| Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis
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Author:
Date:
2020-10-05
[Abstract] Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward α-SMA+ myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i.e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-β could be used as marker for renal pericytes characterization. ...
[摘要] [摘要]越来越多的证据表明，肾小管周围的毛细血管周细胞是慢性肾脏病（CKD）以及急性肾损伤（AKI）早期形成疤痕的成纤维细胞的主要来源。在败血症和I / R（缺血再灌注）损伤诱导的AKI的猪模型中，我们证明了肾周细胞能够向α- SMA +肌成纤维细胞转分化，从而导致间质纤维化。即使精确周细胞识别需要透射电子显微镜和几个标志物联合免疫（即。，的Gli ，NG2硫酸软骨素蛋白聚糖，CD146，结蛋白或CD73）和新兴的新的标志物（CD248或TEM1，唾液酸蛋白），以往的研究表明，PDGFR-β可用作肾周细胞表征的标志物。最近，对PDGFR-β和α-SMA进行了双重免疫荧光染色，以鉴定在纤维化发展的早期受损激活的周细胞（PDGFR-β + /α-SMA +细胞）。我们的数据强调了肾周细胞在败血症和I / R相关性AKI的生理病理中的关键作用。在该协议中，我们描述了猪福尔马林固定石蜡包埋（FFPE）肾脏活检中PDGFR-β和α-SMA双重免疫荧光染色的程序以及图像分析和定量方法。
[背景】肾脏纤维化被认为是主要负责肾脏疾病的进展，其与肾的损伤后的容量有限，再生有关。进行性肾脏疾病中间质纤维化的主要来源（Simone等人，2014 ; ...
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| Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
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Author:
Date:
2018-04-20
[Abstract] Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.
[摘要] 肠道集聚性大肠杆菌（EAEC）是全球儿童和成人急性腹泻的公认原因。 EAEC菌株的特征在于存在聚集粘附菌毛（AAF），其通过介导与肠粘膜的附着和通过引发宿主炎症反应而在发病机制中起关键作用。 聚合粘附菌毛II（AAF / II）是EAEC原型菌株042（EAEC042）对肠细胞最重要的粘附因子。 已经鉴定了上皮细胞上AAF / II的多种受体，包括跨膜信号传导粘蛋白Muc1。 该协议描述了测量EAEC菌株对表达Muc1糖蛋白的HEK293细胞的依从性的方法。
【背景】EAEC是世界范围内地方性和流行性腹泻病的重要原因。尽管发展中国家儿童腹泻最常见，但EAEC还与免疫受损成人腹泻，旅行者和工业化国家的食源性疾病有关，例如由志贺毒素（Stx）2a型产生的大致致命爆发2011年在北欧的血清型O104：H4的EAEC菌株（Harrington等人，2006; Rasko等人，2011）。 EAEC发病机制由生物体粘附肠细胞，产生肠毒素和细胞毒素并最终诱导炎症的能力决定（Harrington等，2006）。 EAEC对肠细胞的依从性由AAF菌毛粘附素介导（Czeczulin等人，1997）。迄今为止，已经描述了至少5种AAF菌毛的变体，全部编码在范围为55至65MDa的毒力质粒中（Jonsson等人，2015）。 ...
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| Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
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Author:
Date:
2017-08-05
[Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of ...
[摘要] 集群定期间隔短回归重复（CRISPR）和CRISPR相关（Cas）系统作为细菌和古菌中的适应性免疫系统，用于防御入侵病毒和质粒（Barrangou和Marraffini，2014）。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向（Jinek et al。，2012; Cong等人，2013; Mali ，2013; Zetsche等人，2015）。然而，真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9（SpCas9）靶向DNA接近的障碍，并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ （Knight等人，，2015; Hinz等人，2015; Horlbeck等人，2016年） ; ...
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