Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment

The DnaE intein of Synechocystis sp. PCC6803 (Ssp DnaE intein) is the first split intein identified in nature. Its N-terminal fragment (Int-n) is attached to the end of the N-terminal half of the DnaE protein (DnaE-n) to form the precursor DnaE-n/Int-n, while the C-terminal fragment (Int-c) precedes the C-terminal half of the DnaE protein (DnaE-c) to form the precursor Int-c/DnaE-c. Int-n and Int-c fragments in the separate precursors catalyze, in concert, a protein trans-splicing process to splice the flanking DnaE-n and DnaE-c into a functional catalytic subunit of DNA polymerase III. They then release themselves from the precursors. Previously, the Ssp DnaE intein has been used to reconstitute a protein trans-splicing mechanism in stably transformed Arabidopsis thaliana, resulting in successful reassembly of an intact and functional GUS from two halves of a split GUS protein. In this report, transient expression using a biolistic particle bombardment approach is described for functional analysis of Ssp DnaE intein. Analyses confirmed that the Ssp DnaE intein could catalyze protein trans-splicing not only in model plants but also in monocot and dicot crops. It also demonstrated that when up to 45 amino acid residues were removed from the C-terminus of the Int-n fragment, the Int-n fragment was still able to function in the protein trans-splicing process.     

Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono‐orientation to bi‐orientation.     

Amino acid prodrugs of NVR3-778: Design,synthesis and anti-HBV activity

A series of amino acid prodrugs of NVR3-778, a potent anti-HBV candidate currently under phase II clinical trial, were designed and synthesized as new anti-HBV agents. Except for 1e, all of them displayed roughly comparable anti-HBV activity (IC₅₀, 0.28–0.56 µM) to NVR3-778 (IC₅₀, 0.26 µM). Compound 1a, a l-valine ester prodrug of NVR3-778, was found to show significantly improved water solubility (0.7 mg/mL, pH 2) as we expected, and lower cytotoxicity (CC₅₀ > 10 µM) than NVR3-778 (CC₅₀, 4.81 µM). Moreover, 1a also exhibited acceptable PK properties and comparable in vivo efficacy in HBV DNA hydrodynamic mouse model to that of NVR3-778, suggesting it may serve as a promising lead compound for further anti-HBV drug discovery.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Identification of a cyanobacterial CRR6 protein,Slr1097, required for efficient assembly of NDH‐1 complexes in Synechocystis sp. PCC 6803

Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH‐1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH‐1 complexes are poorly understood. By transformation of wild‐type cells with a transposon‐tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH‐1‐mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH‐1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH‐1 complexes using a yeast two‐hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ?slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH‐1M complex, whereas almost half the wild‐type levels of these subunits were present in NDH‐1L complex; similar results were observed in the NdhI‐less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH‐1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH‐1L complex.     

高寒草甸植物物种丧失对草原毛虫的影响

植物多样性是调控食物网结构和生态系统功能最重要的生物因素, 植物多样性丧失深刻影响食草动物, 但由于小型食草动物种群数量波动明显、统计随机性较大等困难, 我们对植物多样性丧失如何影响小型食草动物依然知之甚少。基于在青藏高原高寒草甸设置的长期植物物种剔除试验, 本研究于2016-2020年7-8月连续调查了植物物种剔除各处理中草原毛虫(Gynaephora alpherakiif)的数量, 分析了植物物种及功能群丧失对草原毛虫的影响。结果表明, 虽然时空差异及统计随机性是影响草原毛虫数量变化的主要因素, 但植物物种剔除介导的群落差异对草原毛虫数量的影响依然显著: (1)在各观测时段, 优势种线叶嵩草(Kobresia capillifolia)的丧失导致群落中草原毛虫数量显著减少; 禾草类物种丧失也会减少草原毛虫数量, 但其影响仅在8月显著; (2)杂类草物种丧失通过增加群落中禾草物种多度, 可增加草原毛虫数量; 豆科物种丧失使莎草增多, 也会增加草原毛虫数量; (3)各植物功能群部分物种剔除并未显著影响草原毛虫数量。本研究证实了高寒草甸中草原毛虫数量会因优势植物嵩草和禾草的多度减少或禾草物种丧失而显著减少, 但群落总生物量、个体数和物种丰富度、豆科多度以及各功能群植物同比减少, 都对草原毛虫数量没有明显影响。这些结果说明在随机作用主导下, 植物群落中的特定功能群相对多度(而非物种多样性)变化深刻影响草原毛虫适合度, 进而影响生态系功能及服务; 未来生物多样性研究及草地虫害生物防控中应更多考虑统计随机性及植物功能多样性对小型食草动物的影响。     

A simple quantitative chiral analysis of amino acid esters by fluorine‐19 nuclear magnetic resonance using the modified James–Bull method

A simple chiral analysis of amino acid esters by fluorine‐19 nuclear magnetic resonance (¹⁹F NMR) through the modified James–Bull method is described. Thus, amino acid ester acid salt was treated with 5‐fluoro‐2‐formylphenylboronic acid and (S)‐BINOL in the presence of triethylamine (TEA) and MS4A for 10 minutes. The reaction mixture was analysed by ¹⁹F NMR directly to afford good quantifications.