小鼠休眠胚胎与正常胚胎冻融后体内外发育潜力的比较

目的检验小鼠休眠胚胎冻融后的质量及体内外发育潜力,为胚胎休眠技术的生产应用提供必要的参考。方法采用常规冷冻方法将正常孵化期胚胎和休眠胚胎进行冷冻,之后分别进行体外复苏培养实验和胚胎移植实验。随后利用双重荧光染色的方法分别对冻融前后的小鼠休眠胚胎与正常孵化期胚胎进行细胞计数,观察两种胚胎冻融前后的质量变化。结果休眠胚胎的冷冻解冻回收率、发育率均极显著高于孵化期胚胎(72.1%vs 50.2%,P<0.01;94.2%vs 73.9%,P<0.01)。休眠胚胎的移植妊娠率显著高于孵化期胚胎(40.8%vs 30.1%,P<0.05)。休眠胚胎的内细胞团细胞数显著高于孵化期胚胎(27.83 vs 19.53,P<0.05),滋养层细胞数差异不显著。冻融培养后休眠胚胎的内细胞团数,滋养层细胞数均显著高于孵化期胚胎(25.18 vs 14.68,P<0.05;114.09 vs 73.88,P<0.05)。结论小鼠休眠胚胎冻融后胚胎质量及体内外发育潜力均优于小鼠正常孵化期胚胎。     

重组人源性胶原蛋白的制备及表征

为了获得可实现工业化生产的重组人源性胶原蛋白,根据人I型胶原蛋白Gly-X-Y序列,优选亲水性的Gly-X-Y胶原肽段设计人源性胶原蛋白氨基酸序列及对应的核苷酸序列,利用酶切技术构建pPIC9K-COL表达载体,电转化毕赤酵母获得人源性胶原蛋白毕赤酵母工程菌,并对其进行发酵罐发酵、纯化及鉴定。结果显示,获得表达量达4.5 g/L,纯度大于95%的人源性胶原蛋白,经氨基酸N端测序、分子量测定、氨基酸分析及胶原酶降解试验,确定获得的蛋白与理论的人源性胶原蛋白一级结构一致;同时胶原经冷冻干燥后进行扫描电镜分析及细胞毒性试验,确定人源性胶原蛋白冻干品具有多孔纤维网状结构及优良的细胞相容性,预示其具备作为生物医学材料的潜质。     

沙蒿AQP基因结构预测及干旱胁迫下的时空表达模式研究

为探究水通道蛋白(AQP)在沙蒿响应干旱胁迫中的作用机制,该研究以青海省柴达木盆地沙蒿为试验材料,采用RACE技术对其AQP基因进行扩增,获得沙蒿AQP全长克隆并对AQP蛋白进行结构预测和分析;采用qRT-PCR对沙蒿AQP基因在不同程度干旱胁迫以及不同组织部位的表达模式进行分析。结果表明:(1)成功克隆获得沙蒿AQP基因长746 bp的片段1和长534 bp的片段2,经拼接后得到全长cDNA序列,沙蒿AQP基因总长为864 bp。(2)亚细胞定位表明沙蒿AQP基因定位于细胞膜上;同源比对显示沙蒿与向日葵、莴苣、橡胶树等植物的AQP基因具有较高的相似性;结构预测表明AQP蛋白含6个跨膜螺旋结构且亲水性较弱,α螺旋和无规则卷曲为AQP蛋白二级结构的主要构成元件。(3)qRT-PCR分析表明,沙蒿AQP基因随着干旱胁迫的加重呈现有规律的变化,根、茎、叶中表达均上调,且叶中AQP基因表达量上调幅度最大。研究表明沙蒿AQP基因结构特征及其表达模式都是沙蒿对干旱胁迫的一种适应。     

Stratigraphic record of U37K in Sanmen Bay: the effect of SST and EI Niño on life activity in macrobenthos communities

Sea surface temperature (SST) from 1916–2003 was estimated by using the index of U₃₇^K of organic compounds in Sanmen Bay (SMB) sediment. It fluctuated between 15.97°C and 18.00°C with an average of 17.03°C. The estimated SST was 3.52°C lower than the annual average temperature measured in the same year but was close to the temperature measured in fall. The research suggested that the climate events (EI Ni?o) on large scale in Sanmen Bay were affected by the climate, similar to that in the East Pacific Ocean (EPC). The change scale was also affected by geographical location and local climate such as monsoon. According to the chronological basis, the events in SMB corresponded to those in EPC, but the scale of variation is not so strong as that in EPC. By comparing the community, biomass, individual density and biodiversity of macrobenthos during EI Ni?o (between 2002 and 2003) and non-EI Ni?o (between 2005 and 2006) periods, it was indicated that certain macrobenthos communities in SMB responded to EI Ni?o events. During the EI Ni?o period, Taiwan warm current (TWC) is much stronger than that in normal years. The diversity of macrobenthos during 2002 and 2003 was relatively low owing to invasion of the warm current that caused salinity change. The macrobenthos and zooplankton in Sanmen Bay responded to EI Ni?o events significantly. The diversity of macrobenthos decreased significantly because TWC invaded into the shallow bay from the floor and thus changed the temperature and salinity in the bay. On the other hand, zooplankton species, biomass and abundance increased in the period of EI Ni?o since the invaded warm current brought abundant species of zooplankton.     

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC₅₀ of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.     

受脱落酸调节的拟南芥F-box基因的差异表达分析

脱落酸(Abscisic acid,ABA)是一种重要的植物激素,在种子休眠的建立、种子萌发、根发育和非生物胁迫反应过程中发挥作用。F-box蛋白是E3泛素连接酶SCF复合体的组成部分,通过特异识别和调节底物蛋白水平而调控植物生长发育过程。通过分析GEO基因芯片,筛选到38个受ABA调节的拟南芥候选F-box基因。选择其中6个F-box基因,进行实时荧光定量PCR分析。研究结果与基因芯片结果基本一致。分析启动子,发现候选基因含有大量ABA、干旱和胁迫相关的顺式作用元件。分析基因表达谱,发现部分基因在保卫细胞、种皮、花粉和衰老叶片中呈现高表达;大部分基因在ABA处理、胁迫和种子吸胀过程中表达量改变显著。这些分析结果为深入研究ABA调节植物生长发育和抗逆的分子机制提供了线索。     

MiR-323a regulates ErbB3/EGFR and blocks gefitinib resistance acquisition in colorectal cancer

The rapid onset of resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) limits its clinical utility in colorectal cancer (CRC) patients, and pan-erb-b2 receptor tyrosine kinase (ErbB) treatment strategy may be the alternative solution. The aim of this study was to develop a possible microRNA multi-ErbB treatment strategy to overcome EGFR-TKI resistance. We detect the receptor tyrosine kinase activity in gefitinib-resistant colorectal cancer cells, ErbB3/EGFR is significantly activated and provides a potential multi-ErbB treatment target. MiR-323a-3p, a tumor suppressor, could target both ErbB3 and EGFR directly. Apoptosis is the miR-323a-3p inducing main biological process by functional enrichment analysis, and The EGFR and ErbB signaling are the miR-323a-3p inducing main pathway by KEGG analysis. MiR-323a-3p promotes CRC cells apoptosis by targeting ErbB3-phosphoinositide 3‐kinases (PI3K)/PKB protein kinase (Akt)/glycogen synthase kinase 3 beta (GSK3β)/EGFR-extracellular regulated MAP kinase (Erk1/2) signaling directly. And miR-323a-3p, as a multi-ErbBs inhibitor, increase gefitinib sensitivity of the primary cell culture from combination miR-323a-3p and gefitinib treated subcutaneous tumors. MiR-323a-3p reverses ErbB3/EGFR signaling activation in gefitinib-resistant CRC cell lines and blocks acquired gefitinib resistance.Subject terms: Colorectal cancer, Cancer therapeutic resistance     

利用CRISPR/Cas9系统检测果蝇细胞中组蛋白甲基化修饰对20E信号传导的调控

[目的]利用CRISPR/Cas9基因敲除系统在黑腹果蝇Drosophila melanogaster细胞中筛选并检测组蛋白甲基化修饰对蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)信号传导的调控.[方法]通过Flybase网站分析并总结黑腹果蝇组蛋白甲基转移酶及其修饰位点;将5个组蛋白甲基转...