Three pairs of weak interactions precisely regulate the G‐loop gate of Kir2.1 channel

Kir2.1 (also known as IRK1) plays key roles in regulation of resting membrane potential and cell excitability. To achieve its physiological roles, Kir2.1 performs a series of conformational transition, named as gating. However, the structural basis of gating is still obscure. Here, we combined site‐directed mutation, two‐electrode voltage clamp with molecular dynamics simulations and determined that H221 regulates the gating process of Kir2.1 by involving a weak interaction network. Our data show that the H221R mutant accelerates the rundown kinetics and decelerates the reactivation kinetics of Kir2.1. Compared with the WT channel, the H221R mutation strengthens the interaction between the CD‐ and G‐loops (E303‐R221) which stabilizes the close state of the G‐loop gate and weakens the interactions between C‐linker and CD‐loop (R221‐R189) and the adjacent G‐loops (E303‐R312) which destabilizes the open state of G‐loop gate. Our data indicate that the three pairs of interactions (E303‐H221, H221‐R189 and E303‐R312) precisely regulate the G‐loop gate by controlling the conformation of G‐loop. Proteins 2016; 84:1929–1937. © 2016 Wiley Periodicals, Inc.     

A LC-MS/MS method for the specific, sensitive, and simultaneous quantification of 5-aminolevulinic acid and porphobilinogen

Accurate determinations of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiologic fluids are required for the diagnosis and therapeutic monitoring of acute porphyrias. Current colorimetric methods are insensitive and over-estimate ALA and PBG due to poor specificity, while LC-MS/MS methods increase sensitivity, but have limited matrices. An LC-MS/MS method was developed to simultaneously determine ALA and PBG concentrations in fluids or tissues which were solid phase extracted, butanol derivatized, and quantitated by selective reaction monitoring using (13)C(5), (15)N-ALA and 2,4-(13)C(2)-PBG internal standards. ALA was separated from interfering compounds on a reverse phase C8-column. For ALA and PBG, the matrix effects (87.3-105%) and process efficiencies (77.6-97.8% and 37.2-41.6%, respectively) were acceptable in plasma and urine matrices. The assay was highly sensitive for ALA and PBG (LLOQ=0.05 μM with 25 μL urine or 100 μL plasma), and required ～4 h from extraction to results. ALA and PBG accuracy ranged from 88.2 to 110% (n=10); intra- and inter-assay coefficients of variations were <10% for urine and plasma. In clinical applications, patients with mutation-confirmed acute porphyrias had normal to slightly increased urinary ALA and PBG levels when asymptomatic, and high levels during acute attacks, which decreased with hemin therapy. In AIP mice, baseline ALA and PBG levels in urine, plasma, and liver were increased after phenobarbital induction 28-/63-, 42-/266-, and 13-/316-fold, respectively. This LC-MS/MS method is rapid, specific, highly sensitive, accurate, and simultaneously measures ALA and PBG in urine, plasma, and tissues permitting porphyria clinical diagnoses, therapeutic monitoring, and research.     

基于生态系统服务供需的景感尺度特征分析和应用

自然生态系统与人类感知的交互作用是景感生态学关注的焦点,与生态系统服务的供需紧密关联。生态系统、生态过程、生态系统服务以及人的需求均具有一定的时空尺度特征,因此,由这些过程产生的"景感"也具有时空尺度特征,并依赖于这个过程的尺度特征相互作用。梳理生态系统服务供需关系与景感营造规划的尺度特征,探索二者相互联系,进而提出基于景感生态学理论,通过景感营造实现生态系统服务供需平衡的多尺度实现路径。从空间维度,体现从"社区-城市-流域-区域"的多尺度景感规划框架,从应用维度,体现从生态系统服务需求探索-关键指标体系识别-多源谜码数据感知-景感营造与规划设计的具体流程。通过多尺度的联合景感营造,调整自然供给水平与人类不同尺度的生态系统服务需求以期达到平衡,从而实现人类生存环境福利与居民福祉提升的目标。在此基础上,结合雄安新区规划建设实例,从多尺度景感营造的角度为城市绿色生态建设与可持续发展提出优化建议。主要结论:(1)基于"社区-城市-流域-区域"的多尺度景感分析框架,将景感要素与生态系统服务供需思想融入城市规划,在不同尺度上趋善优化,对于实现可持续发展城市规划和管理具有重要指示意义;(2)雄安新...     

Circular RNAs expression profiles in plasma exosomes from early-stage lung adenocarcinoma and the potential biomarkers

This study aimed to identify differential circular RNA (circRNA) in the plasma exosomes of patients with lung adenocarcinoma (LUAD) using high-throughput sequencing. First, exosomes were isolated using an exosome isolation kit and confirmed by Western blotting, transmission electron microscopy, and NanoSight Assay. Subsequently, plasma circRNA expression profiles were screened by high-throughput sequencing and confirmed by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR) and Sanger sequencing. Finally, the circRNA-miRNA-mRNA network was performed to forecast the potential function of circRNAs. The result of high-throughput sequencing data documented that 182 differentially expressed exosomal circRNAs in all were screened, which included 105 that were upregulated and 78 that were downregulated in LUAD patients plasma compared with controls. The four upregulated circRNAs including circ_0001492, circ_0001346, circ_0000690, and circ_0001439 were identical to the sequencing data by qRT-PCR, and their latent circRNA-miRNA-mRNA interactions were exhibited. Taken together, our study firstly revealed the altered exosomal circRNA expression from plasma samples in patients with LUAD and supports the need for exploring their potential as biomarkers and the pathological effects of lung cancer.     

An Excellent Delivery System for Improving the Oral Bioavailability of Natural Vitamin E in Rats

This study set out to develop a novel and stable nanoemulsion formulation of natural vitamin E with increased oral bioavailability. The natural vitamin E nanoemulsion was prepared by a modified emulsification technique. The physicochemical characteristics of natural vitamin E nanoemulsion were characterized and its pharmacokinetics study was performed as well. The experimental results showed droplet diameters ranging from 20 to 400 nm (average, 87.7 nm) with a negative electrostatic potential (−23.5 ± 1.5 mv). The pharmacokinetics study of this nanoemulsion and corresponding soft capsule was carried out using noncompartment model method. Compared with the marketed soft capsule, the C_max of the natural vitamin E nanoemulsion was higher, while the T_max was shorter. Thus, plasma concentration–time profiles in rats dosed with nanoemulsion showed a 1.6-fold enhancement in the area under the curve of natural vitamin E compared with the marketed soft capsule. The antioxidative effects of the natural vitamin E nanoemulsion and the marketed soft capsule were also evaluated by the levels of superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration in serum and liver tissue. According to the SOD activity and the MDA concentration determined, the nanoemulsion was superior to the marketed soft as an antioxidative agent. The overall results demonstrated that the nanoemulsion drug delivery system could be a promising strategy for the delivery of natural vitamin E, which showed great potential for clinical application.Key word: Antioxidation, Nanoemulsion, Natural vitamin E, Oral bioavailability     

Expressed proteome analysis of human hepatocellular carcinoma in nude mice (LCI-D20) with high metastasis potential

We report for the first time an expressed proteome for human hepatocellular carcinoma (HCC) in nude mice model. Most cases of human liver cancer are HCC with highly metastatic ability. Therefore, the early prediction or diagnosis and effective treatment are the key points of research. We have previously successfully established a human HCC nude mice model (LCI-D20) with high metastasis potential. To understand better the tumor biology of HCC it is worth to explore the relativity of all expressed protein profiles in the LCI-D20 HCC nude mice model. With advanced proteomics technologies, we have carried out a proteomic analysis with following stages: protein sample preparation of cancer tissue, including total cellular extraction and sequential fractionation, 2-DE and 2-D LC separation, ESI/MALDI-MS/MS identification, as well as data-dependent bioinformatics. The identified proteins were classified bioinformatically respective to their function, biological process and intracellular localization. Some important proteins found in HCC, e.g. metabolism enzymes, proteins regulating cell motility, signaling proteins, and heat shock proteins, are discussed in terms of their metastasis.     

Depletion of Acidic Phosphopeptides by SAX To Improve the Coverage for the Detection of Basophilic Kinase Substrates

The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.     

不同放牧强度对人工草地牧草生殖分配及种子重量的影响

不同放牧强度对白三叶,红三叶,鸭茅的生殖分配及种子千粒重影响极显著,一定强度的放牧干扰有利于提高休牧后三叶草的花序密度,但鸭茅花序密度却随放牧强度却随放牧强度的提高而出现明显下降,放牧草地三种牧草种群的生殖分配均低于对照草地,在放牧草地,随放牧强度的提高,白三叶生殖分配逐渐增加,红三叶及鸭茅生殖分配逐渐减少,提高放牧强度后,三叶草千粒重出现递减趋势,而鸭茅却出现递增趋势,在无放牧干扰草地,三叶草种群趋于生产数量较少的大粒种子,在放牧干扰下,趋于生产数量较多的小粒种子;鸭茅种群则出现相反的变化。     

K30, H150, and H168 Are Essential Residues for Coordinating Pyridoxal 5′-Phosphate of O-Acetylserine Sulfhydrylase from Acidithiobacillus ferrooxidans

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV–vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5′-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.     

Cys92, Cys101, Cys197, and Cys203 Are Crucial Residues for Coordinating the Iron–Sulfur Cluster of RhdA from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron–sulfur clusters. But so far, there were no articles to confirm the existence of iron–sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV–Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron–sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron–sulfur cluster binding.