Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

玉米开花期相关的Indeterminate domain(IDD)蛋白家族基因的鉴定

开花期是影响玉米产量的重要因子之一。Indeterminate1 (ID1)编码玉米Indeterminate domain (IDD)家族蛋白,是玉米开花期的重要调控因子。然而,其他玉米IDD蛋白家族基因及其生物学功能有待深入研究。本文利用生物信息学技术在玉米基因组中鉴定并分离了37个IDD家族基因,记作ZmIDD。表达分析发现这些ZmIDD基因在8个玉米组织中显示出多种表达模式。为进一步探讨ZmIDD基因在调控玉米开花期上的作用,检测了37个ZmIDD在172个自交系中的遗传多样性,发现35个ZmIDD基因在自交系间具有多态性,平均每个基因具有37.8个多态性位点。关联分析鉴定到包含ID1在内的7个ZmIDD基因在多个环境下与开花期性状显著关联。对Zm00001d020683基因2 kb的启动子区和600 bp编码区重测序,共鉴定到64个多态性位点。候选基因关联分析鉴定到2个启动子区的插入缺失(In/Del)位点与开花期显著关联,其中2个位点分别插入3 bp和2 bp的单倍型为一种提早开花的基因型。研究结果为玉米开花期相关基因的分离和利用研究提供了候选基因和选择靶点。     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

A Lymphoid Organ Specific Anti-Lipopolysaccharide Factor from Litopenaeus vannamei Exhibits Strong Antimicrobial Activities

Different shrimp species are known to possess apparent distinct resistance to different pathogens in aquaculture. However, the molecular mechanism underlying this finding still remains unknown. One kind of important antimicrobial peptides, anti-lipopolysaccharide factors (ALF), exhibit broad-spectrum antimicrobial activities. Here, we reported a newly identified ALF from the shrimp Litopenaeus vannamei and compared the immune function with its counterpart in the shrimp Fenneropenaeus chinensis. The ALF, designated as LvALF8, was specifically expressed in the lymphoid organ of L. vannamei. The expression level of LvALF8 was apparently changed after white spot syndrome virus (WSSV) or Vibrio parahaemolyticus challenges. The synthetic LBD peptide of LvALF8 (LvALF8-LBD) showed strong antibacterial activities against most tested Gram-negative and Gram-positive bacteria. LvALF8-LBD could also inhibit the in vivo propagation of WSSV similar as FcALF8-LBD, the LBD of LvALF8 counterpart in F. chinensis. However, LvALF8-LBD and FcALF8-LBD exhibited apparently different antibacterial activity against V. parahaemolyticus, the main pathogen causing acute hepatopancreatic necrosis disease (AHPND) of affected shrimp. A structural analysis showed that the positive net charge and amphipathicity characteristics of LvALF8-LBD peptide were speculated as two important components for its enhanced antimicrobial activity compared to those of FcALF8-LBD. These new findings may not only provide some evidence to explain the distinct disease resistance among different shrimp species, but also lay out new research ground for the testing and development of LBD-originated antimicrobial peptides to control of shrimp diseases.     

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val¹¹⁵, ASP¹¹⁶, Val¹¹⁷, and Lys¹²²) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.     

面向养殖水体分布差异的COD光谱法检测研究

面向养殖水体，传统光谱法对化学需氧量（Chemical Oxygen Demand，COD）检测模型构建的基础：源域（现有样本库）与目标域（检测地水体）间光谱数据独立同分布。但是当源域与目标域分布间存在差异时，由源域得到的低误差模型常在目标域上表现下滑。针对该问题，提出面向UV Vis光谱的域对抗训练网络（DAUVwpNet），将分布不同的源域和目标域数据映射至相同分布的特征空间中，使其在该空间的分布距离尽可能接近，从而在特征空间中对源域训练的目标函数也可以迁移至目标域上，以降低模型在目标域的误差。试验表明：面向同一批测试数据，DAUVwpNet的预测误差为0.78，要低于传统模型的预测误差（0.85）；DAUVwpNet预测值与实测值间相关系数为0.95，要高于传统模型的相关系数（0.89）。表明了该网络能够较好对齐两域特征空间数据分布，降低因分布差异带来的COD检测误差。     

WSSV envelope protein VP51B links structural protein complexes and may mediate virus infection

White spot syndrome virus (WSSV), an enveloped double‐stranded DNA virus, is the causative agent of a disease that has led to severe mortalities of cultured shrimps in Taiwan and many other countries. In the previous study, Penaeus monodon chitin‐binding protein (CBP) and glucose transporter 1 (Glut1), two cell membrane proteins, were found to at least interact with other 10 WSSV envelope proteins including VP51B. These envelope proteins might form a protein complex. According to the known information, VP51B was used to identify its role in the protein complex. Western blotting of the intact viral particles and fractionation of the viral components confirmed that VP51B is one of WSSV envelope proteins. In this study, the protein–protein interaction between VP51B and other WSSV envelope proteins was identified by far‐western blot experiment and VP51B was found to interact with VP24, VP31, VP32, VP39B and VP41A. Furthermore, the in vivo neutralization experiment using recombinant VP51B plus with VP39B showed the best inhibition. These data indicate that VP51B participates in the WSSV protein complex and plays an important role in WSSV infection.     

昆虫嗅觉相关蛋白的研究进展

昆虫对自然环境中复杂化学信号的识别多依赖于其灵敏的嗅觉系统,选择寄主、觅食、寻找配偶等行为的发生都以嗅觉识别为基础,而完成嗅觉识别还需要多种嗅觉相关蛋白的参与。嗅觉相关蛋白主要包括6种,即气味结合蛋白、化学感受蛋白、气味受体、感觉神经元膜蛋白、离子型受体和气味降解酶。不同种类和性别的昆虫中,嗅觉蛋白的种类、数量和分布各不相同。由于嗅觉蛋白在昆虫识别外界气味分子中的重要作用,国内外近年来对其展开了广泛、深入的研究。本文从几种嗅觉相关蛋白的生化特性、分子结构、生理功能、分布表达部位和研究概况等角度,较详细地综述了近年来国内外昆虫嗅觉相关蛋白的研究进展。     

微波处理条件对蛋清结构和IgG结合能力的影响

为探究降低蛋清致敏性的新方法,在不同的微波条件下处理蛋清蛋白,通过SDS-PAGE(十二烷基磺酸钠-聚丙烯酰氨凝胶电泳)分析分子量变化、圆二色光谱分析二级结构变化、紫外光谱分析三级结构变化、外源性荧光光谱分析疏水性变化、间接ELISA(酶联免疫吸附试验)分析IgG(免疫球蛋白G)结合能力的变化,研究微波对蛋清的影响。研究证实,微波处理降低蛋清蛋白的潜在致敏性,其中400 W/30 s处理后的蛋白显示出最低的IgG结合能力,所有条件下处理后的蛋清蛋白的二、三级结构均发生显著改变。80 W的微波功率下,蛋白质的结构先折叠,该条件下处理30 s后,蛋白结构又展开;当微波功率达到640 W时,处理时间较长的蛋白分子产生堆叠。蛋白质分子的结构变化导致了其抗原性的变化。