| A Robust Mammary Organoid System to Model Lactation and Involution-like Processes
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Author:
Date:
2021-04-20
[Abstract] The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 ...
[摘要]
[摘要]乳腺是一种高度动态的组织，在整个生殖生活中都会发生变化，包括青春期的生长以及怀孕和进化的重复周期。乳腺肿瘤诊断代表在世界女性最常见的癌症宽。研究的监管机制乳腺的发育是至关重要的理解荷兰国际集团d如何YS调节可导致乳腺癌的发生和发展。三维（3D）乳腺组织体提供了许多令人激动的可能性的研究的组织发育和乳腺癌。在第E存在衍生自协议Sumbal等人，我们描述一个简单的3D类器官系统的研究的泌乳和复古体外。我们使用成纤维细胞生长因子2 （FGF2）和催乳素刺激的原代和传代小鼠乳腺类器官来模拟小鼠乳腺泌乳和内卷过程的三个周期。这种3D模型类器官代表一个有价值的工具来研究后期产后乳腺的发育和乳腺癌，尤其是产后-相关性乳腺癌。
图形摘要：
乳腺类器官的分离和培养程序
[背景技术]的Th e是乳腺的主要功能是提供营养吨经由牛奶产量Ò新生儿。牛逼乳腺他的发展是主要发生在出生后，由几个因素，包括激素和生长因子调控的一个高度动态的过程（Brisken和拉贾拉姆，2006;斯特恩利希特，2006年）。在青春期，激素和生长因子调节基本的胚胎导管树的导管形态发生（Brisken and ...
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| Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
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Author:
Date:
2021-01-05
[Abstract] All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze c-di-AMP to the linear phosphoadenylate adenosine 5′-pApA or to AMP. Different methods including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and ion exchange chromatography (IEX) can be used to determine activities of c-di-AMP-synthesizing and degrading enzymes. Here, we describe in detail the TLC and IEX methods adapted for characterization of the diadenylate cyclase DisA and the phosphodiesterase AtaC from ...
[摘要] [摘要]所有活细胞均使用环状核苷酸作为第二信使，以进行信号传感和转导。环状二-3 ' ，5 ' -腺苷一磷酸（C-二-AMP）主要涉及细菌和控制euryarcheal osmoadaptation并且由产生diadenylate环化酶从ATP的两个分子。特定的磷酸二酯酶将c-di-AMP水解为线性磷酸腺苷腺苷5'-pApA或AMP。可以使用包括高效液相色谱（HPLC），薄层色谱（TLC）和离子交换色谱（IEX）在内的各种方法来确定c-di-AMP合成和降解酶的活性。在这里，我们详细描述了TLC和IEX方法适合于表征diadenylate环化酶DISA和磷酸二酯酶ATAC从链霉菌venezuelae 。TLC可以快速，轻松地分离放射性标记的底物和产品，而IEX避免了潜在危险的放射性底物的利用，如果没有HPLC系统，则可以用作良好的替代品。与TLC分析不同，无法使用IEX分析并行分析样品，因此更加耗时。
[背景]环核苷酸第二信使是原核和真核信号通路中的关键分子。环状二-3 ' ，5 ' -腺苷一磷酸（三二- AMP）是一种细菌第二信使与许多重要的功能，如渗压剂动态平衡调节，细胞壁代谢，生物膜的形成，DNA完整，孢子形成，毒力和生长（Fahmi et ...
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| Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue
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Author:
Date:
2020-10-20
[Abstract] Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from ...
[摘要] [摘要] 转录组分析可以为不同遗传背景或不同条件下的生物学过程提供线索。RNA测序（RNA-seq）的价格已经下降到足够低的程度，因此在各种条件下进行中大规模转录组分析是可行的。然而，对于那些希望第一次使用RNA-seq或进行单个实验的人来说，RNA-seq库制备的价格和各种选择仍然是令人望而生畏的。选择文库制备方案的标准包括RNA分离方法、核苷酸片段化以获得所需的大小范围，以及文库索引以汇集测序样本进行多路复用。在这里，我们提出了一个高质量和高通量的选择，从多聚腺苷酸mRNA制备文库用于转录组分析。高质量和高通量的方案选择都包括通过磁珠使poly-A尾部沉淀，cDNA合成，然后通过Tn5介导的“标记”同时裂解和添加适配器的步骤。该方案的所有步骤均已通过拟南芥叶片和幼苗组织的验证，并简化为协同工作，在资金和时间上成本最低，因此旨在为转录组分析提供一个初学者友好的从开始到完成的RNA序列库制备。
[背景] 通过Southern印迹、expressed sequence ...
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