limited flight career of the pilot, the vertebral tissues would not be fractured due to the accumulation of fatigue damage.Conclusions To a certain extent, the results can contribute to formulating pilot training and flight plans, and also provide data support for the development of its protective equipment.

Objective:To establish a colloidal gold technique assay for the rapid detection of immunoglobulin(Ig)M and IgG antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance.Methods:A total of 278 patients who were respectively treated at Wuhan Hankou Hospital and the People′s Hospital of Honghu from February 12, 2020 to February 20, 2020 were collected. According to the diagnostic criteria, 89 patients were confirmed with positive 2019-nCoV nucleic acid, and 189 were 2019-nCoV nucleic acid-negative suspected patients. A total of 273 medical examiners from Nanfang Hospital, Southern Medical University from 2015 to 2018 were selected as controls. The serum samples of patients were collected. 2019-nCoV nucleic proteins were obtained from prokaryotic expression vectors. Indirect IgM and IgG colloidal gold techniques were established by using recombinant nuclear protein. 2019-nCoV nucleic acid detection by reverse transcription-polymerase chain reaction (RT-PCR) was used as control. Serum specimens were tested for 2019-nCoV IgM and IgG. The specificity and sensitivity of colloidal gold assay were analyzed.Results:The positive rates of IgM and IgG with the colloidal gold detection in confirmed patients with positive 2019-nCoV nucleic acid were 78.7%(70/89) and 73.0%(65/89), respectively. The positive rates of IgM and IgG in medical examiners were 1.8%(5/273) and 0.7%(2/273), respectively. The sensitivity and specificity of IgM detection reagents were 78.7% and 98.2%, respectively, those of IgG detection reagents were 73.0% and 99.3%, respectively, and those of IgM combined with IgG detection were 87.6% and 98.2%, respectively. For suspected patients with negative 2019-nCoV nucleic acid, the positive rates of IgM and IgG were 59.8%(113/189) and 52.9%(100/189), respectively, and the positive rate of IgM combined with IgG detection was 66.1%(125/189).Conclusion:This reagent of 2019-nCoV antibodies detection (colloidal gold technique) fulfills the requirement for clinical application with high specificity and sensitivity, which can be served as a supplementary detection method for 2019-nCoV nucleic acid detection by RT-PCR.

[Abstract] Objective: To investigate the effect of miR-93/EphA4 (Eph receptor A4) axis on the proliferation and migration of nonsmall cell lung cancer (NSCLC) H460 and H1299 cells via regulating extracellular regulated protein kinases (ERK) pathway. Methods: The expression levels of miR-93 in H460 and H1299 cells was detected by qPCR. miR-93 mimics and EphA4 overexpression plasmids were transfected into H460 cells and miR-93 inhibitor was transfected into H1299 cells respectively, after which MTT assay and Transwell assay were used to detect the effects of miR-93 on proliferation and migration of transfected cells. The targeted regulatory relationship betweenmiR-93andEphA4wasverifiedbyDual-luciferasereportergeneassay.Theexpression levels of PCNA(proliferating cell nuclear antigen), EphA4, ERK and p-ERK were detected by Westernblotting.The effects of simultaneous overexpression of miR-93 and EphA4 on proliferation and migration of H460 cells were detected by MTT assay and Transwell assay. Results: The expression of miR-93 in H1299 cells was higher than that in H460 cells (P<0.01). Overexpression of miR-93 promoted proliferation and migration of H460 cells (all P<0.01), and knockdown of miR-93 inhibited proliferation and migration of H1299 cells (all P<0.01). The Dualluciferase reporter gene assay confirmed that miR-93 could target EphA4. Overexpression of miR-93 down-regulated the mRNA and protein expression levels of EphA4(allP<0.05), and promoted proliferation and migration of H460 cells through targeted regulation of EphA4 and activation of ERK pathway (all P<0.01). Conclusion: miR-93 promotes the proliferation and migration of NSCLC cells, and its mechanism may be related to the targeted regulation of EphA4 and activation of the ERK pathway.