Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.     

Phenotypic effects of the nurse Thylacospermum caespitosum on dependent plant species along regional climate stress gradients

Contrasting phenotypes of alpine cushion species have been recurrently described in several mountain ranges along small‐scale topography gradients, with tight competitive phenotypes in stressful convex topography and loose facilitative phenotypes in sheltered concave topography. The consistency of phenotypic effects along large‐scale climate stress gradients have been proposed as a test of the likely genetic bases of the differences observed at small‐scale. Inversely, plastic phenotypic effects are more likely to vanish at some points along climate stress gradients. We tested this hypothesis for two phenotypes of the alpine cushion species Thylacospermum caespitosum at four points along regional gradients of cold and drought stress in northwest China. We measured the traits of the two cushion phenotypes and quantified their associated plant communities and environmental variables along the regional temperature and aridity gradients. Cushion height, convexity and stem density overall showed significant effect of phenotypes. Difference in tightness of cushions between phenotypes was consistent across climate conditions, whereas differences in cushion convexity and height between phenotypes increased with increasing cold stress. Phenotypic effects on species richness and abundance were consistent along both climate gradients but not effects on species composition, while there were no phenotypic effects on environmental variables. Additionally, RII (relative interaction index) curves were linear along the drought gradient but unimodal along the temperature gradient, likely due to the occurrence of contrasting species pools at the different sites. We conclude that the consistency of phenotypic effects of T. caespitosum was high for species richness and abundance and mainly explained by differences in interference mediated by likely heritable differences in cushion tightness. Additionally, our study shows that the shapes of the relationship between plant responses to neighbours and environmental stresses are not necessarily driven by niche‐based deterministic factors.     

表达乙型肝炎病毒表面抗原和Epstein-Barr病毒膜抗原的双价痘苗病毒的组建

从痘苗病毒天坛株分离了晚期11k蛋白编码基因的启动子,以痘苗病毒天坛株为载体,构建了双价的重组痘苗病毒。分别在7.5k和11k蛋白基因启动子的控制下,表达乙型肝炎病毒表面抗原和EB病毒的膜抗原。用重组痘苗病毒免疫的家兔,同时产生对这两种抗原的抗体。免疫电镜下观察到乙型肝炎病毒表面抗原颗粒。     

Highly Reproducible Sn‐Based Hybrid Perovskite Solar Cells with 9% Efficiency

The low power conversion efficiency (PCE) of tin‐based hybrid perovskite solar cells (HPSCs) is mainly attributed to the high background carrier density due to a high density of intrinsic defects such as Sn vacancies and oxidized species (Sn⁴⁺) that characterize Sn‐based HPSCs. Herein, this study reports on the successful reduction of the background carrier density by more than one order of magnitude by depositing near‐single‐crystalline formamidinium tin iodide (FASnI₃) films with the orthorhombic a‐axis in the out‐of‐plane direction. Using these highly crystalline films, obtained by mixing a very small amount (0.08 m ) of layered (2D) Sn perovskite with 0.92 m (3D) FASnI₃, for the first time a PCE as high as 9.0% in a planar p–i–n device structure is achieved. These devices display negligible hysteresis and light soaking, as they benefit from very low trap‐assisted recombination, low shunt losses, and more efficient charge collection. This represents a 50% improvement in PCE compared to the best reference cell based on a pure FASnI₃ film using SnF₂ as a reducing agent. Moreover, the 2D/3D‐based HPSCs show considerable improved stability due to the enhanced robustness of the perovskite film compared to the reference cell.     

Biophysical and Molecular-Dynamics Studies of Phosphatidic Acid Binding by the Dvl-2 DEP Domain

The Wnt-dependent, β-catenin-independent pathway modulates cell movement and behavior. A downstream regulator of this signaling pathway is Dishevelled (Dvl), which, among other multiple interactions, binds to the Frizzled receptor and the plasma membrane via phosphatidic acid (PA) in a mechanism proposed to be pH-dependent. While the Dvl DEP domain is central to the β-catenin-independent Wnt signaling function, the mechanism underlying its physical interaction with the membrane remains elusive. In this report, we elucidate the structural and functional basis of PA association to the Dvl2 DEP domain. Nuclear magnetic resonance, molecular-dynamics simulations, and mutagenesis data indicated that the domain interacted with the phospholipid through the basic helix 3 and a contiguous loop with moderate affinity. The association suggested that PA binding promoted local conformational changes in helix 2 and β-strand 4, both of which are compromised to maintain a stable hydrophobic core in the DEP domain. We also show that the Dvl2 DEP domain bound PA in a pH-dependent manner in a mechanism that resembles deprotonation of PA. Collectively, our results structurally define the PA-binding properties of the Dvl2 DEP domain, which can be exploited for the investigation of binding mechanisms of other DEP domain-interacting proteins.     

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled‐2 sulfatide‐binding motif

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea‐equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled‐2 (Dab2) sulfatide‐binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane‐binding, recombinant peptides that co‐purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S‐transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea‐equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at ?80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co‐purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis

Liquid–liquid phase separation is considered a generic approach to organize membrane-less compartments, enabling the dynamic regulation of phase-separated assemblies to be investigated and pivotal roles of protein posttranslational modifications to be demonstrated. By surveying the subcellular localizations of human deubiquitylases, USP42 was identified to form nuclear punctate structures that are associated with phase separation properties. Bioinformatic analysis demonstrated that the USP42 C-terminal sequence was intrinsically disordered, which was further experimentally confirmed to confer phase separation features. USP42 is distributed to SC35-positive nuclear speckles in a positively charged C-terminal residue- and enzymatic activity-dependent manner. Notably, USP42 directs the integration of the spliceosome component PLRG1 into nuclear speckles, and its depletion interferes with the conformation of SC35 foci. Functionally, USP42 downregulation deregulates multiple mRNA splicing events and leads to deterred cancer cell growth, which is consistent with the impact of PLRG1 repression. Finally, USP42 expression is strongly correlated with that of PLRG1 in non-small-cell lung cancer samples and predicts adverse prognosis in overall survival. As a deubiquitylase capable of dynamically guiding nuclear speckle phase separation and mRNA splicing, USP42 inhibition presents a novel anticancer strategy by targeting phase separation.Subject terms: Non-small-cell lung cancer, Deubiquitylating enzymes     

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10^?13 M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity.