A Novel Class of Enzyme Sensors to Elucidate the Biochemical Responses of Human Nasal Epithelial Cells to Heavy Metals

一类新型酶传感器可阐明人鼻上皮细胞对重金属的生化反应

• 批准号: 9755727
• 负责人: Matthew M Anttila
• 金额: $ 3.68万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755727
• 关键词: Asthma; Biochemical; Biochemical Reaction; Biological; Biological Assay; Capillary Electrophoresis; Cell Membrane Permeability; Cell Survival; Cells; Cellular Assay; Cellular Stress; Chemical Exposure; Chemicals; Cytometry; Detection; Development; Disease; Enzymes; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Exposure to; Fluorescence; Fostering; Heavy Metals; Heterogeneity; Human; Individual; Inflammation; Inhalation; Investigation; Irrigation; Kinetics; Knowledge; Malignant Neoplasms; Measurement; Measures; Metal exposure; Metal fever; Methods; Modification; Morbidity - disease rate; Mucous body substance; Normal Range; Nose; Occupational; Pathway interactions; Peptides; Permeability; Phosphorylation Site; Phosphotransferases; Physical Performance; Population; Population Heterogeneity; Public Health; Reaction; Reagent; Recovery; Reporter; Research; Route; Sample Size; Sampling; Signal Transduction; Sputum; Stimulus; Structure of respiratory epithelium; Technology; Testing; Time; Tissues; Toxic effect; UV Radiation Exposure; Work; base; cell behavior; cell type; design; enzyme activity; hydrophilicity; improved; innovation; insight; lead ion; mortality; novel; novel strategies; photoactivation; resilience; respiratory; response; sample fixation; sensor; sensor technology; single cell analysis

7. Project Summary Heavy metals comprise a major class of chemical exposure agents and have a significant impact on public health causing morbidity and mortality following environmental, occupational, and/or ambient exposures.1 Inhalation is one of the most common routes for heavy metal exposures, and is known to cause respiratory inflammation, cancers, metal fume fever, asthma, and reduced physical performance.2-6 Importantly, it is known that aberrant activity of Akt kinase and/or epidermal growth factor receptor kinase (EGFR) occurs within respiratory epithelial tissue following exposures to heavy metals.6-9 However, due to the inherent cellular heterogeneity of cells within the respiratory epithelium and the dynamic responses of single cells to chemical stimuli;10 the effects of heavy metal exposures on the signaling dynamics on single cells across different cell types within the respiratory epithelium is unknown.11 Therefore, a single cell analysis technology that can obtain multiplexed measurements of Akt & EGFR activity in ultra-small samples of primary respiratory epithelial cells, would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures. Importantly, such a technology can potentially enable clinicians to identify early warning signs of heavy metal induced toxicity and/or disease induction in individuals from very small, heterogeneous primary samples. I aim to improve biochemical investigations of the respiratory epithelium, by employing sensor based chemical cytometry. Sensor based chemical cytometry is a single cell analysis method in which biomolecular sensors are used to measure signaling dynamics in small populations of single cells.12-16 Here, I propose the development of a novel set of fluorescent enzyme sensors to obtain multiplexed measurements of Akt & EGFR within single cells using capillary electrophoresis employing fluorescence detection (CE-F). I also aim to improve the design and functionality of the proposed enzyme sensors; by installing photoactivatable moieties on the phosphorylation sites, I expect to improve membrane permeability of the sensors, and gain control over the kinase reaction start time within cells.16 Additionally, I plan to control the kinase reaction stop time in cells by developing a novel chemo-selective reagent which halts intracellular reactions, and facilitates reporter recovery for analysis via CE-F. Studies made possible using these novel enzyme sensors will bolster our understanding of the biochemical mechanisms that govern the induction of disease and/or resilience from ultra-small populations of primary respiratory epithelial cells. Additionally, the knowledge gained from this proposal would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures, while identifying novel strategies to develop cell permeable sensors to achieve temporally controlled reactions within single cells.

7。项目摘要 重金属包括主要的化学暴露剂类别，并对公众产生重大影响 在环境，职业和/或环境暴露后，健康导致发病率和死亡率。1 吸入是重金属暴露的最常见路线之一，已知会引起呼吸系统 炎症，癌症，金属烟气，哮喘和身体表现降低。2-6重要的是，已知 Akt激酶和/或表皮生长因子受体激酶（EGFR）的异常活性发生在 暴露于重金属后的呼吸道上皮组织。6-9然而，由于固有的细胞 细胞在呼吸道上皮内的异质性以及单细胞对化学的动态反应 刺激; 10重金属暴露对信号动力学对单个细胞跨不同细胞的影响 11因此，一种单个细胞分析技术，可以 在原发性上皮的超小样品中获得AKT＆EGFR活性的多路复用测量值 细胞将提高我们对重金属暴露基础的生化机制的理解。 重要的是，这样的技术可以使临床医生能够确定重金属的预警信号 来自非常小的，异质的主要样本的个体引起的毒性和/或疾病诱导。 我旨在通过采用基于传感器的化学物质来改善呼吸上皮的生化研究 细胞仪。基于传感器的化学细胞仪是一种单细胞分析方法，其中生物分子传感器 用于测量小单元群中的信号动力学。12-16在这里，我提出了 开发一组新型的荧光酶传感器，以获得Akt＆EGFR的多路复用测量 在单个细胞中，使用毛细管电泳采用荧光检测（CE-F）。我也打算 改善所提出的酶传感器的设计和功能；通过安装光活化部分 在磷酸化位点，我希望提高传感器的膜渗透性，并控制 激酶反应在细胞内开始。16我计划控制细胞中的激酶反应停止时间 通过开发一种新型的化学选择性试剂，该试剂停止细胞内反应并促进报告基因 通过CE-F恢复进行分析。 使用这些新型酶传感器进行的研究将增强我们对生化的理解 主导疾病诱导疾病和/或弹性的机制 呼吸上皮细胞。此外，从这一提议中获得的知识将改善我们的 对重金属暴露基础的生化机制的理解，同时识别新型 开发可渗透传感器的策略以实现单个细胞内的时间控制反应。