Malaysian weedy rice shows its true stripes: wild Oryza and elite rice cultivars shape agricultural weed evolution in Southeast Asia

Weedy rice is a close relative of domesticated rice (Oryza sativa) that competes aggressively with the crop and limits rice productivity worldwide. Most genetic studies of weedy rice have focused on populations in regions where no reproductively compatible wild Oryza species occur (North America, Europe and northern Asia). Here, we examined the population genetics of weedy rice in Malaysia, where wild rice (O. rufipogon) can be found growing in close proximity to cultivated and weedy rice. Using 375 accessions and a combined analysis of 24 neutral SSR loci and two rice domestication genes (sh4, controlling seed shattering, and Bh4, controlling hull colour), we addressed the following questions: (i) What is the relationship of Malaysian weedy rice to domesticated and wild rice, and to weedy rice strains in the USA? (ii) To what extent does the presence of O. rufipogon influence the genetic and phenotypic diversity of Malaysian weeds? (iii) What do the distributions of sh4 and Bh4 alleles and associated phenotypes reveal about the origin and contemporary evolution of Malaysian weedy rice? Our results reveal the following: independent evolutionary origins for Malaysian weeds and US strains, despite their very close phenotypic resemblance; wild‐to‐weed gene flow in Malaysian weed populations, including apparent adaptive introgression of seed‐shattering alleles; and a prominent role for modern Malaysian cultivars in the origin and recent proliferation of Malaysian weeds. These findings suggest that the genetic complexity and adaptability of weedy crop relatives can be profoundly influenced by proximity to reproductively compatible wild and domesticated populations.     

New insight into the stereoselective interactions of quinine and quinidine,with bovine serum albumin

Quinine (QN) and quinidine (QD), the chief quinoline alkaloids of various species of cinchona bark, are stereoisomers to each other. In this study, a series of appropriate and efficient methods have been applied to compare the binding modes of QN and QD with bovine serum albumin (BSA). The isothermal titration calorimetry and room temperature phosphorescence results show that both QN and QD can interact with BSA at one binding site to form drug–protein complexes, mainly through enthalpic driving force with the binding affinity order: QN > QD. The fluorescence resonance energy transfer and time‐resolved fluorescence spectroscopy exhibits that QN has a larger energy transfer and more intensified binding capacity for BSA than QD. Data of dynamic light scattering reveal that the aggregate state of BSA is changed during this binding process, and the particle size distribution of QN‐BSA bioconjugate is larger than that of QD. Nuclear magnetic resonance analysis indicates that aromatic protons make more contribution during ligand‐protein complexation than that of aliphatic protons. The circular dichroism spectra exhibit different degrees of changes in BSA secondary structures in the presence of QN and QD, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Peptide Scaffold-Based Discovery of Nonpeptide Natural Medicines to Target PI3K p85 SH2 Domain

Targeting phosphoinositide 3-kinase (PI3K) has been recognized as an attractive strategy for anticancer therapy. The PI3K is a heterodimer composed of a catalytic subunit p110 and a regulatory subunit p85. Here, instead of targeting the catalytic p110 that has been considered previously, we purposed targeting the peptide-recognition domain SH2 of regulatory p85 with natural medicines obtained by using a peptide scaffold-based screening scheme. In the procedure, a core binding motif was extracted from the cocrystallized complex of a cognate phosphopeptide with the domain, which was considered as basic scaffold to perform high-through virtual screening against a structurally diverse, nonredundant library of natural products. A number of hit compounds with high binding potency to the domain and significant conformational similarity with the peptide scaffold were identified; in vitro affinity assay confirmed that five hits have moderate or high affinity for the domain with measured dissociation constants K_d range between 25 and 360 μM, which are comparable to or even better than that of the cognate phosphopeptide SDpYMNMTP and its core motif peptide pYMNM (K_d?=?15 and 32 μM, respectively). Structural analysis and nonbonded comparison of SH2 interactions with phosphopeptides and potent hit compounds revealed that only negatively charged phosphate and, sometime, sulfate can confer domain-binding capability to small-molecule compounds, but carboxylate cannot. A similar binding mode of compounds with phosphopeptide is important for the compounds to have high affinity and specificity.     

Atrial overexpression of microRNA-27b attenuates angiotensin II-induced atrial fibrosis and fibrillation by targeting ALK5

Atrial fibrosis influences atrial fibrillation (AF) development by transforming growth factor beta 1 (TGF-β1)/Smad pathway. Although microRNAs are implicated in the pathogenesis of various diseases, information regarding the functional role of microRNAs in atrial dysfunction is limited. In the present study, we found that microRNA-27b (miR-27b) was the dominant member of miR-27 family expressed in left atrium. Moreover, the expression of miR-27b was significantly reduced after angiotensin II (AngII) infusion. Masson’s trichrome staining revealed that delivery of miR-27b adeno-associated virus to left atrium led to a decrease in atrial fibrosis induced by AngII. The increased expression of collagen I, collagen III, plasminogen activator inhibitor type 1 and alpha smooth muscle actin was also inhibited after miR-27b upregulation. In isolated perfused hearts, miR-27b restoration markedly attenuated AngII-induced increase in interatrial conduction time, AF incidence and AF duration. Furthermore, our data evidence that miR-27b is a novel miRNA that targets ALK5, a receptor of TGF-β1, through binding to the 3′ untranslated region of ALK5 mRNA. Ectopic miR-27b suppressed luciferase activity and expression of ALK5, whereas inhibition of miR-27b increased ALK5 luciferase activity and expression. Additionally, miR-27b inhibited AngII-induced Smad-2/3 phosphorylation without altering Smad-1 activity. Taken together, our study demonstrates that miR-27b ameliorates atrial fibrosis and AF through inactivation of Smad-2/3 pathway by targeting ALK5, suggesting miR-27b may play an anti-fibrotic role in left atrium and function as a novel therapeutic target for the treatment of cardiac dysfunction.     

Rapid determination of amino acids in biological samples using a monolithic silica column

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm × 3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.     

Identification of differentially expressed microRNAs and their PKC-isoform specific gene network prediction during hypoxic pre-conditioning and focal cerebral ischemia of mice

We previously reported the involvement of conventional protein kinase C (cPKC) βII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6?h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6?h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6?h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6?h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCβII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCβII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.     

基于光激活定位显微镜的膜蛋白鉴定技术

病原菌膜蛋白的原位标记和定位追踪是一项非常繁琐的工作。为了建立一种可以用于快速鉴定膜蛋白、且分辨率达到纳米级别的膜蛋白荧光定位技术,将结核分枝杆菌外膜蛋白OmpA与具有光敏活性的蛋白mEos2m在无致病性的耻垢分枝杆菌中进行融合表达。重组菌固定在载玻片上后,利用405 nm激光激活mEos2m。利用普通荧光体视显微镜、正置荧光显微镜和超分辨率光激活定位显微镜观察OmpA-mEos2m,分析融合蛋白在细菌内的分布情况,并捕获光激活蛋白在细胞膜上释放的光子信号。通过超分辨率光激活定位显微镜,发现OmpA-mEos2m融合蛋白在细胞膜上呈"带"状环绕分布,这是观察到膜蛋白在细胞膜上定位的最直接证据。mEos2m与OmpA融合表达后,并不改变OmpA的膜蛋白属性和定位特征。因此可以将mEos2m用于其他非多聚体膜蛋白的融合和定位研究。可以应用非致病性的耻垢分枝杆菌作为模型,采用高分辨率活菌成像技术,研究致病性的结核分枝杆菌蛋白结构、定位和功能。这是目前为止国内采用光激活定位显微成像技术研究膜蛋白的首次报道。     

The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the β domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.