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Stable isotope-resolved metabolomics (SIRM) pipelines. Redrawn from ref (15). SIRM approach is implemented by administering stable isotope tracers such as uniformly 13C-labeled glucose (13C6-Glc) or uniformly 13C,15N-labeled glutamine (13 C5,15N2-Gln) to cell cultures, excised tissues, animals, and even human subjects. Therapeutic agents can be included in the treatment to observe their impact on the metabolic networks. The tracers are allowed to be metabolized in situ for a period appropriate to the experimental design, followed by cell harvest or tissue resection via surgery. Polar and lipophilic metabolites are extracted and analyzed by NMR and MS for labeling patterns of various metabolites. Illustrated are the 1-D 1H-11-47 HSQC NMR spectra of polar extracts acquired from control and cisplatin-treated human lung adenocarcinoma A549 cells (A) and the high-resolution exact mass FT-ICR-MS spectra of lipid extracts obtained from control and selenite-treated A549 cells grown in 13C6-Glc tracer (B). M+3: represents excess of three 13C neutron masses, which corresponds to 13C3-glycerol backbone; PI: phosphatidylinositol; curved dashed arrows: cleavage of fatty acyl chains by tandem MS to confirm C18:0 (stearate) and C20:4 (arachidonate) acyl chain composition. Peaks with exact mass higher than M+3 in the insets are derived from 13C-labeled fatty acyl chains. They are greatly reduced under selenite treatment.
Fate of 13C and 15N from fully labeled glutamine through the TCA cycle and ACL-ME pathway. Redrawn from ref (25). Red: 13C fate from glutamine in the first forward TCA turn; Light green: 15N; light blue: 13C derived from the glutamine tracer via the ATP-citrate lyase (ACL)-malic enzyme (ME) pathway in the cytoplasm; orange: 13C labeling of citrate+5 from reductive carboxylation (RedC); Green: 13C labeling of TCA cycle intermediates catalyzed by pyruvate carboxylase (PC); double headed arrows: reversible reactions; dashed arrows: multiple step reactions; not all possible 13C isotopologues of the cycle intermediates are shown; the 13C isotopologues of malate, fumarate, and succinate depicted include those from the reverse direction of OAA to succinate.
This work was supported in part by National Science Foundation EPSCoR grants# EPS-0447479 (TWMF) and EPS-0132295 (R.J. Wittebort), NIH NCRR 5P20RR018733 (D.M. Miller, ANL), 1R01CA118434-01A2, 1RO1CA101199-01, R01ES022191-01 and 3R01CA118434-02S1 (TWMF), R21CA133668-02, and P01CA163223-01A1 (ANL) from the National Cancer Institute, the University of Louisville CTSPGP/ARRA grants 20044, the Kentucky Lung Cancer Research Program (OGMB090354B1 and OGMB101380) (TWMF and ANL), the Robert W. Rounsavall Jr. Family Foundation, the Kentucky Challenge for Excellence. We thank Jin Lian Tan, Alex Belshoff, Katherine Sellers, and Radhika Burra for technical assistance and/or comments on the manuscript.
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