Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis

Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.     

Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway

Background  

Carcinogenicity of nickel compounds has been well documented. However, the carcinogenic effect of metallic nickel is still unclear. The present study investigates metallic nickel nano- and fine particle-induced apoptosis and the signal pathways involved in this process in JB6 cells. The data obtained from this study will be of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles.     

Current challenges in distinguishing climatic and anthropogenic contributions to alpine grassland variation on the Tibetan Plateau

Quantifying the impact of climate change and human activities on grassland dynamics is an essential step for developing sustainable grassland ecosystem management strategies. However, the direction and magnitude of climate change and human activities in driving alpine grassland dynamic over the Tibetan Plateau remain under debates. Here, we systematically reviewed the relevant studies on the methods, main conclusions, and causes for the inconsistency in distinguishing the respective contribution of climatic and anthropogenic forces to alpine grassland dynamic. Both manipulative experiments and traditional statistical analysis show that climate warming increase biomass in alpine meadows and decrease in alpine steppes, while both alpine steppes and meadows benefit from an increase in precipitation or soil moisture. Overgrazing is a major factor for the degradation of alpine grassland in local areas with high level of human activity intensity. However, across the entire Tibetan Plateau and its subregions, four views characterize the remaining controversies: alpine grassland changes are primarily due to (1) climatic force, (2) nonclimatic force, (3) combination of anthropogenic and climatic force, or (4) alternation of anthropogenic and climatic force. Furthermore, these views also show spatial inconsistencies. Differences on the source and quality of remote sensing products, the structure and parameter of models, and overlooking the spatiotemporal heterogeneity of human activity intensity contribute to current disagreements. In this review, we highlight the necessity for taking the spatiotemporal heterogeneity of human activity intensity into account in the models of attribution assessment, and the importance for accurate validation of climatic and anthropogenic contribution to alpine grassland variation at multiple scales for future studies.     

成年黄山短尾猴雌性等级结构稳定性

等级结构稳定性是衡量群体凝聚力的重要指标,雌性等级结构稳定对于母系社会群体具有重要的生物学意义。本文采用目标动物取样和全事件记录相结合的方法,研究黄山短尾猴鱼鳞坑A1 群(YA1 群)和鱼鳞坑A2 群(YA2 群)的成年雌性分别在交配季节(2011 年9 ～ 12 月)和非交配季节(2012 年2 ～ 5 月)内的攻击-屈服行为。采用等级结构中雌性间攻击- 屈服频率、社会顺位和等级梯度3 个量化指标,首次定量探讨了短尾猴雌性等级结构及其稳定性。研究期间,YA1 群和YA2 群雌性间攻击- 屈服频率、社会顺位和等级梯度均无季节性变化(P >0. 05)。结果表征短尾猴雌性等级结构趋于稳定,该结果支持了短尾猴雌性等级结构稳定性的定性判断。     

Characterization of three distinct cDNA clones encoding cysteine proteinases from maize (Zea mays L.) callus

In previous work, a 33 kDa cysteine proteinase was found in callus initiated from maize (Zea mays L.) resistant to fall armyworm feeding. A callus cDNA library from the maize inbred Mp708 was screened with oligonucleotides derived from the N-terminal amino acid sequence of the 33 kDa proteinase and several cDNA clones were isolated and sequenced. A cDNA clone encoding the 33 kDa cysteine proteinase, mir1, was identified. Two additional clones, mir2 and mir3, encoding putative cysteine proteinases were also identified. mir2 and mir3 are distinct from mir1 and each other, but show a high degree of homology. All of the mir cDNA clones map to distinct sites on the maize genome. Amino acid sequences encoded by the mir clones are similar to other known cysteine proteinases and are most closely related to the oryzain- and - precursors. The ERFNIN motif and a 12 amino acid conserved sequence are present in the propeptide region of the putative proteinases encoded by mir clones. mir2 and mir3 appear to have C-terminal extensions. The phylogenetic tree of nucleotide sequences of mir1, mir2, mir3 and other representative cysteine proteinases from protozoa, plants and animals was constructed.     

洞庭湖区灾害性洪水对生态灾害群发的复合效应

灾害性洪水的能量传播与它挟带的泥沙淤积,是诱发洞庭湖区生态灾害的直接和潜伏条件,从而形成了灾害链和灾害网,而不同类型洲滩地的机械阻挡,永生植物阻流促淤及长江洪水顶托的综合作用,改变了湖泊水动力条件与泥沙之间互为因果的关系,导致了洪水泛滥、泥沙淤积,洲滩地浮涨,生态灾害群发的恶性循环,其复合效应是水灾、沙灾、血吸虫病、鼠灾等生态灾害群发频繁且灾情日趋严重。     

外显子捕捉法快速分离新基因的原理和方法

从大片段基因组DNA中快速分离转录序列是疾病基因定位克隆(positional cloning)中的关键步骤.外显子捕捉法是一种较为成功的方法.文章对该法的基本原理、方法步骤及应用成果等作了较为详细的介绍.     

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.     

Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.