Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

Narrowband Light Total Antireflection and Absorption in Metal Film–Array Structures by Plasmonic Near-Field Coupling

We study the cooperative effects between plasmon gap modes and optical cavity modes of a novel triple-layer structure consisting of double continuous gold films separated by a gold nanosphere array. Narrowband near-perfect antireflection of optical field is achieved for the first time due to the strong near-field light–matter interaction within the deep sub-wavelength gaps between adjacent nanospheres combined with the spatial field confinement effects of the optical cavity built by the double gold films. The coexistence cooperation of near-field dipole plasmon resonances and spatial optical field confinement presents more efficient light modification than that of the individual subsystem and may open a new approach to manage light flow. By varying the period of nanosphere array, the diameter of nanospheres, and the distance between the array and the film, optical behaviors of the proposed structure can be tuned in a wide range. High environmental sensitivity and large figure of merit factor are obtained using this structure as the detecting substrate. Furthermore, ultra-compact structure and high conduction suggest the proposed structure being a good candidate for potential applications in highly integrated optoelectronic devices, such as plasmonic filters and sensors.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Acacetin antagonized lipotoxicity in pancreatic β-cells via ameliorating oxidative stress and endoplasmic reticulum stress

Molecular Biology Reports - During the pathogenesis and progression of diabetes, lipotoxicity is a major threat to the function and survival of pancreatic β-cells. To battle against the...     

Characteristics and function of a low-molecular-weight compound with reductive activity from Phanerochaete chrysosporium in lignin biodegradation

A novel reductive compound with molecular weight of about 1000 Da, named Pc reducer, was purified from the liquid culture of a white-rot basidiomycete Phanerochaete chrysosporium. It was likely to have an alkene-ester structure according to Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectra. Pc reducer reduced the hydroxyl radical HO and the stable nitroxide radical under certain conditions. It inhibited the repolymerization of the products from the oxidation of phenolic lignin-model compounds by reducing certain intermediate radicals. The activity of manganese peroxidases was promoted by Pc reducer at certain concentrations. Pc reducer could also weaken the repolymerization of fragments from the oxidation of Na-lignosulfonate by lignin peroxidases and manganese peroxidases. It has potential ability to improve the ligninolytic efficiency of peroxidases in P. chrysosporium.