microRNA-1-3p and T-synthase mRNA have high diagnostic efficacy on intestinal mucosal barrier dysfunction in patients with severe acute pancreatitis.

Acute pancreatitis (AP) is an inflammatory disorder of the pancreas that can be complicated by intestinal mucosal barrier dysfunction (SAP&IBD). The current study sought to examine the diagnostic efficacy of miR-1-3p and T-synthase mRNA in SAP&IBD patients. First, SAP patients were assigned to SAP&IBD and SAP groups. Serum miR-1-3p expression and T-synthase mRNA expression patterns in peripheral blood B lymphocytes were measured using RT-qPCR. Pearson tests, ROC curve analysis, and multivariate logistic regression were used to analyze the correlation between miR-1-3p/T-synthase mRNA and clinical data, their diagnostic efficiency, and independent risk factors for SAP&IBD patients, respectively. The results showed that serum miR-1-3p in the SAP&IBD group was elevated, and T-synthase mRNA expression in peripheral blood B lymphocytes was diminished. Additionally, serum miR-1-3p expression in SAP&IBD patients was negatively correlated with T-synthase mRNA expression, and positively correlated with their Ranson score, CRP, IL-6, DAO, and D-Lactate levels. Meanwhile, T-synthase mRNA level was negatively correlated with IL-6, DAO, and D-Lactate levels. Both, serum miR-1-3p, T-synthase mRNA, and their combination were found to exhibit diagnostic efficiency for SAP&IBD patients, and were independently associated with IBD in SAP patients. Collectively, our findings suggest that miR-1-3p and T-synthase serve as independent risk factors for SAP&IBD patients and can aid the diagnosis of IBD in SAP patients.

Drug susceptibility and molecular epidemiology of Escherichia coli in bloodstream infections in Shanghai, China, 2011-2013.

BACKGROUND: Prevention and management of Escherichia coli bloodstream infections (EC-BSIs) have become increasingly complicated by antimicrobial resistance and rapid dissemination. We investigated the antimicrobial epidemiology and phylogenetic background of clinical E. coli isolates from patients with bloodstream infections in Shanghai from 2011 to 2013. METHODS: Escherichia coli isolates causing bloodstream infections were consecutively collected between June 2011 and June 2013. Antimicrobial susceptibility testing was performed by disk diffusion. Drug resistance genes coding for extended-spectrum ß-lactamases (ESBLs) and carbapenemases, and phylogenetic groups were detected by polymerase chain reaction. eBURST was used for multilocus sequence typing. RESULTS: Of the strains 128 collected, 80 produced ESBLs. No carbapenem-resistant isolates were found. The resistance rates to penicillins, fluoroquinolone, folate pathway inhibitors, tetracyclines and second generation cephalosporins were high. Molecular analysis showed that CTX-M-14 (40/80) was the most common ß-lactamase, followed by CTX-M-55 (17/80) and CTX-M-15 (14/80). Phylogenetic group B2 predominated (37.5%), but phylogenetic group D exhibited the highest rates of ESBL production. ST131 (17/128) was the most common sequence type, followed by ST69 (12/128) and ST648 (10/128). CONCLUSIONS: The antimicrobial resistance rate was high among EC-BSI isolates, but amikacin, piperacillin-tazobactam and carbapenem could be options for empiric therapy. Genetic diversity showed no correlation with the nosocomial origin of the isolates.

Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS.

Ractopamine glucuronides have been identified in cattle urine sampled by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter- and intra-assay variation tests for RR-ractopamine. The concentration levels of parent ractopamine and ractopamine glucuronide metabolites as the main components of total ractopamine in cattle and sheep urine showed similar depletion trends, in which the concentration curves increased and reached a climax during the feeding period, and then dropped quickly when entering the withdrawal period. Data from the three methods had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R(2)) for parent ractopamine between the ELISA and the LC-MS/MS or GC-MS results were 0.93 or 0.92; R(2) values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677, respectively. All R(2) values for data gained from sheep urine samples were >0.95. The study indicated that the close levels of RR-ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.

VEGF, not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia.

The purpose of this study was to determine the relationship between VEGF and mini-TyrRS/mini-TrpRS in angiogenesis in hypoxic culture and to begin to comprehend their mechanism in angiogenesis. We designed a VEGF gene silencing assay by using lentivirus vectors, and then western blotting was used to determine the protein expression of VEGF, VEGFR2 and pVEGFR2 in three groups in hypoxic culture at 3, 6, 12, or 24 h: (1) untransfected human umbilical vein endothelial cells (HUVECs) (Control); (2) pGCSIL-GFP lentivirus vector-transduced HUVECs (Mock); and (3) pGCSIL-shVEGF lentivirus vector-transduced HUVECs (Experimental). We also detected the effects of mini-TyrRS/mini-TrpRS peptides on HUVEC proliferation, migration and tube formation after lentivirus vector transfection and VEGFR2 antibody injection. The results indicated that expression of the mini-TyrRS protein was increased, whereas that of mini-TrpRS was specifically decreased in hypoxic culture both in control and mock groups. However, this trend in protein levels of mini-TyrRS and mini-TrpRS was lost in the experimental group after transduction with the pGCSIL-shVEGF lentivirus vector. The protein expression of VEGF was increased in hypoxic culture both in control and mock groups. After transduction with the pGCSIL-shVEGF lentivirus vector, the protein level of VEGF was noticeably decreased in the experimental group; however, for VEGFR2, the results showed no significant difference in VEGFR2 protein expression in any of the groups. For pVEGFR2, we found a distinct trend from that seen with VEGF. The protein expression of pVEGFR2 was sharply increased in hypoxic culture in the three groups. The addition of mini-TyrRS significantly promoted proliferation, migration and tube formation of HUVECs, while mini-TrpRS inhibited these processes in both control and mock groups in hypoxic culture. However, these effects disappeared after transduction with the pGCSIL-shVEGF lentivirus vector in the experimental group, but no significant difference was observed after VEGFR2 antibody injection. The protein expression of VEGF is similar to that of mini-TyrRS in hypoxic culture and plays an important role in the mini-TyrRS/mini-TrpRS-stimulated proliferation, migration and tube formation of HUVECs in hypoxia. These results also suggest that the change in mini-TyrRS and mini-TrpRS expression in hypoxic culture is not related to VEGFR2 and that some other possible mechanisms, are involved in the phosphorylation of VEGFR2.

Two new lignans from the stems of Schisandra bicolor.

Two new lignans, named zuihonins E (1) and F (2), were isolated from the stems of Schisandra bicolor Cheng var. tuberculata Law. The structures of the new lignans were elucidated on the basis of extensive spectroscopic analysis, including 1D, 2D NMR, and MS experiments, and their absolute stereochemistry was determined by circular dichroism spectrum. Compounds 1 and 2 did not inhibit the growth of hepatoma carcinoma cell (HepG2), lung carcinoma cell (A549), and human breast carcinoma (MCF-7) cell lines.

Effect of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase on ischemic angiogenesis in rats: proliferation and migration of endothelial cells.

The purpose of this study was to determine the mechanism of mini-tyrosyl-tRNA synthetase/mini-tryptophanyl-tRNA synthetase (mini-TyrRS/mini-TrpRS) on ischemic angiogenesis in rats with acute myocardial infarction and proliferation, migration, potential signaling pathways of rat coronary venular endothelial cells (RCVECs). The effects of mini-TyrRS/mini-TrpRS on RCVECs proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The potential involvement of Erk and PI3K signaling pathways was explored using selective chemical inhibitor or Western-blot analysis. Left coronary artery ligation was used to establish the model of acute myocardial infarction in rats (Sprague-Dawley male rats, 200-250 g, 2-3 months old), 20 µl of mini-TyrRS, mini-TrpRS, or PBS (vehicle) was injected subcutaneously every 12 h. The rats were randomly divided into four experimental groups: sham operated group; coronary artery ligation (CAL); CAL + mini-TyrRS (20 µl, twice daily, 600 µg kg(-1) day(-1)); and CAL + mini-TrpRS (20 µl, twice daily, 600 µg kg(-1) day(-1)). The experiment was carried out at four time points on the 3rd, 7th, 14th, and 28th day after ligation. To determine whether mini-TyrRS/mini-TrpRS affected the angiogenesis activity of rats with myocardial infarction, we measured the myocardial infarction size by TTC staining, and microvessel density (MVD) was determined by CD34 staining. The results show that proliferation and migration in RCVECs could be promoted by mini-TyrRS at concentrations of 1-100 µg/ml, and inhibited by mini-TrpRS. Phospho-PI3-kinase and Erk expression increased significantly when mini-TyrRS was added, but could be attenuated by mini-TrpRS. Compared to the CAL group, the myocardial infarction size of the mini-TyrRS group at the 3rd, 7th, 14th, and 28th day were decreased, while mini-TrpRS increased, but only in days 14 and 28 was there a significant difference. Except that, the microvessel density of RCVECs was promoted in mini-TyrRS group but inhibited in the mini-TrpRS group. These results indicated that angiogenesis could be either stimulated by mini-TyrRS or inhibited by mini-TrpRS.

Frequent hyperphosphorylation of AS160 in breast cancer.

Enhanced cellular glucose uptake is a frequent characteristic of malignant cells. The Akt substrate of 160 kDa (AS160) is a newly discovered substrate for the protein kinase AKT and phosphorylation of AS160 (p-AS160) was recently recognized to play an important role in glucose transport. However, studies on AS160 in cancer do not yet exist. The aim of the present study was to investigate the p-AS160 level and its correlation with clinicopathological parameters and various biological markers in breast cancer. Results showed that in breast cancer, phosphorylation of AS160 at the key residue T642 was significantly (p < 0.001) higher than that in normal adjacent tissues. P-AS160 staining was positive in 75 of 81 cases (92.6%), including 32 with weak-(score 1), 31 with moderate-(score 2) and 12 with strong immunoreactivity (score 3). P-AS160 was inversely correlated with patient age (p = 0.041) and positively correlated with tumor size (p = 0.013) and the cell proliferation marker Ki-67 (MIB-1) (p < 0.001). This is the first study of AS160 in cancer. Our results show that AS160 phosphorylation level is frequently increased in breast cancer. These results implicate a possible role of AS160 in human breast tumorigenesis and suggest that p-AS160 might be useful as a marker and a potential novel treatment target for breast cancer.

[Effects of hypoxia on the expression of angiotensin-converting enzyme 2 in human umbilical vein endothelial cells].

OBJECTIVE: To study the influence and mechanism of hypoxia on the expression of angiotensin-converting enzyme 2 (ACE2) in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured as adherent cells and identified by immunohistochemical study of Factor VIII. Then the cells were cultured in 37 degrees C incubators under hypoxia condition (1% O2 + 94% N2 + 5%C O2) or regular 5% CO2 condition for 3 h, 6 h, 12 h, 24 h, respectively, and the culture medium and cells were collected at each time point. ACE2 protein was measure by immunohistochemical assay, ACE2 mRNA was quantified by real-time RT-PCR. RESULTS: (1) ACE2 protein expression decreased after hypoxia treatment for 3 h, 6 h, 12 h, and 24 hour compared with regular oxygen condition (chi2 = 825.078, 768.141, 623.931, 350.260, P < 0.00625), the maximum reduction came at 3 h after hypoxia exposure, then raised gradually after 6 h, 12 h, and 24 h exposure; (2) ACE2 mRNA expression show a similar tendency to ACE2 protein. After hypoxia disposal, its relative expression rate reduced to 0.281 at 3 h (P = 0.031), then increased gradually at 6 h (0.445, P = 0.034), 12 h (0.794, P = 0.434), 24 h (0.891, P = 0.692). CONCLUSION: Our study show hypoxia down-regulate the protein and gene expression of ACE2 in HUVECs in 24 hours. The down-regulation effect of hypoxia reached maximum level at 3 h, and then attenuated gradually at 6 h, 12 h and 24 h, which indicate that ACE2 might play an important role in the regulation of RAS in HUVECs after hypoxia injury.

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture.

Aim We studied the role of mini-TyrRS and mini-TrpRS in angiogenesis by using small interfering RNA-mediated mini-TyrRS/mini-TrpRS knockout in hypoxic culture of human umbilical vein endothelial cells. Methods SiRNA was used as the main method to inhibited the gene function. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction and western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and Matrigel-induced capillary tube formation in hypoxic culture. Cell proliferation was determined by crystal violet staining. Results The results showed that levels of the mini-TyrRS/mini-TrpRS gene and protein in mock transfection group and negative control group were higher, but noticeably decreased in experimental group. However, no significant difference was detected between mock transfection group and negative control group, but there was a statistically significant difference compared with experimental group. For mini-TyrRS-siRNA group, the cell migration, tube formation and the rate of cell proliferation were respectively inhibited by (47.4, 56.3, 65.4, 73.7%), (60.5, 69.1, 75.9, 83.6%) and (40.4, 56.2, 61.2, 68.0%). For mini-TrpRS-siRNA, were respectively increased by (18.0, 33.8, 45.1, 56.4%), (18.3, 31.2, 40.3, 45.7%) and (8.4, 26.4, 38.2, 46.6%). Conclusion These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in matrigel models in hypoxic culture, raising the possibility that mini-TyrRS stimulates a common downstream signaling event. Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell angiogenesis in the matrigel assays. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and anti-angiogenic stimuli.

[Analysis of the main components of coptis-evodia herb couple by HPLC-DAD-MS].

A comprehensive HPLC-DAD-MS method was developed to study the chemical components of semi-bionic extract of Coptis-Evodia herb couple. The extract was isolated on a Hypersil BDS C18 column (4.6 mm x 200 mm, 5 microm) using acetonitrile-ammonium formic buffer as mobile phase by gradient elution. Detection was performed on DAD and MS equipped with an electrospray ionization (ESI) source by full scan and product full scan on positive mode. The chromatogram of Coptis-Evodia showed seventeen main peaks, eight of which were from Evodia while the others were from Coptis. By comparison of the retention time, the on-line UV spectra and MS spectra, four peaks were identified as jatrorrhizine, hydroxevodiamine, palmatine and berberine, and three peaks were deduced as epiberberine, columbamine and coptisine. In addition, berberine and palmatine were quantitatively determined. No new component was created in the semi-bionic extract of the herb couple, yet the solubilities of berberine and palmatine decreased.

[AQP1 expression in vascular endothelial cells in vitro under hypoxia conditions].

OBJECTIVE: To investigate the expression of AQP1 in endothelial cells under hypoxia conditions in vitro. METHODS: The cultured human umbilical vein endothelial cells were randomly assigned to hypoxia groups (exposed to 1% of O2 for 3 h, 6 h, 12 h and 24 h respectively), and control group (ordinary culture). The expressions of AQP1 mRNA and protein were detected by real time PCR and immunohistochemistry respectively. RESULTS: The expressions of AQP1 protein and mRNA increased after exposure to hypoxia initially and then decreased afterwards. CONCLUSION: Hypoxia regulates the expression of AQP1 in vascular endothelial cells.