DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells.

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R2 = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-13C2, 15N, and Cys-15N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

Curcumin suppresses epithelial-to-mesenchymal transition of peritoneal mesothelial cells (HMrSV5) through regulation of transforming growth factor-activated kinase 1 (TAK1).

OBJECTIVE: Peritoneal fibrosis remains a serious complication of long-term peritoneal dialysis (PD) leading to peritoneal membrane ultrafiltration failure. Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Curcumin has been previously shown to inhibit EMT of renal tubular epithelial cells and prevent renal fibrosis. There are only limited reports on inhibition of PMCs-EMT by curcumin. This study aimed to investigate the effect of curcumin on the regulation of EMT and related pathway in PMCs treated with glucose-based PD. METHODS: EMT of human peritoneal mesothelial cells (HMrSV5) was induced with glucose-based peritoneal dialysis solutions (PDS). Cells were divided into a control group, PDS group, and PDS group receiving varied concentrations of curcumin. Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability, and a transwell migration assay was used to verify the capacity of curcumin to inhibit EMT in HMrSV5 cells. Real-time quantitative PCR and western blot were used to detect the expression of genes and proteins associated with the EMT. RESULTS: High glucose PDS decreased cell viability and increased migratory capacity. Curcumin reversed growth inhibition and migration capability of human peritoneal mesothelial cells (HPMCs). In HMrSV5 cells, high glucose PDS also decreased expression of epithelial markers, and increased expression of mesenchymal markers, a characteristic of EMT. Real-time RT-PCR and western blot revealed that, compared to the 4.25% Dianeal treated cells, curcumin treatment resulted in increased expression of E-cadherin (epithelial marker), and decreased expression of α-SMA (mesenchymal markers) (P < 0.05). Furthermore, curcumin reduced mRNA expression of two extracellular matrix protein, collagen I and fibronectin. Curcumin also reduced TGF-ß1 mRNA and supernatant TGF-ß1 protein content in the PDS-treated HMrSV5 cells (P < 0.05). Furthermore, it significantly reduced protein expression of p-TAK1, p-JNK and p-p38 in PDS-treated HMrSV5 cells. CONCLUSIONS: Our results demonstrate that curcumin showed an obvious protective effect on PDS-induced EMT of HMrSV5 cells and suggest implication of the TAK1, p38 and JNK pathway in mediating the effects of curcumin in EMT of MCs.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Validity and reliability of the Uighur version of Chinese Soldier Personality Questionnaire / 中国心理卫生杂志

Objective:To test the validity and reliability of the Uighur version of Chinese Soldier Personality Questionnaire (CSPQ) in Uygur ethnic group of recruited youtt.Methods:Using the two-way checklist,all the items were judged by professors as their items belonging to test the content validity.Totally 101 Uygur population of permanent residents and totally 102 patients with schizophrenia in remission in sample 1 were tested for discrimination validity analysis.Totally 460 Uygur youths were recruited to complete the Uygur form of CSPQ for subscale normal distribution analysis and reliability analysis in sample 2.Totally 118 students of Urumqi College of Land Army from sample 3 were selected and retested for test-retest reliability with three weeks interval.Results:Uygur form of CSPQ had 283 items and 8 dimensions.Classification and recognition rate judged by professors ranged from 74.6％ to 91.5％.Patients with schizophrenia scored higher than normal people in all scales.Reliability coefficients of the 8 dimensions ranged from 0.69 to 0.91,and the test-retest reliability ranged from 0.85 to 0.92.Conclusion:It suggests that Uighur version of Chinese Soldier Personality Questionnaire is of good validity and reliability.

Proteomic characteristics of circulating microparticles in patients with newly-diagnosed type 2 diabetes.

OBJECTIVE: This study aimed to evaluate the proteomic characteristics of plasma microparticles (MPs) from patients with newly diagnosed type 2 diabetes (T2DM). METHODS: The subjects comprised eight male T2DM patients recruited between December 2013 and March 2014, as well as eight age and sex-matched healthy controls enrolled during the same period. Plasma microparticles (MPs) were extracted from the blood of each subject, and subjected to proteomics analysis using label-free methods. Bioinformatic analyses were performed using specialized software. RESULTS: 3,148 unique peptides and 496 proteins were identified, among these, 46 proteins were differentially expressed between the two groups. Among these 46 candidates, 20 proteins had higher expression in T2DM group compared with the control group, whereas 3 proteins displayed lower expression. There were 17 proteins only detected in T2DM group, and 6 proteins only detected in the control group. Gene ontology (GO) analysis revealed significant differences between the two groups in some functional nodes, including neutrophil accumulation, chemokine production, platelet activation, and blood coagulation. Pathway analysis showed that proteins involved in platelet activation, cell adhesion, focal adhesion, and extracellular matrix-receptor interaction were differentially expressed between the 2 groups. Network analysis indicated that ubiquitin was the protein with the highest degree of connectivity. CONCLUSIONS: Blood MPs from T2DM patients are enriched in proteins involved in platelet activation, cell adhesion, and inflammation. Therefore, MPs in T2DM patients might be associated with hypercoagulable state in diabetic patients and the development of diabetic complications.

[In vitro Metabolism of Volatile Methyl Siloxanes].

Volatile methyl siloxanes (VMSs) are of great concern in the past few years due to their high production volume, ubiquitousness in the environment and toxicities. The bioaccumulation of VMSs is reported to be sensitive to their metabolism rates, however, little is known about their metabolic rates in aquatic organisms. The present study measured intrinsic clearance rates of 16 common VMSs (D3-D6 and L3-L14) incubated in liver microsomes of weever and quail. The intrinsic clearance rates of VMSs in weever microsomes were 0-0.031 mL·(h·mg)-1. The fact that D5 exhibited no significant decline trend in the incubations with liver microsomes of weever corresponded with its trophic magnification behaviour in aquatic food webs. L4-L14, similar to D5, were also persistent in microsomes, suggesting the high bioaccumulation potentials of the chemicals. The intrinsic clearance rates of VMSs incubated in quail microsomes [0.25-1.7 mL·(h·mg)-1] were significantly higher than those incubated in weever microsomes. The relatively high biotransformation rates of D3-D6 and L3-L14 suggested that these compounds may not exhibit high biomagnification potentials in birds. B[a]P was used as a benchmark chemical to normalize variations in determining the metabolism rates among batches, and the structure-activity relationships were explored for the normalized intrinsic clearance rates of quail microsomes. Hydrophobicity and electric properties were found to be major factors influencing the bird microsomal intrinsic clearance rates of VMSs.

[Cloning and expression analysis of GGPPS gene from Panax notoginseng].

According to the transcriptome dataset of Panax notoginseng, the key geranylgeranyl pyrophosphate synthase gene (GGPPS) in terpenoid backbone biosynthesis was selected to be cloned. Using specific primer pairs combining with RACE (rapid amplification of cDNA ends) technique, the full-length cDNA sequence with 1 203 bp, which containing a 1 035 bp open reading frame, was cloned and named as PnGGPPS. The corresponding full-length DNA sequence contained 2 370 bp, consisted of 1 intron and 2 exons. The deduced protein PnGGPPS contained 344 amino acids and shared more than 73% identity with GGPPS from Ricinus communis and Salvia miltiorrhiza. PnGGPPS also had specific Aspartic acid enrichment regions and other conserved domains, which belonged to the Isoprenoid-Biosyn-C1 superfamily. The quantitative real-time PCR showed that PnGGPPS expressed in different tissues of 1, 2, 3 years old root, stem, leaf and 3 years old flower, and the expression level in 3 years old leaf was significant higher than that in other organs, which suggested that it might not only be involved in the regulation of the growth and development, but also be associated with the biosynthesis of chlorophyll and carotenoids, the development of chloroplast, the shade habit and the quality formation of P. notoginseng.

[Cloning and functional characterization of pathogenesis-related PR10-1 gene in Panax notoginseng].

With homology cloning approaches coupling with RACE (rapid-amplification of cDNA ends) techniques, the full-length coding sequence of pathogenesis-related protein PR10-1 with differential expression was cloned from the total RNA of the root of Panax notoginseng, and its function was explored furtherly. As a result, the longest 465 bp ORF (named as PnPR10-1 with the Accession No. KJ741402 in GenBank) was detected from the cloned sequence with full-length of cDNA of 863 bp. The corresponding peptide encoded consisted of 155 amino acids, contained some domains such as Bet-v-I, and showed high similarity with that from Panax ginseng by analysis of phylogenetic trees created from the alignments. Real-time quantitative PCR showed that the expression of PnPR10-1 gene was constitutive in different tissues of 1-3 year old plant, suggesting that it might be involved in growth, development, and secondary metabolism; yet it was up-regulated significantly with the infection of Fusarium oxysporum in root, suggesting that it might be involved in defense against many diseases including root rot in P. notoginseng.

Quantitative evaluation in the stage of chronic nephrosis by shear-wave elasticity technology / 中华超声影像学杂志

Objective To investigate the diagnostic value of the shear-wave elasticity (SWE) imaging technology on the quantitative diagnosis of chronic nephrosis stage.Methods Sixty patients with nephrosis (nephrosis group) were evaluated with SWE and the renal function test.The Young's modulus value and the renal function were measured,and the results were compared with those of twenty healthy subjects (control group).Results Twenty cases of healthy control group were definited as R0.Sixty patients of nephrosis group were divided into four groups according to renal function:R1-R4.The Young's modulus of the nephrosis group was significantly higher than the control group (P ＜0.01).There were also statistically significant differences among each stage of the nephrosis group (except R1 and R2 of nephrosis group)(P ＜ 0.01).According the ROC curve,the cut-off value of the Young's modulus was 5.53 kPa when maximum area under the curve equal to 0.886,the sensitivity and specificity were 81.70％ and 80.40％.The Young's modulus value and renal function were positively correlated with the stage of nephrosis.The areas under the ROC curves for the Young's modulus,urea nitrogen and csytatin C were 0.965,0.950,0.965 for ≥R3,0.978,0.912,0.961 for =R4,respectively.Conclusions SWE imaging technology provided a new quantitative index for the stage of nephrosis through quantizing the elasticity of the tissue.

Evaluation of shear wave elastography in diffuse thyroid disease / 中华超声影像学杂志

Objective To explore the value of shear wave elastography (SWE) in diffuse thyroid disease.Methods The elastic modulus were detected by SWE in 41cases of diffuse thyroid disease [including 16 cases of Graves' disease (GD),16 cases of Hashimoto' s thyroiditis (HT) and 9 cases of subacute thyroiditis(SAT)] and 30 cases of healthy volunteers.The elastic modulus,including Emean,Emin and Emax,were measured and compared.Results Compared with the normal group[Emean(15.7-± 2.5)kPa,Emin(11.6 ± 2.4)kPa and Emax (20.2 ± 3.0)kPa],the Emean[(20.4 ± 4.7)kPa],Emin[(14.4-± 3.8)kPa] and Emax [(27.8 ± 7.3)kPa] of GD,the Emean [(18.4-± 5.0)kPa] and Emax [(25.2 ± 5.8)kPa] of HT,and the Emean[(11.0 ± 2.9)kPa] and Emin [(6.0 ± 2.7)kPa] of the SAT were different significantly(P =0.001,0.007,0.001 ; P =0.045,0.001 ; P =0.000,0.000).There were significant differences between the SAT and the other two groups,namely GD and HT (P ＜0.05).Such differences,however,were not found between GD and HT (P ＞0.05).Conclusions SWE can be used to measure the elastic modulus of the thyroid tissue quantitatively and objectively,serving as a useful technique to predict the diffuse thyroid disease.

[Cloning and expression of ORF146 gene of Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII)].

OBJECTIVE: To study the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) ORF146 gene. METHODS: The primers were designed according to the sequence of SpltMNPVII genome. The promoter of ORF146 was amplified by PCR. The promoter activities and the time course of mRNA transcription were analyzed. The fragment of the ORF146 gene was then cloned into the vector of pET28a(+) and expressed. The polyclonal antibody was prepared by using the purified fusion protein. Titration determination of anti-ORF146 antibody was evaluated by ELISA. RESULT: Nucleotide sequence analysis demonstrated that this gene has a 1383 bp ORF, encoding 460 amino acids with a predicted molecular weight of 50.4 kDa. Analysis of both promoter activities and the time course of mRNA transcription of the ORF146 gene showed that ORF146 was transcripted in early stage as well as in late stage. The transcription began at 2 h post infection (hpi) and reached two peaks at 8 and 18 hpi and then the transcription level was slightly decreased from 24 hpi. pET-28a-ORF146 fusion protein expressed in prokaryotic and purified polyclonal antibody was with good specificity, with the titer above 1:3200. CONCLUSION: SpltMNPV II ORF146 gene is a composition structure protein, which was expressed at both early and late stage. ORF146 might be involved in viral DNA replication. The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.

[Studies on speaker recognition based on complexity].

OBJECTIVE: To analyze the speech characters with computation complexity. METHOD: Voices of 51 testees spoke the same paragraph were recorded and the same sentence of voice waveform was intercepted as source data. There were two kinds of sample voices: same testee speaking the same sentence at different time and different testee speaking the same sentence. RESULT: The computation complexity curves of the different testee were obviously distinguishing, while those of the same testee were almost the same. In a 2D embedded space the computation complexity features of individual testee differs with others even if testees speak the same sentences. CONCLUSION: This complexity features might be applied to speaker recognition system and using complexity method to analyze speech signals has wide application prospect.