这项研究得到了部分赠款援助具有挑战性的探索性研究（JK），科研（一）（JK和SM），从教育，文化，体育，科学部，科研和技术，日本。一个RIKEN的玻璃钢奖学金“（RCE），提供额外的支持。作者表达自己的谢意博士。英介Chikayama Yasuyo Sekiyama和冈本真身用核磁共振和统计分析的技术援助。

1。过滤删除萃取物的制备<ol><li>使用25毫米直径0.22微米孔径Durapore PVDF（Millipore公司）亲水性过滤器。放置在一个干净的500毫升耐热烧杯过滤器，使用镊子。前用蒸馏水冲洗三次。漩涡以及你冲洗过滤器，以防止从坚持彼此。加入300毫升的Milli-Q（Millipore公司）或相当于高品质的水。釜萃取物彻底清除，以方便从过滤器。 </li><li>倒掉的Milli-Q和再次三重冲洗过滤器，这个时候用Milli-Q。使用镊子个人清洁干燥的表面（如铝箔），要么在一个合理的温度（例如37℃）或空气干燥干燥过滤器。现在可以使用过滤器。 </li></ol> 2。过滤材料的样品<ol><li>玻璃支持这一协议，微孔不锈钢3就地过滤多方面展示与25毫米的微量过滤列使用机械泵。使用无菌操作技术，将一个单一的25毫米的过滤器，适用于过滤柱基列和取缔一起。 </li><li>列装入15毫升的样品，打开过滤器流形上的截止阀，并打开泵。温柔的压力下，过滤器，以减少细胞破损（&lt;5千帕）。如手或蠕动泵，其他泵，可适应于本议定书。对于密度较低的样品，连续增加的水可能是必要的，不要让去添置水之间的时间间隔较长时间的干燥过滤器。 </li><li>海洋样品，你过滤你的样品后，您可以执行一个可选的淡水冲洗，以减少您的样品，并在过滤器上的残余顺磁离子。这可能有助于更精确调谐谱仪磁铁。只是轻轻地补充水分，体积小，并在年底通过您收集的样本进行筛选。 </li><li>一次过滤完成后，关闭泵，并留下阀门OPE列印因此仍有负压下过滤器。取出钳和过滤器列。 </li><li>用一只手，用干净的镊子，采取保留在过滤器中。双臂交叉本身的过滤器，但没有折痕。使用无菌的2 ml离心管唇用另一只手按住过滤器。然后松开镊子，用他们重新夹住过滤器的两边。 regrip在45°角对折。 </li><li>放入2毫升管和释放过滤器，所以它与样品的一面朝内打​​开。您可以将高达两成无菌的2 ml离心管这种方式过滤。如果使用两个过滤器，确保他们尽可能少的重叠。立即冻结（至少-30℃）。 </li></ol> 3。提取溶于水的代谢<ol><li>冻干的样品过夜或至少10个小时。 </li><li>冻干后，添加到每个管（Tokken）不锈钢破碎机。加入750μL标准化钾磷酸盐在氧化氘（2H&gt; 90％）与2,2 -二甲基-2-silapentane-5-磺酸钠（DSS）的标准（KPI核磁共振缓冲区; 38.3毫米KH 2 PO 4计 ，61.7毫米K 2 HPO 4，决策支持系统0.1毫米，pH值7.0，90％D 2 O）2。 </li><li>超声在4样品°水sonicator的℃（Bioruptor，Diagenode）的电池材料从过滤器中删除5分钟。用干净的镊子取出过滤器。 </li><li>使用一个磨式破碎机（1600转）5分钟，破坏细胞。 </li><li>在65°C孵育摇晃在台式振动筛（Eppendorf公司）为15分钟（1400转）。 </li><li>用干净的镊子取出金属猪，离心13,000Ğ样本为5分钟。 </li><li>绘制了核磁共振光谱，核磁共振管上清液直接。 </li></ol> 4。核磁共振光谱和数据分析<ol><li>加载您的样品（在这里，在Bruker DRX-500光谱仪配备到核磁共振仪TXI探头由计算机正在运行的XWIN-NMR）控制的三轴梯度。 </li><li>获得1D 1 H NMR谱，采用适当的方法原样XWIN-NMR接口2,15。在本研究中均录得1 H NMR谱在500.03兆赫在298 K的残留水信号被抑制水门事件的脉冲序列重复了1.2秒的时间，在DRX-500光谱仪的工作。 128的瞬态采集，获得了32000个频谱数据点。 </li><li> NMR数据目录转移NMRPipe软件16安装一台PC。处理的原始数据和一套决策支持系统，为0 ppm的参考，然后手动相位谱。数字化成一套整合软件，如rNMR，Automics或“分级”，或使用公开可用FT2B包ECOMICS网站（离散值光谱数据<a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/的</a> ）3,17。在日例如，被谱进行了整合之间0.5和10.5 ppm的0.032 ppm的积分使用ECOMICS地区，要么DSS或总信号强度和规范化。输出数据，现在可以用于下游的统计分析，如主成分分析（PCA），使用免费的软件包， 如 R 18。 </li></ol> 5。代表结果例如1 H NMR谱的取得，使用上述方法如图1所示。这些样本，从两个时间点的一个缩影实验显示明显的差异，由于藻类的代谢活动。 4天的频谱显示相当丰富，尤其是在3-4 ppm范围内的高峰相比，天1个样本。这些山峰，可以归结为绽放在缩影硅藻产生的糖。在一个类似的实验，比较自然的浮游生物群落生长在人工或天然海水，统计方法小号主成分分析（PCA）的得分分级核磁共振光谱所得情节UCH可以用来表明两者之间的处理（图2）明确的代谢差异，而负荷地块内可以识别的光谱峰形状分布的数据。可以与其他组学的水平，从基因组的指纹识别方法，如（图3）的数据相比，这样的结果。这些核磁共振峰可以单独查询（例如在BMRB; <a href="http://www.bmrb.wisc.edu/">http://www.bmrb.wisc.edu/</a> ）19，或整个光谱可以进行统计分析（与在SpinAssign如<a href="http://prime.psc.riken.jp/?action=nmr_search">http://prime.psc.riken。 JP /？行动= nmr_search</a> ）2。在这个例子中，处理之间的差异是由于糖的地区丰富的山峰，从天然浮游生物群落代谢产物的光谱（3.39 ppm到4.04 ppm）的人工海水社区特点的几座山峰，并初步IDE作为乳酸ntified和使用SpinAssign的甲酸。 <img src="/files/ftp_upload/3163/3163fig1.jpg" alt="图1" /> 图1代表1 H NMR谱从处理的样品，使用此过程中获得。前（1天）和（4日）激烈的硅藻水华期间的缩影样本。 NMR实验在Bruker DRX-500信号与内标峰高（决策支持系统; 0 ppm）的正常化。 <img src="/files/ftp_upload/3163/3163fig2.jpg" alt="图2" /> 图2。主成分分析（PCA）的得分为自然衍生的浮游微生物的成长与自然的缩影社区（开放的钻石）或人工（黑眼圈）海水metabolomes分级核磁共振光谱图。散点图中可以观察到清晰的代谢差异。从这样的分析，加载情节可以被用来确定不同的重要性峰系统;这些山峰可以进一步分析需要。 <img src="/files/ftp_upload/3163/3163fig3.jpg" alt="图3" /> 图3。与基因组数据相结合的多组学分析的一个例子核磁共振。根据群落组成的18S变性梯度凝胶电泳（左）和（右）从16S rRNA基因相同的样本分析图2也显示出不同的微生物之间的自然（开放的钻石）及人工（黑圈）海水的缩影社区模式。从自然生态系统之间的代谢组学和基因组等通信演示了这种方法的有效性。

<ol>
	<li>Bundy, J. G., Davey, M. P., Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+metabolomics:+a+critical+review+and+future+perspectives.">Environmental metabolomics: a critical review and future perspectives.</a> Metabolomics. 5, 3-21 (2008).</li><li>Chikayama, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+indices+for+simultaneous+large-scale+metabolite+detections+for+a+single+NMR+spectrum.">Statistical indices for simultaneous large-scale metabolite detections for a single NMR spectrum.</a> Anal. Chem. 82, 1653-1658 (2010).</li><li>Lewis, I. A., Schommer, S. C., Markley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rNMR:+open+source+software+for+identifying+and+quantifying+metabolites+in+NMR+spectra.">rNMR: open source software for identifying and quantifying metabolites in NMR spectra.</a> Magn. Reson. Chem. 47, S123-S126 (2009).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+gut+microbes+modulate+human+metabolic+phenotypes.">Symbiotic gut microbes modulate human metabolic phenotypes.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2117-2122 (2008).</li><li>Mochida, K., Furuta, T., Ebana, K., Shinozaki, K., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+exploration+of+metabolic+and+genomic+diversity+in+rice.">Correlation exploration of metabolic and genomic diversity in rice.</a> BMC Genomics. 10, 568 (2009).</li><li>Fukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacteria+can+protect+from+enteropathogenic+infection+through+production+of+acetate.">Bifidobacteria can protect from enteropathogenic infection through production of acetate.</a> Nature. 469, 543-547 (2011).</li><li>Kikuchi, J., Hirayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+stable+isotope+labeling+of+higher+plants+for+a+hetero-nuclear+multi-dimensional+NMR-based+metabolomics.">Practical aspects of stable isotope labeling of higher plants for a hetero-nuclear multi-dimensional NMR-based metabolomics.</a> Methods Mol. Biol. 358, 273-286 (2007).</li><li>Martin, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+top-down+systems+biology+view+of+microbiome-mammalian+metabolic+interactions+in+a+mouse+model.">A top-down systems biology view of microbiome-mammalian metabolic interactions in a mouse model.</a> Mol. Syst. Biol. 3, 112 (2007).</li><li>Mahrous, E. A., Lee, R. B., Lee, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+approach+to+lipid+profiling+of+mycobacteria+using+2D+HSQC+NMR+maps.">A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps.</a> J. Lipid Res. 49, 455-463 (2008).</li><li>Fukuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+characterization+of+bacterial+metabolic+dynamics+with+a+novel+profiling+technique,+real-time+metabolotyping.">Evaluation and characterization of bacterial metabolic dynamics with a novel profiling technique, real-time metabolotyping.</a> PloS ONE. 4, e4893 (2009).</li><li>Date, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+monitoring+approach+for+metabolic+dynamics+in+microbial+ecosystems+using+stable-isotope-labeling+technologies.">New monitoring approach for metabolic dynamics in microbial ecosystems using stable-isotope-labeling technologies.</a> J. Biosci. Bioeng. 110, 87-93 (2010).</li><li>Nakanishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+omics+approach+identifies+nutrition-mediated+microbial+interactions.">Dynamic omics approach identifies nutrition-mediated microbial interactions.</a> J. Proteome Res. 10, 824-836 (2011).</li><li>Falkowski, P., Barber, R., Smetacek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+controls+and+feedbacks+on+ocean+primary+production.">Biogeochemical controls and feedbacks on ocean primary production.</a> Science. 281, 200-207 (1998).</li><li>Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+of+aquatic+organisms:+the+new+'omics'+on+the+block.">Metabolomics of aquatic organisms: the new 'omics' on the block.</a> Mar. Ecol. Prog. Ser. 332, 301-306 (2007).</li><li>Sekiyama, Y., Chikayama, E., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+semipolar+solvent+system+as+a+step+toward+heteronuclear+multidimensional+NMR-based+metabolomics+for+13C-labeled+bacteria,+plants,+and+animals.">Evaluation of a semipolar solvent system as a step toward heteronuclear multidimensional NMR-based metabolomics for 13C-labeled bacteria, plants, and animals.</a> Anal. Chem. 83, 719-726 (2011).</li><li>Delaglio, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMRPipe:+A+multidimensional+spectral+processing+system+based+on+UNIX+pipes.">NMRPipe: A multidimensional spectral processing system based on UNIX pipes.</a> J. Biomol. NMR. 6, 277-293 (1995).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automics:+an+integrated+platform+for+NMR-based+metabonomics+spectral+processing+and+data+analysis.">Automics: an integrated platform for NMR-based metabonomics spectral processing and data analysis.</a> BMC Bioinformatics. 10, 83 (2009).</li><li>Eldon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BioMagResBank.">BioMagResBank.</a> Nucleic Acids Res. 36, D402-D408 (2007).</li><li>Sekiyama, Y., Chikayama, E., Kikuchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+polar+and+semipolar+plant+metabolites+throughout+extraction+processes+using+a+combined+solution-state+and+high-resolution+magic+angle+spinning+NMR+approach.">Profiling polar and semipolar plant metabolites throughout extraction processes using a combined solution-state and high-resolution magic angle spinning NMR approach.</a> Anal. Chem. 82, 1643-1652 (2011).</li></ol>

没有利益冲突的声明。

这里展示的过滤和代谢产物提取方法允许在收集足够数量的核磁共振代谢微生物浮游生物。虽然只溶于水的使用KPI和1D 1 H核磁共振的代谢产物提取证明，其他的提取溶剂和光谱方法可以使用。一个有用的例子是使用氘代甲醇作为一个半极性溶剂，这已被证明产生优越的核磁共振光谱，从异构样品及顺磁离子污染不敏感，在15个海洋样品发现。在这种情况下，从上面提取的颗粒应保?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 0.22μm的亲水性Durapore PVDF的过滤器，25毫米</td><td> Millipore公司</td><td> GVWP02500 </td><td></td></tr><tr><td>微观滤镜托架，25毫米，熔块玻璃支持</td><td> Millipore公司</td><td> XX1002500 </td><td></td></tr><tr><td> 3地方多方面的，47毫米，不锈钢</td><td> Millipore公司</td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>瓦科</td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td>瓦科</td><td> 164-04295 </td><td></td></tr><tr><td>氧化氘，2 H&gt; 90％; </td><td> campridge同位素Laboratoties </td><td> DLM - 4 </td><td></td<html> &gt; </tr><tr><td>决策支持系统</td><td> Fluka公司</td><td> 92754 </td><td></td></tr><tr><td> automill </td><td> tokken </td><td> TK-AM4 </td><td>包括不锈钢粉碎机</td></tr><tr><td> thermomixer舒适</td><td> Eppendorf公司</td><td> 5355 000.011 </td><td></td></tr><tr><td> bioruptor </td><td> diagenode </td><td> UCD-200 </td><td></td></tr><tr><td>真空蒸发器</td><td> EYELA </td><td> CVE - 3100 </td><td></td></tr><tr><td>核磁共振</td><td>布鲁克</td><td> DRX-500 5毫米TXI探头</td><td></td></tr><tr><td>光谱分选工具</td><td>最初开发</td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td>代谢产物的注释工具和数据库</td><td>最初开发</td><td> SpinAssign </td><td>= nmr_search“&gt; http://prime.psc.riken.jp/?action=nmr_search </td></tr></tbody></table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

环境代谢组学是一个新兴的领域有了新的认识，促进生物体是如何应对和交互环境和对方在生化水平1。核磁共振（NMR）光谱是多种技术，包括气相色谱 - 质谱（GC-MS法）之一，此类研究大有前途。核磁共振的优点是，它是适用于不相关的分析，提供结构化信息和光谱可以对最近2,3个人代谢物谱数据库查询数量和统计的举止。此外，核磁共振光谱数据可与其他组学水平（如转录，基因组学）的数据，以提供更全面的了解类群的生理反应，彼此和环境4,5,6。然而，核磁共振是比其他代谢组学技术的敏感，使其难以AP铺设天然微生物系统，样本人群低密度及代谢物浓度比较低的代谢物的定义和随时提取来源，如整个组织，biofluids或细胞培养。因此，一些直接环境日期进行微生物的代谢物的研究一直局限于文化为基础的或容易定义的高密度的生态系统，如主机的共生系统，构建联合培养或肠道内环境的稳定同位素标记可以操作此外用于增强核磁共振信号7,8,9,10,11,12。核磁共振适合浓度的方法，促进环境代谢物的浓度和收集所缺乏的。由于近期关注已在水生环境，其中大部分能量和物质流介导由的浮游社区13,14生物体对环境的代谢，我们已经开发出一种浓度的方法TION和全社会的代谢产物从浮游微生物系统过滤提取。市售亲水性的聚-1 ,1-difluoroethene（PVDF）过滤器是经过特殊处理，以彻底清除萃取物，可作为后续分析中的污染物，否则会出现。然后，这些治疗的过滤器用于过滤利益的环境或实验样品。湿样品材料的过滤器含有冻干和使用一个标准化的钾磷酸盐提取液常规核磁共振光谱直接溶于水的代谢产物中提取。可以从这些方法得出的数据进行统计分析，找出有意义的模式，或集成水平与其他组学为社会和生态系统功能的全面了解。

Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

从微生物浮游社区的代谢产物提取方法。抽样实现整个社会上特意准备的过滤器过滤。冷冻干燥后，溶于水，代谢产物中提取。这种方法用于环境代谢的自然或实验的微生物群落的跨组学研究中的应用。

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

concentration-metabolites-from-low-density-planktonic-communities-for

Research

JoVE Journal

Biology

12.7K 次观看.  RIKEN Advanced Science Institute. 从微生物浮游社区的代谢产物提取方法。抽样实现整个社会上特意准备的过滤器过滤。冷冻干燥后，溶于水，代谢产物中提取。这种方法用于环境代谢的自然或实验的微生物群落的跨组学研究中的应用。

视频: Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

科研

生物学

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

社区环境转录从海洋微生物基因表达分析

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

量化混合使用磁共振成像

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

双正电子发射层析成像用 68 ga 芯掺杂氧化铁纳米粒子的合成/(t1) 磁共振成像

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;