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*

Objective:To investigate the efficacy of self-prescribed Huoxue Shengjing Prescription as adjuvant therapy for varicocele-induced infertility and its impact on the outcomes of in vitro fertilization-embryo transfer/intracytoplasmic sperm injection (IVF-ET/ICSI).Methods:A randomized controlled trial was conducted. A total of 99 patients with varicocele-induced infertility in our hospital from January 2022 to July 2023 were selected as observation subjects and divided into three groups using a random number table method, with 33 patients in each group. The low ligation group received low ligation of varicocele under a microscope, the low ligation + conventional Western medicine treatment group received low ligation + conventional Western medicine therapy, and the combined group received low ligation + conventional Western medicine therapy + a self-prescribed Huoxue Shengjing Prescription. Among them, the low ligation of varicocele under a microscope was followed by IVF-ET/ICSI assisted reproductive technology 3 months after surgery; the conventional Western medicine therapy involved continuous administration of L-carnitine oral solution for 3 months; the self-prescribed Huoxue Shengjing Prescription was started on the first day after surgery and continued for 3 months. TCM syndrome scores were assessed before and after treatment, and semen routine analysis was performed using an automated semen quality analyzer. Mitochondrial activity of granulosa cells was measured using the Hrudka extraction method, and sperm nuclear DNA integrity was assessed using a modified alkaline single-cell gel electrophoresis method. Follow-up was conducted for 1 year to observe and record the outcomes of IVF-ET/ICSI and evaluate the clinical efficacy.Results:The total effective rate was 93.9% (31/33) in the combined group, 69.7% (23/33) in the low ligation group, and 75.8% (25/33) in the low ligation + conventional Western medicine treatment group, with statistical significance ( χ 2=6.52, P=0.039). After treatment, the scores for mild abdominal pain, testicular heavy pain, impotence, mental fatigue, and the total score in the combined group were lower than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 89.29, 97.51, 136.36, 155.06, and 311.13, respectively, P<0.001). The sperm survival rate, sperm concentration, normal morphology rate, and progressive motility rate in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 19.23, 11.85, 35.97, and 52.21, respectively, P<0.001). Mitochondrial grade I cell activity of granulosa cells was higher than that of the low ligation + conventional treatment group and low ligation group ( F=23.23, P<0.001), and grade Ⅲ cell activity was lower than that of the low ligation + conventional treatment group and low ligation group ( F=20.28, P<0.001). After treatment, the detection of grade Ⅰ and Ⅱ sperm nuclear DNA integrity in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 17.73 and 18.39, respectively, P<0.001), while grades Ⅲ and Ⅳ were lower than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( F values were 29.07 and 10.36, respectively, P<0.001). During follow-up, the excellent embryo rate and the spouse's clinical pregnancy rate in the combined group were higher than those in the low ligation + conventional Western medicine treatment group and the low ligation group ( χ2 values were 14.92 and 8.38, respectively; P values were 0.001 and 0.015, respectively). Conclusion:The adjuvant treatment with a self-prescribed Huoxue Shengjing Prescription can enhance sperm quality in patients with varicocele-related infertility, maintain DNA integrity, regulate seminal plasma mitochondrial function, increase the rate of high-quality embryos, and improve the spouse's pregnancy outcomes.

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 explore the diagnostic value of laboratory examination in bronchoalveolar lavage fluid(BALF) for interstitial lung disease (ILD) and its application value in assessing the degree of fibrosis in the disease.Methods:Retrospective analysis. The clinical data of ILD patients treated in Shanghai First People′s Hospital from January 1, 2021 to December 31, 2023[104 cases, male︰female=48︰56, (62.79±1.24) years] were collected. According to the imaging scores, they were divided into a mild fibrosis ILD group [53 cases, male︰female=26︰27, (61.32±1.71) years] and a moderate to severe ILD fibrosis group [51 cases, male︰female=22︰29, (64.31±1.88) years]. Patients with community-acquired pneumonia without fibrotic lesions by HRCTduring the same period were selected as the control group [49 cases, male︰female=25︰24, (65.37±1.65)years]. The clinical information of all study subjects, as well as BALF lymphocyte subset analysis, cytokine and cytology count detection results were collected. Furthermore, the Kruskal-Wallis H test was used to screen the differential indexes, and the receiver operating characteristic (ROC) curve was used to evaluate the differential indexes to assess the degree of ILD pulmonary fibrosis.Results:Compared with the non-fibrotic pneumonia group, IL-6, IL-8, CD4+CD45RO+cells and macrophages (M%) were significantly upregulated in the mild fibrosis ILD group (P<0.05), and significantly higher in the moderate to severe fibrosis ILD group ( P<0.05). Compared with the non-fibrotic pneumonia group, IL-1β and white blood cell (WBC) count were significantly upregulated only in the moderate to severe fibrotic ILD group ( P<0.05). The correction model was constructed by stepwise logistic regression analysis, and the differential indexes were combined, and the proportion of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages was finally screened as the optimal combined diagnostic mode, with an area under the curve of 0.925, sensitivity of 92.3%, and specificity of 80.0%. Conclusion:Compared with the non-fibrotic pneumonia group, BALF-derived IL-1β, IL-6, IL-8, CD4+CD45RO+cells, WBC count and M% can be used as potential biomarkers to assess the degree of fibrosis, and the combination of IL-1β+IL-6+IL-8+CD4+CD45RO+cells+macrophages has a better diagnostic efficacy for moderate to severe fibrotic ILD.

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

Objective : To construct folate modified D ⁃α ⁃tocopheryl polyethylene glycol 1000 succinate ( TPGS) oparticles on the cytotoxicity and spliced X ⁃box binding protein 1 ( XBP1s) of mouse leukemia cells of monocyte macrophage (RAW264. 7) . Methods : Mixed FA⁃TPGS and rhodamine B (RhB) labeled XBP1 siRNA solution in a proportion of 5 ∶ 1 and obtained the nano⁃complex coated with XBP1 siRNA(FT@ XBP1) . FT@ XBP1 nanocarriers were characterized by transmission electron microscope , dynamic light scattering , ultraviolet visible spectrum analysis and/or fluorescence analysis. And the release of siRNA from FA⁃TPGS nano⁃carriers was calculated simultaneously. The cell cytotoxicity of FT@ XBP1 nanomaterials were detected by scanning electron microscopy (SEM) , CCK⁃8 and flow cytometry. And the inhibited effect of XBP1 s of RAW264. 7 cells was checked by Western blot. Results FA modified TPGS could effectively bind XBP1 siRNA. And the average particle size of FT@ XBP1 nanocarriers were(200 ± 20) nm. The relative release assays showed that acidic environments (pH 5. 0) was beneficial for siRNA to release from FT@ XBP1 . Both CCK⁃8 and apoptosis assay showed that the effects of FT@ XBP1 on the proliferation and apoptosis of RAW264. 7 cells were relatively small , and FT@ XBP1 could significantly inhibit the expression of XBP1 s protein in RAW264. 7(P < 0. 001) . Conclusion TPGS nanoparticles modified with folic acid can effectively encapsulate XBP1 siRNA and inhibit XBP1 s expression of RAW264. 7 cells with good cellular compatibility.

Objective : To construct folate modified D ⁃α ⁃tocopheryl polyethylene glycol 1000 succinate ( TPGS) nanomaterials (FA⁃TPGS) , which can stably load and effectively release siRNA , and to observe the effects of nanoparticles on the cytotoxicity and spliced X ⁃box binding protein 1 ( XBP1s) of mouse leukemia cells of monocyte macrophage (RAW264. 7) . Methods : Mixed FA⁃TPGS and rhodamine B (RhB) labeled XBP1 siRNA solution in a proportion of 5 ∶ 1 and obtained the nano⁃complex coated with XBP1 siRNA(FT@ XBP1) . FT@ XBP1 nanocarriers were characterized by transmission electron microscope , dynamic light scattering , ultraviolet visible spectrum analysis and/or fluorescence analysis. And the release of siRNA from FA⁃TPGS nano⁃carriers was calculated simultaneously. The cell cytotoxicity of FT@ XBP1 nanomaterials were detected by scanning electron microscopy (SEM) , CCK⁃8 and flow cytometry. And the inhibited effect of XBP1 s of RAW264. 7 cells was checked by Western blot. Results : FA modified TPGS could effectively bind XBP1 siRNA. And the average particle size of FT@ XBP1 nanocarriers were(200 ± 20) nm. The relative release assays showed that acidic environments (pH 5. 0) was beneficial for siRNA to release from FT@ XBP1 . Both CCK⁃8 and apoptosis assay showed that the effects of FT@ XBP1 on the proliferation and apoptosis of RAW264. 7 cells were relatively small , and FT@ XBP1 could significantly inhibit the expression of XBP1 s protein in RAW264. 7(P < 0. 001) . Conclusion TPGS nanoparticles modified with folic acid can effectively encapsulate XBP1 siRNA and inhibit XBP1 s expression of RAW264. 7 cells with good cellular compatibility.

Objective:To develop an objective structured clinical examination (OSCE) case base suitable for emergency nursing team training, and evaluate the quality of the case base.Methods:From June to August 2021, based on the literature research, we initially prepared the OSCE case base for emergency nursing team training through the discussion of the research group. From September to December 2021, eight experts were selected for expert group discussion, and the case base were revised and improved according to expert opinions to form a standardized OSCE case base for emergency nursing team training. The quality of cases and case base were evaluated by consulting experts.Results:The effective recovery rate of expert consultation questionnaire was 100%, and the expert authority coefficient was 0.913. A total of 32 OSCE training cases for emergency nursing team were formed. Each case included six parts, namely, training objectives, training content, training preparation, standardized patient information and plot content, scenario evolution diagram, and references. The quality evaluation score of 32 cases was (44.50±1.85) , with the lowest score 40 and the highest score 48. The case base quality evaluation score was (45.26±0.35) , with the lowest score 42 and the highest score 47.Conclusions:The overall quality of the compiled OSCE case base for emergency nursing team training is high, which is suitable for emergency nursing team training.

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 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 2019-nCoV nucleic acid positive diagnosis, 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 N 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 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.