SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor‐like proteins

Receptor‐like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine‐rich repeats (LRRs) and, in contrast with receptor‐like kinases (RLKs), lack a cytoplasmic kinase required for the initiation of downstream signalling. Recent studies have revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1‐1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf‐4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf‐4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked down by virus‐induced gene silencing, showed that the LRR domain as well as the kinase activity of SOBIR1 are required for the Cf‐4/Avr4‐triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitation of Cf‐4 accumulation. Together, these results suggest that, in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for the initiation of downstream signalling through its kinase domain.     

Characterization of the binding of paylean and DNA by fluorescence,UV spectroscopy and molecular docking techniques

The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant K_a between PL and DNA were 5.11 × 10³, 2.74 × 10³ and 1.74 × 10³ L mol^–1 at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern–Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL–DNA decreased with increasing ionic strength. The value of K_a for PL with double‐stranded DNA (dsDNA) was larger than that for PL with single‐stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A–T‐rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non‐radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd.     

Study on the interaction between pelargonidin‐3‐O‐glucoside and bovine serum albumin using spectroscopic,transmission electron microscopy and molecular modeling techniques

The aim of this study is to evaluate the binding behavior between pelargonidin‐3‐O‐glucoside (P3G) and bovine serum albumin (BSA) using multi‐spectroscopic, transmission electron microscopy (TEM) and molecular docking methods under physiological conditions. Fluorescence spectroscopy and time‐resolved fluorescence showed that the fluorescence of BSA could be quenched remarkably by P3G via a static quenching mechanism, and there is a single class of binding site on BSA. In addition, the thermodynamic functions ΔH and ΔS were –21.69 kJ/mol and 24.46 J/mol/K, indicating that an electrostatic interaction was a main acting force. The distance between BSA and P3G was 2.74 nm according to Förster's theory, illustrating that energy transfer occurred. In addition, the secondary structure of BSA changed with a decrease in the α‐helix content from 66.2% to 64.0% as seen using synchronous fluorescence, UV/vis, circular dichroism and Fourier transform infrared spectroscopies, whereas TEM images showed that P3G led to BSA aggregation and fibrillation. Furthermore, site marker competitive experiments and molecular docking indicated that P3G could bind with subdomain IIA of BSA. The calculated results of the equilibrium fraction showed that the concentration of free P3G in plasma was high enough to be stored and transported from the circulatory system to its target sites to provide therapeutic effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014–2015

A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015. Based on the genetic and phylogenetic analyses, the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA, a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin. The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain (A/Alberta/01/2014). Further analysis showed that similar viruses have been circulating in wild birds in China, Russia, Dubai (Western Asia), Bulgaria and Romania (Europe), as well as domestic poultry in some regions of Africa. The affected areas include the Central Asian, East Asian-Australasian, West Asian-East African, and Black Sea/Mediterranean flyways. These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds, thus posing a serious threat to wild birds and potentially humans.

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MicroRNA‑224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1

Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR‐224 in the development and progression of osteosarcoma. We demonstrated that miR‐224 was down‐regulated in osteosarcoma cell lines and tissues. Lower miR‐224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR‐224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG‐63 cells to cisplatin. We identified Rac1 as a direct target gene of miR‐224 in osteosarcoma. Rac1 expression was up‐regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR‐224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR‐224‐overexpressing MG‐63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR‐224‐overexpressing MG‐63 cells. These data suggest that miR‐224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin.     

Identification of the active site of human mitochondrial malonyl‐coenzyme a decarboxylase: A combined computational study

Malonyl‐CoA decarboxylase (MCD) can control the level of malonyl‐CoA in cell through the decarboxylation of malonyl‐CoA to acetyl‐CoA, and plays an essential role in regulating fatty acid metabolism, thus it is a potential target for drug discovery. However, the interactions of MCD with CoA derivatives are not well understood owing to unavailable crystal structure with a complete occupancy in the active site. To identify the active site of MCD, molecular docking and molecular dynamics simulations were performed to explore the interactions of human mitochondrial MCD (HmMCD) and CoA derivatives. The findings reveal that the active site of HmMCD indeed resides in the prominent groove which resembles that of CurA. However, the binding modes are slightly different from the one observed in CurA due to the occupancy of the side chain of Lys183 from the N‐terminal helical domain instead of the adenine ring of CoA. The residues 300 ? 305 play an essential role in maintaining the stability of complex mainly through hydrogen bond interactions with the pyrophosphate moiety of acetyl‐CoA. Principle component analysis elucidates the conformational distribution and dominant concerted motions of HmMCD. MM_PBSA calculations present the crucial residues and the major driving force responsible for the binding of acetyl‐CoA. These results provide useful information for understanding the interactions of HmMCD with CoA derivatives. Proteins 2016; 84:792–802. © 2016 Wiley Periodicals, Inc.     

毛竹的花药发育研究

竹类植物因有着较长的开花周期,其生殖生物学研究的报道相对较少。该研究采用石蜡切片与野外观察的方法,对毛竹花药的发育以及花药发育与花序的关系进行了研究。结果表明:毛竹的花药壁结构包括4层细胞:表皮细胞、药室内壁细胞、中层细胞和绒毡层细胞。药室内壁和中层都只有一层细胞,而且细胞形状较扁,花药发育后期药室内壁会逐渐降解,而中层则会完全解体消失。花药壁的发育为单子叶型,绒毡层为腺质型,而且只有一层,细胞径向较长,最后也会消失。小孢子母细胞减数分裂时,胞质分裂方式为连续型。形成的小孢子经一次有丝分裂后逐渐形成成熟花粉粒,大多为二细胞型,很少产生三细胞型。此外,还发现毛竹花药的发育与花序形态变化存在着相对应的关系。野外连续观察和切片发现,随着花序形态的不断发育变化,首先花药开始形成并不断分化,药壁备层也逐渐形成;接着小孢子逐渐成熟,备层也慢慢随之解体、消失;最后花药逐渐开裂并开始散粉。该研究结果不仅丰富了毛竹和竹类生殖生物学的研究内容,而且对毛竹种质的研究也具有重要意义。     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.