Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry.

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.

A New Approach to Deliver Anti-cancer Nanodrugs with Reduced Off-target Toxicities and Improved Efficiency by Temporarily Blunting the Reticuloendothelial System with Intralipid.

We have developed a new strategy to temporarily blunt the reticuloendothelial system uptake of nanodrugs, a major challenge for nanodrug delivery and causing off-target toxicities, using an FDA approved nutrition supplement, Intralipid. We have tested our methodology in rats using an experimental platinum-containing anti-cancer nanodrug and three FDA approved nanodrugs, Abraxane, Marqibo, and Onivyde, to determine their toxicities in liver, spleen, and kidney, with and without the addition of Intralipid. Our method illustrates its potentials to deliver nanodrugs with an increase in the bioavailability and a decrease in toxicities. Our study shows that Intralipid treatment exhibits no harmful effect on tumor growing and no negative effect on the anti-tumor efficacy of the platinum-containing nanodrug, as well as animal survival rate in a HT-29 xenograft mouse model. Our methodology could also be a valuable complement/supplement to the "stealth" strategies. Our approach is a general one applicable to any approved and in-development nanodrugs without additional modification of the nanodrugs, thus facilitating its translation to clinical settings.

Effect of surface Si redistribution on the alignment of Ge dots grown on pit-patterned Si(001) substrates.

Thermally activated redistribution of Si surface atoms is found to be a crucial factor for the growth of aligned Ge dots on pit-patterned Si(001) substrates. A phenomenon of Si accumulation around the edge of pits significantly alters the substrate surface morphology. As the pit spacing is reduced to below 100 nm, a convex morphology developed between adjacent pits causes a chemical potential distribution that drives the Ge dots into the pits. In addition, the pits of an etching depth greater than 60 nm will evolve into truncated inverted pyramids with sharp base corners that provide deep potential wells for the confinement of Ge dots. Perfectly aligned Ge dots are obtained on pit-patterned Si substrates with this range of pit spacing and etching depth. We also find that the initial geometric shape of the pits does not affect the spatial arrangement of Ge dots.

Thermally induced morphology evolution of pit-patterned Si substrate and its effect on nucleation properties of Ge dots.

We demonstrate the effect of the pre-growth heat treatment process on the nucleation properties of Ge dots grown on pit-patterned Si(001) substrates. The prefabricated 200 nm diameter pits inherently evolve into truncated inverted pyramids (TIPs) with (110) base edges and a 7°-9° sidewall slope during heat treatment; this morphology transformation is robust against variations in shape and orientation of the pit patterns. Uniform Ge dots with an areal density of 4 × 10(9) cm(-2) were obtained on the Si substrates having TIPs. Each TIP contains four aligned Ge dots locating symmetrically with respect to (110). These dots exhibit an elliptical dome shape with major axis oriented along (100). The nucleation position, shape and spatial orientation of these Ge dots coincide with the calculated surface chemical potential distribution of the TIP.

Studying the formation of large cell aggregates in patterned neuronal cultures.

Patterned neuronal cultures could be produced by plating cells dissociated from rat cortices on glass coverslips, the surface of which was printed with poly-L-lysine (PLL)-positive micropatterns. Large cell aggregates, which greatly disrupted the patterned distribution of neurons, were also generated. To investigate how large cell aggregates were formed, dissociated rat cortical neurons were plated on PLL-coated coverslips in a Petri dish, the surface of which was non-adherent to cells. The cell and cell aggregate densities found later on the coverslip surface increased significantly when larger dishes were used. Most of the neurons not attaching to substratum were able to survive for at least 24h without entering apoptosis. During this time they formed floating spherical aggregates in the medium. These aggregates subsequently were able to attach to PLL-coated coverslips and produced large aggregates resembling those found within our patterned neuronal cultures. The results suggest a causative relationship between the generation of large numbers of neurons unattached to substratum and the formation of large cell aggregates on the patterned neuronal cultures. It was further demonstrated here that patterned neuronal cultures free of large cell aggregates could be prepared by a procedure employing both stencil patterning and microcontact printing technologies.