A Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR↓STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.     

A transversely isotropic constitutive model of excised guinea pig spinal cord white matter

Narrowing of the spinal canal generates an amalgamation of stresses within the spinal cord parenchyma. The tissue’s stress state cannot be quantified experimentally; it must be described using computational methods, such as finite element analysis. The objective of this research was to propose a compressible, transversely isotropic constitutive model, an augmentation of the isotropic Mooney–Rivlin hyperelastic strain energy function, to describe the guinea pig spinal cord white matter. Model parameters were derived from a combination of inverse finite element analysis on transverse compression experiments and least squared error analysis applied to quasi-static longitudinal tensile tests. A comparison of the residual errors between the predicted response and the experimental measurements indicated that the transversely isotropic constitutive law that incorporates an offset stretch reduced the error by a factor of four when compared to other commonly used models.     

Production of marker-free transgenic rice expressing tissue-specific Bt gene

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3′ untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P _Actin1 :Cry1Ab/Ac:T _Nos gene in the control Bt rice line T51-1, the P _Pepc :Cry1Ab/Ac:T _Nos gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.     

紫菀属植物化学成分及药理作用研究进展

紫菀属(Aster L.)植物为多年生草本、亚灌木或灌木,该属植物中紫菀(Aster tataricus)为常用中药,具有润肺下气、止咳祛痰之功效,主治气逆咳嗽、痰吐不利、肺虚久咳、痰中带血等症,是临床常用的润肺祛痰止咳药。该属所含化学成分较丰富,已对该属中16种植物做过化学成分研究,分离出的化合物124个,其类型有各种萜类及其苷、肽类、黄酮类、蒽醌类、甾醇类、香豆素、有机酸类及挥发油等。对国内外有关该属植物的化学成分、药理作用和临床应用的最新研究进行了综述,为该药用植物资源的进一步研究和开发利用提供依据。     

Non-stop mRNA decay initiates at the ribosome

The translation machinery deciphers genetic information encoded within mRNAs to synthesize proteins needed for various cellular functions. Defective mRNAs that lack in-frame stop codons trigger non-productive stalling of ribosomes. We investigated how cells deal with such defective mRNAs, and present evidence to demonstrate that RNase R, a processive 3'-to-5' exoribonuclease, is recruited to stalled ribosomes for the specific task of degrading defective mRNAs. The recruitment process is selective for non-stop mRNAs and is dependent on the activities of SmpB protein and tmRNA. Most intriguingly, our analysis reveals that a unique structural feature of RNase R, the C-terminal lysine-rich (K-rich) domain, is required both for productive ribosome engagement and targeted non-stop mRNA decay activities of the enzyme. These findings provide new insights into how a general RNase is recruited to the translation machinery and highlight a novel role for the ribosome as a platform for initiating non-stop mRNA decay.     

肽链释放因子和核糖体蛋白L11在八肋游仆虫细胞中的定位

原生动物纤毛虫是一类单细胞真核生物,其蛋白质合成终止过程中密码子使用的特殊性使其成为研究蛋白质合成终止机制的一个经典模型。为了能够有效地分析生物大分子在该细胞中的功能作用位点,本研究根据该生物染色体结构的特征,构建了含有红色荧光蛋白基因的大核人工染色体EoMAC_R,并与之前构建的含绿色荧光蛋白基因的大核染色体EoMAC_G一起,对蛋白质合成终止有关的3个重要因子核糖体大亚基蛋白L11、多肽链释放因子eRF1和eRF3在八肋游仆虫细胞中进行了荧光共定位分析。结果显示,在八肋游仆虫细胞中,蛋白质翻译过程主要位于"C"形大核内侧区域。构建的人工染色体能够作为一种有效的工具,对目的蛋白质在八肋游仆虫细胞中进行定位分析。     

高分子人工血管材料大鼠肌肉内的急性期反应

本实验研究临床常用的几种人造血管生物材料在大鼠体内引起的急性期组织反应,并与自主研发的丝素蛋白改性聚氨酯(Silk fibroin-polyurethane(1:1),SF-PU(1:1))材料相比较,以期找出组织相容性最佳的材料。将涤纶(Dacron)材料、膨化聚四氟乙烯(Expanded polyterafluoroethylene,e-PTFE)材料、聚氨酯(Polyurethane,PU)材料、以及丝素蛋白改性聚氨酯复合材料(SF-PU(1:1))埋植入大鼠肌肉内,通过大鼠急性毒性实验、肌肉植入局部组织反应实验、局部组织切片染色、白细胞及血小板计数,探讨几种材料对大鼠的局部及全身影响,研究比较各组材料的组织相容性。结果表明:涤纶材料的组织相容性较差;其余各组材料的局部组织炎性反应较轻,且白细胞及血小板计数与假手术组无显著性差异。故认为涤纶作为临床上常用的人造血管材料组织相容性最差,所研发的SF-PU(1:1)材料及另两种临床上常用的e-PTFE材料和PU材料的组织相容性较好,尤以SF-PU(1:1)材料的组织相容性最好,结合SF-PU(1:1)优异的物理性能,在小口径人造血管的研制方面有很大的研究前景。     

Ecosystems patterns and dynamics in Haihe river basin

Research on ecosystems patterns and dynamics is critical for understanding the regional problems of resources and environment. In this study, based on the technology of RS and GIS, the composition, patterns and dynamics of the ecosystems in Haihe river basin during 1990–2005 were analyzed using landscape indices including the area of average patches, aggregation index and Shannon–Weaver diversity index. According to the development situation of economy and society in Haihe river basin, some driving factors for dynamics of ecosystems patterns were concluded based on correlation analysis. Results showed that the ecosystems patterns in Haihe river basin changed greatly from 1990 to 2005. The area of urban and grassland increased, but farmland, forest and wetland decreased. As for landscape indices, the area of average patches, value of aggregation index, clump index and diversity index all increased, indicating the trend of evenness for the whole ecosystems. Growth of population, economy growth together with urbanization and policy were the main driving factors for dynamics of ecosystem pattern in this basin.     

Shape-dependent adhesion and endocytosis of hydroxyapatite nanoparticles on A7R5 aortic smooth muscle cells

The interaction between nanohydroxyapatite (HAP) and smooth muscle cells is an important step in vascular calcification. However, the effect of the shape of HAP on adhesion and endocytosis to aortic smooth muscle cells has been rarely reported. Four different morphological HAP crystals (H-Rod, H-Needle, H-Sphere, and H-Plate) were selected to interact with rat aortic smooth muscle cells (A7R5). Fluorescence-labeled HAP was used to detect crystal adhesion and endocytosis and then pretreated with different endocytic inhibitors to explore the pathway of endocytotic crystals. The distribution of crystals inside and outside the cells and the crystal localization in lysosomes was observed through laser confocal microscopy. The effect of crystal on the cell cycle and the changes in the expression of phosphatidylserine, osteopontin, α-actin, core binding factor alpha 1, and osterix on the surface of A7R5 cells were detected. The adhesion and endocytosis of HAP on A7R5 cells were closely related to crystal shapes and ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. H-Sphere and H-Needle were internalized into the cells mainly via the clathrin-mediated pathway, whereas H-Plate and H-Rod were internalized into the cells mainly via macropinocytosis. The endocytosed nano-HAP was mainly distributed in the cell lysosome. The adhesion and endocytosis of HAP to A7R5 cells were positively correlated with the specific surface area, and contact area of HAP and negatively correlated with the absolute value of Zeta and contact angle of HAP. This study provided insights into the effect of crystal morphology on vascular calcification and its mechanism.     

罗霄山脉地区鱼类物种多样性

罗霄山脉是赣江和修水流域与湘江流域的分水岭, 是中国生物多样性保护的关键地区之一。然而, 罗霄山脉地区的鱼类缺乏系统性的研究, 其鱼类物种组成、分布以及受威胁因素尚不清楚。为此, 我们于2014-2018年对罗霄山脉地区11条河流的鱼类进行了系统的调查。结果表明, 该地区共有鱼类5目17科64属113种, 山脉东坡鱼类108种, 高于西坡的72种。从生态类型看, 罗霄山脉鱼类以肉食性、底栖性、定居性类群为主。区系组成上以东亚江河平原类群为主。从物种多样性看, 遂川江、袁水、蜀水和修河的鱼类物种多样性较高, 锦江和富水的鱼类物种多样性较低; β多样性指数揭示遂川江与锦江、禾水、富水间鱼类物种出现一定的分化现象。