| Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
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Author:
Date:
2020-10-20
[Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily ...
[摘要] [摘要] 比率可能因多种因素而变化，但最主要的因素是复制应力。一些研究应用了不同的方法来估计复制源在不同生物体中的数量和位置。然而，如果没有一个参数来限制必要起源的最小值，那么不太敏感的技术可能会产生相互矛盾的结果。估计每个染色体的最小复制源数量（MO）是一种创新的方法，它允许建立一个阈值，作为绘制起源的基因组方法的参数。为此，MO可以很容易地通过一个公式得到，这个公式需要作为参数：染色体大小、S期持续时间和复制率。在基因组数据库（如NCBI）中可以获得任何生物体的染色体大小，通过监测DNA复制来估计S期的持续时间，并通过DNA组合方法获得复制率。 提供了一种简单、快速的估算MO的方法一个新的方法学框架来协助研究任何有机体。
关键词： DNA复制，复制来源，复制率，S期持续时间，染色体大小
[背景] ...
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| An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens
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Author:
Date:
2020-09-05
[Abstract] Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming.
Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were ...
[摘要] [摘要 ] 组织内真核细胞或病原体的运动性是通过与其他细胞或细胞外基质特异性相互作用的转换来介导的。这些配体-受体粘附的生物物理特征有助于揭示驱动迁移的分子机制。牵引力显微镜或光学镊子通常用于测量细胞在基质上施加的细胞力。但是，牵引力显微镜的空间分辨率仅限于〜2 µm，使用光阱进行实验非常耗时。
在这里，我们介绍了仿生表面的生产，该表面能够通过合成配体实现特定的细胞粘附，同时通过使用分子张力传感器监控传递的力。将配体与双链DNA探针偶联，该探针具有确定的DNA解链力阈值。从而将pN范围内的受体介导力半定量转换为荧光信号，可以通过标准荧光显微镜在分辨率极限（〜0.2 µm）上检测到。
该测定的模块化设计允许改变所呈现的配体和DNA探针的机械强度，这为探测不同的真核细胞类型和病原体的粘附提供了多种可能性，此处以骨肉瘤细胞和伯氏疟原虫子孢子体为例。
[背景 ] 运动细胞和病原体以多种不同方式与环境相互作用（Parsons 等，2010； Nan ，2017； Muthinja 等，2018 ）。例如，跨膜受体将单个细胞锚定在其环境中，并使其与其他细胞相互作用（Hynes ，1992）。整联蛋白是将细胞连接到细胞外基质的主要受体，它以双向方式传递力（Schoen et ...
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| SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
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Author:
Date:
2020-08-05
[Abstract] DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single ...
[摘要] [摘要] DNA双链断裂（dsb）是影响基因组完整性的最具毒性的损伤之一。dsb主要通过非同源末端连接（NHEJ）和同源重组（HR）进行修复。HR过程的一个关键步骤是通过DNA末端切除，产生一个长的3′单链DNA链，这是在DNA链入侵后，以同源区域为模板进行DNA合成所必需的。DNA末端切除抑制NHEJ并触发同源定向的DSB修复，最终保证DNA的可靠修复。已建立的评价DNA末端切除过程的方法是免疫荧光法分析磷酸化S4/8rpa32蛋白病灶（DNA末端切除的标志物）或磷酸化S4/8rpa32蛋白水平。近年来，切除轨迹单分子分析（SMART）被认为是一种可靠的方法，可以通过免疫荧光法观察S期特异性DNA损伤剂（如喜树碱）处理细胞后产生的长3′单链DNA尾。然后，通过图像分析软件（如Photoshop）测量DNA束长度，评价DNA末端切除机的处理能力。DNA纤维的制备是在非变性条件下进行的，因此免疫荧光只检测DSB处理产生的特定的3′单链DNA尾。
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