Fluorescent visualization and evaluation of NPC1L1- mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1L1)is a major membrane protein responsible for cholesterol absorption,in which the physiological role of vesicular endocytosis is still controversial,and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo.Here,we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model,and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption.The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets.The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine,and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol.Of note,the vesicles colocalize with the early endosomal marker early endosome antigen 1(EEA1)and the filipin-stained free cholesterol.Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles.Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process.NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.

Hot Rolled Strip Re-reddening Temperature Changing Law during Ultra-fast Cooling

Temperature deviation between surface and the center of hot rolled strip is formed during ultra-fast cooling （UFC）. Surface temperature would rise when temperature deviation goes up to an extent, and strip re-reddening phenomenon will appear. Strip re-reddening affects the stability of strip microstructure, property and temperature control precision. Thus, it is necessary to conduct research on re-reddening temperature changing law to improve strip property and temperature control precision. Strip temperature trends for various strip thicknesses and ultra-fast cooling rates were obtained by numerical calculation method. Re-reddening temperature, temperature deviation between surface and center, and boundary layer position changing law were obtained. By comparison, some conclusions were obtained： UFC re-reddening temperature and laminar cooling （LC） re-reddening temperature were linear to ultra-fast cooling rate respectively. Ultra-fast cooling rate affected UFC re-reddening temperature greatly, but it had little effect on LC re-reddening temperature. Equations which were used to calculate UFC re-reddening temperature, LC re-reddening temperature and maximum temperature deviation were obtained. The position of boundary layer stayed in 1/4 strip thickness.