| Intestinal Co-culture System to Study TGR5 Agonism and Gut Restriction
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Author:
Date:
2021-03-20
[Abstract] The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5 agonists as type 2 diabetes therapeutics. However, the clinical application of TGR5 agonists has been hampered by side effects of these compounds that primarily result from their absorption into circulation. Here we describe an in vitro screening protocol to evaluate the TGR5 agonism, GLP-1 secretion, and gut-restricted properties of small molecules. The protocol involves differentiating gut epithelial and endocrine cells together in transwells to ...
[摘要] [摘要]肠内分泌L细胞中的Takeda G蛋白受体5（TGR5，也称为G蛋白偶联胆汁酸受体1，GPBAR1）的激活导致抗糖尿病激素胰高血糖素样肽1的分泌。 （GLP-1）进入全身循环。因此，最近的研究集中于鉴定和开发TGR5激动剂作为2型糖尿病治疗剂。但是，TGR5激动剂的临床应用受到这些化合物的副作用的阻碍，这些副作用主要是由于它们吸收进入循环系统所致。这里我们描述一个体外 筛选协议以评估TGR5激动剂，GLP-1分泌和小分子的肠道限制性特性。该协议涉及在跨孔中将肠道上皮细胞和内分泌细胞一起分化，以评估TGR5激动剂的药效学和化合物对肠道单层的毒性。作为概念的证明，我们证明了该协议在评估有效的TGR5激动剂自然存在的胆汁酸代谢物的性质中的应用。该协议改编自Chaudhari等人。（202 1 ）。
[背景和d ] GI道的肠壁是由几个不同类型的细胞，每一个特定的和，有时独有的功能的（阿莱尔等人。，2018） ...
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| Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin
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Author:
Date:
2014-06-05
[Abstract] ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive ...
[摘要] ROS诱导的DNA损伤在时间和空间背景下在活细胞中修复，并且染色质结构对于原位DNA修复过程的考虑是关键的。众所周知，染色质重塑因子参与许多DNA损伤修复途径，表明染色质重塑促进DNA损伤修复的重要性。到目前为止，还没有方法诱导活细胞中的位点特异性氧化性DNA损伤。因此，不知道DNA修复机制在活性或凝集的染色质中是否不同。我们最近建立了一种新的方法，DTG（损害靶向一个基因组位点），研究活性氧（ROS）诱导的DNA损伤活动细胞中的DNA损伤反应在一个基因组活性或无活性转录。为此，我们在U2OS细胞中在X染色体上整合了四环素响应元件（TRE）盒（〜90kb）（Lan等人，2010），然后融合KillerRed（KR）刺激的ROS诱导物，其可以特异性产生ROS诱导的DNA损伤，tet-阻遏物（tetR-KR，OFF）或转录激活物（TA-KR，ON）（Lan等人， ，2014）（图1）。 TetR-KR或TA-KR分别结合TRE盒并在异源或真核细胞状态下诱导ROS损伤。如何染色质状态调节DNA损伤反应过程可以通过使用这种强大的方法来检查。
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