AIM　To develop a sensitive, specific and reliable reversed-phase high performance liquid chromatographic method(RP-HPLC) to determine the total and unbound(free) concentrations in human plasma of amitriptyline and its major metabolite, nortriptyline. METHODS The assay involved a simple extraction procedure. The mobile phase consisted of acetonitrile and distilled water(30∶70, V/V), containing triethylamine(0.5%) and orthophosphoric acid(0.3%), pH 3.1. Separation was achieved on the C18 ODS column and the effluent was measured for UV absorption at 240 nm. RESULTS　The calibration curves were linear in the range of 4～400 μg*L-1 for total concentration, and in the range of 4～64 μg*L-1 for free concentration for both amitriptyline and nortriptyline. The lowest limits of detection were 4 μg*L-1 for both compounds. The absolute recovery rates were 102.0%±3.77% for amitriptyline and 99.3%±7.13% for nortriptyline. The precision values(RSD) of intra-day and inter-day for both amitriptyline and for nortriptyline were determined to be <5%, and <8%, respectively. The method was applied to determine the total and free concentrations in plasma of the healthy volunteers after a single oral dose of 50 mg amitriptyline. CONCLUSION　The assay was simple, repid, highly selective and sensitive. It is suitable for the routine analysis of total and free drug concentrations in plasma using readily available instruments with lower cost.

Cephalosporins are widely used antibiotics owing to their broad activity spectra and low toxicity. Many of these medically important compounds are made chemically from 7-aminodeacetoxycephalosporanic acid. At present, this intermediate is made by synthetic ring-expansion of the inexpensive penicillin G to form G-7-ADCA, followed by enzymatic removal of the side chain to obtain 7-ADCA. The chemical synthetic process is expensive, complicated and environmentally unfriendly. Environmentally compatible enzymatic process is favorable compared with chemical synthesis. In our previous research, metabolic engineered Escherichia coli strain (H7/PG15) was constructed and used as whole-cell biocatalyst for the production of G-7-ADC with penicillin G as substrate. The whole-cell biocatalysis was studied by single factor experiment, including the composition of substrates and the conversion conditions (OD600, pH, concentration of penicillin G, MOPS, glucose, time and FeSO4). After optimization, 15 mmol/L of G-7-ADCA was obtained. The process is convenient, efficient and economic. This work would facilitate the industrial manufacturing and further product research.
Anti-Bacterial Agents ; biosynthesis ; Biocatalysis ; Cephalosporins ; biosynthesis ; Escherichia coli ; metabolism ; Metabolic Engineering

AIM To develop a sensitive, specific and reliable reversed phase high performance liquid chromatographic method(RP\|HPLC) to determine the total and unbound(free) concentrations in human plasma of amitriptyline and its major metabolite, nortriptyline. METHODS\ The assay involved a simple extraction procedure. The mobile phase consisted of acetonitrile and distilled water(30∶70, V/V ), containing triethylamine(0 5%) and orthophosphoric acid(0 3%), pH 3 1. Separation was achieved on the C18 ODS column and the effluent was measured for UV absorption at 240 nm. RESULTS The calibration curves were linear in the range of 4～400 ?g?L -1 for total concentration, and in the range of 4～64 ?g?L -1 for free concentration for both amitriptyline and nortriptyline. The lowest limits of detection were 4 ?g?L -1 for both compounds. The absolute recovery rates were 102 0%?3 77% for amitriptyline and 99 3%?7 13% for nortriptyline. The precision values(RSD) of intra day and inter day for both amitriptyline and for nortriptyline were determined to be

Objective To detect the expression of long non-coding RNA (lncRNA) CCAT1 in human papillary thyroid cancer, and to observe the effect of CCAT1 down-regulation on the invasion and migration of human papillary thyroid cancer.Methods The expression of CCAT1 was detected in human normal thyroid Nthy-ori 3-1 cells and human papillary thyroid cancer TPC-1 cells.CCAT1 siRNA plasmid was transfected into TPC-1 cells.The effect of CCAT1 down-regulation on cell invasion and migration was observed by Transwell chamber assay and scratch test, and the expressions of BRAF, MUC15 and RKIP proteins were detected by Western blot.Results The level of CCAT1 in human papillary thyroid cancer TPC-1 cells was significantly higher than that in human normal thyroid Nthy-ori 3-1 cells.CCAT1 down-regulation significantly inhibited the invasion and migration of TPC-1 cells.The Transwell invasion assay revealed that the number of migrated TPC-1 cells in the CCAT1 down-regulation group was significantly lower than that in the control group.The scratch test showed an increased distance between cells in the CCAT1 down-regulation group compared to the control group, suggesting a reduced cell motility.The expressions of BRAF and MUC15 proteins were decreased in the CCAT1 down-regulation group, while that of RKIP protein was increased.Conclusions The expression of CCAT1 in papillary thyroid cancer cells is significantly higher than that in normal human thyroid cells.Down-regulation of CCAT1 in papillary thyroid cancer cells may inhibit the cell invasion and migration by regulating the expression of BRAF, MUC15 and RKIP proteins.

In order to successfully develop the effective population pharmacokinetic model to predict the concentration of propofol administrated intravenously, the data including the concentrations across both distribution and elimination phases from five hospitals were analyzed using nonlinear mixed effect model (NONMEM). Three-compartment pharmacokinetic model was applied while the exponential model was used to describe the inter-individual variability and constant coefficient model to the intra-individual variability, accordingly. Covariate effect including the body weight on the parameter CL, V1, Q2, V2, Q3 and V3 were investigated. The performance of final model was assessed by Bootstrapping, goodness-of-fit and visual predictive checking (VPC). The context-sensitive half-times and the infusion rates necessary to maintain the concentration of 1 microg x mL(-1) were simulated to six subpopulations. The results were as follows: the typical value of CL, V1, Q2, V2, Q3 and V3 were 0.965 x (1 + 0.401 x VESS) x (BW/59)(0.578) L x min(-1), 13.4 x (AGE/45)(-0.317) L, 0.659 x (1 + GENDER x 0.385) L x min(-1), 28.8 L, 0.575 x (1 + GENDER x 0.367) x (1 - 0.369 x VESS) L x min(-1) and 196 L respectively. Coefficients of the inter-individual variability of CL, V1, Q2, V2, Q3 and V3 were 29.2%, 46.9%, 35.2%, 40.4%, 67.0% and 49.9% respectively, and the coefficients of residual variability were 24.7%, 16.1% and 22.5%, the final model indicated a positive influence of a body weight on CL, and also that a negative correlation of age with V1. Q2 and Q3 in males were higher than those in females at 38.5% and 36.7%. The CL and Q3 were 40.1% increased and 36.9% decreased in arterial samples compared to those in venous samples. The determination coefficient of observations (DV)-individual predicted value (IPRED) by the final model was 0.91 which could predict the propofol concentration fairly well. The stability and the predictive performance were accepted by Bootstrapping, the goodness-of-fit and VPC. The context-sensitive half-times and infusion rates necessary to maintain the concentration of 1 microg x mL(-1) were different obviously among the 6 sub-populations obviously. The three-compartment model with first-order elimination could describe the pharmacokinetics of propofol fairly well. The involved fixed effects are age, body weight, gender and sampling site. The simulations in 6 subpopulations were available in clinical anesthesia. The propofol anesthesia monitor care could be improved by individualization of pharmacokinetic parameter estimated from the final model.

Objective To investigate the MRI features of supratentorial solid hemangioblastoma and to improve its diagnostic accuracy.Methods MRI image manifestations of 7 cases with supratentorial solid hemangioblastoma confirmed by operation and pathology were analyzed retrospectively.Results 7 cases of supratentorial solid hemangioblastoma were solitary.All tumors appeared as round or quasi-round masses with clear boundary, and showed equal or long T1 signal and slight long or long T2 signal,the obvious enhancement was seen in all tumors.There was mild to moderate edema around the tumors.The blood vessel was found within or around the tumors.Conclusion MRI manifestations of supratentorial solid hemangioblastoma have certain characteristics.

Purpose To investigate the c1inicopatho1ogica1 and immunohistochemica1 characteristics of enteropathy-associated T-ce11 1ymphoma( EATL)and to eva1uate the criteria of diagnosis and differentia1 diagnosis. Methods There were enteropathy-associated T-ce11 1ymphoma patients co11ected with c1inica1 data(n=9). Histo1ogica1 features were observed under microscope by HE staining and by immunohistochemstry. EBV was tested by in situ hybridization. Results EATL type Ⅰ showed a variab1e histo1ogy consisting of medium-sized to 1arge 1ymphoid ce11s with round or po1ygona1 nuc1ei,containing remarkab1e nuc1eo1i. EATL typeⅡshowed that tumor ce11s were medium-sized,with round,hyperchromatic nuc1ei. Nuc1ear debris and necrosis cou1d be seen easi1y. A 1arge number of his-tiocytes and neutrophi1s formed the inf1ammatory background. Immunohistochemica1 findings showed that tumor ce11s of two types were diffuse1y positive for CD3,CD43 and TIA-1,whi1e negative for CD4,CD5,CD20,CD79a. Tumor ce11s of EATL type II expressed CD56 and CD8,but negative in EATL typeⅠ. A high pro1iferation index was demonstrated by Ki-67. EBER was negative detection. There were seven patients with fo11ow-up data from 0 to 18 months. Four patients died within 10 months and three patients died within 18 months. Conclusions EATL is a rare type of 1ymphoma with intestina1 invo1vement. Patients often present with chronic abdomina1 pain,diarrhea,persistent fever and abdomina1 mass for a 1ong time. Intestina1 perforation occurs in some cases. Diagnosis shou1d be corre1ated to c1inica1 symptoms whi1e the fina1 diagnosis is main1y based on the patho1ogica1 features and the immune phenotypes.

Objective To investigate the expression of TAZ in peripheral blood and tumor tissue of the patients with colorectal cancer and to analyze their relationship and clinical significance.Methods Eighty-five cases of pathologically verified colorectal cancer treated by radical operation were selected as the operation group,and 35 cases with postoperative relapse served as the recurrent group.Meanwhile 50 age and sex-matched healthy persons were selected as the control group.The immunohistochemical(SP)technique was adopted to detect the expression of TAZ in tumor tissues and expression of TAZ in peripheral blood was tested with enzyme linked immunosorbent assay(ELISA).Results The level of preoperative TAZ in peripheral blood of the operation group was significantly higher than that of the control group(P＜0.05).The level of peripheral blood TAZ after operation in the operation group had no significant change compared with the control group (P＞0.05).The level of peripheral blood TAZ in the recurrent group was significantly higher than preoperative level of operation group(P＜0.05).The expression of tumor tissue TAZ was significantly higher than that of normal mucosal tissue(P＜0.05);the expression of tumor tissue TAZ in the recurrent group was higher than that in the operation group(P＜0.05).The tumor tissue TAZ level in the operation group and recurrent group had obvious correlation with peripheral blood TAZ level(P＜0.05).Conclusion Monitoring the TAZ level has obvious reference value for predicting colorectal cancer staging and timely understanding tumor recurrence.

【Objective】 To explore the effects of long non-coding RNA (lncRNA) small nucleolar molecule RNA host gene 12 (SNHG12) targeting inhibition of miR-495-3p/ phospholipinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway on the proliferation, migration and invasion of prostate cancer cells. 【Methods】 The expressions of SNHG12 and miR-495-3p in prostate cancer tissues and cells (LNCaP, C4-2, DU145) were detected with real-time fluorescence quantitative PCR (qRT-PCR).After DU145 cells were divided into si-NC, si-SNHG12, si-SNHG12+anti-miR-NC and si-SNHG12+anti-miR-495-3p groups, the expressions of SNHG12 and miR-495-3p were detected with qRT-PCR; the targeting relationship between SNHG12 and miR-495-3p was determined with dual luciferase assay; cell proliferation was assessed with MTT assay; cell migration and invasion were evaluated with Transwell assay; the protein expressions of proliferating cell nuclear antigen (PCNA), N-cadherin, and E-cadherin were detected with Western blot. 【Results】 The expressions of SNHG12 were significantly increased, while the expression of miR-495-3P was significantly decreased in prostate cancer tissues and cells (LNCaP, C4-2, DU145) (P<0.05).Knockdown of SNHG12 decreased DU145 cell activity, lowered the protein expressions of PCNA and N-cadherin, reduced the number of migrating and invading cells, but increased the protein expression of E-cadherin (P<0.05).SNHG12 targeted and negatively regulated miR-495-3p, and down-regulation of miR-495-3p reversed the effects of SNHG12 knockdown on the proliferation, migration and invasion of prostate cancer cells.Compared with the si-NC group, the si-SNHG12 group had significantly decreased expressions of p-PI3K and p-Akt (P<0.05).Compared with the si-SNHG12+anti-miR-NC group, the si-SNHG12+anti-miR-495-3p group had significantly increased protein expressions of p-PI3K and p-Akt (P<0.05). 【Conclusion】 lncRNA SNHG12 can promote the proliferation, migration and invasion of prostate cancer cells through targeted inhibition of miR-495-3p/PI3K/Akt signaling pathway.

OBJECTIVES: To establish an experimental method for evaluating material permeability of type I collagen hydrogels. METHODS: Using BSA-FITC as an indicator, by combining BSA-FITC with PBS they were used as permeability media, and using transwell load hydrogen sample to detect BSA-FITC transparent rate. RESULTS: In the concentration range of 100 μg·mL~0.781 μg·mL, the standard curve ≥ 0.99, Lower Limit of Quantity (LLOQ) is 3.125 μg·mL, RSD <5%, detection recovery rate is in the range of 80%~120%. CONCLUSIONS In this study, we established an experimental method for evaluating material permeability of hydrogel. The BSA-FITC transparent rate of type I collagen hydrogel was 100% at 28 h.
Collagen Type I ; chemistry ; Hydrogels ; chemistry ; Materials Testing ; Permeability