Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1

Background

Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats, such as geothermal hot springs and manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life.

Results

We report here the complete genome sequence of A. flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres.

Conclusions

Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.     

新疆雪莲体细胞胚胎发生

通过体细胞胚胎发生途径实现了新疆雪莲(Saussurea involucrata Kar.et Ki r.)的植株再生。选用新疆雪莲子叶为外植体, 接种于MS+0.5 mg.L-1 2,4-D+0.05-1 mg.L-1 BA的固体培养基上, 进行愈伤组织的诱导。从第1次继代培养的愈伤组织中挑选出黄绿色、颗粒状、质地致密的胚性愈伤组织, 转移到含0.05-0.1 mg.L-1 2,4-D 的MS液体培养基中进行悬浮培养,20天后可分化产生大量球形胚。继代过程中相继加入PEG和GA3 , 可以促进体细胞胚的分化和生长。体细胞胚在含有5 mg.L-1 GA3 的MS固体培养基上, 可发育成完整的植株。     

P13 K、p-AKT及HIF-1α在胰腺癌中的表达及意义

目的 检测P13K、p-AKT及HIF-1α三种蛋白在胰腺癌组织中的表达并探讨其与临床病理因素的关系及三者之间的相关性.方法 采用免疫组织化学SP法检测43例胰腺癌组织、9例胰腺炎组织和8例正常胰腺组织中P13K、p-AKT及HIF.1a三种蛋白的表达.结果 P13K、p-AKT的阳性表达主要位于肿瘤细胞胞浆,HIF-1α的阳性表达主要位于细胞核或细胞浆.P13K、p-AKT及HIF-1α在胰腺癌组织中的阳性表达率分别为62.79%、67.44%和69.77%,三者均显著高于在正常胰腺组织和慢性胰腺炎组织中的表达,差异有统计学意义(P<0.01).P13K、p-AKT及HIF-1α的异常表达均与胰腺癌的淋巴结转移和TNM分期有关(P<0.05),与患者的性别、年龄、部位、分化程度和病理分型无关(P>0.05).P13K、p-AKT及HIF-1α三者间的表达呈正相关.结论 P13K、p-AKT及HIF-1α的表达在胰腺癌的生长、浸润转移中起重要作用,P13K-AKT信号通路激活可能促进HIF-1α的表达.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

To Control False Positives in Gene-Gene Interaction Analysis: Two Novel Conditional Entropy-Based Approaches

Genome-wide analysis of gene-gene interactions has been recognized as a powerful avenue to identify the missing genetic components that can not be detected by using current single-point association analysis. Recently, several model-free methods (e.g. the commonly used information based metrics and several logistic regression-based metrics) were developed for detecting non-linear dependence between genetic loci, but they are potentially at the risk of inflated false positive error, in particular when the main effects at one or both loci are salient. In this study, we proposed two conditional entropy-based metrics to challenge this limitation. Extensive simulations demonstrated that the two proposed metrics, provided the disease is rare, could maintain consistently correct false positive rate. In the scenarios for a common disease, our proposed metrics achieved better or comparable control of false positive error, compared to four previously proposed model-free metrics. In terms of power, our methods outperformed several competing metrics in a range of common disease models. Furthermore, in real data analyses, both metrics succeeded in detecting interactions and were competitive with the originally reported results or the logistic regression approaches. In conclusion, the proposed conditional entropy-based metrics are promising as alternatives to current model-based approaches for detecting genuine epistatic effects.     

Mutation Inactivation of Nijmegen Breakage Syndrome Gene (NBS1) in Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin), involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC). Eight missense NBS1 mutations were identified in six of 64 (9.4%) HCCs and two of 18 (11.1%) ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.     

TMaCS: A hybrid template matching and classification system for partially-automated particle selection

Selection of particle images from electron micrographs presents a bottleneck in determining the structures of macromolecular assemblies by single particle electron cryomicroscopy (cryo-EM). The problem is particularly important when an experimentalist wants to improve the resolution of a 3D map by increasing by tens or hundreds of thousands of images the size of the dataset used for calculating the map. Although several existing methods for automatic particle image selection work well for large protein complexes that produce high-contrast images, it is well known in the cryo-EM community that small complexes that give low-contrast images are often refractory to existing automated particle image selection schemes. Here we develop a method for partially-automated particle image selection when an initial 3D map of the protein under investigation is already available. Candidate particle images are selected from micrographs by template matching with template images derived from projections of the existing 3D map. The candidate particle images are then used to train a support vector machine, which classifies the candidates as particle images or non-particle images. In a final step in the analysis, the selected particle images are subjected to projection matching against the initial 3D map, with the correlation coefficient between the particle image and the best matching map projection used to assess the reliability of the particle image. We show that this approach is able to rapidly select particle images from micrographs of a rotary ATPase, a type of membrane protein complex involved in many aspects of biology.     

Identification of Gene Clusters Associated with Host Adaptation and Antibiotic Resistance in Chinese Staphylococcus aureus Isolates by Microarray-Based Comparative Genomics

A comparative genomic microarray comprising 2,457 genes from two whole genomes of S. aureus was employed for the comparative genome hybridization analysis of 50 strains of divergent clonal lineages, including methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and swine strains in China. Large-scale validation was confirmed via polymerase chain reaction in 160 representative clinical strains. All of the 50 strains were clustered into seven different complexes by phylogenetic tree analysis. Thirteen gene clusters were specific to different S. aureus clones. Ten gene clusters, including seven known (vSa3, vSa4, vSaα, vSaβ, Tn5801, and phage ϕSa3) and three novel (C8, C9, and C10) gene clusters, were specific to human MRSA. Notably, two global regulators, sarH2 and sarH3, at cluster C9 were specific to human MRSA, and plasmid pUB110 at cluster C10 was specific to swine MRSA. Three clusters known to be part of SCCmec, vSa4 or Tn5801, and vSaα as well as one novel gene cluster C12 with homology with Tn554 of S. epidermidis were identified as MRSA-specific gene clusters. The replacement of ST239-spa t037 with ST239-spa t030 in Beijing may be a result of its acquisition of vSa4, phage ϕSa1, and ϕSa3. In summary, thirteen critical gene clusters were identified to be contributors to the evolution of host specificity and antibiotic resistance in Chinese S. aureus.