Activiteit

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC’s superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.The effect of structured triacylglycerols [1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL), 3-dilinoleoyl-2-palmitoylglycerol (LPL), and 1,3-dioleoyl-2-palmitoylglycerol (OPO)] in human milk on the lipid metabolism was unclear. Hence, this study investigated the effects of different structured triacylglycerols and their mixtures (M) (OPL/LPL/OPO in M1, M2, and M3 were 1.50.51, 1.21.21, and 0.50.21, respectively) on lipid and expression levels of some critical proteins involved in lipid metabolism in LO2 cells. selleck products Results showed that there was more lipid accumulation in the LO2 cells exposed to 2,3-dioleoyl-1-palmitoylglycerol (POO) than OPL, LPL, and OPO (p less then 0.05), and more lipid accumulation was observed in the OPL group compared to LPL and OPO groups (p less then 0.05). Moreover, there was more lipid accumulation in the M3 group compared to M1 and M2 groups. The expression level of diacylglycerol acyltransferase was highest in the POO group compared to LPL, OPO, and OPL groups and was higher in the M3 group than M1 and M2 groups. The expression levels of acetyl-CoA carboxylase 1 and long-chain acyl-CoA synthetase 1 were highest in the OPL group compared to OPO and LPL groups. In comparison to OPO and LPL, OPL seemed to be more likely to increase the content of triacylglycerols and cholesterol in LO2 cells; therefore, whether this was beneficial to the growth and development of infants needs further verification.We demonstrate for the first time a fast aptamer generation method based on the screen-printed electrodynamic microfluidic channel device, where a specific aptamer selectively binds to a target protein on channel walls, following recovery and separation. A malaria protein as a model target, Plasmodium vivax lactate dehydrogenase (PvLDH) was covalently bonded to the conductive polymer layer formed on the carbon channel walls to react with the DNA library in a fluid. Then, the AC electric field was symmetrically applied on the channel walls for inducing the specific binding of the target protein to DNA library molecules. In this case, the partitioning efficiency between PvLDH and DNA library in the channel was attained to be 1.67 × 107 with the background of 5.56 × 10-6, which was confirmed using the quantitative polymerase chain reaction (qPCR). The selectively captured DNAs were isolated from the protein and separated in situ to give five aptamers with different sequences by one round cycle. The dissociation constants (Kd) of the selected aptamers were determined employing both electrochemical impedance spectroscopy (EIS) and the fluorescence method. The sensing performance of each aptamer was evaluated for the PvLDH detection after individual immobilization on the screen-printed array electrodes. The most sensitive aptamer revealed a detection limit of 7.8 ± 0.4 fM. The sensor reliability was evaluated by comparing it with other malaria sensors.Spraying solutions of serine under a wide variety of conditions results in unusually abundant gaseous octamer clusters that exhibit significant homochiral specificity, but the extent to which these clusters exist in solution or are formed by clustering during droplet evaporation has been debated. Electrospray ionization emitters with tip sizes between 210 nm and 9.2 μm were used to constrain the number of serine molecules that droplets initially contain. Protonated octamer was observed for all tip sizes with 10 mM serine solution, but the abundance decreases from 10% of the serine population at the largest tip size to ∼5.6% for the two smallest tip sizes. At 100 μM, the population abundance of the protonated serine octamer decreases from 1% to 0.6% from the largest to the smallest tip size, respectively. At 100 μM, fewer than 10% of the initial droplets should contain even a single analyte molecule with 210 nm emitter tips. These results indicate that the majority of protonated octamer observed in mass spectra under previous conditions is formed by clustering inside the electrospray droplet, but ≤5.6% and ∼0.6% of serine exists as an octamer complex in 10 mM and 100 μM solutions, respectively. These results show that aggregation occurs in large droplets, but this aggregation can be eliminated using emitters with sufficiently small tips. Use of these emitters with small tips is advantageous for clearly distinguishing between species that exist in solution and species formed by clustering inside droplets as solvent evaporation occurs.