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*

BACKGROUND AND AIM: Remnant cholesterol (remnant-C) mediates the progression of major adverse cardiovascular events. It is unclear whether remnant-C, and particularly cumulative exposure to remnant-C, is associated with nonalcoholic fatty liver disease (NAFLD). This study aimed to explore whether remnant-C, not only baseline but cumulative exposure, can be used to independently evaluate the risk of NAFLD. METHODS: This study included 1 cohort totaling 21,958 subjects without NAFLD at baseline who underwent at least 2 repeated health checkups and 1 sub-cohort totaling 2,649 subjects restricted to those individuals with at least 4 examinations and no history of NAFLD until Exam 3. Cumulative remnant-C was calculated as a timeweighted model for each examination multiplied by the time between the 2 examinations divided the whole duration. Cox regression models were performed to estimate the association between baseline and cumulative exposure to remnant-C and incident NAFLD. RESULTS: After multivariable adjustment, compared with the quintile 1 of baseline remnant-C, individuals with higher quintiles demonstrated significantly higher risks for NAFLD (hazard ratio [HR] 1.48, 95%CI 1.31-1.67 for quintile 2; HR 2.07, 95%CI 1.85-2.33 for quintile 3; HR 2.55, 95%CI 2.27-2.88 for quintile 4). Similarly, high cumulative remnant-C quintiles were significantly associated with higher risks for NAFLD (HR 3.43, 95%CI 1.95-6.05 for quintile 2; HR 4.25, 95%CI 2.44-7.40 for quintile 3; HR 6.29, 95%CI 3.59-10.99 for quintile 4), compared with the quintile 1. CONCLUSION Elevated levels of baseline and cumulative remnant-C were independently associated with incident NAFLD. Monitoring immediate levels and longitudinal trends of remnant-C may need to be emphasized in adults as part of NAFLD prevention strategy.
Adult ; Humans ; Cohort Studies ; Non-alcoholic Fatty Liver Disease/etiology* ; Cholesterol ; Proportional Hazards Models ; Risk Factors

OBJECTIVE: To assess the value of using flat-sided culture tubes for preparing chromosomes through chorionic villi (CV) and amniotic fluid (AF) cell cultures during prenatal diagnosis. METHODS: From February to March 2020, 157 CV samples and 147 AF samples subjected to prenatal diagnosis at the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region were selected as the study subjects. For each sample, one flat-sided tube and one flask culture were set up by following the standard protocols. The methods were evaluated by comparing the cell growth, experimental process, quality of chromosome preparation and costs. RESULTS: The success rates for the culturing of CV and AF samples by the flat-sided culture tube method were 97.45% (153/157) and 97.96% (144/147), respectively. By contrast, the success rates for the conventional flask method were 98.72% (155/157) for CV and 98.64% (145/147) for AF samples. No significant difference was found between the two methods (P > 0.05). The average harvest time required by the flat-sided culture tube method was 8.45 days for CV and 9.43 days for AF cultures, whilst the average harvest time for conventional flask method was 9.05 days and 9.54 days, respectively. The flat-sided culture tube method for CV had required significantly shorter average harvest time than the conventional method (P < 0.001). No statistical significant difference was found in the average harvest time for AF by the two methods (P > 0.05). The conventional culturing method had required three containers with two sample transfers. By contrast, the flat-sided culture tube method was carried out in one tube without any sample transfer. The average total amount of medium used was 3.91 mL for each flat-sided culture tube and 6.26 mL for each conventional flask. CONCLUSION The flat-sided culture tube method can provide a simple, cost-effective and error-reducing procedure for the CV and AF samples culture during prenatal diagnosis.
Child ; Female ; Pregnancy ; Humans ; China ; Prenatal Diagnosis ; Chorionic Villi Sampling ; Amniotic Fluid ; Cell Proliferation

Objective:To explore the key factors affecting the formulation of treatment and prognosis of medullary thyroid carcinoma.Methods:Patient data, clinical characteristics and the results of follow-up of typical cases of 23 patients with medullary thyroid carcinoma admitted to Hunan Provincial People’s Hospital Breast and Thyroid Surgery from Apr. 2007 to Mar. 2020 were retrospectively analyzed. The therapeutic schedule and prognosis of medullary thyroid carcinoma were discussed in combination with ATA guidelines and others.Results:Of the 23 patients with MTC, 22 (95.65%) had elevated serum calcitonin, 15 (65.22%) had elevated carcinoembryonic antigen, 3 (13.04%) had suspected abnormal lymph nodes, and 2 (8.70%) had capsule invasion. Thyroid lobectomy, thyroid lobectomy with lateral lymph node dissection in level VI, total thyroidectomy, total thyroidectomy with lateral lymph node dissection in level VI, total thyroidectomy with bilateral lymph node dissection in level VI, total thyroidectomy with bilateral lymph node dissection in level VI with lymph node dissection in level I, II, III, IV, V or VII were performed in 1, 2, 3, 1, 13, 3 cases respectively. 8 cases had postoperative recurrence (34.78%) , of which 7 cases were caused by the first operation. The level of Ctn increased significantly in 2 cases before operation, who underwent total thyroidectomy with bilateral lymph node dissection in level VI, and no recurrence was found after operation.Conclusions:The key to the biological cure of medullary thyroid carcinoma is standardized surgical treatment. The surgery method cannot be determined simply by calcitonin. The modern treatment of medullary thyroid carcinoma needs to follow the principle of standardization and individualization at the same time.

Objective:Based on the analysis of case report and case series studies involving COVID-19 pregnant women in China, we aimed to discuss the updated management requirements for publication of scientific papers at hospitals in the new circumstances and context.Methods:The case report and case series studies published by domestic scholars in the early stage of COVID-19 outbreak were retrieved. Bibliometrics were used to analyze the research time, research content and research objects of the included literatures.Results:Nearly 200 cases (55%) of the 360 cases of COVID-19 pregnant women could not be clearly excluded from the possibility of repeated reporting. However, all these cases were not disclosed in the paper for possible repeated report.Conclusions:Potential repeated report problem may cause trouble for later meta-analysis, which also may have negative impact for the development of clinical guidelines. The departments of scientific research management at hospital should pay more attention to the management of patient′s data in scientific papers, so that it can better serve the accurate assessment of disease characteristics and epidemic situation, as well as the scientific formulation of clinical guidelines and control strategies.

Objective:To collect and analyze the non-compliance/protocol violation or deviation reported to the hospital Ethics Committee from 2017 to 2019, to explore the pattern of such events, as well as the causes and related logic. Empirical data evidence, lessons learned and preliminary advice were proposed to improve the understandings, management and implementation of clinical trials regarding to the non-compliance/protocol violation or deviations.Methods:The chi-square test and non-parameter test were conducted using SPSS 22.0.A total number of 754 reports received were analyzed, compared and summarized to provide the preliminary advice.Results:From 2017 to 2019, 754 reports on non-compliance/protocol violation or deviation, which covered 219 projects from 29 clinical departments, were received. 88% of these projects were drug/medical device clinical trials, while other 12% were investigator initiated trials. The reportrd events could be divided into 7 categories. The distribution of project resources, trial types and clinical specialties are significantly different. Meanwhile, the reports submitted by investigators have a lot of space for improvement.Conclusions:There were significant differences in the type and number of reported events on non-compliance/protocol violation or deviation between different trial types, as well as clinical trial specialties. The application report of protocol violation is generally poor. Combined with the working practice, the common reasons for the protocol violation are " the low overall compliance of the subjects" and " the insufficient compliance of the researchers" . Preliminary suggestions for management reform, such as increasing the training for researchers, improving the reporting mode, and regularly issuing follow-up and review research reports are recommended.

Objective: To observe the effect of quercetin on myelin regeneration in desyelinating mice induced by cuprizone (CPZ). Methods: A total of 50 C57BL/6J mice were randomly divided into normal control group, model group, low, medium and high dose quercetin groups, 10 mice in each group. In addition to the normal control group, mice demyelinating model was induced by feeding with 0.2% CPZ rodent feed. Quercetin solution was administered to the low, medium and high dose quercetin groups at 25, 50 and 100 mg/kg, and to the normal control group and the model group at 1 time/d. After 5 weeks of continuous gavage, the weight of mice was recorded every week, and the experiment of rotating bar was carried out. After 5 weeks, the changes of the myelin sheath in the corpus callosum of mice were observed by luxol fast blue (LFB) and transmission electron microscope (TEM). Immunofluorescence method was used to determine the protein expressions of myelin basic protein (MBP) and oligodendrocyte transcription 2actor (Olig2) in mouse brain tissue. The expression of MBP and cyclic nucleotide-3'phosphate hydrolase (CNPase) in mouse corpus callosum was determined by Western blot. Results: After 2-5 weeks, compared with the model group, the body mass of the medium and high dose quercetin groups significantly increased (P<0.01), and the score of turning rod significantly increased (P<0.01). The LFB observation showed that the demyelination score of corpus callosum in low, medium and high dose quercetin groups (2.23 ± 0.25, 1.50 ± 0.15, 1.14 ± 0.97 vs. 2.83 ± 0.18) significantly decreased (P<0.01). TEM observation showed that the G-ratio value in low, medium and high dose quercetin groups (0.75 ± 0.05, 0.75 ± 0.08, 0.73 ± 0.08 vs. 0.87 ± 0.05) significantly reduced (P<0.01). Immunofluorescence observation showed that the positive expression of MBP (37.40 ± 2.41, 37.40 ± 1.14 vs. 24.40 ± 3.65) and Olig2 (7.40 ± 1.14, 4.60 ± 1.14 vs. 2.80 ± 0.84) in the medium and high dose quercetin groups significantly increased (P<0.05). WB showed that the expression of MBP protein (1.32 ± 0.12, 0.80 ± 0.34 vs. 0.21 ± 0.07) and CNPase protein (0.72 ± 0.13, 1.06 ± 0.36 vs. 0.36 ± 0.21) in the medium and high dose quercetin groups significantly increased (P<0.05). Conclusions Quercetin (50-100 mg/kg) can reduce myelin injury and promote myelin regeneration in CPZ mice, as a neuroprotective effect on CPZ mice.

Objective To translate and revise the form of rheumatoid arthritis (RA) self-efficacy scale (RASE),and evaluate the revised scale.Methods Totally 101 patients with RA in one hospital were selected for investigation.Through the translation-back to the source scale and the cultural adjustment,the Chinese version of RASE (C-RASE) was formed.Exploratory factor analysis was conducted to acquire the construct validity while the Pearson correlation analysis was used to obtain the test-retest reliability.Results Factor analysis was carried out on 28 items,and the loading was 0.496,0.490,0.482 and 0.493 of item 5,and item 15 on two factors respectively.Exploratory factor analysis was conducted secondarily after deleting items of 5,15,and 17,considering the item contents,and 9 factors were extracted,respectively named for exercise,comprehensive treatment,relaxation,activity,sleep,social relationship,fatigue,cognition and active planning,which could explain 68.36％ of total variation;aud the correlation coefficient between each dimension and total table ranged 0.344 to 0.679 (P＜0.01);The Cronbach's alpha coefficient of the C-RASE was 0.751,and the retest reliability was 0.731.After deleting their own items,the Cronbach's alpha value range was 0.730 to 0.756.Conclusion C-RASE has sound reliability and validity and could provides a measurement of the self-efficacy among rheumatoid arthritis patients.

Objective:To explore the correlation between cerebrovascular hemodynamic index (CVHI) accumulative score and subclinical arteriosclerosis indicators.Methods:A total of 27 184 cases were collected from the Health Management Center,the Third Xiangya Hospital,Central South University.Linear regression analysis was carried out to confirm the correlations between CVHI accumulative score and the modified Framingham stroke profile (FSP),as well as between CVHI accumulative score and cerebrovascular diseases (ICVD) scale.The correlation between CVHI accumulative score and brachial-ankle pulse wave velocity (baPWV),carotid plaque orcarotid intima-media thickness (CIMT) was analyzed by multifactor logistic regression model in 11 580 cases.Moreover,the correlation between CVHI accumulative score and microalbuminuria or serum cystatin C was performed by multifactor logistic regression model in 9 860 cases.Results:In this study,the people whose CVHI accumulative score was less than 75 accounted for 12.98％.The CVHI accumulative score was negatively related with the modified FSP score (r=-0.484,P＜0.01) or ICVD score (r=-0.455,P＜0.01).The multifactor logistic regression model found that the baPWV,carotid plaque,microalbuminuria and serum cystatin C were independent predictors for CVHI accumulative score.Conclusion:The CVHI accumulative score is correlated with the modified FSP score,ICVD score and indexes of subclinical arteriosclerosis (baPWV,carotid plaque,microalbuminuria and serum cystatin C).The CVHI accumulative score could be used as a tool for zero-level and primary prevention of cerebral stroke.

Objective Ethical review is an important safeguard for the development of human subject research.The post approval review during the research process is particularly vital because it is the core method in the ethical review to protect participants' safety and right.In this article,current problems about post approval review were analyzed deeply to explore how to improve the mechanism of post approval review from the combination of management and technical aspect,particularly for the plan of not-for-cause visit.Methods According to the work experiences,literature review,identifying problems,to explore the collaboration to strengthen post approval review with experts,pharmacological institutions and scientific research management department.Results There are more and more frequent ethical issues during the implementation process,besides continuing strengthen the passive continuing review,continuing review guidelines and more detailed active continuing review screening and implementation mechanism should be developed.Conclusions It will prevent the occurrence of injury through establishing more optimal post approval review mechanism and a forehand active prevention strategy,which play an important role in the ethical review of human subject research.