Cyclic Di-AMP Homeostasis in Bacillus subtilis: BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.     

基于MIMIC数据库的多参数数据D/A回放系统

文中分析了MIMIC多参数智能监护数据库存储文件的识读方法,该数据库公开发表于PhysioNET,还研究及基于STM32和DAC8568的多参数数据D/A回放系统,包括系统硬件工作原理和系统软件工作原理。MIMIC多参数智能监护数据库的数据文件对于变异性较大的生理参数采用连续采样的方式,而对于相对稳定的生理参数采用周期采样的方式,因此,多参数数据各信号的抽样频率不同。系统首先通过VC++编程读取一个多抽样频率数据并在PC机上显示其波形,然后经过D/A硬件设备进行D/A转换后使用示波器输出其波形,实现输出的波形与原始的波形能同时回放。系统可作为模拟信号源,为远程多参数生理监护仪的研制奠定了基础。     

Intestinal tube formation in Caenorhabditis elegans requires vang-1 and egl-15 signaling

Understanding how epithelial organs form during morphogenesis is a major problem in developmental biology. In the present paper, we provide a detailed analysis of vang-1, the only homolog of the planar cell polarity protein Strabismus/Van Gogh in Caenorhabditis elegans. We demonstrate that during organogenesis of the intestine, (i) VANG-1 specifically interacts with PDZ 2 domain of DLG-1 (Discs large) and becomes phosphorylated by the kinase domain of the FGF-like receptor tyrosine kinase EGL-15; (ii) VANG-1 is predominantly restrained to the cell cortex but relocates to the apical junction; and (iii) in vang-1 embryos epithelial cells of the intestine are not correctly arranged along the anterior-posterior axis. To investigate what determines the disposition of the VANG-1 protein, either truncated protein forms were expressed in the intestine or RNAi was used to remove the functions of gene products previously shown to be involved in apical junction formation. Removal of the VANG-1 PDZ binding motif “− ESAV” and depletion of dlg-1 or let-413 gene functions interferes with the localization of VANG-1. In addition, egl-15 embryos show a premature relocation of VANG-1 to the apical junction, causing defects that resemble those observed in mutant vang-1 embryos and after intestine-specific overexpression of full-length vang-1. Finally, the localization of VANG-1 depends on DSH-2, a homolog of the planar cell polarity protein Dishevelled and depletion phenocopies vang-1 and egl-15 phenotypes in the embryonic intestine.     

体外反搏机用心电和颞脉波发生器

以病员作为信号源对体外反搏装置的控制系统进行检测既不方便又不实际,本研究利用单片机模拟输出多种心电波形和反搏前、反搏中的颞脉波能解决此问题。实验表明研制出的体外反搏机用心电和颞脉波信号发生器运作良好,具有应用开发价值。     

C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of K(m) and k(cat). Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Driving force of binding of amyloid beta-protein to lipid bilayers

Amyloid β-protein (Aβ) has been reported to interact with a variety of lipid species, although the thermodynamic driving force remains unclear. We investigated the binding of Aβs labeled with the dye diethylaminocoumarin (DAC-Aβs) to lipid bilayers under various conditions. DAC-Aβ-(1-40) electrostatically bound to anionic and cationic lipids at acidic and alkaline interfacial pH, respectively. However, at neutral pH, electroneutral Aβ did not bind to these lipids, indicating little hydrophobic interaction between Aβ-(1-40) and the acyl chains of lipids. In contrast, DAC-Aβ associated with glycolipids even under electroneutral conditions. These results suggested that hydrogen-bonding as well as hydrophobic interactions with sugar groups of glycolipids drive the membrane binding of Aβ-(1-40).     

A combined literature and in silico analysis enlightens the role of the NDRG family in the gut

Background

The N-Myc Downstream-Regulated Gene (NDRG) family comprises four members that function in cellular processes like proliferation and differentiation. While NDRG1 and NDRG2 are extensively studied, knowledge regarding NDRG3 and NDRG4, despite its recognition as a well-established early-detection marker for colorectal cancer (Cologuard®), is sparse.

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

单片机模拟心电图发生器的制作

本文介绍了采用８９Ｇ５１单片机的心电图信号发生器的原理和制作。本仪器小巧,灵便,能模拟产生标准件的十二导联心电图波形信号,可用来调整心电图仪和心电监视器的增益,走纸速度以及检查导联线,病人电极的性能。