Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice
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Author:
Date:
2020-05-20
[Abstract] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in ...
[摘要] [摘要 ] T细胞急性淋巴细胞白血病(T-ALL)是一种侵袭性血液恶性肿瘤,其起源于T细胞引发的造血祖细胞的转化。尽管T-ALL是一种异质且分子复杂的疾病,但超过65%的T-ALL患者在NOTCH1 基因中带有激活突变。大多数与T-ALL相关的NOTCH1 突变要么破坏负调控区,允许在没有配体结合的情况下激活信号,要么导致蛋白酶体降解终止NOTCH1信号终止所涉及的C末端PEST域被截短。迄今为止,逆转录病毒转导模型在很大程度上依赖于侵袭性截短的变种的过度表达。 NOTCH1 (例如ICN1或ΔE-NOTCH1)可导致信号传导的超生理水平,并且在人类T-ALL中很少见。当前方案描述了小鼠骨髓分离,造血干细胞和祖细胞(HSC)富集,然后逆转录病毒转导的致癌突变体形式的NOTCH1受体(NOTCH1-L1601P-ΔP)的方法,该方法与获功能突变最常见于患者样品中。组成型活性NOTCH1的这种强制表达的标志是胸腺外未成熟T细胞发育的瞬时波,此波在致癌性转化为T-ALL之前。此外,该方法通过允许白血病细胞与微环境之间的串扰来模拟体内白血病的转化和进展,这是基于细胞系的体外研究无法解释的一个方面。因此,HSC转导和移植模型更真实地概括了人类疾病的发展,为进一步的体内和离体功能研究提供了高度全面和通用的工具。
[背景 ] ...
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Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry
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Author:
Date:
2018-08-20
[Abstract] Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. This methodology is not amenable to the combinations of multiple markers necessary to unequivocally define discrete immune cell populations. We have developed a multi-parameter, flow cytometric-based approach that addresses the need for panels of cell-specific markers in the identification of immune cell populations, allowing both the accurate detection and quantitation of leukocyte subpopulations from a single, clinical kidney biopsy specimen. In ...
[摘要] 炎症性免疫细胞在急性肾损伤(AKI)和慢性肾病(CKD)的病例中发挥直接的病理作用。然而,人肾活组织检查中不同白细胞群的鉴定和表征已被用于检测它们的基于免疫组织化学(IHC)的技术的局限性所混淆。该方法不适用于明确定义离散免疫细胞群所必需的多种标志物的组合。我们开发了一种多参数,基于流式细胞仪的方法,解决了在鉴定免疫细胞群体时需要细胞特异性标记组,从而允许从单个临床肾活检标本中准确检测和定量白细胞亚群。在该方法中,将新鲜的人肾组织解离成单细胞悬浮液,然后进行抗体标记和基于流式细胞术的采集和分析。这种新技术为鉴定和计数人类肾脏疾病中的免疫细胞亚群提供了重大进步,并且是补充临床肾脏活组织检查的传统组织病理学检查的有力平台。
【背景】 肾脏疾病的发病率和患病率正在上升,给全世界的医疗保健社区带来了沉重的负担,并显着影响了患者的生活质量(世界肾脏日:慢性肾病,2015年)。肾脏损害可能由多种损伤引起,包括感染,缺血,毒素,高血压,遗传和代谢紊乱(Imig和Ryan,2013)。对功能性损伤的即时反应是急性肾损伤(AKI),这是一种临床综合征,其特征在于生理上通过肾功能的快速(数小时至数天)降低和组织形态学上通过炎性免疫细胞浸润到肾小管中,组织隔室邻接小管。肾脏(Basile ...
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Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
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Author:
Date:
2018-03-20
[Abstract] Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.
[摘要] 核小体是真核染色体包装的基本单元,由围绕核心组蛋白H2A,H2B,H3和H4中的每一个的两个分子包裹的147bp DNA组成。 核小体是对称的,一个对称轴以H3分子的C-末端之间的同源二聚体相互作用为中心。 为了探索核小体对称性的功能性后果,我们设计了一对特异性H3异二聚体,称为H3X和H3Y,使我们能够比较具有单一或双重H3改变的细胞。 我们对异二聚体X-Y相互作用的生物化学验证包括使用二甲基琥珀三酸酯(DMS)进行的核内H3交联。 在这里,我们提供了使用DMS来分析酵母核小体的详细方案。
【背景】组蛋白的翻译后修饰影响染色体生物学的各个方面,包括转录,复制,修复和重组。因为核小体包含每个核心组蛋白的两个拷贝,所以修饰可以是对称的(在两个H3尾部上的相同修饰,例如,在核小体内的两个H3尾部上的K27me(Voigt等人
对于单个核小体内H3X-H3Y相互作用的生化验证,我们生成了表达细菌生物素连接酶BirA,N-末端V5-标记的H3X和N-末端生物素接受表位标记的H3Y的酵母菌株(Beckett等人, 1999)。 ...
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