In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.     

AP2M1 Supports TGF-β Signals to Promote Collagen Expression by Inhibiting Caveolin Expression

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.     

基于深度学习的秀丽隐杆线虫显微图像分割方法

利用计算机实现自动、准确的秀丽隐杆线虫(C.elegans)的各项形态学参数分析,至关重要的是从显微图像上分割出线虫体态,但由于显微镜下的图像噪声较多,线虫边缘像素与周围环境相似,而且线虫的体态具有鞭毛和其他附着物需要分离,多方面因素导致设计一个鲁棒性的C.elegans分割算法仍然面临着挑战。针对这些问题,提出了一种基于深度学习的线虫分割方法,通过训练掩模区域卷积神经网络(Mask R-CNN)学习线虫形态特征实现自动分割。首先,通过改进多级特征池化将高级语义特征与低级边缘特征融合,结合大幅度软最大损失(LMSL)损失算法改进损失计算;然后,改进非极大值抑制;最后,引入全连接融合分支等方法对分割结果进行进一步优化。实验结果表明,相比原始的Mask R-CNN,该方法平均精确率(AP)提升了4.3个百分点,平均交并比(mIOU)提升了4个百分点。表明所提出的深度学习分割方法能够有效提高分割准确率,在显微图像中更加精确地分割出线虫体。     

Glucosamine Extends the Lifespan of Caenorhabditis elegans via Autophagy Induction

Glucosamine (GlcN) is commonly used as a dietary supplement to promote cartilage health in humans. We previously reported that GlcN could induce autophagy in cultured mammalian cells. Autophagy is known to be involved in the prevention of various diseases and aging. Here, we showed that GlcN extended the lifespan of the nematode Caenorhabditis elegans by inducing autophagy. Autophagy induction by GlcN was demonstrated by western blotting for LGG-1 (an ortholog of mammalian LC3) and by detecting autophagosomal dots in seam cells by fluorescence microscopy. Lifespan assays revealed that GlcN-induced lifespan extension was achieved with at least 5 mM GlcN. A maximum lifespan extension of approximately 30 % was achieved with 20 mM GlcN (p<0.0001). GlcN-induced lifespan extension was not dependent on the longevity genes daf-16 and sir-2.1 but dependent on the autophagy-essential gene atg-18. Therefore, we suggest that oral administration of GlcN could help delay the aging process via autophagy induction.     

绿春地区断肠草生物总碱提取工艺优化

优选微波法提取断肠草生物总碱的工艺,以断肠草生物总碱的提取率为指标,考察不同提取条件下的提取率。先通过单因素实验对断肠草生物总碱的提取工艺中溶剂种类、提取系统中固液比、浸提时间、浸提温度4个因素进行优选,进而考察多因素协同作用对生物总碱提取率的影响设计了L9（33）的正交实验。结果表明,采用氯仿作为提取溶剂、微波功率500W、固液比1∶8（g/L）、浸提时间240 s（80 s×3）、浸提温度55℃时生物总碱得率最高为1.63%,因此,以氯仿为提取溶剂,经微波辅助技术处理后生物总碱提取量显著得到了提高。     

高温豆粕肽粉的苦味及ACE抑制活性研究

以高温豆粕为原料,Alcalase2．4L酶为催化剂,通过L9（3^4）止交试验,获得制备ACE抑制肽的最佳条件为：底物浓度4％、酶活力1500U／g、pH值8．5、温度50℃,此时ACE抑制活性达到78．85％。在此基础上,采用毛霉蛋白酶酶解3h、6h和l％、2％的β-环糊精（β-CD）包埋进行脱苦处珲,试验结果表明,1％的β-CD包埋对高温豆粕酶解液具有最佳的脱苦和最人保存其ACE抑制沂性的作用。     

腐乳生产过程中自由基清除能力的变化

实验用DPPH法对腐乳的抗氧化能力变化趋势作了测定，前酵和盐腌期迅速上升，后酵期上升比较缓慢，最终产品腐乳的自由基清除能力比发酵前的豆腐坯高了2倍以上。并且发现，用雅致放射毛霉和少根根霉发酵腐乳，前者的自由基清除能力更高。