Indoleamine 2,3-dioxygenase (IDO), an immune checkpoint protein, can cause the depletion of tryptophan (Trp) and accumulation of its metabolite of kynurenine (Kyn) in cancer cells, and generates the immunosuppressive microenvironment that supports tumor cell growth. A novel immunoregulatory prodrug micelle based on polyethylene glycol-derivatized an IDO-selective inhibitor of 1-methyltryptophan (1-MT), PEG-Fmoc-1-MT, was developed for inhibiting the IDO activity of the conversion of Trp to Kyn in tumor microenvironments. To investigate the 1-MT distribution and Trp/Kyn ratios in mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment, a HPLC–MS/MS method for simultaneous determination of 1-MT and IDO biomakers of Trp and Kyn in mouse tumors was developed and validated. Triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple reaction monitoring at m/z 219.0?→?160.1, 205.0?→?118.2, 209.0?→?146.1 and 249.3?→?148.3 was used for determination of 1-MT, Trp, Kyn and matrine (internal standard). The method demonstrated good linearity at the concentrations ranging from 10 to 10,000 ng/mL and lower limits of quantitation of 1 ng/mL for 1-MT, Trp and Kyn, respectively. The validated method was successfully applied to 1-MT tumor biodistribution and Trp/Kyn ratio studies in 4T1 tumor bearing mice i.v. with PEG-Fmoc-1-MT prodrug micelles. The mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment exhibited higher 1-MT accumulation and lower Trp/Kyn ratio, in comparison with those of mice with 1-MT solution treatment. The developed PEG-Fmoc-1-MT prodrug micelles could be a promising IDO immunoregulatory prodrug micelles for cancer immunotherapy.
In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture. 相似文献
Fifteen Chinese fly ashes were converted hydrothermally into zeolites, and phosphate immobilization capacity (PIC) of the synthesized zeolites and the corresponding raw fly ashes were determined using an initial phosphate concentration of 1000 mg/L. Results showed that there was a remarkable increase in PIC (from 1.2 to 7.6 times) following the synthesis process. Fractionation of immobilized phosphorus indicated that Fe+Al-P increased most significantly and consistently among all the phosphorus fractions following the conversion of fly ash to zeolite. The PIC and Ca+Mg-P were closely related to Ca content (with r values of 0.9683 and 0.9651, respectively) rather than Mg content (with r values of 0.3920 and 0.3212, respectively). The r values of PIC and Fe+Al-P with Fe content (with r values of 0.4686 and 0.6385, respectively) were higher than those with Al content (with r values of -0.7857 and -0.3770, respectively). Although calcium and iron components were mainly involved in phosphate immobilization, there was no significant change of Ca and Fe content following the conversion of fly ash to zeolite. Increase in dissociated Fe(2)O(3) and specific surface area probably accounted for the enhancement in PIC of synthesized zeolites compared with corresponding fly ashes. The PIC value of zeolites showed a significant correlation with dissociated Fe(2)O(3) (r=0.6186). The specific surface area increased 26.0-89.4 times as a result of the conversion of fly ash to zeolite. The maximum removal of phosphate occurred within different pH ranges for zeolites which were synthesized from high, medium and low calcium fly ashes and this behavior was explained by the reaction of phosphate with calcium and iron components. 相似文献