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

我的实验室专注于生化适应，以发现支持动物在极端环境压力(如寒冷和冰冻温度、氧气限制、脱水)中存活的分子机制。我们的主要模型包括在全身冰冻中存活的青蛙，冬眠数月的小型哺乳动物，以及在没有氧气的情况下可以在水下生活数周的海龟。我们探索了有助于生存的独特和统一的分子特征，特别是代谢率抑制的机制，通过将动物的能量需求降低到正常水平的一小部分，这种机制允许在应激条件下延长寿命。这项工作不仅扩大了我们对新陈代谢是如何调节的，以及它可以伸展到的极限的理解，而且还具有多种潜在的生物医学应用；例如，用于组织/器官的冷冻保存或作为一种治疗选择开发可诱导的麻木。我们目前的研究计划有四个主要方向：(A)microRNAs在控制基因转录物的命运和抑制低代谢过程中蛋白质翻译的调节作用，(B)表观遗传机制(如组蛋白修饰，DNA甲基化)在基因转录应激反应控制中的作用，(C)翻译后对酶的修饰在适应中间代谢以实现胁迫耐受和延长低代谢过程中的多方面作用，以及(D)验证和解释我们最近在基因组、转录组和蛋白质组项目中所涉及的“大数据”。我们的许多研究现在都来自微小的组织样本(&lt；100毫克)，依赖于对mRNA和microRNA的qPCR定量，高精度的移液进样以准备微小体积的聚合酶链式反应，酶和Luminex分析，以及核苷酸和蛋白质浓度的精确量化。本应用需要3台仪器组合，以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)用于将微小体积准确地移液到微孔板中的液体处理机器人，以及(3)用于1-2微米L定量的DNA/RNA/蛋白质定量的微型体积分光光度计。这个包将极大地提高所有HQP(包括9名博士生和3名硕士学生)的研究产出，支持新的合作机会，如我们最近在灵长类动物麻木方面的工作，并允许我们通过使用微观分析方法从小样本中最大限度地获取数据。

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.