Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)₃-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.     

Knocking down Wnt9a mRNA levels increases cellular proliferation

Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.     

抑卵激素对家蝇卵巢周期性发育的调控

抑卵激素是调控家蝇Musca dorncstica vicina卵巢周期性发育的关键因子之一。在家蝇中,当第一个周期的卵母细胞处于卵黄发生期或卵黄发生后期时,其第二个周期的卵母细胞的发育不进入卵黄发生期。本文建立了家蝇抑卵激素的生物测定方法,即用一对卵巢提取物注射1头羽化后12h家蝇,并在羽化后60h观察卵母细胞的发育及卵黄蛋白的沉积情况。抑卵激素的作用首先是延缓了卵母细胞在卵黄发生前期的发育;其次,抑卵激素抑制脂肪体中卵黄蛋白的合成,导致血淋巴中卵黄蛋白含量的下降,从而抑制了卵母细胞的发育。抑卵激素并不抑制卵母细胞对卵黄原蛋白的摄取。卵发育神经激素可以颉抗抑卵激素的抑制作用。抑卵激素无种属特异性。     

An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples

As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.     

Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC₅₀) or effective concentration (EC₅₀), as well as its range of cytotoxicity (CC₅₀). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.     

应用微卫星标记分析23个中国地方马种的遗传多样性

为了调查中国马种的群体遗传分化与遗传结构状况, 本研究应用FAO和ISAG推荐的25对微卫星引物, 结合荧光标记PCR分析技术, 对中国23个马群体和1个英纯血马群体进行了分子遗传学研究。结果表明: 中国地方马群体的遗传多样性比较丰富, 23个中国马群体的等位基因数、多态信息含量和遗传杂合度等都高于英国纯血马。根据群体遗传距离构建的系统进化树, 能够清晰地将英纯血马从中国马中独立出来, 同时可将中国马群体分成不同的支, 基本上与其地理分布格局相符。我们用MVSP软件进行群体遗传分化分析可见, 前三个主成分的三维散点图可以明显地把英纯血马从所有群体中独立出来, 并把中国的马群体分成几个相对独立的支。进一步分析第一、二主成分的二维散点图, 可将中国的马群体分化成南方支系、藏马支系、新疆和青海支系、内蒙古支系及东北支系等5个部分。根据Structure软件分析, 推测中国马群体含有5个潜在的支系, 基本上代表了我国现在主要家马来源的基础遗传支系。这些信息可以为我国现有马种类型的划分与马种资源遗传多样性的保护提供科学依据。     

JUVENILE HORMONE BINDING PROTEIN IN THE OVARIES OF HOUSEFLY MUSCA DOMESTICA VACINA

Abstract  By using charcocal binding assay, the juvenile hormone binding protein (JHBP) was determined in the ovaries of houseflies. This ovarian JHBP possesses high affinity with juvenile hormone III (JH III) and has a K_d of 2.1 III 10^--⁸ M. The binding of ³H-juvenile hormone III (³H-JH III) to this protein was inhibited by unlablled JH III, but not by juvenile hormone analog ZR 512 or ZR 515. The level of this ovarian JHBP reached the highest in houseflies 48 h after emergence, and was 6. 5-fold and 15. 5-fold higher than that in housefIies 60 h and 72 h after emergence, respectively. No binding activity was detected in the ovaries of houseflies 24 h or 36 h after emergence. The absence of JHBP in the ovaries of houseflies 36 h after emergence could be reversed by applying JH III to newly emerged houseflies. The data suggest that the fluctuation of the JHBP concentration might associate with the action of juvenile hormone (JH) on housefly vitellogenesis.     

边际多样性方法在中国猪种保护资金分配中的应用

农业动物种质资源面临着严重危机, 有必要构建一个科学、有效的保护方案, 合理分配有限的资金, 以期保护最大的多样性。本文应用边际多样性方法, 以中国18个地方猪种为例, 通过对灭绝概率、现实多样性、边际多样性和保护潜力的分析, 应用3种模型分析了未来群体期望多样性变化趋势, 并提出了相应的资金优化配置方案。在文中所涉及的模型中, 加性模型(Model A)被评定为资金优化配置的最佳模型; 共有10个品种获得保护资金, 其中杭猪获得最高比例(16％)的保护资金; 其次为赣中南花猪(万安猪)、嵊县花猪, 各获得14％; 阳新猪、乐平猪各获得11%; 五指山猪、玉山乌猪、赣中南花猪(冠朝猪)、清平猪、武夷黑猪和嘉兴黑猪分别获得10％、9％、9％、2％、2％和2％的保护资金。结果表明, 分配保护资金需综合考虑群体的受威胁程度、群体遗传多样性和经济重要性等。