Metabolic Discrimination of Unknown Bacterial Pathogens

描述（由申请人提供）：范德比尔特大学和位于阿伯丁试验场的美国陆军埃奇伍德化学和生物中心（ECBC）之间的合作将开发一种采用多相传感的广谱活动检测技术，以提供生化武器制剂、未知药物或其他威胁的生物功能特征。这些特征将与先进的算法一起使用，以区分作用于一组目标细胞系的不同药物。所提议的方法非常通用，因为我们正在测量毒素的生物影响，而不仅仅是它们的存在。这将解决诊断工具的一个至关重要且尚未满足的需求，该工具能够识别未知或重新设计的威胁剂的作用机制，这些威胁剂无法被现有的特定代理传感器准确检测到。将创建诊断工具，以建立对炭疽、蓖麻毒素、葡萄球菌肠毒素B （SEB）和肉毒杆菌毒素有反应的细胞系中代谢和信号通路关键变化的特征。毒素与细胞的相互作用导致多种代谢和信号事件，因为毒素以尚未完全了解的特异性和非特异性方式破坏正常细胞功能。为了提供一种方法来表征未知毒素的影响，所提出的诊断系统将在时间和体积尺度上监测特定代谢和信号通路的关键参数。新开发的终端代谢率孔板方案将与信号事件的商业荧光分析相结合。此外，同样的代谢和信号事件将被捕获为动态生物特征，使用改进的细胞传感器，同时监测多种分析物在分钟的时间尺度。这种方法将被缩小，以在微制造纳米物理计中实现秒级的动态分辨率。这种方法应该在发现新药和意外和/或不希望的生理活动中得到广泛应用；环境和工业毒理学；代谢组学和信号传导；以及对致病菌和毒素的对策、治疗和预防措施的筛选。

DESCRIPTION (provided by applicant): A collaboration between Vanderbilt University and the U.S. Army Edgewood Chemical and Biological Center (ECBC) at the Aberdeen Proving Grounds will develop a wide-spectrum, activity-detection technology that employs multiphasic sensing, in order to provide a biofunctional signature of a CBW agent, unknown drug, or other threat. The signatures will be used with advanced algorithms to discriminate between different agents acting on a set of target cell lines. The proposed approach is extraordinarily versatile and general, because we are measuring the biological impact of the toxins, rather than simply their presence. This will address a critically important and as-yet-unmet need for diagnostic tools capable of identifying the mechanism of action of unknown or reengineered threat agents that defy accurate detection by existing, agent-specific sensors. The diagnostic tools will be created to establish signatures of key changes in metabolic and signaling pathways that occur in cell lines responsive to Anthrax, Ricin, Staphylococcal Enterotoxin B (SEB), and Clostridium Botulinum toxins. The interaction of the toxins with cells leads to a multitude of metabolic and signaling events, as toxins disrupt normal cellular functions in specific and non-specific ways that are not yet fully understood. To provide a means to characterize the effects of unknown toxins, the proposed diagnostic system will monitor key parameters of specific metabolic and signaling pathways over a spectrum of time- and volume-scales. Newly developed well-plate protocols for end-point metabolic rates will be coupled with commercial fluorescence assays for signaling events. Additionally, the same metabolic and signaling events will be captured as dynamic biosignatures using a modified Cytosensor that simultaneously monitors multiple analytes on the time scale of minutes. This approach will be scaled down to achieve dynamic resolution on the order of seconds in a microfabricated NanoPhysiometer. This approach should find wide application in the discovery of new drugs and unexpected and/or undesired physiological activity; environmental and industrial toxicology; metabonomics and signaling; and screening of countermeasures, therapies, and prophalaxis for pathogenic bacteria and toxins.