Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

雄性黄山短尾猴(Macaca thibetana)攻击支持雌性以换取交配回报

生物市场理论认为动物个体之间通过某种协定交换有价值的商品,使双方均受益。该研究采用目标动物法、行为取样法和连续记录法,对浮溪黄山野生猴谷鱼鳞坑短尾猴(Macaca thibetana)A1群(YA1群)和A2群(YA2群)成年个体在非繁殖季节(2011年8月—12月)和繁殖季节(2012年2月—5月)的雄性攻击支持雌性行为和交配行为进行研究,探讨雄性攻击支持雌性与交配之间的关系。两猴群在繁殖季节和非繁殖季节雄性攻击支持雌性与交配行为均呈显著正相关;YA2群繁殖季节与非繁殖季节攻击支持后交配频次均显著高于随机交配;YA1群在繁殖季节攻击支持后交配频次与随机交配频次差异不显著,但在非繁殖季节攻击支持后交配频次显著高于随机交配,说明短尾猴成年雄性攻击支持雌性可以换取与该雌性个体的交配回报。本研究验证了生物市场理论中社会行为存在交换,首次证明了雄性攻击支持可以换取雌性的交配回报,为进一步研究雄性性竞争与雌性选择提供了实例。     

树鼩IL-2全长编码序列的克隆及分子特征分析

树鼩作为多种人类疾病模型已受到广泛关注,而免疫因子对于树鼩模型评价至关重要,但目前对其白细胞介素-2(IL-2)的研究鲜有报道。该实验以经ConA(concanavalin)诱导培养的树鼩淋巴细胞总RNA为模板,RT-PCR克隆出465bp的树鼩IL-2全长编码序列,并采用ClustalW软件分析其序列和分子特征。结果表明树鼩IL-2cDNA编码一个由154个氨基酸组成的蛋白质,其cDNA及氨基酸序列与人的同源性分别为93%及80%,且其整体结构与人IL-2相似。MEGA5.0软件构建的进化树表明,树鼩与人及恒河猴的亲缘关系较近。Pymol软件对树鼩和人IL-2氨基酸序列进行的三维结构模建表明,两者的IL-2分子三维空间结构基本相似,表面大部分区域所带电荷相同,但在某些区域差异较大,且树鼩多出一个糖基化位点,这些差异对抗体的结合可能存在影响。该研究为今后树鼩IL-2单克隆抗体的制备及功能研究奠定了基础。     

巴氏醋杆菌高酸度醋发酵过程的能量代谢分析

【目的】初步分析了Acetobacter pasteurianus CICIM B7003-02在醋酸发酵过程中的能量代谢状况, 通过强化细胞能量代谢水平以提升菌株高酸发酵的产酸强度。【方法】探明A. pasteurianus CICIM B7003-02在高酸度醋发酵的不同阶段中三羧酸循环底物含量、乙醇呼吸链酶活及能量代谢酶基因的转录水平等代谢特点, 分析用于醋酸发酵的产能代谢途径及其作用。【结果】发现A. pasteurianus CICIM B7003-02在醋酸发酵初期, 主要通过苹果酸/琥珀酸回补偶联有氧呼吸途径产能。进入醋酸快速积累阶段, 乙醇呼吸链为主要供能代谢途径。发酵后期苹果酸/琥珀酸回补途径配合乙醇呼吸链供能。基于上述研究, 采取添加琥珀酸和苹果酸强化细胞产能, 促进高酸度醋发酵强度。【结论】能量供给影响醋杆菌耐酸能力和醋酸生产能力。确定乙醇呼吸链为醋酸发酵的主要供能系统。强化细胞产能手段可达到提高醋酸发酵强度的目的。     

Coxsackievirus A16 infection triggers apoptosis in RD cells by inducing ER stress

Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot and mouth disease (HFMD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the unfolded protein response (UPR), which may lead to apoptotic cell death and influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8 and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection leads to ER stress, which in turn results in prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.     

Novel indoline-2,3-dione derivatives as inhibitors of aminopeptidase N (APN)

Aminopeptidase N (APN/CD13), as a zinc-containing ectoenzyme, plays a critical role in the process of tumor angiogenesis, invasion and metastasis. Through the docking-based virtual screening of chemical databases and the further activity assay, we discovered that compound 10c exhibits potent and selective inhibitory ability towards APN. In addition, a series of indoline-2,3-dione derivates have been designed and synthesized as APN inhibitors. The results of preliminary activity evaluation showed that compound 12a (IC₅₀ = 0.074 ± 0.0026 μM) exhibited the best inhibitory activity against APN, which could be used for further anticancer agent research.     

Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors

Atrial fibrillation (AF) is one of the common arrhythmias that threaten human health. Kv1.5 potassium channel is reported as an efficacious and safe target for the treatment of AF. In this paper, we designed and synthesized three series of compounds through modifying the lead compound RH01617 that was screened out by the pharmacophore model we reported earlier. All of the compounds were evaluated by the whole-patch lamp technology and most of them possessed potent inhibitory activities against Kv1.5. Compounds IIIi and IIIl were evaluated for the target selectivity as well as the pharmacodynamic effects in an isolated rat model. Due to the promising pharmacological behavior, compound IIIl deserves further pharmacodynamic and pharmacokinetic evaluations.     

光照和生长阶段对菖蒲根系泌氧的影响

以自然湖泊沉积物为研究基质,利用微型电机控制溶氧微电极实现纵向精确微位移,在照光与遮光条件下,对典型湿地植物菖蒲幼苗、成株根系根基部起总根长1/4处(根1/4)、根系中部(根1/2)、从根基部起总根长3/4处(根3/4)及根尖(根1)处根系微界面径向溶氧浓度变化进行原位精确测定。结果表明:无论有无光照,菖蒲幼苗、成株根系不同部位均存在从根表面至沉积物氧饱和度由高到低的氧扩散层,其厚度0.18—0.68 mm;根1/2、3/4、1处氧扩散能力菖蒲成株较幼苗显著增强(P<0.01),根1/4处二者则无显著差异(P>0.05);光照对菖蒲幼苗、成株根系不同部位氧扩散能力的影响存在差异,光照对菖蒲幼苗根1/2及菖蒲成株根1/2、根3/4处影响显著(照光组显著高于遮光组,P<0.01),而对菖蒲幼苗根1/4、根3/4、根1及菖蒲成株根1/4、根1处无显著影响(P>0.05);从根系泌氧空间差异上看,照光条件下菖蒲幼苗、成株分别表现为根1/2>根3/4≈根1≈根1/4(P<0.01,P>0.05)和根1/2>根3/4>根1>根1/4(P<0.01),遮光条件下菖蒲幼苗、成株分别表现为根1/2≈根3/4≈根1≈根1/4(P>0.05)和根1/2>根3/4≈根1>根1/4(P<0.01,P>0.05)。     

PFOS对斑马鱼胚胎及仔鱼的生态毒理效应

全氟辛烷磺酸（Perfluorooctane sulfonate, PFOS）是一种广泛存在于水生生态系统的新型持久性有机污染物（Persistent Organic Pollutants, POPs）,其对鱼类健康的影响以及水生生态系统安全的潜在威胁是当前人们高度关注的水环境问题。为探究PFOS对斑马鱼（Danio rerio）胚胎及仔鱼的生态毒理效应,本文研究了不同浓度（0,0.1,1,10 mg/L）PFOS暴露对斑马鱼胚胎孵化率、仔鱼畸形率与死亡率、仔鱼心率、仔鱼运动行为以及生长的影响。结果表明：PFOS暴露对斑马鱼胚胎孵化率、孵出仔鱼死亡率与畸形率的影响显著（P﹤0.05）,10 mg/L PFOS暴露导致胚胎孵化率下降,孵化延迟,仔鱼死亡率与畸形率升高;PFOS暴露4 dpf（day post-fertilization,dpf）或8 dpf 对斑马鱼仔鱼心率影响显著（P﹤0.05）,心率随PFOS暴露浓度升高而增加;PFOS 暴露6 dpf 或9 dpf 对斑马鱼仔鱼的运动行为影响显著（P﹤0.05）,10 mg/L PFOS暴露6 dpf 导致运动斑马鱼仔鱼比例和仔鱼最大持续运动距离增加（P﹤0.05）,PFOS暴露9 dpf,单位时间内仔鱼的运动距离、停顿频率、平均每次运动距离随PFOS暴露浓度升高而减少（P﹤0.05）,最大持续运动距离随PFOS暴露浓度升高而增加（P﹤0.05）,呈剂量依赖的毒理学效应;PFOS暴露导致斑马鱼仔鱼体长和吻宽下降（P﹤0.05）或有下降的趋势,并对吻宽/体长、吻宽/头长影响显著（P﹤0.05）。以上研究结果提示：PFOS对斑马鱼胚胎及仔鱼具有显著的发育与行为毒性,仔鱼心率、运动行为、吻宽/体长以及吻宽/头长等是评估水体PFOS污染敏感而有效的生物标志物。     

不同林龄胡杨克隆繁殖根系分布特征及其构型

以中龄林和成熟林胡杨为研究对象,采用挖剖面和根窗的方法,研究胡杨繁殖根系分布、根系构型,以及胡杨根蘖与繁殖根系构型之间的关系。结果表明:(1)细根(d<2 mm)的根长密度、根表面积密度,随深度增加呈现指数函数分布;(2)中龄林细根的根长密度、根表面积密度在0—90 cm各层都是显著大于成熟林的对应指标(P<0.05),成熟林的中等粗根(5 mm0.05),且两种林龄的一级侧根数、分枝角度亦无显著差异(P>0.05);(5)对比两种林龄不同根序上的根蘖芽发现,二级根上不定芽个数均是同组一级根上不定芽个数的3—4倍;基于以上对胡杨根系的功能权衡的分析,得出:细根对胡杨根系构型有重要的影响,在胡杨根系功能权衡中扮演重要角色。