Monitoring diacylglycerols in biofluids by non-isotopically paired charge derivatization combined with LC-MS/MS.

Diacylglycerols (DAGs) are important lipid mediators in cellular signaling transduction and metabolism. Imbalanced production or consumption of DAGs has a negative impact on the physiological functions of the body. However, comprehensive monitoring of structurally diverse DAGs remains a daunting task due to the rapid metabolism and ion suppression characteristics in biofluids. These bottlenecks call for developing a method that enables sensitive quantification of DAGs in biological sample. In this work, a straightforward charge derivatization strategy was developed to insert a series of structure analogs charge label, i.e., N, N-dimethylglycine (DMG) and N, N-dimethylalanine (DMA), on the free hydroxyl group of the DAGs. Owing to the existence of tertiary amino groups in charge label, the mass spectrometry ionization response of the derivatized DAGs was significantly increased in comparison with traditional metal ion adducts. After charge derivatization, the specific neutral loss diagnostic ions (DMG, 103 Da and DMA, 117 Da) were captured by mass spectrometry. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the characteristic diagnostic ions of the derivatized DAGs have been developed as sensitive methods for the detection (detection limit = 16 aM) and quantification (quantification limit = 62.5 aM) of DAGs in serum. Moreover, the tagged 1,2-DAGs and 1,3-DAGs sn-isomers have been well separated on the reversed-phase column in combination with ultra-performance liquid chromatography. Finally, metabolic characterizations of the tagged DAGs were further explored in L-Arginine-induced acute pancreatitis mice and resveratrol treated model mice. The results indicated that 1,2-DAGs were increased in the serum of model mice relative to normal controls and resveratrol significantly altered this metabolic abnormality. The currently established DMG/DMA-oriented paired charge derivatization strategy is promising for depicting DAGs changes more accurately in metabolic studies of lipid-related diseases and accurately evaluating drug treatment strategies.

Experimental Study on Danggui Shaoyao San Improving Renal Fibrosis by Promoting Autophagy.

Renal fibrosis could lead to chronic kidney disease (CKD) developing into the end-stage with its pathological manifestation is the deposition of extracellular matrix (ECM). Danggui Shaoyao San (DSS) is one of the widely used herbal formulas in ancient China, which has been proven to have efficacy in the treatment of CKD. The experiment employed TGF-ß1 to stimulate the NRK-52E cells to establish a renal fibrosis model. With rapamycin (RAPA) used as the positive control, we detected the expression of fibronectin (FN), caspase-3, and autophagy-related proteins in the NRK-52E cells treated with DSS by Western blot and immunofluorescence assay. In order to further verify autophagy-promoting effects of DSS, we adopted 3-MA to inhibit autophagy. The experiment has found that DSS can lower the protein levels of FN and caspase-3 in the NRK-52E cells induced by TGF-ß1. After TGF-ß1 stimulation, the expression of LC3 II/I and Beclin 1 has decreased, and the protein levels of mTOR and p62 have increased. Consistent with rapamycin, DSS has significantly reduced these effects of TGF-ß1. It has also been found that DSS can increase the expression of LC3 II/I and Beclin 1 proteins and can reduce the level of mTOR in cells treated with 3-MA, suggesting that DSS can promote autophagy. In conclusion, DSS has been proved to reduce the apoptosis and fibrosis of NRK-52E cells induced by TGF-ß1, which may be achieved by promoting autophagy.

The changes in the levels of IL-6, IL-17, and IL-21 in the acute stage of childhood asthma.

BACKGROUND: Asthma is the most common disease in children, and its pathogenesis is highly complicated. METHODS: CD4+ cells and CD4+ IL-17+ cells were analysed by flow cytometry, and the ratio of CD4+ cells to the peripheral blood mononuclear cells (PBMCs) and CD4+ IL-17+ cells to PMBCs were calculated from the control group, acute group, and moderate group. The levels of protein expression of IL-6, IL-17, and IL-21 were detected by ELISA in the plasma and culture supernatants of PBMCs of the three groups. The correlations between IL-7 and IL-6, IL-7 and IL-21 were analysed in culture supernatants of PBMCs of the three groups. RESULTS: The mean ratio of CD4+ IL-17+ cells/peripheral blood mononuclear cells (PBMCs) was 4.32% in the acute group, which was higher than in the control group and moderate group. The levels of IL-6 and IL-17 were elevated, and the levels of IL-21 were decreased in the acute group. The levels of IL-17 and IL-6 were positively correlated (r = 0.182, p = 0.03), and the levels of IL-17 and IL-21 were negatively correlated (r = -0.834, p = 0.02). CONCLUSIONS: The results suggest that the levels of IL-17 and IL-6 are related to the severity of asthma. Furthermore, IL-17 may play a role in the development of childhood asthma.

ALK-positive extramedullary plasmacytoma with expression of the CLTC-ALK fusion transcript.

Anaplastic lymphoma kinase (ALK)-positive non-Hodgkin lymphoma (NHL) was long thought to be a disease occurring uniquely in T or null-cell lymphomas. More recently, however, a small number of B-lineage lymphomas have been reported to express ALK fusion genes. These tumors often exhibit a plasmablastic morphology, a finding which prompted our interest in looking for ALK fusions in plasma cell neoplasms. We studied 46 cases of extramedullary plasmacytoma by immunostaining with anti-ALK antibody and fluorescence in situ hybridization (FISH) analysis using an ALK break-apart probe and found one case to be ALK protein-positive and demonstrated the disruption of the ALK gene in this case. Immunohistochemistry showed that the tumor cells were strongly positive for CD138, VS38c, and epithelial membrane antigen, but lacked expression of CD20, CD79a, CD45, and CD30. Both RT-PCR and genomic DNA-PCR confirmed the CLTC-ALK fusion. This finding expands the lists of the ALK-positive tumors, and ALK-positive extramedullary plasmacytoma may benefit from the treatment of ALK inhibitor in the future.

2,5-Dibromo-terephthalic acid dihydrate.

The asymmetric unit of the title compound, C(8)H(4)Br(2)O(4)·2H(2)O, contains one half-mol-ecule of 2,5-dibromo-terephthalic acid (DBTA) and one water mol-ecule. The DBTA mol-ecule is centrosymmetric. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the mol-ecules, forming a three-dimensional framework.