这项工作是由能源部大湖生物能源研究中心（DE-FC 02-07ER64494科学能源部的BER办公室）赠款支持。张旭东博士和他的小组成员有用的技术讨论和宝贵意见，最后敲定协议，我们对此表示感谢。

1。样品制备和酸的提取<ol><li>冷冻干燥的土壤样品收集后场。 </li><li>使用球磨机，土壤粉碎机，或杵臼研磨，均质土壤样品。 </li><li> 25毫升水解瓶称取土壤样品（含0.3毫克ñ）。 </li><li>到每个水解瓶中，加入10毫升6M盐酸，填补紧紧氮2瓶气，帽。 </li><li>在孵化器105°Ç水解为8小时，使用自动定时开关。 </li></ol> 2。样品纯化<ol><li>删除从孵化器的烧瓶，冷却至室温。 </li><li>加入100μL的内部标准，每个烧瓶肌肌醇（1毫克毫升水-1）;纷飞组合。 </li><li>成立到200毫升梨ST /生理盐水24/40个关节，稳定的塑料杯形瓶塑料排水渠道。 </li><li>倍滤纸＃2定性到宿舍和集成漏斗圆（直径11厘米）。 </李&gt; <li>漩涡每个水解瓶中，倒入漏斗浆过滤（您可能会进一步〜3毫升水冲洗每个瓶）。 </li><li> 〜45℃，采用真空旋转蒸发干燥过滤。 </li><li>干渣悬浮梨瓶从每个3〜5毫升水（如果需要的话，使用超声波浴），倒入40 mL的聚四氟乙烯管;第二等份水冲洗烧瓶。 </li><li> pH值调整到6.6 - 6.8使用1M KOH溶液中析出的金属离子和其他有机分子。 </li><li>取出离心沉淀10分钟，在2000 RCF。 </li><li>上清倒入40毫升的玻璃试管，覆盖，然后用封口膜管开放，冻结在-20°C间戳孔的封口膜。 </li><li>冷冻干燥冷冻上清，去除所有的液体。 </li><li>干用3 mL甲醇溶解残渣，彻底涡旋（如果需要的话，使用超声波浴）;管帽，然后在2000离心力离心10分钟解决出盐。 </li><li>将上清转移至3 mL锥形反应瓶; RapidVap机蒸发干燥，在45°C（或温和的干燥氮气流，如果需要的话）。 </li><li>每个小瓶，100μL，恢复标准的N-methylglucamine（1毫克毫升水-1）和1 mL H 2 O，覆盖小瓶嘴巴用封口膜，冻结在-20°C间，穿孔的封口膜，然后冷冻干燥。 </li><li>标准：添加100μL，胞壁酸（0.5毫克毫升甲醇-1），100μL，氨基葡萄糖（1毫克毫升水-1），100μL，肌醇（1毫克毫升水-1），100μL列印methylglucamine（1毫克毫升水-1），1毫升氢澳，封口膜每小瓶，于-20℃冻结，穿孔封口膜，然后冷冻干燥。 </li></ol> 3。衍生<ol><li>含有32毫克毫升-1盐酸羟胺和40 mg mL -1的 4 -二甲-PY准备衍生试剂在ridine吡啶甲醇（4:1 V / V）。 </li><li>加入300μL衍生试剂的每个reactivials，盖紧瓶盖，涡旋彻底。 </li><li>小瓶放在75-​​80℃水浴35分钟（以及密封的小瓶，以避免让任何水进入小瓶）。 </li><li>从水浴，冷却至室温，取出小瓶。 </li><li>加入1 mL醋酸酐到每个3毫升reactivials，盖紧瓶盖，涡彻底，然后再加热75-80℃水浴25分钟。 </li><li>从水浴，冷却至室温，取出小瓶。 </li></ol> 4。分离和测量<ol><li>加入1.5 mL二氯甲烷每个小瓶，盖紧瓶盖，涡旋彻底。 </li><li>加入1 mL 1M盐酸每个小瓶，盖紧瓶盖，涡彻底的解决方案允许坐在原状，直到独立的两个短语，吸，并放弃使用1000微升移液器顶部（水）相。 </li><li>在相同的Fashion，提取有机相的3倍，但与1毫升H2O（最后的洗涤步骤，采取特殊照顾，以确保水的顶级阶段已被删除）。 </li><li>干燥的最终解决方案，使用RapidVap 45°C间（或干使用氮气，如果需要的话）。 </li><li>溶于300μL乙酸乙酯正己烷（1:1 V / V），并转移到2 mL的琥珀色的螺丝帽，小批量的插入样品瓶，盖紧瓶盖。 </li><li>量化，使用熔融硅极性毛细管柱：长30米，内径为0.25 mm，0.25微米的薄膜厚度;固定相，5％苯基，95％甲基聚硅氧烷（DB-5或同等学历）与氢的GC-FID分析或氦气作为载气，在0.5毫升分钟-1不断流动。色谱法是更好的溢价“惰性”阶段和氢气载气，但接受是较常见的品种和氦。探测器的设置是300°C，400毫升分钟-1空气和氮为30毫升分钟-1氢妆气体。注射和烤箱参数如下：1μL分流进样GC进样口设置在250°C（30:1）;初始烤箱温度120°C，保持1分钟，烤箱温度增加10°C最低。 -1至250°C，保持2.5分钟。坡道270°C时，20°C最低-1;保持2分钟，坡道290°C时，20°C最低-1，保持5分钟。调整载气流量，以便在250°C，肌醇，葡萄糖和胞壁酸衍生物洗脱，在270的N-methylglucamine流出°C。 </li></ol> 5。衍生验证<ol><li>使用软电离的LC-ESI-TOF-MS的识别分子离子，并确定分子量的衍生。 </li><li>使用的GC-EI-MS-SIM卡或GC-EI-MSMS调查衍生的目标离子的敏感性增强。 </li><li>使用多个同位素标记试剂的衍生工具的准备，然后使用大规模转移在MS同位素标记衍生物的研究分子离子和碎片离子。 </li></ol> 6。代表结果该方法的协议主要包括四个步骤：酸消化，样品净化，衍生化，气相色谱法测定（ 图1）。在图2所示为葡萄糖和胞壁酸标准股票和从土壤样品的分析的一个例子。除了葡萄糖和胞壁酸，甘露和半乳糖（葡萄糖的同分异构体），也可以决定同时使用的方法。内部标准肌醇标准响应因素的基础上，我们可以量化的土壤样品中的这些生物标志物。恢复标准已用于定性监察衍生过程。该计划的aldononitrile醋酸衍生物氨基葡萄糖和胞壁酸的形成如图3所示</strong&gt;。 拟议的衍生工具结构测定气相色谱-质谱-SIM卡的敏感性增强，或软电离的LC-ESI-TOF-MS的8;电子电离后形成的碎片离子的拟议结构进行了比较离子光谱研究样品制备与各种同位素团11。 图4显示的大规模转移，主导离子米/ 187 aldononitrile醋酸衍生物氨基葡萄糖ž准备了三种方案，代理，非标记的D-醋酸硬石膏和U -13的C-葡萄糖。其他详细的资料及解释，可以被称为我们最近的出版物8，11。这一战略可以作为一个模型来研究衍生化学。 <img alt="图1" src="/files/ftp_upload/3767/3767fig1.jpg" /> 图1。测量分析土壤中的葡萄糖和胞壁酸的协议流程图样品。该协议主要包含4个步骤：酸消解，净化，衍生化和气相色谱法测定。 <img alt="图2" src="/files/ftp_upload/3767/3767fig2.jpg" /> 图2。的GC-FID色谱标准和土壤的葡萄糖，肌醇，胞壁酸，甘露，半乳糖和methylglucamine aldononitrile醋酸。 <img alt="图3" src="/files/ftp_upload/3767/3767fig3.jpg" /> 图3。形成的aldononitrile醋酸衍生物氨基葡萄糖和胞壁酸的计划。数字1代表腈反应。数字2代表的乙酰化。 <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="图4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> 质量aldononitrile醋酸衍生物氨基葡萄糖，主导离子结构的光谱图4。ES EI模式下，与准备（一）非标记的代理，（乙）的D-醋酸硬石膏（C），ü-13的C-葡萄糖。星代表重同位素原子或同位素组。

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	<li>Zelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+patterns+of+phospholipids+and+lipopolysaccharides+in+the+characterisation+of+microbial+communities+in+soil:+a+review.">Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review.</a> Biology and Fertility of Soils. 29, 111-129 (1999).</li><li>Kirk, 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=Methods+of+studying+soil+microbial+diversity.">Methods of studying soil microbial diversity.</a> Journal of Microbiological Methods. 58, 169-188 (2004).</li><li>Joergensen, R. 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我们什么都没有透露。

提出的GC基于衍生aldononitrile乙酸氨基葡萄糖和胞壁酸的分析方法，提供了一个相对快速的方法来量化这些氨基酸糖，从土壤中提取。衍生品是化学性质稳定，可以在一次分析确定。该方法不仅限于土壤样品，并可以简化从非土壤基质的样品。 在这种方法中使用的真空泵是建立耐酸。我们进一步建议设立一个基本的陷阱，以保护泵，蒸发6M HCl溶液时。必须小心在衍生步骤，严?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>胞壁酸</td><td> Sigma-Aldrich公司</td><td> M2503-25毫克</td></tr><tr><td> D - （+）-氨基葡萄糖盐酸盐</td><td> Sigma-Aldrich公司</td><td> G1514-100G </td></tr><tr><td> N-甲基-D葡萄糖胺</td><td> Sigma-Aldrich公司</td><td> M2004-500G </td></tr><tr><td>肌醇</td><td> Fisher Scientific则</td><td> A307003G025 </td></tr><tr><td>甲醇（干） </td><td> Acros Organics公司</td><td> AC3269500​​10 </td></tr><tr><td> 4 - 二甲氨基吡啶</td><td> Acros Organics公司</td><td> AC148270050 </td></tr><tr><td>乙酸乙酯</td><td> VWR国际</td><td> BJGC100-4 </td></tr><tr><td> hydroxlamine盐酸</td><td> Fisher Scientific则</td><td> H330-100 </td></tr><tr><td>吡啶</td><td> Fisher Scientific则</td><td> P368-500 </td></tr><tr><td>醋酸酐</td> &lt;TD&gt;费舍尔科学</td><td> A10-100 </td></tr><tr><td>二氯甲烷（二氯甲烷） </td><td> Fisher Scientific则</td><td> D37-500 </td></tr><tr><td>正己烷</td><td> Fisher Scientific则</td><td> H303-4 </td></tr><tr><td>盐酸（6M） </td><td> Fisher Scientific则</td><td> S456-4 </td></tr><tr><td>盐酸羟胺-15列印</td><td>图标服务</td><td> IN5280 </td></tr><tr><td>醋酸酐-2的H（6号D C 4 O 3的 ） </td><td> Acros Organics公司</td><td> AC174670050 </td></tr><tr><td> D-葡萄糖- U -13ç </td><td>剑桥同位素实验室</td><td> CLM的-1396-1 </td></tr><tr><td>铵sufate -15列印</td><td>剑桥同位素实验室</td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 设备名称 </td><td> 公司 </td><td> 类型 </td></tr><tr><td>快速VAP </td&gt; <td> LabConco </td><td> 790002 </td></tr><tr><td> vacum泵</td><td> KNF纽伯格</td><td> D - 79112 </td></tr><tr><td>水解瓶</td><td> Fisher Scientific则</td><td> 06 423A </td></tr><tr><td>衍生microvial </td><td> Fisher Scientific则</td><td> 06-100E </td></tr><tr><td>气相色谱</td><td>惠普</td><td> 6890 </td></tr><tr><td>质谱</td><td>惠普</td><td> 5972 </td></tr><tr><td>的LC-ESI-TOF-MS的</td><td>安捷伦</td><td>加上安捷伦1200系列高效液相色谱系统安捷伦LC / MSD TOF </td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

表征微生物的定量方法是为更广泛地了解微生物生态系统内的地位和功能的关键。微生物分析当前的策略包括传统的实验室培养相关的技术和基于直接提取和测定某些生物标志物1，2。居住在土壤中的微生物物种多样性之间很少可以培养的，所以依赖于文化的方法引进重大偏差，缺席的生物标志物分析的限制。 葡萄糖，半乳糖，甘露和胞壁酸已担任活着和死去的微生物质量的措施，这些葡萄糖最丰富的胞壁酸（唯一从细菌细胞）是最重要的成分在土壤系统3 4。然而，分析知识缺乏制约的科学同行之间的广泛普及。其中所有EXI蜇分析方法，到aldononitrile乙酸酯衍生GC为基础的分析，作为不错的选择，以最佳的平衡精度方面出现的，灵敏度，简单，良好的色谱分离，样品储存5时的稳定性。 在这里，我们提出了一个详细的协议为葡萄糖和胞壁酸的可靠和相对简单的分析，从土壤后，他们转换到aldononitrile醋酸。该协议主要包括四个步骤：酸消化，样品净化，衍生化和气相色谱法测定。根据修改前的出版物6，7分步过程。此外，我们提出了一项战略，以结构验证的分子离子和电子电离后形成的衍生离子碎片。我们采用的GC-EI-MS-SIM卡，的LC-ESI-TOF-MS的同位素标记试剂来确定分子量aldononitrile醋酸衍生物氨基葡萄糖和MUR聚酰胺酸;我们用同位素标记的衍生工具的大规模转移，在调查每个衍生物8离子碎片离子谱。在另外的理论澄清，验证衍生的分子离子和离子碎片将是有益的研究人员使用的δ13 C或离子碎片，这些生物标志物在生物地球化学研究，9，10。

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

我们描述了一个协议aldonitrile从土壤中提取的葡萄糖和胞壁酸醋酸衍生物GC为基础的分析方法。对于澄清的化学机制，我们也提出一项战略，以确认衍生工具及电子电离后形成的碎片离子的结构。

衍生Aldononitrile乙酸氨基葡萄糖和胞壁酸的微生物在土壤中残留量的测定气相色谱的检测

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.8K 次观看.  University of Wisconsin, Madison. 我们描述了一个协议aldonitrile从土壤中提取的葡萄糖和胞壁酸醋酸衍生物GC为基础的分析方法。对于澄清的化学机制，我们也提出一项战略，以确认衍生工具及电子电离后形成的碎片离子的结构。

视频: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

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

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

一种快速的疾病抑制初步评估的板块竞争分析法

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.