Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase rlsr5 was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of rlsr5 was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)₈-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize R-TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 °C. It was independent of metal ions, with the activity enhanced by Mn²⁺. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.
Animals ; Rabbits ; Alcohol Oxidoreductases/biosynthesis* ; Recombinant Proteins/metabolism* ; Escherichia coli/metabolism* ; Liver/enzymology*

BACKGROUND: Rheumatoid arthritis (RA), a chronic systemic autoimmune disease, is characterized by synovitis and progressive damage to the bone and cartilage of the joints, leading to disability and reduced quality of life. This study was a randomized clinical trial comparing the outcomes between withdrawal and dose reduction of tofacitinib in patients with RA who achieved sustained disease control. METHODS: The study was designed as a multicenter, open-label, randomized controlled trial. Eligible patients who were taking tofacitinib (5 mg twice daily) and had achieved sustained RA remission or low disease activity (disease activity score in 28 joints [DAS28] ≤3.2) for at least 3 months were enrolled at six centers in Shanghai, China. Patients were randomly assigned (1:1:1) to one of three treatment groups: continuation of tofacitinib (5 mg twice daily); reduction in tofacitinib dose (5 mg daily); and withdrawal of tofacitinib. Efficacy and safety were assessed up to 6 months. RESULTS: Overall, 122 eligible patients were enrolled, with 41 in the continuation group, 42 in the dose-reduction group, and 39 in the withdrawal group. After 6 months, the percentage of patients with a DAS28-erythrocyte sedimentation rate (ESR) of <3.2 was significantly lower in the withdrawal group than that in the reduction and continuation groups (20.5%, 64.3%, and 95.1%, respectively; P  < 0.0001 for both comparisons). The average flare-free time was 5.8 months for the continuation group, 4.7 months for the dose reduction group, and 2.4 months for the withdrawal group. CONCLUSION: Withdrawal of tofacitinib in patients with RA with stable disease control resulted in a rapid and significant loss of efficacy, while standard or reduced doses of tofacitinib maintained a favorable state. TRIAL REGISTRATION Chictr.org, ChiCTR2000039799.
Humans ; Quality of Life ; China ; Arthritis, Rheumatoid/drug therapy* ; Piperidines/therapeutic use* ; Treatment Outcome ; Antirheumatic Agents/therapeutic use* ; Pyrroles/therapeutic use*

Objective:To analyze the current situation of Investigator-Initiated Trials in medical and health institutions in Shandong Province, the problems in the process of conducting clinical research, and put forward proposals for the establishment of a clinical research management system with effective supervision, sound systems and supporting services, taking into account the progress of the projects since the pilot work was carried out.Methods:A questionnaire was created, an online survey was conducted, a database was set up, a status analysis was conducted and a post-launch analysis of the progress of the pilot was carried out using the National Medical Research Registry Information System, culminating in recommendations using the literature summary method and empirical analysis.Results:Statistical analysis of the questionnaire found that 29.39% of the institutions have a dedicated clinical research management department, and 75.97% of the institutions have a management approach. 25.52%, 40.30%, and 43.07% of institutions established biobanks, clinical research centers, and follow-up centers. There was a statistically significant difference in the establishment of clinical research centers, biobanks, and follow-up centers in secondary and tertiary medical institutions ( P<0.05). The number of general clinical research projects filed, the number of submissions and the number of ethics committees filed in the filing system have all increased significantly after the pilot work, with growth percentages of 126%, 141% and 62% respectively. Conclusions:Shandong Province clinical research pilot work has begun to bear fruit, the current clinical research project still exists in the lack of special funding support, perfect service platform and system support and training system to be improved and other issues.

The potential medicinal value of Ma bamboo (Dendrocalamus latiflorus), one of the most popular and economically important bamboo species in China, has been underestimated. In the present study, we found that D. latiflorus leaf extract (DLE) reduced fasting blood glucose levels, body weight, and low-density lipoprotein cholesterol with low liver toxicity in db/db mice. In addition, gene expression profiling was performed and pathway enrichment analysis showed that DLE affected metabolic pathways. Importantly, DLE activated the AKT signaling pathway and reduced glucose production by downregulating glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) expression. Moreover, network pharmacology analysis identified rutin as an active component in DLE through targeting insulin growth factor 1 receptor (IGF1R), an upstream signaling transducer of AKT. Due to its hypoglycemic effects and low toxicity, DLE may be considered an adjuvant treatment option for type 2 diabetes patients.

Background: Thyroid diseases are highly prevalent worldwide, but their diagnosis remains a challenge. We established reference intervals (RIs) for thyroid-associated hormones and evaluated the prevalence of thyroid diseases in China. Methods: After excluding outliers based on the results of ultrasound screening, thyroid antibody tests, and the Tukey method, the medical records of 20,303 euthyroid adults, who visited the Department of Health Care at Peking Union Medical College Hospital from January 2014 to December 2018, were analyzed. Thyroid-associated hormones were measured by the Siemens Advia Centaur XP analyzer. The RIs for thyroid-associated hormones were calculated according to the CLSI C28-A3 guidelines, and were compared with the RIs provided by Siemens. The prevalence of thyroid diseases over the five years was evaluated and compared using the chi-square test. Results: The RIs for thyroid stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), total thyroxine (TT4), and total triiodothyronine (TT3) were 0.71–4.92 mIU/L, 12.2–20.1 pmol/L, 3.9–6.0 pmol/L, 65.6–135.1 nmol/L, and 1.2–2.2 nmol/L, respectively. The RIs of all hormones except TT4 differed significantly between males and females. The RIs of TSH increased with increasing age. The prevalence of overt hypothyroidism, overt hyperthyroidism, subclinical hypothyroidism, and subclinical hyperthyroidism was 0.5% and 0.8%, 0.2% and 0.6%, 3.8% and 6.1%, and 3.3% and 4.7% in males and females, respectively, which differed from those provided by Siemens. Conclusions Sex-specific RIs were established for thyroid-associated hormones, and the prevalence of thyroid diseases was determined in the Chinese population.

Objective:To observe the clinical efficacy and adverse reactions of rituximab in the treatment of systemic sclerosis (SSc).Methods:Eight SSc patients who received rituximab treatment in the Department of Rheumatology of Shanghai Ruijin Hospital from November 2016 to May 2020 were treated with rituximab at week 0, week 2, week 4, week 24 and week 48. The clinical symptoms and laboratory parameters were evaluated at baseline, week 4, week 24 and week 48 respectively. All data were analyzed by Wilcoxon test.Results:All the patients were diagnosed as diffuse SSc, including seven females and one male, with a median disease course of 2.5 years. At week 0, week 24 and week 48, the modified Rodnon skin scores (MRss) were 16.5 (11.8, 29.5) , 14.5 (9.5, 27) ( Z=0.841) and 10.5 (7, 24.3) ( Z=0.420) respectively, which were significantly improved as compared with the baseline ( P<0.05). The patients' self-scores were 60(50, 77.5), 52.5(41.3, 67.5)( Z=0.113) and 47.5(36.3, 57.5)( Z=0.474) respectively, which were significantly improved at week 24 and week 48, and the High Resolution CT (HRCT) scores at baseline and week 48 were 2.7(1.02, 3.7) and 1.6(0.65, 2.95)( Z=0.964) respectively, significantly improved after treatment ( P<0.05). The pulmonary aterial hypertension (PAH) values were 48(41, 58.5) mmHg and 47(38.5, 57) mmHg ( Z=0.315) respectively. There was no significant difference between the two groups. Clinical observation showed that the condition was improved and no adverse reaction occurred at the same time period. Conclusion:The improvement of skin sclerosis, pulmonary interstitial lesion and pulmonary artery pressure can be observed during the treatment with rituximab, which may be a new choice for the treatment of SSc. There is no serious adverse reaction during the treatment, and the patients are well tolerated and safe.

Objective:To report our experience in diagnosis and treatment of posterior atlantoaxial dislocation with odontoid retrolisthesis.Methods:A retrospective study was conducted of the 5 patients who had been treated from July 2012 to August 2018 at Department of Orthopaedics, General Hospital of Southern Theater Command for posterior atlantoaxial dislocation. They were 4 men and one woman, aged from 34 to 67 years (average, 47 years). All of them had a history of trauma. Of them, 4 were complicated with odontoid fracture and one with congenital free os odontoideum. Their posterior atlantoaxial dislocation ranged from 3 to 9 mm (average, 6 mm). By the American Spinal Injury Association (ASIA) grading system, their preoperative spinal injury was rated as grade B in one, as grade C in 3 cases and as grade D in one. All the 5 patients underwent skull traction at 10° flexion. Surgical trans-oralpharyngeal atlantoaxial reduction and internal fixation was performed for the one patient whose reduction had not been achieved by traction while posterior atlantoaxial screw-rod fixation or anterior odontoid screwing was conducted for the 4 patients whose reduction had been achieved by traction. The distance of posterior atlantoaxial dislocation was measured to evaluate their reduction and ASIA grade system was used to assess their spinal function after operation.Results:The postoperative distance of posterior atlantoaxial dislocation was 0 mm, showing a reduction rate of 100%. The 5 patients were followed up for 6 to 36 months (average, 15 months). By the ASIA grade system, the postoperative functional recovery of the spine was grade D in 4 cases and grade C in one. No implant loosening or breakage occurred.Conclusion:As a kind of high-energy hyperextension injury, posterior atlantoaxial dislocation is rare in clinic, but an appropriate treatment can be adopted to deal with its different clinical types to achieve good outcomes.

Objective To explore the clinical application effect of multiple end-to-side anastomosis in free anterolateral thigh perforator flap transplantation.Methods From January,2013 to October,2017,29 cases were applied the technology of multiple end-to-side anastomosis to the same recipient vessel in anterolateral thigh perforator flap transplantation for wound repair.Fifteen cases treated with multiple end-to-side anastomosis on flap arteries,and 2-3 arteries were anastomosed,with the average of 2.13.Nine cases were treated with venous multiple end-to-side anastomosis,and 2-4 veins were anastomosed,with the average of 3.11.Five cases treated with arterial and venous multiple end-to-side anastomosis,and 4-6 vessels were anastomosed,with the average of 5.20.Distribution of wounds:10 cases with hands and wrists wounded,15 cases with forearms and elbow joints wounded,and 4 cases with lower legs and feet wounded.The soft tissue defect size was 5 cm×13 cm-11 cm×27 cm,and the flap area was 6 cm× 15 cm-12 cm×29 cm.Postoperative followed-up was performed every 3-6 months to review flap survival.Results All flaps of 29 cases survived.Venous congestion occurred in 2 flaps within 48 h after the operation,among which,1 was overcomed after released the dressing and sutures,and the other underwent surgical exploration.The venous end-to-side anastomotic stomas were unobstructed,and hematoma was formed.After the hematoma compression was removed,circulation was recovered and the flap survived.With followed-up for 6 months to 2.5 years,both donor site and recipient site of the flaps healed well and the injured distal limbs had no hemodynamic disorder.Conclusion The application of multiple end-to-side anastomosis to the same recipient vessel for free transplantation of anterolateral thigh perforator flaps is safe and reliable.

Objective To discuss the clinical value of bile reinfusion via nasojejunal tube on liver function after biliary tract surgery. Methods Eighty patients with biliary tract surgery and bile outer drainage were divided into bile reinfusion group and control group by random digits table method with 40 cases each. The clinical data concerning the liver function and volume of biliary drainage were collected. Results The patients were well tolerated for bile reinfusion, and abdominal distension, nausea and vomiting occurred in some patients. The symptoms improved significantly after symptomatic treatment. The alanine aminotransferase (ALT) and total bilirubin (TBIL) levels at the fifth day after operation in bile reinfusion group were significantly reduced than those in control group:(31 ± 18) U/L vs. (48 ± 32) U/L and (51 ± 32)μmol/L vs. (76 ± 38)μmol/L, the aspartate aminotransferase (AST) and ALT levels at the seventh day after operation in bile reinfusion group were significantly reduced than those in control group: (32 ± 19) U/L vs. (43 ± 26) U/L and (20 ± 19) U/L vs. (31 ± 22) U/L, and there were statistical differences (P<0.05). The volume of biliary drainage in the bile reinfusion group was significantly increased compared with that in control group at the third and fourth day after operation:(485 ± 52) ml vs. (428 ± 96) ml and (509 ± 62) ml vs. (458 ± 59) ml, and there was statistical difference (P<0.01). Conclusions Bile reinfusion via the nasojejunal tube may facilitate the recovery of liver function after biliary tract surgery.

Background and purpose:Multiple microRNAs (miRNAs) are abnormally expressed in breast cancer and play an important role in the regulation of breast cancer. miRNAs may be a new target for the treatment of breast cancer. This study aimed to investigate the expression of miR-199a-3p in breast cancer and the effect of miR-199a-3p on proliferation and apoptosis of breast cancer.Methods:Real-time PCR was used to test the expression of miR-199a-3p in breast cancer tissues, normal breast tissues, breast cancer cells and normal breast cells. Overexpression (or silencing the expression) of miR-199a-3p was conducted by transfecting MDA-MB-231 with miR-199a-3p mimics (or inhibitors). The proliferation of MDA-MB-231 was detected by MTT method. The apoptosis of MDA-MB-231 was investigated by Hoechst staining and caspase-3 activity assay kit.Results:Compared to corresponding non-tumor breast tissues (or normal breast cell HBL-100), lower levels of miR-199a-3p were expressed in breast cancer tissues or breast cancer cells. Overexpression of miR-199a-3p induced by miR-199a-3p mimic inhibited the proliferation and promoted the apoptosis of MDA-MB-231, while silencing the expression of miR-199a-3p induced by miR-199a-3p in-hibitor increased the proliferation and suppressed the apoptosis of MDA-MB-231.Conclusion:The expression of miR-199a-3p is lower in breast cancer, which shows its tumor suppression effect by regulating the proliferation and apoptosis of breast cancer cells.