This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

In this work,near infrared carbon quantum dots(NIR-CDs)were synthesized by hydrothermal method using biomass material Clausena lansium leaves.The synthesized NIR-CDs emitted maximum fluorescence signal at 677 nm,which was independent of excitation wavelength.The characterization results showed that there were abundant groups on the surface of NIR-CDs.Pd2+could form non-fluorescent compounds with the surface groups of NIR-CDs,resulting in fluorescence quenching(Fluorescence signal was denoted as F0).Because chlorpromazine hydrochloride(CPZ)parent nucleus contained unoxidized S atom,CPZ could form stable colored complex with Pd2+under acidic conditions.In the presence of CPZ,Pd2+dissociated from the surface of NIR-CDs and bonded with CPZ,so that the fluorescence signal could be restored(Fluorescence signal was denoted as F).An"on-off-on"fluorescence sensor was thus constructed.The fluorescence signal recovery value of NIR-CDs(△F=F-F0)showed a good linear relationship with the concentration of CPZ in the range of 5.68-28.43 μg/mL,and the detection limit(3σ)was 0.078 μg/mL.The sensor was applied to determination of CPZ in pharmaceutical preparations,and the recoveries were 94％-106％.The developed fluorescence sensor was expected to be used in quality control of actual pharmaceutical preparations.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

OBJECTIVE: To compare the clinical efficacy of acupuncture with different frequencies in the treatment of patients with functional dyspepsia (FD). METHODS: A total of 90 patients with FD were randomly divided into a 3-time acupuncture treatment per week group (3-A group, 31 cases, 2 cases dropped off), a 1-time acupuncture treatment per week group (1-A group, 30 cases, 2 cases dropped off) and a control group (29 cases, 2 cases dropped off). In the two acupuncture groups, the acupoints were Zhongwan (CV 12) and bilateral Tianshu (ST 25), Neiguan (PC 6), Liangqiu (ST 34), Yanglingquan (GB 34), Zusanli (ST 36) and Taichong (LR 3), stimulated 3 times a week and once a week, respectively; and the treatment was given consecutively for 4 weeks. In the control group, no intervention was adopted, but the compensatory therapy was provided after the end of follow-up. The scores of the symptom index of dyspepsia (SID), self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were compared among the 3 groups before treatment, after 4 weeks of treatment and in 4 and 8 weeks after treatment completion separately. The score of Nepean dyspepsia life quality index (NDLQI) was evaluated before treatment, after 2 and 4 weeks of treatment and in 4 and 8 weeks after treatment completion. RESULTS: After 4 weeks of treatment and in 4 and 8 weeks after treatment completion, the scores of SID, SAS and SDS were all reduced in the 3-A group and the 1-A group when compared with the scores before treatment (P<0.000 1, P<0.05). After 4 weeks of treatment, the scores of SID, SAS and SDS in the two acupuncture groups were lower than those in the control group (P<0.000 1). After 2 and 4 weeks of treatment, the increased values of NDLQI score in the two acupuncture groups were all higher than those in the control group (P<0.05). In 4 and 8 weeks after treatment completion, the scores of SID, SAS and SDS in the 3-A group were lower than those in the 1-A group (P<0.001, P<0.05), and the increased values of NDLQI score in the 3-A group were higher than those in the 1-A group (P<0.000 1). CONCLUSION Acupuncture given 3 times per week is superior to the treatment given once per week in the aspects of relieving the clinical symptoms, improving the quality of life and regulating the emotional state in patients with FD. This efficacy is persistent for 8 weeks after treatment completion.
Humans ; Dyspepsia/therapy* ; Quality of Life ; Acupuncture Therapy ; Acupuncture Points ; Emotions

This study aims to compare the prevalence of sexually transmitted infections (STIs) with semen quality in men from couples with primary and secondary infertility. Semen samples were collected from 133 men who requested fertility evaluation. Seminal tract infection with Ureaplasma spp. (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and herpes simplex virus-2 (HSV-2) was assessed by PCR-based diagnostic assays. Among all patients, the prevalence of STIs was higher in men from couples with primary infertility than that in men from couples with secondary infertility (39.7% vs 21.7%, P = 0.03). The prevalence of UU was 28.8% and 13.3% in men from couples with primary and secondary infertility, respectively. Men from couples with primary infertility were more likely to be positive for UU than men from couples with secondary infertility (P = 0.04). Regarding the UU subtype, the prevalence of Ureaplasma urealyticum (Uuu) and Ureaplasma parvum (Uup; including Uup1, Uup3, Uup6, and Uup14) did not differ between the two groups. No associations between the prevalence rates of MH, MG, and CT were found in men from either infertility group. A lower sperm concentration was associated with STI pathogen positivity in men with primary infertility according to the crude model (P = 0.04). The crude and adjusted models showed that semen volume (both P = 0.03) and semen leukocyte count (both P = 0.02) were independently associated with secondary infertility. These findings suggest the importance of classifying the type of infertility during routine diagnosis of seminal tract infections.
Female ; Humans ; Infertility, Male/epidemiology* ; Male ; Mycoplasma genitalium ; Mycoplasma hominis ; Prevalence ; Semen ; Semen Analysis ; Sexually Transmitted Diseases/epidemiology* ; Ureaplasma urealyticum

An ultra-high performance liquid chromatography method for the determination of 8 constituents in Qingzao Jiufei Decoction was established and the basis of related chemical substances with antioxidant activity in Qingzao Jiufei Decoction was explored. The separation was performed on a Waters Cortecs RP Shield C18 (150 mm × 2.1 mm, 1.6 μm) using UHPLC-DAD as the mobile phase was water (containing 0.1% phosphoric acid) – acetonitrile with flow rate of 0.30 mL·min^-1 by gradient elution ① determining 5 constituents (amygdalin, liquiritin, liquiritin apioside, rutin and isoquercitrin) at the wavelength of 210 nm, 237 nm and 358 nm. Under gradient elution ②, 3 constituents (glycyrrhizin, glycyrrhizic acid and sesamin) were determined at the wavelength of 210 nm and 265 nm. The IC₅₀ of 10 batches of Qingzao Jiufei Decoction scavenging 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS⁺) free radicals obtained through test and Probit model was analyzed for correlation with the contents of 8 constituents. The established methods had a good linear relationship (r > 0.999), good repeatability and stability. The recovery rate was between 82.8% and 112.4%. In a series of concentration range, the higher the concentration of Qingzao Jiufei Decoction, the stronger the free radical scavenging effect. There was a significant correlation between the content of rutin and glycyrrhizic acid and the IC₅₀ of scavenging free radicals. The content determination methods established in this experiment provide a basis for a reasonable and scientific evaluation of the quality of Qingzao Jiufei Decoction. Qingzao Jiufei Decoction has antioxidant activity, which is significantly positively correlated with the content of rutin and glycyrrhizic acid.

A UHPLC method for the simultaneous determination of multiple constituents in QingJinHuaTan Decoction was established. The separation was performed on a Waters cortecs T3 column (150 mm×2.1 mm, 1.6 μm); the mobile phase was acetonitrile-water (containing 0.04% phosphoric acid) with gradient elution at a flow rate of 0.30 mL·min^-1, the column temperature at 25 ℃ and the wavelengths at 238 nm and 280 nm. The results showed that all peaks were well separated and all components had a good linear relationship in the investigative range, (r > 0.999). The repeatability and stability were good and the recovery was between 92.5%-104.7%. The method is simple, accurate and reliable and provides a basis for quality control of QingJinHuaTan Decoction and for further development of methods for its standardization.

For qualitative and quantitative analysis of related substances in clotrimazole cream, HPLC-Q-TOF spectrometer was used to analyze the fragmentation pathways and identify structures of the related substances. Five related substances named by BP (2018) were identified as impurity A ((2-chlorophenyl)-diphenylmethanol), impurity B (para-clotrimazole isomer), impurity E (2-chlorobenzophenone), impurity F (1-tritylimidazole) and impurity 4 (9-(2-chlorophenyl)-fluorene), respectively, by using impurity references matching and comparison with the literature data. Four related substances were detected in clotrimazole cream except impurity E, and 9-(2-chlorophenyl)-fluorene is the first identified impurity in this preparation. To establish an HPLC method for determination of the related substances in Clotrimazole Cream, the Agilent Poroshell Bonuns RP column was used (100 mm×4.6 mm, 2.7 μm) with UV detection at 215 nm. The mobile phase was acetonitrile-10 mmol·L^-1 dipotassium phosphate buffer (adjusted with phosphoric acid to pH of 5.80) with a flow rate of 1.0 mL·min^-1. Gradient elution was used. The column temperature was maintained at 40℃. A good linear behavior was achieved between component's concentrations and peak area for impurity A, B, E, F within the range of 0.20-10.02 μg·mL^-1, 0.20-10.00 μg·mL^-1, 0.20-10.10 μg·mL^-1, 0.10-5.01 μg·mL^-1 with the correlation coefficients were 0.999 7, 1.000 0, 1.000 0, 0.999 9, respectively. The average recoveries were 94.3%, 95.0%, 100.0%, 99.6% with RSDs were 2.8%, 2.2%, 1.1%, 2.7%, respectively (n=9). LOQ were 200.4, 200.0, 202.0, 100.2 ng·mL^-1, respectively. LOD were 57.25, 57.14, 57.71, 28.63 ng·mL^-1, respectively. The developed method was simple, rapid, accurate and effective for testing related substances in clotrimazole cream to control its quality, ensuring the safety of clinical medication.

Objective To investigate the effects of a training program based on virtual reality on static and dynamic balance perfor-mance in patients with Parkinson's disease (PD). Methods From June, 2014 to June, 2016, 46 patients with PD were randomly divided into control group (n=23) and experimental group (n=23). The control group received routine balance training, while the experimental group re-ceived balance training of virtual reality, for six weeks. They were assessed with Unified Parkinson's Disease Rating Scale part 3 (UP-DRS3), Berg Balance Scale (BBS), TimedUp and GoTest (TUGT) and Hamilton Depression Scale (HAMD) before and after training. The envelope area, anteroposterior standard deviation (AP-SD), mediolateral standard deviation (ML-SD) of centre of pressure (COP) were also measured with posturography. Results The scores of BBS, TUGT and HAMD improved in both groups after training (t>2.657, P<0.05), and improved more in the experimental group than in the control group (t>2.426, P<0.05). The score of UPDRS3 and the parameters of pos-turography improved in the experimental group (t>2.626, P<0.05), and improved more than that in the control group (t>2.112, P<0.05). Con-clusion Virtual reality rehabilitation is more effective than routine balance training on the static and dynamic balance function in patients with PD, and may release their depression.

PURPOSETo investigate the effects of salvianolic acid B (SAB) on tumor necrosis factor a (TNF-α) induced alterations of cerebral microcirculation with a bone-abrading model.

METHODSThe influences of craniotomy model and bone-abrading model on cerebral microcirculation were compared. The bone-abrading method was used to detect the effects of intracerebroventricular application of 40 μg/kg·bw TNF-α on cerebral venular leakage of fluorescein isothiocyanate (FITC)- albulmin and the rolling and adhesion of leukocytes on venules with fluorescence tracer rhodamine 6G. The therapeutical effects of SAB on TNF-α induced microcirculatory alteration were observed, with continuous intravenous injection of 5 mg/kg·h SAB starting at 20 min before or 20 min after TNF-α administration, respectively. The expressions of CD11b/CD18 and CD62L in leukocytes were measured with flow cytometry. Immunohistochemical staining was also used to detect E-selectin and ICAM-1 expression in endothelial cells.

RESULTSCompared with craniotomy method, the bone-abrading method preserved a higher erythrocyte velocity in cerebral venules and more opening capillaries. TNF-α intervention only caused responses of vascular hyperpermeability and leukocyte rolling on venular walls, without leukocyte adhesion and other hemodynamic changes. Pre- or post-SAB treatment attenuated those responses and suppressed the enhanced expressions of CD11b/CD18 and CD62L in leukocytes and E-selectin and ICAM-1 in endothelial cells induced by TNF-α.

CONCLUSIONSThe pre- and post-applications of SAB during TNF-α stimulation could suppress adhesive molecular expression and subsequently attenuate the increase of cerebral vascular permeability and leukocyte rolling.

Animals ; Benzofurans ; pharmacology ; Blood Flow Velocity ; Cerebrovascular Circulation ; drug effects ; Craniotomy ; Disease Models, Animal ; E-Selectin ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Mice ; Mice, Inbred C57BL ; Microcirculation ; drug effects ; Random Allocation ; Reference Values ; Tumor Necrosis Factor-alpha ; administration & dosage