这项工作是由癌症的主动性，5U24CA126477 - 04的临床蛋白质组学技术支持。

1）准备凝集素结合POROS列<ol><li>在步骤1.1 - 1.2，戴上口罩，防止吸入孔隙AL珠保护。称取所需POROS珠的量（100毫克beads/300μL终体积），并转移到一个干净的Eppendorf管。 </li><li>用1毫升磷酸缓冲液（PBS）除珠。在以最大速度离心3分钟离心颗粒珠。取出上清液，重复清洗。 </li><li>称取所需数量未结合凝集素（1 - 4毫克/ 200μL珠），并转移到一个干净的Eppendorf管。加入PBS，形成5 - 20毫克/毫升的溶液。 25μL储备这个解决方案。 </li><li>转移POROS珠其余的外源凝集素的解决方案。添加到终浓度为50毫米的钠氰基。广场上的摇杆管，并在室温下反应过夜。 （注：钠氰基是有毒的，必须在通风柜处理污染废物必须处理适当。） </li><li>颗粒的步骤1.2 POROS珠。取出上清液并保存后共轭解决方案。 </li><li>用1毫升淬火液（1米，pH值7.4的Tris - CL）珠。在步骤1.2和安全的颗粒珠弃上清。 </li><li> 1毫升淬火液座POROS珠其余的活性位点。添加到终浓度为50毫米的钠氰基。广场上的摇杆管，并在室温下孵育30分钟。 </li><li>步骤1.2颗粒珠，弃上清。 </li><li>洗净，用1 mL 1 M氯化钠珠。颗粒珠，弃上清。共5洗，重复四次。珠现正准备收拾。 </li><li>为了确定蛋白质的量，结合POROS珠，进行预共轭后结合凝集素的解决方案中的蛋白浓度测定。浓度不同的是，结合珠蛋白量。每珠卷结合蛋白量等于凝集素珠的浓度。目标凝集素浓度2 -20毫克/毫升之间。 </li></ol> 2）包装成一个PEEK柱凝集素结合POROS珠<ol><li>组装的包装系统（描述如下），并支持它在一个金属环的立场。包装系统组成，从底部到顶部，压力限流器，耦合器1，熔块，最终列列（2 × 50毫米），2月底列耦合，列连接器，结束列耦合器3，柱（4.5 × 50毫米），年底列耦合4月底装修。上部列作为包装材料水库。 </li><li>在理想的缓冲液重悬POROS珠一“（10毫米Tris缓冲液，pH值7.4，氯化钠150毫米，10毫米氯化钙，10毫米氯化镁）。如果包装一列（〜200μL床体积），在400μL缓冲液A悬浮</li><li>转移到上层列共轭POROS珠（藏）。添加缓冲水库，直到缓冲区达到列的顶部。轻轻地放到列的顶部接头，试图避免在列的气泡。上层柱接头连接到HPLC系统。 </li><li>包流经系统的一个缓冲区列。开始用0.5毫升/分的流量。直到4毫升/分钟的最大压力（3000 psi）的已达到增加0.5毫升/分钟，每分钟的流量。在最大可能的流量继续，直到至少35毫升的缓冲区通过列通过。 </li><li>关闭泵，并允许在列上的压力下降到&lt;20 PSI。 </li><li>轻轻地，拆开包装系统，从顶部开始。当最终列耦合器2，仔细去除。一些包装材料应挤出列的顶部。用刀片或类似的锋利的边缘，轻轻擦去挤出的珠子，留下了珠表面，甚至是与列的顶部。不要施加压力，而这样做的珠子。 </li><li>放开柱从环站。放入一个新的终端耦合器的一个新的釉料和填充柱转开放（前顶部）柱端熔块/结束耦合连接。 </li><li>适当的标签列。现在准备使用。在不使用时，列可能存储长达6个月0.02％的叠氮化钠在4 ° C的PBS。 </li></ol> 3）计划的高效液相色谱法<ol><li>根据制造商的软件的具体编程的高效液相色谱法的细节会有所不同。我们使用一个Michrom范式MG4高效液相色谱法。这台机器上，方法是建立和在屏幕上方的“设置”标签下访问。计划的以下方法： <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>时间</td><td>流量（μL/ min的） </td><td> ％A </td><td> ％B级</td></tr><tr><td> 0:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 9:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 9:01 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td>十三时50分</td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13时51分</td><td> 3000 </td><td> 100 </td><td> 0 </td></tr><tr><td>十九点50分</td><td> 3000 </td><td> 100 </td><td> 0 </td></tr></tbody></table> 缓冲答：10毫米Tris缓冲液，pH值7.4，150毫米氯化钠，10毫米氯化钙，10毫米氯化镁 缓冲液B：0.5 M的醋酸 紫外检测器应设定在280 nm处读取从0:00到19时50。应调整Y轴，检测低吸光度的水平 ，例如，0 - 50非盟。 </li><li>如果自动进样器，程序馏分收集以下的时间表。否则，收集的分数，表示在表演色谱时。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>分数</td><td>从进样延迟集合</td><td>采集时间（分钟） </td></tr><tr><td>流通过</td><td> 2.8 </td><td> 4.1 </td></tr><tr><td>展</td><td> 9 </td><td> 2.6 </td></tr></tbody></table></li></ol> 4）准备样品的HPLC <ol><li>在使用之前，在本议定书，应枯竭使用一个火星的列（美国安捷伦，美国加州圣克拉拉，）14最丰富的蛋白质，人体血浆。贫血浆，人乳铁蛋白和酵母的转化应单独胰蛋白酶消化，脱盐作为先前所描述的 1 。 </li><li>要准备的乳铁蛋白凝集素的具体标准，执行超过50微克胰蛋白酶消化的乳铁蛋白AAL的或使用的方法在第3部分的SNA列色谱。绑定的分数将包含特定的外源凝集素的乳铁蛋白糖肽。中在第6部第4.4的样品，并淡化。悬浮在1 mL缓冲液A的样品，造成样品准备使用。 </li><li>复制将进行三个实验。要准备三个样本复制，添加3 pmol胰蛋白酶消化，转化和1μL凝集素特有的乳铁蛋白（无论是AAL的LAC或SNA - 拉丁美洲和加勒比）30μL火星耗尽，胰蛋白酶消化，血浆现金等价物（PE）。一个PE的定义是non-processed/intact/original从中派生一个处理血浆（MARS耗尽和胰蛋白酶消化）量的血浆量。添加缓冲样品，以达到最终体积330μL。 </li><li>添加和缓冲液（1米的Tris pH值8.0），样品瓶，将收集洗脱液。的数量和你一起工作的小瓶量将取决于您的自动进样器的约束。对于范式MG4，一小瓶收集的流量通过和两个收集洗脱液。这将需要约1.5 mL的Tris缓冲液中1毫升洗脱液。目标瓦解pH值7.0-8.0; pH试纸测试测试的最终pH值。 </li></ol> 5）执行色谱<ol><li>所有的空白和样品的运行，将使用第1部分中描述的方法。列和缓冲区是在室温下，同时在使用中。列存储在4 ° C，而缓冲区是在室温下储存。 </li><li>附加的AAL或SNA凝集素列的高效液相色谱系统，并运行两个空白的方法（注一）只能缓冲。确保凝集素列对应的外源凝集素特有的乳铁蛋白（2）。 </li><li>三个血浆样品色谱仪，注射在彼此之间注入样品的空白，共7高效液相色谱法运行。从空白运行的分数不会被收集。 </li></ol> 6）脱盐收集馏分对于每个部分1毫升水域绿洲HLB SPE柱使用如下： <ol><li>将所需的墨盒数量真空歧管。 </li><li>湿用1毫升80％，1％甲酸乙腈（上气歧管的真空压力表应改为5 - 20 lnHg）每个墨盒。 </li><li>平衡与1.5 mL 0.1％的甲酸（上气歧管的真空压力表应读5 - 20 lnHg）墨盒</li><li>一个样品的整个卷装到一个墨盒（歧管的真空压力表应读2 - 2.5 lnHg，流速不应超过1毫升/分钟） </li><li>用3 × 1 mL 0.1％的甲酸（上气歧管的真空压力表应读5 - 20 lnHg）墨盒</li><li>洗脱肽/糖肽标记的1 × 1毫升80％，0.1％甲酸乙腈的Eppendorf管中。 （歧管上的真空计应改为2 - 2.5 lnHg，流速不应超过1毫升/分钟。）一个集合管应为每个碳粉盒使用。 </li><li>中加入60μL0.5 M的碳酸氢铵，每个收集管洗脱液。目标neutralized pH值是7.0-8.0，pH试纸测试的最终pH值。 </li><li>集中真空离心机运行（ 例如，一个热的学者SpeedVac）〜2小时@ 35 ° C〜50μL洗脱肽/糖肽</li></ol> 7）PNGase F -消化<ol><li>测试样品的pH值，以确保它是在7.0 - 8.0之间。如果需要，增加50毫米碳酸氢铵，以提高pH值。如果最终样本量为100μL，speedvac样品，直到它在50-100微升。 </li><li>在37℃过夜，加入0.5μL（250 U）的甘油PNGase F每个样品管和孵化。 </li></ol> 8）邮编提示样本<ol><li> PNGase F -消化样品开始，每一个如下ziptip。 </li><li>湿ziptip 10μL100％乙腈，10μL80％乙腈，0.1％甲酸。 </li><li> 20μL0.1％甲酸平衡ziptip。 </li><li>样品加载到ziptip吹打起来，并通过ziptip矩阵的10倍。 </li><li>吸取10微升0.1％甲酸，5次清洗ziptip。 </li><li> 10μL80％乙腈，0.1％甲酸，到一个干净的Eppendorf管流出的样品。 </li><li>重复洗脱，并添加到同一管内的第二次10μL。 </li><li> Speed​​vac体积&lt;2μL的样品。 </li><li>悬浮样品20μL0.1％甲酸。样品现已准备的LC-MS/MS分析。 </li></ol> 9）代表性的成果： POROS共轭通常产生在珠上的外源凝集素的浓度范围为2 - 20毫克/毫升。这是为亲和层析的最佳选择。 胰蛋白酶消化MARS耗尽人血浆中的凝集素层析通常在约束分数丰富的糖肽。由于糖肽PNGase F -治疗前到的LC-MS/MS，他们被认定为与N -连接的共识序列肽，NXS / T（其中X是任何残留物，但脯氨酸），在其中的天冬酰胺已被转换为通过天冬氨酸PNGase楼肽具有这些特性的行动，被认为是deglycosylated肽（或“deglycopeptides”）。平均流量通过（FT）的分数从AAL或SNA色谱包含2-4％deglycopeptides，而30-50％的肽在绑定的分数追回deglycopeptides。通常情况下，使用QSTAR精英，1000年至1300年肽将确定在“金融时报”的一小部分，和200-400肽将在绑定的分数确定。两个或多个不同的蔗糖deglycopeptides应遵守在FT分数和绑定的比例（见表1）两个或两个以上不同的乳铁蛋白deglycopeptides。 <img src="/files/ftp_upload/1398/1398table.jpg" alt="表1" /> 表1。

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

该协议提供了一种快速分离鉴定糖肽在聚糖特有的方式方法。作为广泛的范围内，特别是在发展和发病机制的有机体，糖基化变化，这种技术可能会适用于解决一个问题的多种。特别是，我们正在使用的方法，选择性地丰富糖肽已作为第一步对生物标志物发现与癌症相关的抗原表位修改。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
		</div>

a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

聚糖是一类重要的翻译后修饰。通常分泌和细胞外分子中，糖链结构的细胞信号的内部状态。聚糖对肿瘤细胞往往有丰富的唾液酸和岩藻糖基团。我们建议，旨在早期疾病诊断的生物标志物开发利用这些癌症相关聚糖变种。因此，我们开发了质谱为基础的工作流程，采用色谱凝集素，特定的糖链结构的蛋白质结合形成的亲和力矩阵。凝集素接骨木黑（SNA）和Aleuria aurantia（AAL），结合唾液酸和岩藻糖，分别，共价键耦合POROS珠（美国应用生物系统公司），并包装成高压液相色谱（HPLC）的PEEK列。简单地说，等离子消耗使用多重亲和去除系统（MARS - 14;安捷伦）的14个最丰富的蛋白质。贫血浆胰蛋白酶消化成流过约束分数和SNA或AAL高效液相色谱法分隔。的分数均与PNGaseF删除N -连接聚糖，和上一个QSTAR精英的LC-MS/MS分析。使用Mascot软件对数据进行分析。实验设计包括积极控制，fucosylated和唾液酸人乳铁蛋白糖肽和阴性对照组高甘露糖肽酿酒酵母，用于监测特异性的凝集素捕获。这个工作流程的主要功能包括格式的高效液相色谱法，deamidated Asn-Xxx-Ser/Thr图案的捕获和PNGaseF处理的糖肽的积极识别和使用糖蛋白标准质量评估所得的重复性。协议优化还包括确定适当比例的起始原料，柱容量，找出最有效的捕获和洗脱缓冲液，并监测PNGaseF治疗，以确保充分deglycosylation。未来的方向，包括使用此工作流程执行从乳腺癌患者的血浆和控制个人质谱为基础的发现实验的。

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

凝集素结合POROS珠被雇用的高效液相色谱法。糖肽类标准担任阳性和阴性对照。 MARS的14枯竭，胰蛋白酶消化人体血浆层析和流过（FT）和约束分数ESI-LC-MS/MS分析收集。糖肽的富集在绑定的分数相比，FT。

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

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15.9K 次观看.  University of California, San Francisco - UCSF. 凝集素结合POROS珠被雇用的高效液相色谱法。糖肽类标准担任阳性和阴性对照。 MARS的14枯竭，胰蛋白酶消化人体血浆层析和流过（FT）和约束分数ESI-LC-MS/MS分析收集。糖肽的富集在绑定的分数相比，FT。

视频: A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

科研

生物学

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过<em&gt; E.大肠杆菌</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

从复杂的动物相关样品测序宏基因组和Metatranscriptomes：净化不纯

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

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 ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

准备厚生物样品的观察扫描透射电子断层扫描

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.