No abstract

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. Thus, a systematic understanding of cellular systems requires methods that allow the activation of more than one specific signaling molecule or pathway within a cell. However, the construction of tools suitable for such multiplexed signal control remains challenging. In this work, we aimed to develop a platform for chemically manipulating multiple signaling molecules/pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). SLIPT is an emerging chemogenetic tool that controls protein localization and cell signaling using synthetic self-localizing ligands (SLs). Focusing on the inner leaflet of the plasma membrane (PM), where there is a hub of intracellular signaling networks, here we present the design and engineering of two new PM-specific SLIPT systems based on an orthogonal eDHFR and SNAP-tag pair. These systems rapidly induce translocation of eDHFR- and SNAP-tag-fusion proteins from the cytoplasm to the PM specifically in a time scale of minutes upon addition of the corresponding SL. We then show that the combined use of the two systems enables chemically inducible, individual translocation of two distinct proteins in the same cell. Finally, by integrating the orthogonal SLIPT systems with fluorescent reporters, we demonstrate simultaneous multiplexed activation and fluorescence imaging of endogenous ERK and Akt activities in a single cell. Collectively, orthogonal PM-specific SLIPT systems provide a powerful new platform for multiplexed chemical signal control in living single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.

The transition temperature of high-density polyethylene (PE), low-density PE, and hydrogenated poly(butadiene), which was detected by the spin-labeled ESR method, was evaluated. The detected transition corresponded to the β transition of semicrystalline polymer PE. The transition temperature was found to depend on the crystallinity, the molecular weight of the sample, and labeled site. The β relaxation around chain-end segments was observed by the selective spin-labeling method. The mobility of the end segment as well as the inner segments was revealed to depend on the molecular weight (M n) of the sample. The motional length scale (cooperative segment length) ξ of amorphous PE at Tg was estimated to be around 2 nm, which was the same order as the reported ξ for various polymers and the Kuhn length of PE.

In order to evaluate computed tomography (CT) in the diagnosis of liver disease, 90 cases of diffuse parenchymal diseases and 37 cases of mass lesions were examined with GE 8800 CT scanner. Abnormal CT findings in liver cirrhosis were characterized by splenomegaly, uneven liver margin and asites. Atrophy of right lobe and enlargement of left lobe could not be easily recognized on CT scan, compared with nuclear imaging. CT values of the liver were decreased and the ratios of CT values of the liver to those of the spleen were less than 0.9 in all cases with fatty liver. Jaundice in acute viral hepatitis can be easily differentiated from obstructive jaundice on CT scan because of observing no dilatation of intrahepatic bile duct. CT was superior in detecting space-occupying lesions to nuclear imaging and was more specific in that it was able to differentiate cystic from solid lesions.However, it was almost impossible to make a histological diagnosis of solid lesions even on CT scan.