Molecular Determinants of Calcitic Biomineralization.

鸡蛋壳制造中的缺陷是一个人类健康和食品安全问题；营养丰富的餐用鸡蛋是全球亿万贫困人口S的蛋白质、矿物质和维生素的重要来源。在加拿大，据估计，每年约有1万起沙门氏菌中毒病例是由于污染了进入食物链的破裂蛋壳。从这个意义上说，蛋壳钙质生物矿化的可重复性是人类社会关注的关键问题。在过去的20年里，我的研究项目调查了在这种生物矿化屏障上发挥作用的综合防御策略。我的研究揭示了蛋壳基质中的蛋白质成分如何调节碳酸钙晶体的成核和生长，以了解蛋壳机械性能的调节。此外，我们还证明了抗微生物蛋壳基质蛋白(EMPs)的双重功能，它调节蛋壳屏障的矿化，从而维持鸡蛋的无病原体状态。 目的是研究蛋壳矿化的启动和蛋白质的调节作用。将确定特定的EMPs和无定形碳酸钙(ACC)在蛋壳形成(矿化)和再吸收(脱矿)中的作用。抗菌EMPS的双重作用将被进一步研究。 具体目标： 1.我们对输卵管峡部特异基因表达的转录分析表明，胶原X(COL10A1)、纤维蛋白-1(FBN1)和富含半胱氨酸的蛋壳膜蛋白(CREMP)是主要的结构成分。它们在天然蛋壳膜中的结合和相对组织将使用特定的抗体进行研究。这些蛋白质组装成稳定的纤维将在体外用纯化的天然和/或重组蛋白质进行研究。 2.我的假设是，哺乳动物锥体含有一个稳定的ACC池，作为胚胎在生长过程中访问的钙库，我的假设将通过调查CaCO3的状态和确定哺乳动物锥体底部钙储备体中的相关蛋白质来验证。 3.拟在蛋壳形成过程中调节矿化的候选蛋白质(如OC17、NUCB)将通过体外结晶研究进行研究。调控分子将通过重组实验得到验证，利用纯化的重组蛋白来调节ACC到方解石的转化，并复制哺乳动物锥体的成核。 4.研究OCX32的抗菌活性和CaCO3成核/晶体生长特性，以评估这一重要的基质蛋白的双重功能。 这些研究将为严重的食品安全问题提供解决方案，例如食用鸡蛋受到沙门氏菌污染。

Flaws in chicken eggshell fabrication are a human health and food safety issue; the nutritious table egg is an important source of protein, minerals and vitamins for 100’s of millions of individuals living in poverty worldwide. In Canada it is estimated that about 10,000 annual cases of salmonella poisoning are due to contamination of cracked eggshells that find their way into the food chain. In this sense, the reproducible nature of calcitic biomineralization of the eggshell is of critical concern to human society. My research program during the past 20 years has investigated the integrated defense strategies that function at this biomineralized barrier. My studies have revealed how protein components of the eggshell matrix modulate calcium carbonate crystal nucleation and growth in order to understand regulation of eggshell mechanical properties. Moreover, we have demonstrated the dual function of antimicrobial eggshell matrix proteins (EMPs) that regulate mineralization of the shell barrier which maintains the pathogen-free status of the egg. The goals are to investigate the initiation of eggshell mineralization and the regulatory role of proteins. The role of specific EMPs and amorphous calcium carbonate (ACC) in eggshell formation (mineralization) and resorption (demineralization) will be determined. The dual role of antimicrobial EMPs will be further investigated. Specific objectives: 1. Our transcriptomic analysis of specific gene expression in the isthmus segment of the oviduct identified collagen X (COL10A1), fibrillin-1 (FBN1) and cysteine rich eggshell membrane protein (CREMP) as key structural constituents. Their incorporation and relative organization in the native eggshell membranes will be investigated using specific antibodies. The assembly of these proteins into stable fibres will be investigated in vitro with purified native and/or recombinant proteins. 2. My hypothesis that the mammillary cone contains a stabilized pool of ACC as a Ca reservoir that the embryo accesses during growth will be tested by investigating the status of CaCO3 and identifying associated proteins in the calcium reserve body at the mammillary cone base. 3. Candidate proteins (such as OC17, NUCB) proposed to modulate mineralization during eggshell formation will be investigated with in vitro crystallization studies. Regulatory molecules will be validated by reconstitution experiments utilizing purified recombinant proteins to modulate the ACC to calcite transformation, and to reproduce nucleation of mammillary cones. 4. The antimicrobial activity and CaCO3 nucleation / crystal growth properties of OCX32 will be investigated to assess the dual function of this important matrix protein. These studies will provide solutions to serious food safety issues such as salmonella contamination of the table egg.