生活磁共振波谱果蝇</em用魔角旋转
Summary
这项技术使活的分子特征的高分辨率魔角旋转的质子磁共振波谱（1H – MRS HRMAS）<em>果蝇</em与传统的14.1特斯拉光谱仪配备HRMAS探头。
Abstract
高分辨魔角旋转（HRMAS）质子核磁共振光谱（<sup> 1</sup> H – MRS）是一种新型的非破坏性技术，提高了光谱线的宽度和允许提取物，完整的细胞，细胞培养，更重要的是完整的组织获得高分辨率光谱研究代谢产物和细胞过程之间的关系。<em>在体内</em> HRMAS<sup> 1</sup> H – MRS的研究还没有要在活的果蝇报告<em>果蝇。</em><em>果蝇，</em>作为一个简单的基因生物体，允许多种生物学功能和各种进化上保守的信号转导通路在整个生物体水平的检验，它是一个有用的模型研究遗传学和生理学。为此，我们制定并实施了<em>在体内</em> HRMAS<sup> 1</sup> H – MRS方法来调查现场<em>果蝇</em> 14.1 T.这里，我们介绍一个HRMAS<sup> 1</sup> H – MRS协议的分子特性<em>果蝇</em与传统的磁共振光谱仪配备HRMAS探头。这种技术是一种新型的，<em>在体内，</em>非破坏性<em>果蝇</em>代谢产物的测量方法，从而使疾病的生物标志物的鉴定，从而可能导致新的治疗发展。
Protocol
Discussion
随着最近在体内 MRI水果中的可行性报告的异常苍蝇6，在果蝇体内MRS研究尚未报道。在本议定书中，我们描述了在体内HRMAS 1 H – MRS检测生物的重要分子方法的小说实施。具体来说，我们检测血脂和小活果蝇的代谢产物在14.1 T苍蝇在约45分钟，它允许有足够的采集时间，同时实现零飞死亡率。使用转子同步WURST 8绝热脉冲（C9 1月 15日 ）在TOBS…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持部分由一个国家AI063433卫生研究院（NIH）的资助机构，一个国立卫生研究院（NIH）的中心格兰特（P50GM021700）罗纳德G.汤普金斯（A.咏叹调Tzika，主任劳伦斯G. Rahme核磁共振核心），并为儿童的研究经费（＃8893）A.咏叹调Tzika一个Shriner的医院。我们感谢Dionyssios Mintzopoulos博士在本议定书和奥维迪乌C. Andronesi博士开发的初始阶段的援助TOBSY脉冲序列的援助。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Deuterium oxide | Reagent | Sigma-Aldrich | 7789-20-0 | |
| 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid | Reagent | Sigma-Aldrich | 24493-21-8 | |
| agar, sucrose, yeast, cornmeal | Food | Genesee Scientific Corporation | http://www.flystuff.com/ | |
| Oregon RS or Canton-S flies | Adult fly lines | Bloomington Stock Center | http://flystocks.bio.indiana.edu/ | |
| Paintbrush | Equipment | (size 0) | ||
| 2ml tubes | Equipment | Fisher Scientific | K749521-1590 | |
| Fly incubators | Equipment | high humidity capacity (60-75%), adjustable temperature, and a 12 h:12 h light: dark cycle. | ||
| Bruker Bio-Spin Avance NMR spectrometer (600.13 MHz) 4mm triple resonance (1H, 13C, 2H) HRMAS probe | Equipment | Bruker Bio-Spin | ||
| BTO-2000 unit in combination with a MAS pneumatic unit | Equipment | Bruker Bio-Spin | ||
| 4mm zirconium oxide rotor (capacity 50 ul) | Equipment | Bruker Bio-Spin | B3829 (Bruker store) | |
| MestReC (Mestrelab Research) | Software | 1D NMR spectra analysis http://mestrelab.com/ |
||
| SPARKY 3, USCF | Software | 2D NMR spectra
analysis
http://www.cgl.ucsf.edu/home/sparky/ |
References
- Apidianakis, Y., Rahme, L. G. Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection. Nat Protoc. 4, 1285-1294 (2009).
- Meiboom, S., Gill, D. Modified spin-echo method for measuring nuclear relaxation time. Rev Sci Instrum. 29, 688-691 (1958).
- Andronesi, O. C., Mintzopoulos, D., Struppe, J., Black, P. M., Tzika, A. A. Solid-state NMR adiabatic TOBSY sequences provide enhanced sensitivity for multidimensional high-resolution magic-angle-spinning 1H MR spectroscopy. J Magn Reson. 193, 251-258 (2008).
- Astrakas, L. G. Proton NMR spectroscopy shows lipids accumulate in skeletal muscle in response to burn trauma-induced apoptosis. Faseb J. 19, 1431-1440 (2005).
- Fan, T. W. M. Metabolite profiling by one- and two dimensional NMR analysis of complex mixtures. Prog Nuc Magn Reson Spec. 28, 161-219 (1996).
- Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla. PLoS One. 3, e2817-e2817 (2008).
- Zektzer, A. S. Improved signal to noise in high-resolution magic angle spinning total correlation spectroscopy studies of prostate tissues using rotor-synchronized adiabatic pulses. Magn Reson Med. 53, 41-48 (2005).
