Leptin, the product of ob gene, is an endocrine hormone that regulates adipose tissue mass. Recently, leptin has been found to generate a growth signal involving a tyrosine kinase-dependent intracellular pathway and promote angiogenic processes via activation of leptin receptor (Ob-R) in endothelial cells. However, it is not clear how leptin functions to promote multi-step processes involved in the neovascularization at the atherosclerotic plaque. We have examined the expression of matrix metalloproteinases (
In this study, an analytical method for the simultaneous separation and characterization of various molecular species of lysophospholipids (LPLs) and phospholipids (PLs) is introduced by employing nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). Since LPLs and PLs in human plasma are potential biomarkers for cancer, development of a sophisticated analytical method for the simultaneous profiling of these molecules is important. Standard species of LPLs and PLs were examined to establish a separation condition using a capillary LC column followed by MS scans and data-dependent collision-induced dissociation (CID) analysis for structural identification. With nLC-ESI-MS/MS, regioisomers of each category of LPLs were completely separated and identified with characteristic CID spectra. It was applied to the comprehensive profiling of LPLs and PLs from a human blood plasma sample and yielded identifications of 50 LPLs (each regioisomer pair of 6 lysophosphatidylcholines (LPCs), 7 lysophosphatidylethanolamines (LPEs), 9 lysophosphatidic acid (LPAs), 2 lysophosphatidylglycerols (LPGs), and 1 lysophosphatidylserine (LPS)) and 62 PLs (19 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 3 phosphatidylserines (PSs), 16 phosphatidylinositols (PIs), 8 phosphatidylglycerols (PGs), and 5 phosphatidic acids (PAs)).
The efficiencies of four different methods for the extraction of phospholipids (PLs) and lysophospholipids (LPLs) from human plasma samples were examined by comparing extraction recovery values using nanoflow liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS). For recovery measurements, six PL and six LPL standards of different head groups were spiked into a human plasma sample, and the peak areas of each individual species after extraction were measured from the chromatograms of the nLC-ESI-MS runs. Recovery was calculated by comparing the peak area of an extracted standard species with that of the same species' spike after extraction of the same plasma sample. For lipid extraction, four different extraction methods were examined: three based on the Folch method with different organic solvents such as CHCl(3), methyl-tert-butyl ether (MTBE), and MTBE/CH(3)OH, and one relatively fast method involving CH(3)OH only. Evaluations of recovery showed that the modified Folch method with MTBE/CH(3)OH proposed in this study was effective for extracting most PL and LPL standards. Then, the four extraction methods were compared with the identified numbers of plasma PLs and LPLs, of which molecular structures can be confirmed by data-dependent, collision-induced dissociation experiments during nLC-ESI-MS-MS. These results demonstrated that the proposed method yielded the identification of 54 LPLs and 66 PLs from a plasma sample, which was the highest identification rate among the four methods.
Nanoparticles (NPs) tend to adsorb matrix molecules like proteins and lipids incubated with biological fluids, forming a biological corona. While the formation and functions of protein corona have been studied extensively, little attention has been paid to lipid adsorption on NPs. However, lipids are also abundantly present in biological fluids and play important roles in processes like cell signaling and angiogenesis. Therefore, in this study, we established the analytical procedure for study of lipid adsorption on three different types of NPs in two matrices: human serum and heavy cream, using nanoflow liquid chromatography-mass spectrometry (nanoflowLC-MS). Serum was chosen to represent the common environment the NPs would be present once entering human body, and heavy cream was the representative food matrix NPs may be added to improve the color or taste. Steps of liquid-liquid extraction were established and optimized to achieve maximum recovery of the adsorbed, standard lipids from the NPs. Then, the LC-MS/MS method was developed to attain base-line separation of the standard lipids that represent the major lipid classes. At last, the lipid adsorption profiles of the three NPs were compared. We found that the lipid adsorption profile on the same type of NP was significantly different between the two matrices. The established method will help us investigate lipid adsorption on additional NPs and reveal how it could be affected by the physiochemical properties of NPs and the presence of proteins and other components in the biological matrix.
We present a novel method for the fabrication of void-free copper-filled through-glass-vias (TGVs), and their application to the wafer-level radio frequency microelectromechanical systems (RF MEMS) packaging scheme. By using the glass reflow process with a patterned silicon mold, a vertical TGV with smooth sidewall and fine pitch could be achieved. Bottom-up void-free filling of the TGV is successfully demonstrated through the seedless copper electroplating process. In addition, the proposed process allows wafer-level packaging with glass cap encapsulation using the anodic bonding process, since the reflowed glass interposer is only formed in the device area surrounded with silicon substrate. A simple coplanar waveguide (CPW) line was employed as the packaged device to evaluate the electrical characteristics and thermo-mechanical reliability of the proposed packaging structure. The fabricated packaging structure showed a low insertion loss of 0.116 dB and a high return loss of 35.537 dB at 20 GHz, which were measured through the whole electrical path, including the CPW line, TGVs and contact pads. An insertion loss lower than 0.1 dB and a return loss higher than 30 dB could be achieved at frequencies of up to 15 GHz, and the resistance of the single copper via was measured to be 36 mΩ. Furthermore, the thermo-mechanical reliability of the proposed packaging structure was also verified through thermal shock and pressure cooker test.
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