Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.     

N-Glycan structure annotation of glycopeptides using a linearized glycan structure database (GlyDB)

While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision-induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.     

Synthesis of closo-Dodecaboryl Lipids and their Liposomal Formation for Boron Neutron Capture Therapy

High accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve efficient neutron capture therapy of cancers. We focused on liposomal boron delivery system to achieve a large amount of boron delivery to tumor. We succeeded in the synthesis of the double-tailed boron cluster lipids 4a–c and 5a–c, which has a B₁₂H₁₁S-moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols. Size distribution of liposomes prepared from the boron cluster lipid 4b, dimyristoylphosphatidylcholine, polyethyleneglycol-conjugated distearoylphosphatidylethanolamine, and cholesterol was determined as 100 nm in diameter by an electrophoretic light scattering spectrophotometer. Calcein-encapsulation experiments revealed that these boronated liposomes are stable at 37 °C in fetal bovine serum solution for 24 h.     

Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants

KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.     

A novel technique for loading of paclitaxel-PLGA nanoparticles onto ePTFE vascular grafts

The major cause of hemodialysis vascular access dysfunction (HVAD) is the occurrence of stenosis followed by thrombosis at venous anastomosis sites due to the aggressive development of venous neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective in inhibiting hyperplasia without causing systemic side effects. We have previously demonstrated that paclitaxel-coated expanded poly(tetrafluoroethylene) (ePTFE) grafts, by a dipping method, could prevent neointimal hyperplasia and stenosis of arteriovenous (AV) hemodialysis grafts, especially at the graft-venous anastomoses; however, large quntities of initial burst release have remained a problem. To achieve controlled drug release, paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-NPs) were prepared by the emulsion-solvent evaporation method and then transferred to the luminal surface and inner part of ePTFE vascular grafts through our micro tube pumping and spin penetration techniques. Scanning electron microscope (SEM) images of various stages of Ptx-PLGA-NPs unequivocally showed that micro tube pumping followed by spin penetration effectively transferred Ptx-PLGA-NPs to the inner part, as well as the luminal surface, of an ePTFE graft. In addition, the in vitro release profiles of paclitaxel demonstrated that this new system achieved controlled drug delivery with a reduced initial burst release. These results suggest that loading of Ptx-PLGA-NPs to the luminal surface and the inner part of an ePTFE graft is a promising strategy to ultimately inhibit the development of venous neointimal hyperplasia.     

一株产酶真菌Phaeophlebiopsis sp.转录组分析

对从药渣中分离的真菌ZYJHYZ254进行鉴定及产酶活性研究,从转录组分析菌株不同生长时期差异表达基因对其生长发育及产酶调控的影响,以筛选高产木质纤维素水解酶真菌,寻找调控关键基因。鉴定ZYJHYZ254为拟暗射脉菌Phaeophlebiopsis sp.,产酶在第5-7天最高。从生长3 d与7 d的菌丝中共检测到1 232个差异基因,以3 d的菌丝为对照,显著上调、下调基因分别有826及406个,基因注释和GO、KEGG功能富集分析结果表明差异表达基因主要与蛋白质合成、代谢及酶合成相关。此外,共有387个CAZymes基因表达,GH数量最多,约占49.61%,其次为AA (97)与GT (62),约占25.06%与16.02%。GH16 (24个)占GH的12.50%,含量最多,主要编码葡萄糖苷酶、木聚糖酶等,AA中AA3 (37个)占比38.14%,编码氧化酶、脱氢酶等。结果表明ZYJHYZ254中生长3 d与7 d的菌丝经功能富集分析发现差异表达基因主要与蛋白质合成、代谢,以及酶合成相关。进一步研究发现在两个生长时期中CAZymes基因表达最多的是GH16与AA3,预示了该菌葡萄糖苷酶、木聚糖酶、β-半乳糖苷酶、氧化酶与脱氢酶含量最丰富,对降解特殊生物质中的木质纤维素具有重要意义。     

研究报告: 急性缺血性卒中患者血清脑红蛋白的动态变化及临床意义

目的 脑红蛋白（neuroglobin,Ngb）又称神经球蛋白,是一种携氧球蛋白。有研究证明Ngb在脑缺血缺氧疾病中具有神经保护作用,对氧具有高亲和力,有助于预防缺氧缺血性脑损伤,并影响急性缺血性卒中（acute ischemic stroke,AIS）的预后。本研究观察了AIS后血清Ngb水平的变化,并评估了Ngb与卒中严重程度及预后的关系。方法 前瞻性地募集AIS患者和健康对照者进行研究。在AIS患者起病后不同时间点（脑梗死发病后72 h内和第14天）及对照组中分别检测血清Ngb水平,并比较AIS患者与对照组之间的血清Ngb水平。对合并与不合并糖尿病的AIS患者的血清Ngb水平进行比较。分析患者血清Ngb水平与梗死体积及国家卫生研究院卒中量表（NIHSS）评分之间的相关性。应用受试者操作特征（ROC）曲线评估Ngb对AIS预后的预测价值。结果 AIS患者的血清Ngb水平在脑梗死后72 h内和第14天时分别为105.7（88.3,123.1）μg/L和72.8（58.7,86.9）μg/L。对照组血清Ngb水平为58.2（35.0,81.6）μg/L。AIS发病后72 h内血清Ngb水平较对照组显著升高（P<0.05）。合并与不合并糖尿病的AIS患者血清Ngb水平在发病后72 h内及第14天均无显著差异（P>0.05）。AIS发病72 h内的血清Ngb水平与NIHSS评分显著相关（Spearman相关系数=0.232,P=0.038）。在各时间点,大体积脑梗死组患者血清Ngb水平与中小体积脑梗死组相比无显著性差异（P>0.05）。ROC曲线分析表明,血清Ngb水平对AIS患者的预后具有良好的预测能力。结论 AIS发病后72 h内血清Ngb水平升高。合并与不合并糖尿病的AIS患者血清Ngb水平无显著差异。发病后72 h内的血清Ngb水平与NIHSS评分显著相关。Ngb可能作为卒中严重程度和预后的预测因子。     

皮肤类器官的构建、功能及其应用研究进展

皮肤类器官作为一种新型的类器官模型,不仅能高度模拟皮肤组织的生理结构和功能,更好地在不同体外环境下还原较真实的皮肤生态,还可以应用于皮肤发育研究、皮肤疾病病理研究及药物筛选等领域。在干细胞研究中,皮肤类器官模型可以在特殊的生境下对具有特定功能的皮肤细胞及其附属物进行重建和改造,以弥补现有体外皮肤模型在结构、功能等方面的不足。基于此,皮肤类器官将会在皮肤再生、组织修复、药物筛选及医学美容等方面扮演越来越重要的角色。本文详述了皮肤类器官构建中所参与的细胞来源及近年来的应用,并对未来皮肤类器官的发展与优化做出了展望。