采用代谢组学分析技术分析工业啤酒发酵过程中风味物质生成规律

啤酒风味是保证啤酒品质的关键因素之一。运用代谢组学的方法,分析工业啤酒发酵过程中酵母胞内代谢物和啤酒风味物质的对应关系,从代谢水平上研究风味物质形成过程中的关键影响因素。在啤酒发酵过程中,同时检测风味物质的含量变化和酵母胞内代谢物的变化,对得到海量的、多维的代谢数据采用主成分分析（PCA）和偏最小二乘分析（PLS）的多元统计分析方法进行处理。由PCA分析结果可知：磷酸、海藻糖、琥珀酸、谷氨酸、天冬氨酸、丙氨酸对主成分贡献比较大,说明这些代谢物在不同发酵阶段含量变化显著。由PLS分析结果可知：对啤酒风味影响最大的物质主要为氨基酸,包括丝氨酸、缬氨酸、苏氨酸、赖氨酸、丙氨酸、亮氨酸和天冬酰胺等,这为啤酒中风味物质的调控提供了一定的理论指导。     

Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.     

Melatonin inhibits triple-negative breast cancer progression through the Lnc049808-FUNDC1 pathway

Melatonin has been reported to have tumor-suppressive effects via comprehensive molecular mechanisms, and long non-coding RNAs (lncRNAs) may participate in this process. However, the mechanism by which melatonin affects the function of lncRNAs in triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, is still unknown. Therefore, we aimed to investigate the differentially expressed mRNAs and lncRNAs in melatonin-treated TNBC cells and the interaction mechanisms. Microarray analyses were performed to identify differentially expressed mRNAs and lncRNAs in TNBC cell lines after melatonin treatment. To explore the functions and underlying mechanisms of the mRNAs and lncRNAs candidates, a series of in vitro experiments were conducted, including CCK-8, Transwell, colony formation, luciferase reporter gene, and RNA immunoprecipitation (RIP) assays, and mouse xenograft models were established. We found that after melatonin treatment, FUNDC1 and lnc049808 downregulated in TNBC cell lines. Knockdown of FUNDC1 and lnc049808 inhibited TNBC cell proliferation, invasion, and metastasis. Moreover, lnc049808 and FUNDC1 acted as competing endogenous RNAs (ceRNAs) for binding to miR-101. These findings indicated that melatonin inhibited TNBC progression through the lnc049808-FUNDC1 pathway and melatonin could be used as a potential therapeutic agent for TNBC.Subject terms: Breast cancer, Non-coding RNAs     

Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.     

Identification of blapsins A and B as potent small-molecule 14-3-3 inhibitors from the insect Blaps japanensis

In this study, we report three novel naturally occurring compounds, blapsins A (1) and B (2), and blapsamide (3) from the ethanol extract of the stink beetle, Blaps japanensis. The structures of these compounds were determined using spectroscopic methods. Compound 3 is a phenolic compound bearing a formamido group in the structure. Functional studies revealed that compounds 1 and 2 potently inhibited 14-3-3 protein-protein interactions (PPIs) with IC(50) values of 9.2 and 10.0 μM as determined by an ELISA assay, and 2.0 and 2.5 μM in an FP assay, respectively. These compounds represent the first example of natural small-molecule 14-3-3 inhibitors.     

花颜色和花气味的量化研究方法

花颜色和花气味是花部构成的重要内容。在已开展的传粉生态学研究中对二者的报道主要是描述性的,而其量化研究可以为揭示传粉机制提供有力的实验证据。本文主要介绍了花颜色的测量和标定方法,包括比色卡、分光色差仪和便携式光谱仪等;花气味的采集方法,包括动态顶空套袋-吸附采集法、吸附-溶剂洗脱法和固相微萃取法等;花气味的检测和分析方法,包括气相色谱-质谱联用仪分析和电子鼻型超速气相色谱仪分析等;以及昆虫行为学实验方法,包括气相色谱-昆虫触角电位联用技术、Y型嗅觉仪和飞行箱实验等。科研人员可以根据实验材料的特点和实验目的选择适合的量化研究方法。     

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ～30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.