Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 μg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 μg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.     

温热带玉米株高的全基因组关联分析

以365份遗传背景丰富的玉米自交系为材料,采用自主研发的55K芯片,在海南三亚、河南新乡和北京顺义3个环境下对玉米自交系株高进行全基因组关联分析(Genome-wide association study,GWAS)。结果表明,株高在3个环境下变异非常丰富,有从低纬度到高纬度逐渐增加的趋势,株高在基因型、环境、基因型与环境互作方面均达极显著差异水平。全基因组关联分析表明,不同环境下均检测到与株高显著关联的位点,在第1、3、5、6、7、8号染色体上均发现了显著关联的SNP。新乡和顺义环境下,在bin5.05区段发现了显著关联的SNP;三亚和顺义环境下,在bin6.05区段发现了显著关联的SNP。对关联分析显著的SNP位点比对到B73参考基因组上,在maizeGDB网站上得到22个最可能的候选基因,为探索玉米株高遗传机制提供参考。     

Expression of biologically active measles virus hemagglutinin glycoprotein by a recombinant baculovirus

In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay.     

Association between ABCB1-T1236C polymorphism and drug-resistant epilepsy in Iranian female patients

Background: One third of epileptic patients are resistant to several anti-epileptic drugs (AED). P-glycoprotein (P-gp) is an efflux transporter encoded by ATP-binding cassette subfamily B member 1 (ABCB1) gene that excludes drugs from the cells and plays a significant role in AEDs resistance. Over-expression of P-gp could be a result of polymorphisms in ABCB1 gene. We studied the association of T129C and T1236C single-nucleotide polymorphisms (SNP) of ABCB1 gene with drug-resistant epilepsy in Iranian epileptics. Methods: DNA samples were obtained from 200 healthy controls and 332 epileptic patients, of whom 200 were drug responsive and 132 drug resistant. The frequencies of the genotypes of the two SNP were determined by polymerase chain reaction followed by restriction fragment length polymorphism. Results: No significant association was found between T129C and T1236C genotypes and drug-resistant epilepsy neither in adults nor in children. However, the risk of drug resistance was higher in female patients with 1236CC (P = 0.02) or CT (P = 0.008) genotype than in those with TT genotype. The risk of drug resistance was also higher in patients with symptomatic epilepsies with 1236CC (P = 0.02) or CT (P = 0.004) genotype than in those with TT genotype. The risk of drug resistance was lower in patients with idiopathic epilepsies with 129TT genotype (P = 0.001) than in those with CT genotype. Conclusion: Our results indicate that T1236C polymorphism is associated with drug resistance in Iranian female epileptic patients. Replication studies with large sample sizes are needed to confirm our results. Key Words: ATP-binding cassette subfamily B member 1 (ABCB1), Drug-resistant epilepsy, Single nucleotide polymorphism     

甘蓝型油菜和甘蓝CRISPR/Cas9编辑效果的快速检测

CRISPR/Cas9系统是目前最常用的基因组定点编辑工具，通过瞬时表达试验提前验证Cas9/sgRNA载体诱导的突变效率，可以提高基因组定点编辑成功的几率，且显著节省费用及时间。本研究开发了一种利用农杆菌介导的操作简单、适用性好、成本低廉的叶片瞬时表达技术，可用于快速检测甘蓝型油菜和甘蓝中CRISPR/Cas9的编辑效果。针对甘蓝型油菜BnaC.WRKY11.a设计了2个靶位点Tgt1（Target 1）和Tgt2（Target 2），并构建了Cas9/sgRNA-Tgt1/2多重突变载体，在甘蓝型油菜叶片中瞬时表达后，2个靶位点都出现突变，突变效率达11.2%～82.2%。针对甘蓝BolPDS3基因设计了1个靶位点Tgt3（Target3），并构建了Cas9/sgRNA-Tgt3敲除载体，在甘蓝叶片瞬时表达后，Tgt3发生了突变，且大部分为碱基缺失突变，缺失的数目为1～18bp不等，同时还存在少量碱基插入以及碱基替换等突变类型。结果表明这种甘蓝型油菜和甘蓝的叶片瞬时表达技术操作简单、适用性好、成本低廉，CRISPR/Cas9的编辑效果快速易检测。     

New Sinularianin Sesquiterpenes from Soft Coral Sinularia sp.

Four new sesquiterpenes, sinularianins C–F (3–6), together with known sinularianins A (1) and B (2) were identified from a South China Sea soft coral Sinularia sp. Compounds 1–6 were evaluated for inhibition of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay. Compounds 1 and 4 were exhibited a potent effect with inhibitory rates of 41.3% and 43.0% at the concentration of 10 µg/mL, respectively.     

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis

Background

Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody – the serological marker of type 2 autoimmune hepatitis (AIH-2)- is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1) – either in association with anti-LKM1, or in isolation- and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients.

Methods

One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls.

Results

Six out of 18 (33%) anti-LKM^pos/HCV^pos patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM^neg/HCV^pos patients and 0/120 (0%) of the anti-LKM1^neg/HBV^pos patients (p < 0.001 for both comparisons). Anti-SLA antibodies were not present in any of the HCV (with or without anti-LKM1) or HBV-infected patients.

Conclusion

We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of anti-LKM1 negative patients with chronic hepatitis B and C infections. Based on these results we cannot find any justification for the application of anti-LC1 and anti-SLA tests in the routine laboratory testing of viral hepatitis-related autoantibody serology with the only potential exception being the anti-LC1 screening in anti-LKM1^pos/HCV^pos patients.

     

综合代谢组学和转录组学分析揭示火龙果不同发育阶段的潜在颜色变化

火龙果的发育可分为幼果期(Y)、转色期(C)和成熟期(M).本研究综合运用代谢组学和转录组学分析方法,探讨火龙果发育过程中理化和结构变化的分子机制,特别关注调节果皮和果肉颜色变化的因素.本研究在每个对照组中发现了大量差异表达的基因和代谢物.在火龙果不同发育阶段观察到大量的生化和生理变化,鉴定出214种代谢物.KEGG分...     

小麦 DA1基因家族成员的鉴定及其表达谱分析

泛素受体DA1在控制种子和器官大小上发挥着重要作用。本研究以中国春小麦为材料,采用生物信息学的方法对小麦 DA1基因家族成员进行了全基因组鉴定和分析,共鉴定到12个小麦 DA1同源基因（ TaDA1s）,分别位于2B、2D、4A、4B、4D、5A、5B和5D染色体上,亚细胞定位预测结果表明其位于细胞核中。系统进化分析发现, TaDA1s可分为 TaDA1、 TaDAR1和 TaDAR2三类。除TaDA1-2D1编码的蛋白只含有DA1-like结构域外,其他成员编码的蛋白均含有DA1-like、UIM和LIM结构域。启动子序列分析发现, TaDA1s的启动子区含有较多的响应激素、逆境以及与生长发育相关的顺式作用元件。通过公共的转录组数据和qRT-PCR试验验证, 发现 TaDA1s在不同组织中都有表达,在籽粒发育过程中,不同的小麦 DA1基因呈现不同的表达模式,暗示存在基因功能分化现象;脱落酸（ABA）能够抑制 TaDA1s的表达;在低温和盐胁迫条件下, TaDA1s基因表达有明显的差异。酵母双杂交和荧光素酶互补试验发现,TaDA1-2B、TaDAR1-5A和TaGW2存在转录自激活现象,且TaDAR1-5A与TaGW2有微弱的互作效应。本研究结果为进一步探究 DA1基因家族在小麦中的生物学功能奠定了基础。     

海南龙血树查尔酮合酶基因(DcCHS)的克隆及表达分析

利用RT-PCR和RACE技术对海南龙血树查尔酮合酶基因进行克隆,得到1条1 456 bp的cDNA序列,命名为DcCHS。DcCHS含有1 173 bp的阅读框架、99 bp的5′非编码区和184 bp的3′非编码区,编码390个氨基酸。DcCHS与其他植物的CHS氨基酸序列同源性高达84%以上,具有高度保守的CHS_like结构域、活性位点以及信号序列。推测DcCHS的分子量为42.7 ku,等电点pI为6.14,稳定性极差,具有15个磷酸化位点,无跨膜结构,无信号肽,亚细胞定位在细胞质的可能性较大,并预测了蛋白质的二级、三级结构。组织特异性分析结果表明,DcCHS在花中的表达远远高于根、茎、叶和果实。     

甘蓝型油菜Δ9硬脂酰ACP脱氢酶（SAD)基因的克隆与表达分析

为改良甘蓝型油菜菜籽油脂肪酸的组分,根据拟南芥Δ9硬脂酰ACP脱氢酶（SAD)核酸序列特征,检索白菜全基因组SAD基因和cDNA的可能序列,通过同源序列法克隆获得6个甘蓝型油菜SAD基因。比对结果显示,这6个基因编码的氨基酸序列同源性达53.2%~96.3%。系统进化分析显示,甘蓝型油菜SAD基因与蓖麻、大豆、芝麻、葵花等6个油料作物SAD基因的序列相似性很高,甘蓝型油菜与这些高等植物的SAD基因在进化上具有较高的保守性。本文还对4个甘蓝型油菜SAD基因BnSAD1:1、BnSAD2:1、BnSAD2:2和BnSAD2:3进行了表达模式分析,发现它们在种子发育过程中表达,并且都在40d的种子中表达量达到最高值,推测这4个基因均参与了硬脂酰ACP (C18:0-ACP)脱氢生成油酰基ACP(Δ9C18:1-ACP)的过程,尤其是BnSAD2:3可能为种子特异表达基因。     

LcMYB1激活荔枝花色苷生物合成关键基因LcDFR和LcUFGT1的启动子区域分析

花色苷含量是荔枝果实呈现鲜红色的重要次生代谢产物,前人研究结果表明,LcMYB1通过调控关键基因的表达而影响荔枝果皮中花色苷的积累,其中LcDFR和LcUFGT1是荔枝果皮花色苷生物合成的关键基因,但是LcMYB1如何影响基因的表达,是否与结构基因启动子结合以及具体结合区段目前还不清楚。本研究通过克隆和分析LcDFR和LcUFGT1的启动子区域并对其进行分段处理,利用双荧光素酶和酵母单杂交实验,探究LcMYB1与LcDFR和LcUFGT1的启动子的结合区域。结果表明,LcDFR起始密码子上游1927 bp和LcUFGT1起始密码子上游1584 bp的启动子上分别有3个可能的MYB结合位点;双荧光素酶和酵母单杂交实验均显示LcMYB1可以结合LcDFR基因起始密码子上游904 bp到1425 bp包含有1个MYB-CORE元件的区域,LcMYB1与LcUFGT1基因启动子结合的区域是起始密码到上游610 bp,此区域包含有2个MYB-CORE元件。研究结果进一步证实了LcMYB1通过与基因LcDFR和LcUFGT1启动子结合发挥调控作用,并缩小了结合位点的范围。     

Immunogenicity Evaluation of a DNA Vaccine Expressing the Hepatitis C Virus Non-Structural Protein 2 Gene in C57BL/6 Mice

Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses. Key Words: Hepatitis C virus (HCV), NS2 protein, DNA vaccine, IL-12     

穗发芽相关基因Vp1B3不同等位变异在山东小麦中的分布与演变

为了探讨穗发芽相关基因Vp1B3不同等位变异在山东小麦中的分布与演变规律,利用Vp1B3的功能标记对山东小麦385份农家品种、101份历史品种和25份当前主栽品种进行鉴定.结果表明,Vp1B3的不同等位变异在不同历史时期的山东小麦中具有不同的分布特点:25份当前主栽品种中有17份含有抗穗发芽类型的等位变异(Vp1B3b、Vp1B3c),占68.0%;101份历史品种中抗穗发芽的类型有58份,占57.4%;而385份农家品种中只有100份属于抗穗发芽类型,仅占26.0%.在山东省四大麦区的地方品种中Vp1B3的等位基因分布规律亦不同:在胶东丘陵晚熟麦区及鲁西北平原冬性、半冬性、晚熟麦区的地方品种中抗穗发芽类型所占比例较小,分别为16.2%和9.9%;在鲁中山丘川半冬性、冬性中熟麦区和鲁西南平原湖洼半冬性早熟麦区的地方品种中抗穗发芽类型所占比例相对较大,分别为44.7%和37.8%.     

Phase II-Inducing,Polyphenols Content and Antioxidant Capacity of Corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) from Phenotypes of White,Blue, Red and Purple Colors Processed into Masa and Tortillas

White, blue, red and purple corns (Zea mays L.) were lime-cooked to obtain masa for tortillas. The total phenolics and anthocyanins content, antioxidant activity expressed as total reducing power (TRP), peroxyl radical bleaching (PRAC), total antioxidant activity (TAA) and quinone reductase (QR) induction in the murine hepatoma (Hepa 1 c1c7 cell line) as a biological marker for phase II detoxification enzymes were investigated. Among the extracts prepared from raw corn varieties the highest concentration of total phenolics, anthocyanins, antioxidant index and induction of QR-inducing activity were found in the Veracruz 42 (Ver 42) genotype. The nixtamalization process (masa) reduced total phenolics, anthocyanins and antioxidant activities and the ability for QR induction when was compared to raw grain. Processing masa into tortillas also negatively affected total phenolics, anthocyanin concentration, antioxidant activities, and QR induction in the colored corn varieties. The blue variety and its corresponding masa and tortillas did not induce QR. Ver 42 genotype and their products (masa and tortilla) showed the greatest antioxidant activity and capacity to induce QR     

甘蓝型油菜脂肪酸碳链延长酶BnaA.FAE1与BnaC.FAE1的底物特异性分析

为提供信息改良油料种子脂肪酸,研究脂肪酸碳链延长酶1（FAE1）的底物特异性,将甘蓝型油菜A、C基因组的BnaA.FAE1和BnaC.FAE1分别在亚麻荠种子中表达。其转基因种子中山嵛酸与芥酸含量的比值（C22:0/C22:1）分别为0.18和0.88,表明BnaA.FAE1对单不饱和脂肪酸亲和力较高,而BnaC.FAE1则对饱和脂肪酸亲和力较高,这种差异在各转基因世代表现稳定。通过分析T3转基因家系种子中酰基辅酶A（Acyl-CoAs）的组成,进一步证明了上述结论。但是在拟南芥中分别异源表达上述基因,并没有观察到类似的底物特异性差异。在甘蓝型油菜祖先种,白菜型油菜和甘蓝种子中调查其C22:0/C22:1比值,同样未发现其FAE1底物特异性存在差异。综上认为BnaA.FAE1和BnaC.FAE1在亚麻荠中存在底物特异性。     

Fucoidan Elevates MicroRNA-29b to Regulate DNMT3B-MTSS1 Axis and Inhibit EMT in Human Hepatocellular Carcinoma Cells

Accumulating evidence has revealed that fucoidan exhibits anti-tumor activities by arresting cell cycle and inducing apoptosis in many types of cancer cells including hepatocellular carcinoma (HCC). Exploring its effect on microRNA expression, we found that fucoidan markedly upregulated miR-29b of human HCC cells. The induction of miR-29b was accompanied with suppression of its downstream target DNMT3B in a dose-dependent manner. The reduction of luciferase activity of DNMT3B 3′-UTR reporter by fucoidan was as markedly as that by miR-29b mimic, indicating that fucoidan induced miR-29b to suppress DNMT3B. Accordingly, the mRNA and protein levels of MTSS1 (metastasis suppressor 1), a target silenced by DNMT3B, were increased after fucoidan treatment. Furthermore, fucoidan also down-regulated TGF-β receptor and Smad signaling of HCC cells. All these effects leaded to the inhibition of EMT (increased E-cadherin and decreased N-cadherin) and prevention of extracellular matrix degradation (increased TIMP-1 and decreased MMP2, 9), by which the invasion activity of HCC cells was diminished. Our results demonstrate the profound effect of fucoidan not only on the regulation of miR-29b-DNMT3B-MTSS1 axis but also on the inhibition of TGF-β signaling in HCC cells, suggesting the potential of using fucoidan as integrative therapeutics against invasion and metastasis of HCC.     

芸薹属油菜种质资源抗(耐)菌核病、病毒病的鉴定

芸薹属油菜种质资源抗(耐)菌核病、病毒病的鉴定结果表明:1.抗菌核病的基因主要存在于B和C染色体组,抗芜菁花叶病毒(TuMV)的基因主要存在于C染色体组。2,农艺性状与抗菌核病性存在相关;B.campestris有效分枝点高度、B.carinata的株高、有效分枝点高度与抗性指标RRA分别呈显著和极显著正相关,B.juncea二次有效分枝数与RRA呈显著负相关,B.napus的上述性状与RRA相关不显著。3.油菜单株产籽量和产油量随病情加重而下降,达显著或极显著程度。     

Svalbamides A and B,Pyrrolidinone-Bearing Lipodipeptides from Arctic Paenibacillus sp.

Two new secondary metabolites, svalbamides A (1) and B (2), were isolated from a culture extract of Paenibacillus sp. SVB7 that was isolated from surface sediment from a core (HH17-1085) taken in the Svalbard archipelago in the Arctic Ocean. The combinational analysis of HR-MS and NMR spectroscopic data revealed the structures of 1 and 2 as being lipopeptides bearing 3-amino-2-pyrrolidinone, d-valine, and 3-hydroxy-8-methyldecanoic acid. The absolute configurations of the amino acid residues in svalbamides A and B were determined using the advanced Marfey’s method, in which the hydrolysates of 1 and 2 were derivatized with l- and d- forms of 1-fluoro-2,4-dinitrophenyl-5-alanine amide (FDAA). The absolute configurations of 1 and 2 were completely assigned by deducing the stereochemistry of 3-hydroxy-8-methyldecanoic acid based on DP4 calculations. Svalbamides A and B induced quinone reductase activity in Hepa1c1c7 murine hepatoma cells, indicating that they represent chemotypes with a potential for functioning as chemopreventive agents.     

Role of XmnIG Polymorphism in Hydroxyurea Treatment and Fetal Hemoglobin Level at Isfahanian Intermediate β-Thalassemia Patients

Background:

β-thalassemia is the most common monogenic disorder in human. The (CT) polymorphism at -158 upstream region of the γ^G-globin gene and pharmacological factors such as hydroxyurea have been reported to influence γ-globin gene expression and the severity of clinical symptoms of β-thalassemia.

Methods:

In the present study, 51 β-thalassemia intermediate patients were studied. Xmn1γ^G polymorphism genotype was determined using Tetra-Primer ARMS-PCR technique. Hemoglobin (Hb) and fetal hemoglobin (HbF) levels were determined by gel electrophoresis.

Results:

Of 51 patients, 35 (68.6%) patients were heterozygous (CT) and 16 (31.4%) patients were homozygous (CC). Of 30 patients under treatment by hydroxyurea, 20 (66.7%) patients were heterozygous (CT) and 10 (33.3%) patients were homozygous (CC). Our results demonstrated that in the heterozygous (CT) genotype, the Hb (9.58 ± 1.25 gm/dl) and HbF (89.30 ± 21.87) levels were significantly higher in comparison with homozygous (CC) genotype (7.94 ± 1.34 gm/dl and 70.32 ± 40.56, respectively). Furthermore, we observed that after drug usage, the Hb and HbF levels in patients with heterozygous (CT) genotype (0.7 ± 1.26 gm/dl and 5.95±14.8, respectively) raised more in comparison with homozygous (CC) genotype (0.26 ± 1.43 gm/dl and 0.8 ± 1.31, respectively).

Conclusion:

Hb and HbF levels in the patients carrying T allele are increased significantly, and they also response to hydroxyurea treatment.Key Words: Fetal hemoglobin (HbF), Hydroxyurea, Intermediate β-thalassemia