Anaerobic Degradation of Normal- and Branched-Chain Fatty Acids with Four or More Carbons to Methane by a Syntrophic Methanogenic Triculture

Syntrophic degradation of normal- and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum T1N, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methylvalerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

皖南尖吻蝮蛇毒出血毒素Ⅳ的纯化与初步鉴定

从皖南尖吻蝮蛇（Ａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）毒液中经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＳｅｐｈａｃｒｙｌＳ－２００两步凝胶柱层析首次纯化出一种中分子量出血毒素（简称ＡａＨⅣ）．经ＳＤＳ－ＰＡＧＥ和等电聚焦凝胶电泳测定其分子量为４４ｋＤ,等电点为ｐＨ５．０．从５００ｍｇ粗毒中可获得２０ｍｇＡａＨⅣ纯品．ＡａＨⅣ有较强的出血活性,最小出血剂量（ＭＨＤ）为０．４μｇ．     

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.     

Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.     

紫金山铜矿酸性矿山废水微生物群落多样性

【背景】为避免环境污染,酸性矿山废水需经处理后才能排放,处理后的废水理化性质会发生显著变化,将影响整个微生物群落的结构。【目的】分析处理前后的细菌和真菌群落变化及其与理化参数的关系,为矿山废水的处理提供参考指标,并为矿山污染场地的修复提供理论基础。【方法】采集福建紫金山铜矿的酸性矿山废水并测定其理化性质。采用基于原核微生物16S rRNA基因V4区和真菌18S rRNA基因ITS的高通量测序技术分析水样的微生物群落结构。【结果】经中和处理后的回水与矿坑水和生物浸出液相比,pH升高,重金属离子含量显著降低。原核微生物的多样性高于真菌,回水的物种多样性高于矿坑水和浸出液。回水中变形菌门的丰度最高,矿坑水和浸出液中分别以广古菌门和硝化螺菌门的丰度最高。回水中噬氢菌属为优势类群,矿坑水和浸出液中的优势菌是钩端螺旋菌属,铁质菌属等古菌也有一定的比例。pH、Al、Mn、Zn与回水中相对丰度较高的菌属显著相关,而矿坑水和浸出液中的高丰度类群与环境因子没有显著的相关性。【结论】研究表明酸性废水的中和沉淀处理对微生物群落产生了较大的影响,微生物群落变化可以作为矿山酸性废水污染处理效果的一个参考指标。     

螺环哌啶类似物作为DOR激动剂的特征结构研究

阿片受体激动剂与特定阿片受体亚型结合,常用来治疗与外伤、癌症或心脏病相关的严重疼痛,是十分有吸引力的药用物质．阿片受体有3种经典亚型(δ, κ, μ),均有与其对应的激动剂．δ阿片受体(DOR)激动剂因其还有明显的抗焦虑、抗抑郁和器官保护作用,是非常有前景的药物．本文研究了一批共102个N-取代螺环哌啶类似物作为δ阿片受体激动剂的分子,采用比较分子力场(CoMFA)和比较分子相似性指数(CoMSIA)两种分析方法对所有分子进行了三维定量构效关系(3D-QSAR)研究,其中基于疏水场和氢键供体场参数建立的CoMSIA模型最佳,其模型结果为：Q²=0.501,R²_ncv=0.787,R²_pre=0.780,证明模型自我吻合良好,同时有较强的内部及外部预测能力．而模型的等势线图分析表明,在R₁处引入疏水性的取代基及在R₂处引入亲水性的取代基或氢键供体基团对提高激动剂活性有利．这些结论有助于更好地理解N-取代螺环哌啶类似物作为DOR激动剂的机理,为新型的δ阿片受体激动剂的设计和优化提供一定的指导．     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

Adenosine A2A receptor deficiency attenuates the somnogenic effect of prostaglandin D2

Sleep and Biological Rhythms -     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.