Osteoimmunology of Retarded Bone Regeneration in Periodontitis

本多样性行政补助金申请书(PA-21-071)是为申请为期一年而编写的 支持西班牙裔后单身女实习生伊丽莎白·利昂女士的父母RO1赠款，题为， 《关于牙周炎骨再生的骨免疫学研究》她将进行基础研究，以 阐明牙周炎病理性骨吸收的机制。通过建议的 在培训期间，她不仅会熟悉操作方法，还会进行与 对于口腔疾病，还要掌握寻找知识鸿沟的概念性方法和解决办法 存在的问题。提高精确度和重复性的基本原则，以及 负责任的研究行为也将被学习。与此同时，她将努力争取在 牙科专业。该培训的目标是支持未来临床医生/科学家的职业发展 在牙科的基础研究和翻译研究方面。 候选人的研究项目背景：我们未发表的数据表明，牙龈假单胞菌的水泡 能促进破骨细胞的生成，而Pad-KO的囊泡能明显抑制破骨细胞的生成 牙龈假单胞菌。此外，牙龈假单胞菌的小泡可上调RANKL-1中OC-STAMP的表达。 刺激RAW264.7破骨细胞前体细胞。 假设：我们假设牙龈假单胞菌的囊泡表达的PAD可以使宿主来源的瓜氨酸化。 波形蛋白反过来作用于破骨细胞表达的OC-STAMP，促进破骨细胞的生成 应聘者的项目目标： 目的：探讨牙龈假单胞菌小泡表达的PAD(PG Vesicle-Pad)在牙周炎中的作用。 在囊泡和破骨细胞的共培养中，瓜氨酸化宿主蛋白，包括Vimentin。 目的：检测PG水泡垫介导的宿主波形蛋白的瓜氨酸化是否会改变 波形蛋白在促进RANKL诱导破骨细胞生成中的作用 囊泡将从PAD-KO PG W83(来自Potempa博士的礼物)和WT PG W83中分离出来 超速离心机。这些分离的囊泡以及整个细菌细胞将与 破骨细胞前体。宿主蛋白的瓜氨酸化水平将通过W-Blot和MS-Spec来确定。 将使用W-印迹技术监测囊泡中Pad的表达。最后，奥委会可能的参与- 破骨细胞表达的STAMP是破骨细胞中瓜氨酸化Vimentin的受体 用抗OC-STAMP单抗或抗瓜氨酸Vimentin单抗封闭测定。 在完成这两个目标后，候选人将掌握细胞培养的方法，W- 印迹、定量聚合酶链式反应、流式细胞仪和破骨细胞生成分析，以及基本的统计分析和 对数据的解读增强了她成为研究生课程候选人的资质。

This application for Diversity Administrative Supplement (PA-21-071) is written to request for the one-year support for Hispanic Post Bachelorette Trainee, Ms. Elizabeth Leon, under the parent RO1 grant, titled, “Osteoimmunology of regarded bone regeneration in periodontitis.” She will perform the basic research to elucidate the mechanism underlying the pathogenic bone resorption in periodontitis. Through the proposed training, she will not only become familiar with the operational approach to perform basic research related to the oral diseases, but also master the conceptual approach to find the knowledge gap and the way to solve the existing problem. The fundamentals for increasing the rigor and reproducibility, as well as the principle for responsible research conduct will be also learned. Parallelly, she will be working toward acceptance in the program of dentistry. The goal of this training is to support the career development of a future clinician/scientist in the basic and translational research in the dental sciences. Candidate’s research project background: Our unpublished data demonstrated that P. gingivalis’ vesicles can promote the osteoclastogenesis which was significantly attenuated by the vesicles isolated from PAD-KO P. gingivalis. Furthermore, P. gingivalis’ vesicles up-regulated the expression of OC-STAMP in the RANKL- stimulated RAW264.7 osteoclast precursor cells. Hypothesis: We hypothesized that PAD expressed by vesicles of P. gingivalis can citrullinate the host derived Vimentin which, in turn, acts on OC-STAMP expressed by osteoclasts and promotes osteoclastogenesis Project Objectives for Candidate: Objective 1: To evaluate the role of PAD expressed by vesicles of P. gingivalis (Pg vesicle-PAD) in citrullinating the host proteins, including Vimentin, in the co-culture of vesicles and osteoclasts. Objective 2: To examine whether Pg vesicle-PAD-mediated citrullination of host vimentin can alter Vimentin’s effect on promoting RANKL-induced osteoclastogenesis. The vesicles will be isolated from PAD-KO Pg W83 (gift from Dr. Potempa) and WT Pg W83 using a ultracentrifuge machine. Those isolated vesicles, as well as whole bacterial cells, will be co-cultured with osteoclast precursors. The level of citrullination of host proteins will be determined by W-blot and Mass-Spec. The expression of PAD by vesicles will be monitored using a W-blot. Finally, possible engagement of OC- STAMP expressed by osteoclasts which is the receptor for citrullinated Vimentin in osteoclastogenesis will be determined by blocking with anti-OC-STAMP mAb or anti-citrullinated Vimentin mAb. After completing these two objectives, the candidate will have mastered the methods of cell culture, W- blotting, qPCR, flow cytometry, and osteoclastogenesis assays, as well as basic statistical analysis and interpretation of data which strengthen her credential to be a candidate for graduate program.