Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli

Journal of Industrial Microbiology & Biotechnology - Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA...     

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.     

MicroRNA-21与平滑肌蛋白22α在哈萨克族食管癌组织中的表达及其临床病理学意义

目的探讨microRNA21与SM22a基因在哈萨克族食管癌发生发展中的作用及临床意义。方法免疫组织化学法检测162例石蜡包埋食管鳞状细胞癌组织及RT-PCR方法检测47例哈萨克族食管癌标本中microRNA21、SM22a表达水平,分析这些基因与临床病理特征的关系。结果SM22a在162例食管鳞癌组织中的阳性表达率(87.0%)显著高于食管正常黏膜组织(36.0%);在47例哈萨克族食管癌组织中,SM22a表达水平较远端无癌组织增高。与远端无癌组织相比,microRNA21在哈萨克族食管癌组织中表达水平增高。MicroRNA21高表达与分化程度、淋巴结转移、临床分期相关,SM22a高表达与临床分期相关、淋巴结转移相关;microRNA21与SM22a的表达呈正相关。结论MicroRNA21、SM22a协同高表达共同参与哈萨克族食管癌的侵袭发展过程。     

药用白及资源再生策略初探

白及为兰科白及属植物。作为著名中药,白及功效多样、用途广泛,具有较高的药用价值和经济价值。随资源利用强度的增加,野生白及资源日渐萎缩,人工规模化种植是实现白及药用资源可持续利用的必由之路。优良种质及繁育技术是种植业的关键问题。为了更好地推动白及资源研究和可持续利用,本文就白及种质及快繁技术研究进展作一综述,并就白及资源再生策略进行初步探讨,为相关研究和生产提供参考。     

Combined Treatment with Cadmium and Zinc Enhances Lateral Root Development by Regulating Auxin Redistribution and Cell-Cycle Gene Expression in Rice Seedlings

Russian Journal of Plant Physiology - Enhanced lateral root (LR) development is of critical importance for rice plants adapting to heavy-metal-stress conditions. LR development is affected by heavy...     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.     

In vivo monitoring of thrombosis in mice by optical coherence tomography

The objective of this study is to establish a novel method for continuously monitoring thrombus progression with various outcome measures and to assess the efficacy of antithrombotic drugs in murine thrombosis model in mice. In the study, thrombus was induced in the femoral vein of mice by FeCl₃ and monitored over time by spectral‐domain optical coherence tomography (OCT). Three‐dimensional images of thrombi with or without heparin as an antithrombotic agent were obtained from OCT angiography. In addition, several parameters of thrombi were analyzed and compared between control and anticoagulant groups. By using OCT, we were able to trace thrombus generation in the same mouse in real time. We found that in our model heparin reduced thrombus size by ~60% and thrombus cross‐sectional area by 50%. OCT results also show that both time to thrombus size (>0.02mm³) and time to occlusion (>30%) were significantly reduced after heparin addition. This study demonstrates that OCT reliably monitors thrombus generation and progression from various aspects including thrombus size. This enables us to measure the kinetic of thrombosis more accurately, and effectively evaluate the efficacy and activities of antithrombotic drugs. This model may represent a useful tool in antithrombotic drug discoveries in preclinical studies.