An electrochemical method to detect gamma glutamyl transpeptidase.

Gamma glutamyl transpeptidase (GGT) is a transferase, which is of great importance in sustaining intracellular cysteine and glutathione levels. The abnormal expression of GGT is significantly associated with features of many metabolic syndromes (e.g., hepatocellular carcinoma). Therefore, it is essential to develop methods to detect GGT so as to monitor the physiological or pathological phenomena related to this species. In this work, by making use of a complex formed by Cu(2+) and glutathione, which may exhibit excellent voltammetric response, we have proposed a novel potential electrochemical method for the detection of the enzyme. Results show that in the presence of GGT, the formation of Cu(2+)-glutathione complex on a working electrode will be disrupted, resulting in greatly depressed electrochemical signals. The primary method exhibits some advantages, such as it being fast, cost-efficient, and conveniently operated. It also has the potential to be further developed as an effective method in the quantitative detection of GGT in real samples.

Comparative Transcriptomics and Metabolites Analysis of Two Closely Related Euphorbia Species Reveal Environmental Adaptation Mechanism and Active Ingredients Difference.

Roots of Euphorbia fischeriana and Euphorbia ebracteolata are recorded as the source plant of traditional Chinese medicine "Langdu," containing active ingredients with anticancer and anti-AIDS activity. However, the two species have specific patterns in the graphic distribution. Compared with E. ehracteolata, E. fischeriana distributes in higher latitude and lower temperature areas and might have experienced cold stress adaptation. To reveal the molecular mechanism of environmental adaptation, RNA-seq was performed toward the roots, stems, and leaves of E. fischeriana and E. ehracteolata. A total of 6,830 pairs of putative orthologs between the two species were identified. Estimations of non-synonymous or synonymous substitution rate ratios for these orthologs indicated that 533 of the pairs may be under positive selection (Ka/Ks > 0.5). Functional enrichment analysis revealed that significant proportions of the orthologs were in the TCA cycle, fructose and mannose metabolism, starch and sucrose metabolism, fatty acid biosynthesis, and terpenoid biosynthesis providing insights into how the two closely related Euphorbia species adapted differentially to extreme environments. Consistent with the transcriptome, a higher content of soluble sugars and proline was obtained in E. fischeriana, reflecting the adaptation of plants to different environments. Additionally, 5 primary or secondary metabolites were screened as the biomarkers to distinguish the two species. Determination of 4 diterpenoids was established and performed, showing jolkinolide B as a representative component in E. fischeriana, whereas ingenol endemic to E. ebracteolate. To better study population genetics, EST-SSR markers were generated and tested in 9 species of Euphorbia. A total of 33 of the 68 pairs were screened out for producing clear fragments in at least four species, which will furthermore facilitate the studies on the genetic improvement and phylogenetics of this rapidly adapting taxon. In this study, transcriptome and metabolome analyses revealed the evolution of genes related to cold stress tolerance, biosynthesis of TCA cycle, soluble sugars, fatty acids, and amino acids, consistent with the molecular strategy that genotypes adapting to environment. The key active ingredients of the two species were quantitatively analyzed to reveal the difference in pharmacodynamic substance basis and molecular mechanism, providing insights into rational crude drug use.

Radiation field feature of 188Re esophageal stent.

OBJECTIVE: To evaluate the radiation field feature of Re esophageal stent and provide scientific basis for clinical application. METHODS: We measure the beta-ray, gamma-ray and bremssfrahlung dose of every selected point on the bionics esophageal stent and then draw out computer software by mathematical formula. RESULTS: The radiation field of Re esophageal stent has its own feature: the max range of beta-ray is 11 mm, 90% dose construction field is within 1.5 mm, 95% dose range is within 2.5 mm, and only 4.21% of total energy of gamma-ray and bremssfrahlung is out of 6.5 mm range. The absorption dose of every direction in same point of the esophageal model was similar (P>0.05). CONCLUSION: Beta ray is the major radiation of Re esophageal stent while gamma-ray and bremssfrahlung are 4.21% among the radiation field. The max dose construction field is within 0.5-1.5 mm, just short at the depth of esophagus mucosa within 0.5-1.5 mm range. So Re stent is a good choice of palliative intracavitary radiotherapy of esophageal carcinoma.

[ESAT-6 inhibits autophagy in macrophages and promotes the growth of BCG].

Objective To explore the interaction between 6 kD early secretory antigenic target (ESAT6) and autophagy in order to provide an experimental basis for the immune evasion mediated by ESAT6. Methods RAW264.7 cell lines (mouse macrophages) were treated with Earle's balanced salt solution (EBSS), and another cells were transfected by control plasmid pCMV-HA or recombinant plasmid pCMV-HA-ESAT6. Then we observed the growth of Bacillus Calmette-Guerin (BCG) in macrophages. Western blotting was used to detect the LC3 levels in RAW264.7 cells at 0, 8, 12, 32 hours after transducted by pCMV-HA-ESAT6. In RAW264.7 cells transfected with PCMV-HA, PCMV-HA-ESAT6, and treated with chloroquine (CQ) and CQ combined with pCMV-HA-ESAT6, which were lysed and cultured in Lowenstein-Jensen culture medium for BCG counting, LC3 was detected by Western blot analysis, and the number and size of lysosomes were observed by LysoTracker Red staining. Results Compared with control plasmid pCMV-HA transfected RAW264.7 cells, the number of BCG significantly increased in PCMV-HA-ESAT6-transfected cells, while decreased in EBSS-treated cells. PCMV-HA-ESAT6 transfection resulted in the increased transition of LC3 I to LC3 II in a time-depended manner. Compared with the controls, LysoTracker Red staining showed PCMV-HA-ESAT6, CQ and CQ plus PCMV-HA-ESAT6 transfections resulted in the increased number and size of lysosomes, and there were no differences among the three groups. Moreover, the growth potential of BCG was strong in the three transfection groups. Conclusion ESAT6 can inhibit the autophagy and promote the growth of BCG in RAW264.7 cells.

[SapM-induced fusion blocking of autophagosome-lysosome is depended on interaction with Rab7].

Objective To study the role of Rab7 in the blockage of autophagosome-lysosome fusion induced by secretory acid phosphatase (SapM), a virulence factor of mycobacterium tuberculosis. Methods The Raw264.7 cells were transfected with siRab7, and the P62 was detected using Western blotting. After transfected with mCherry-SapM, the co-localization of SapM and Rab7 in Raw264.7 cells was detected by immunofluorescence cytochemistry and the interaction of SapM with Rab7 was determined by co-immunoprecipitation. SapM mutants including SapM(δ ARCA), SapM(δ FRED) and SapM(δ CT) were used to transfect Raw264.7 cells, and their associations with Rab7 were analyzed. Results The treatment of siRab7 induced a significant increase of P62 in these cells. Immunofluorescence cytochemistry showed the intracellular co-localization of SapM and Rab7. Co-immunoprecipitation showed that SapM and Rab7 were precipitated by each other. Only SapM(δ CT) failed to interact with Rab7 among the three SapM mutants. Conclusion The inhibition of autophagosome-lysosome fusion induced by SapM is dependent on the interaction between SapM and Rab7.

Sequential transcatheter arterial chemoembolization, three-dimensional conformal radiotherapy, and high-intensity focused ultrasound treatment for unresectable hepatocellular carcinoma patients.

The purpose of this study was to evaluate the outcome of patients with unresectable hepatocellular carcinoma (HCC) treated by sequential therapy of transcatheter arterial chemoembolization (TACE), three-dimensional conformal radiotherapy (3-DCRT) and high-intensity focused ultrasound (HIFU). From October, 2005 to September, 2010, 120 patients with unresectable HCC received the sequential treatments of several courses of TACE followed in 2-4 weeks by 3-DCRT and then a single session of HIFU with a curative intent. The median tumor irradiation dose was 40 Gy. Tumor response, toxicity and overall survival rate were analyzed. Clinicopathologic factors affecting the primary technique effectiveness and overall survival rates were investigated by univariate analysis or multivariate analysis. All 120 HCC patients were followed up by the last follow-up time. Among these patients, hepatic toxicities due to treatment were notable in 9 cases. Gastrointestinal bleeding after the overall treatment occurred in 2 cases, leukopenia of grade III was detected in 1 case, radiation-induced liver disease (RILD) was observed in 2 patients, and first- and second-degree skin burn around the HIFU treatment zone were observed in 2 patients and 1 patient, respectively. Among 120 patients, 23, 83 and 14 cases achieved partial response, stable disease and progressive disease, respectively. The overall survival rates at 1 year, 3 years and 5 years were 70%, 35% and 15%, respectively, with a median survival time of 26 months. Both Child-Pugh liver function grading and radiation dose were determined to be independent predictors for overall survival revealed by the multivariate analysis. It is concluded that the sequential therapy of TACE, 3-DCRT and HIFU is a promising therapeutic regimen for unresectable HCC.