Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts to synthesize a series of valuable esters and lactones. Based on protein sequence alignment and crystal structure analysis, a nonconserved hinge which linked NADPH domain and FAD domain was speculated to play an important role in substrate recognition and catalytic oxidation process. Cyclohexanone monooxygenase (CHMO) was selected as a model. Mutants obtained by homologous replacement of the whole hinge almost completely lost its original catalytic activity, demonstrating that the overall hinge structure was of great importance. Some significant sites were identified to greatly affect the catalytic activity and stereoselectivity by alanine scanning mutagenesis, accompanied by enzyme activity assessments and chiral kinetic resolutions. Altering K153 decreased the activity of the enzyme but enhanced the stereoselectivity. Changing L143 site reduced stereoselectivity but had little effect on enzyme activity. Mutation at L144 site dramatically weakened both activity and stereoselectivity. Subsequently, these corresponding sites in phenylacetone monooxygenase were also illustrated to follow a similar rule, revealing a universal importance of these sites in the BVMO family. These results expanded our understanding of the structure-activity relationship of these enzymes and provided more proofs for future directed evolution of BVMOs.
Amino Acid Sequence ; Catalysis ; Mixed Function Oxygenases ; chemistry ; Models, Molecular ; NADP ; Oxidation-Reduction ; Oxygenases ; chemistry ; Protein Conformation ; Sequence Alignment ; Structure-Activity Relationship ; Substrate Specificity

Bone Diseases ; Diagnosis, Differential ; Femur Head Necrosis ; Humans ; Medicine, Chinese Traditional ; methods ; Osteoporosis

Objective To analyze the merger candida albicans pulmonary tuberculosis irfection risk factors, clinical characteristics,treatment and prognosis,ways of improving the coexistence of the two diseases in the early di-agnosis and treatment methods. Methods 86 cases with pulmonary tuberculosis combined candida albicans infection were analyzed. Results Pulmonary lung infection candida albicans no specific clinical manifestations, in addition to X-ray signs of tuberculosis,mainly for the merger under the shadow of lung flake fuzzy; prediction for risk factors: long-term use of broad-spectrum antibiotics, the multi-antibiotics, glucocorticoid or immunosuppreasanta, long-term bed rest, physical weakness, the length of hospitalization, chronic bronchitis and obstructive pulmonary disease (COPD), respiratory failure, mechanical ventilation, diabetes, hypoproteinemia, blood diseases, multiple organ failure,mechanical ventilation,and other invasive operation is closely related to a higher mortality rate of pulmonary fungal infections. Conclusion Active tuberculosis disease and therapy to reduce risk factors, prevention and early diagnosis of good,reasonable use of antibiotics,hormones,and so un,the key is to improve the cure rate.

BackgroundResearches demonstrated that the levels of soluble CD44 (sCD44)molecule in aqueous is significantly higher in primary open-angle glaucomous(POAG) eye than normal eye,but how the sCD44 would affect the expression of apoptosis protein in trabecular meshwork cells is below understanding.Objective The present study was to investigate the effect of sCD44 on the expression of regulatory proteins bcl-2 associated death factor bad in trabecular meshwork cells in the patients with POAG.MethodsHuman scleral tissue with trabecular meshwork were obtained from POAG patients during the surgery.The trabecular meshwork cells were primarily cultured by explant culture method and identified by immunochemistry.The third generation of cells were incubated with free-serum DMEM/F12 medium added differnt dosages of sCD44 (0,1,5,10,25,50 mg/L) for 48 hours.The expression of bad protein in cultured cells was detected using cell counting kit-8 (CCK-8) as the absorbance values at 490 nm(A,90 value),and the bad protein level in cultured cells was assayed by ELISA.ResultsThe cultured cells showed the positive response for laminin ( LM ),neuron specific enolase ( NSE ),fibronectin ( FN ) monoclonal antibodies.The CCK-8 assay showed that the A490 values of the trabecular meshwork cells in 0,1,5,10,25,50 μg/L of sCD44 groups were 0.2460±0.0019,0.1874±0.0015,0.1570±0.0016,0.1302±0.0019,0.1084±0.0018,0.0940±0.0020 respectively with a statistically significant difference among the 6 groups( F =14.922,P =0.000 ),and the A490 values in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group (P=0.013,0.008,0.011,0.005,0.004).The ELISA assay showed that bad protein levels in 0,1,5,10,25,50 μg/L of sCD44 groups were ( 114.8461 ± 2.9560 ),( 137.8270 ± 2.4259 ),( 161.4194 ± 3.7381 ),( 170.9453 ± 3.2006 ),( 221.2252 ±4.3738 ),( 324.6167±4.4220) ng/L,showing a total difference among them ( F =16.610,P =0.000 ),and the bad protein levels in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group( P =0.017,0.013,0.008,0.007,0.006).ConclusionssCD44 can contribute to the apoptosis of the trabecular meshwork cells in patients with POAG in certain dose range by regulating the apoptosis regulatory proteins bcl-2 associated death factor bad.

Biopsy ; Carcinoma, Squamous Cell ; classification ; metabolism ; pathology ; Epithelial-Mesenchymal Transition ; Humans ; Immunohistochemistry ; Nasopharyngeal Neoplasms ; classification ; metabolism ; pathology ; Nasopharynx ; pathology ; RNA, Viral ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Viral Matrix Proteins ; metabolism

Introduction:Transcatheter arterial chemoembolization (TACE) plus thermal ablation has been widely used recently in the treatment of hepatocellular carcinoma (HCC). In this study, we aimed to compare results of the combination of TACE and percutaneous thermal ablation with those of hepatectomy in patients with HCC. Methods:The clinical data of 137 HCC patients who sequentially received TACE and computed tomography (CT)-guided percutaneous thermal ablation as an initial curative treatment (combination group) and 148 matched HCC patients who received hepatectomy (surgery group) between 2004 and 2011 were collected and analyzed. After TACE, multiphase contrast-enhanced CT was performed to identify the total number of tumors as well as lipiodol deposition in the liver. Survival was calculated by using the Kaplan-Meier method and compared by using the log-rank test. The prognostic factors were assessed with multivariate Cox proportional hazards regression analysis. Results:Of all 285 patients, 225 (79.0%) had cancerous lesions≤5 cm in diameter. In preoperative contrast-enhanced CT or magnetic resonance imaging, the number of tumors was 1–4 for each patient. The 1-, 3-, and 5-year overal survival rates were 95, 74%, and 67%in the combination group and 88, 66, and 47%in the surgery group, respectively (P=0.004);the corresponding recurrence-free survival rates for the two groups were 92, 69, and 61%and 75, 58, and 44%, respectively (P=0.001). In the multivariate analysis, treatment al ocation was an independent prognostic factor for survival. Only 60 patients in the combination group had sufficient imaging data, and 135 new lesions with lipiodol deposition were diagnosed as malignancies in 22 of 60 patients, whereas 20 new lesions were found in 11 of 148 patients in the surgery group. Conclusion:The combination of TACE and CT-guided percutaneous thermal ablation for HCC improves survival of HCC patients compared with hepatectomy.

The clinical efficacy and safety of auricular acupuncture (AA) for treatment of primary insomnia was evaluated. After a comprehensive retrieval in domestic and foreign databases, literatures were strictly screened and Revman 5.2 software was applied to perform a Meta-analysis on eligible randomized controlled trials (RCTs). The evidence quality was assessed with GRADE profiler 3.6 software. As a result, 8 articles were included involving 894 patients. Compared among AA and sham AA, placebo AA, blank control, there was significant difference in Pittsburgh sleep quality index (PSQI) [WMD = -3.48, 95% CI (-3.96, -3.00)], sleep latency LWMD = -10.14, 95% CI (-17.16, -3.12)] and sleep awakening times [WMD = -9.98, 95% CI (-1.10,-0.48)]. Compared between AA and western medication, there was significant difference in PSQI [WMD = -3.62, 95% CI (-4.59, -2.65)]. The evidence quality was moderate in AA vs. sham AA, placebo AA or blank control, while that of the rest was extremely low. No reports of adverse events were described in all studies. In conclusion, for the treatment of primary insomnia, AA could effectively improve sleep quality, but due to the low evidence quality, cautious attitude should be taken on this conclusion, and clinical trials with large sample and high quality were needed in the further.
Acupuncture, Ear ; Humans ; Randomized Controlled Trials as Topic ; Sleep ; Sleep Initiation and Maintenance Disorders ; physiopathology ; therapy

Objective To investigate the role of increased microRNA-21 (miR-21) in the development of renal tubulointerstitial fibrosis secondary to aristolochic acid induced acute kidney injury.Methods C57BL/6J male mice were intraperitoneally injected with aristolochic acid at a dose of 10 mg/kg.Blood samples and kidneys were harvested at day 1,3,7,14,28 after aristolochic acid treatment.To assess the role of miR-21 in aristolochic acid induced acute kidney injury to chronic kidney disease progression,mice were intravenously injected with anti-miR-21 or anti-scramble (10 mg/kg) at 1 h before aristolochic acid dosing,as well as d5 and d10 after aristolochic acid dosing.Results Increased serum creatinine and severe kidney injury were found at d3 after aristolochic acid treatment.Renal tubulointerstitial fibrosis was developed at d14 after aristolochic acid treatment.Protein expression of α-SMA,vimentin and collagen Ⅰ were significantly up-regulated at d7 and peaked at d14 (P ＜ 0.01),while protein abundance of E-Cadherin decreased at d14 and lasted until d28 (P ＜ 0.01).The abundance of miR-21 increased at d7 after aristolochic acid dosing,peaking at d14 and thereafter maintaining at a high level.Anti-miR-21 intervention relieved renal injury with reduced serum creatinine (P ＜ 0.05) and attenuation of renal tubulointerstitial fibrosis.Besides,the protein expression of α-SMA,vimentin,and collagen Ⅰ/Ⅳ was all down-regulated after anti-miR-21 treatment (P ＜ 0.05).PTEN was up-regulated and the ratio of its downstream genes p-AKT/AKT was decreased.(P ＜ 0.05) Conclusions A single high dose of aristolochic acid leads to acute kidney injury and the development of renal tubulointerstitial fibrosis secondary to AKI.Renal tubulointerstitial fibrosis could be partially reversed by inhibiting miR-21 via PTEN/p-AKT pathway.

Objective To investigate the state of endemic fluorosis, running status of water improvement project to reduce fluoride in Jiangsu province, and to provide a scientific basis for prevention and control of endemic fluorosis. Methods In 2008, in the ten key counties of endemic fluorosis (zone), in Xuzhou,Lianyungang and Suqian, a stratified sampling method was employed to select 40 diseased villages according to their past water fluoride survey data. All children aged 8 to 12 were examined dental fluorosis, and all adults over 16 years were examined clinical skeletal fluorosis. Thirty per cent of the 40 diseased villages were selected, and 20 adults over the age of 16 in each selected village were examined by X-ray, respectively;50% of the 40 diseased villages were selected, and 30 any time urine samples of children aged 8 to 12 in each diseased village were tested urine fluoride. In each city, select a county, the status of water improvement project to reduce fluoride, water supply capacity and coverage in the county were investigated. Results A total of 3560 children aged 8 to 12 were examined, the detection rate of dental fluorosis was 38.51% (1371/3560), tooth defect rate was 5.34% (190/3560), and dental fluorosis index was 0.8. Seven hundred and eight urine samples were tested, the median urinary fluoride was 1.47 mg/L and the range was 0.08 ~ 10.08 mg/L. Clinical detection of skeletal fluorosis was 21.3% among adults over the age of 16, and X-ray detection rate of skeletal fluorosis was 39.2% (123/314).Investigated a total of 248 facilities of centralized water improvement projects, no funds to run or damaged 49, the water fluoride > 1.0 mg/L was 18. Conclusions Endemic fluorosis in Northern Jiangsu province has not been controlled completely, but has a rising trend, we should further strengthen the supervision of water fluoride reduction.

Antigens, CD20 ; metabolism ; Epstein-Barr Virus Infections ; virology ; Female ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Lymphoma, Large B-Cell, Diffuse ; pathology ; virology ; Middle Aged ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; virology