Extreme Life: Microvolume analytics to probe animal adaptation to environmental stress

我的实验室专注于生物化学适应，以发现支持动物在极端环境压力下生存的分子机制（例如寒冷和冰冻温度，氧气限制，脱水）。我们的主要模型包括能在全身冰冻中存活的青蛙，能冬眠数月的小型哺乳动物，以及能在水下生活数周而不呼吸氧气的海龟。我们探索了帮助生存的独特和统一的分子特征，特别是代谢率降低的机制，通过将动物的能量需求降低到正常情况下的一小部分，从而延长了应激条件下的寿命。这项工作不仅扩展了我们对代谢是如何调节的理解，以及它可以被拉伸的极限，而且具有多种潜在的生物医学应用；例如，用于组织/器官冷冻保存或开发诱导性麻木作为一种治疗选择。我们目前的研究项目主要有四个方向：(a)在低代谢过程中，microRNAs在指导基因转录本命运和抑制蛋白质翻译中的调节作用，(b)表观遗传机制（如组蛋白修饰、DNA甲基化）在应激反应性基因转录控制中的作用，(c)翻译后修饰对酶的多方面作用，以适应中间代谢，实现应激耐受和延长低代谢。(d)验证和解释我们最近参与的基因组、转录组和蛋白质组项目的“大数据”。我们现在的大部分研究都来自于微小的组织样本（<100 mg），依赖于mRNA和microRNA的qPCR定量，用于制备PCR的高精度移液，小体积的酶和Luminex测定，以及核苷酸和蛋白质浓度的精确定量。目前的申请需要一个3台仪器包来增加我们工作流程的容量和自动化：(1)使用384孔微孔板的qPCR热循环仪，(2)将微量移液到微孔板的液体处理机器人，以及(3)用于1-2µL量的DNA/RNA/蛋白质定量的微体积分光光度计。这个包将大大提高所有HQP（包括9名博士和3名硕士学生）的研究成果，支持新的合作机会，例如我们最近在狐猴灵长类动物冬眠方面的工作，并允许我们通过使用微分析方法最大限度地从小样本中获取数据。

My lab focuses on biochemical adaptation to discover the molecular mechanisms that support animal survival of extreme environmental stresses (e.g. cold & freezing temperatures, oxygen limitation, dehydration). Our main models include frogs that survive whole body freezing, small mammals that hibernate for months, and turtles that can live underwater for weeks without breathing oxygen. We explore both unique and unifying molecular characteristics that aid survival, particularly the mechanisms of metabolic rate depression that permit prolonged life extension under stress conditions by lowering animal energy needs to just a fraction of normal. This work not only extends our understanding of how metabolism is regulated, and the limits to which it can be stretched, but has multiple potential biomedical applications; e.g. for tissue/organ cryopreservation or the development of inducible torpor as a treatment option. Our current research program has four main directions: (a) the regulatory roles of microRNAs in directing the fate of gene transcripts and suppressing protein translation during hypometabolism, (b) the action of epigenetic mechanisms (e.g. histone modifications, DNA methylation) in stress-responsive control of gene transcription, (c) the multi-faceted actions of posttranslational modifications to enzymes in adapting intermediary metabolism to achieve stress tolerance and prolonged hypometabolism, and (d) the validation and interpretation of “big data” from our recent involvements in genome, transcriptome, and proteome projects. Much of our research now derives from tiny tissue samples (<100 mg) and depends on qPCR quantification of mRNA and microRNA, highly accurate pipetting for preparation of PCR, enzymatic and Luminex assays in tiny volumes, and precise quantification of nucleotide and protein concentrations. The present application requests a 3-instrument package to increase capacity and automation of our workflow: (1) a qPCR thermocycler that uses 384-well microplates, (2) a liquid handling robot for accurate pipetting of tiny volumes into microplates, and (3) a micro-volume spectrophotometer for DNA/RNA/protein quantification in 1-2 µL amounts. This package will greatly improve the research output of all HQP (including 9 PhD & 3 MSc students), support novel collaborative opportunities such as our recent work on primate torpor in lemurs, and allow us to maximize data acquisition from small samples via the use of micro-analytic methods.