作者感谢 DBT（生物技术教学计划）、DBT（BUILDER 计划）和 FIST 的资助。MR 感谢 SHODH 的奖学金。惠普感谢CSIR的奖学金。

铜绿假单胞菌的所有细菌分离株均来自印度巴罗达和斋浦尔的医学微生物学实验室。所有选定的临床分离株均在生物安全柜（BSL2）中处理，并且在实验期间处理细菌分离株时要格外小心。所有试剂/溶液的商业细节均在材料表中提供。
1. 天蓝色铬磺酸铬（CAS）染料和琼脂培养基的制备
<ol><li>用以下组合物制备CAS染料（100mL）：<ol><li>将 60 mg CAS 溶解在 50 mL 蒸馏水（溶液 1）中。</li>
		<li>将 2.7 mg FeCl3·6H2O 溶于 10 mL 10 mM HCl（溶液 2）中。</li>
		<li>将 73 mg 西曲溴铵 （HDTMA） 溶解在 40 mL 蒸馏水（溶液 3）中。</li>
		<li>小心混合溶液 1、溶液 2 和溶液 3。将其存放在玻璃瓶中。</li>
	</ol></li>
	<li>按照以下步骤准备CAS琼脂：<ol><li>将 100 mL MM9 盐溶液加入 750 mL 蒸馏水中。</li>
		<li>溶解32.24g哌嗪-N，N'-双（2-乙磺酸）（PIPES）游离酸。</li>
		<li>加入 15 克琼脂。高压灭菌器并使其冷却。</li>
		<li>向混合物中加入 30 mL 无菌酪氨基酸溶液和 10 mL 无菌 20% 葡萄糖溶液。</li>
		<li>加入 100 mL CAS 染料，混合，并在无菌条件下倒入。 注： 补充文件 1 中提供了 MM9 培养基、酪蛋白氨基酸溶液和 8-羟基喹啉的制备。PIPES缓冲液对pH值敏感。在达到 pH 值 5.6 之前，它不会溶解。确保持续监测 pH 值，因为当 PIPES 开始溶解在水中时，它会进一步降低 pH 值。用相同体积的3%8-羟基喹啉溶解在氯仿中提取Casamino酸。将不混溶液在4°C下放置约20分钟，小心地收集溶液的上相，不要干扰下相。</li>
	</ol></li>
</ol>2. 铁载体生产的定性分析
<ol><li>将OD600nm 设置为0.2，用于 铜绿假单胞菌的24小时培养物。</li>
	<li>使用无菌线环，在CAS琼脂平板上划线细菌培养物。</li>
	<li>在30°C孵育24小时。 注意：蛋白胨水培养基或 0.8% 生理盐水可用于稀释细菌培养物。如果在24小时未观察到细菌生长，则将CAS板孵育48至72小时。</li>
</ol>3. 总铁载体的定量估计
<ol><li>将OD600nm调节至0.25后，在蛋白胨水培养基中重新接种24小时生长的铜绿假单胞菌培养物，并在30°C下孵育48小时。</li>
	<li>48小时后，在室温下以4650× g 离心细菌培养物10分钟。</li>
	<li>离心后，向 96 孔微量滴定板中加入 100 μL 无细胞上清液，并向其中加入 100 μL CAS 染料。</li>
	<li>用铝箔盖住板，在室温下孵育20分钟。</li>
	<li>孵育后，在630nm处进行分光光度读数。</li>
	<li>将总铁载体定量的结果计算为铁载体单位百分比（PSU）。 注意： PSU 的计算公式为：[（Ar - As）/Ar] x 100 其中，Ar = 参比在 630 nm 处的吸光度，As = 样品游离上清液的吸光度。作为参考，应将CAS染料添加到未接种的蛋白胨水培养基中。用6M HCl填充所有玻璃器皿，如试管，烧瓶等2小时，并用蒸馏水冲洗两次，以去除其上的任何铁痕迹。</li>
</ol>4. 吡啶的定量估计
<ol><li>将OD600nm调节至0.25并在30°C孵育48小时后，在蛋白胨水培养基中重新接种24小时生长的铜绿假单胞菌培养物。</li>
	<li>48小时后，在进一步进行之前测量细菌生长的OD600nm 。</li>
	<li>在室温下以4650× g 离心细菌培养物10分钟。</li>
	<li>离心后，向 96 孔微量滴定板中加入 100 μL 无细胞上清液，并向其中加入 100 μL 50 mM Tris-HCl （pH 8.0）。</li>
	<li>在OD405 nm处进行分光光度读数。</li>
</ol>5. 脓气素提取和分光光度法
<ol><li>将OD600nm调整为0.25并在30°C孵育24小时后，在King's B培养基（补充文件1）中重新接种24小时生长的铜绿假单胞菌培养物。</li>
	<li>24小时后，取100mL培养物，并在室温下以4650× g 离心10分钟。</li>
	<li>离心后，向上清液中加入 5 mL 1 M 柠檬酸。用 50 mL 乙酸乙酯提取两次。</li>
	<li>通过注射器过滤器用硫酸镁过滤有机相。将过滤后的有机相储存在-20°C。</li>
	<li>在 320 nm 处获取分光光度读数。 注意：由于 pyochelin 在室温下是一种高度不稳定的化合物，因此请在冰上进行提取过程。使用 50 mL 无菌注射器进行过滤器分离。将棉花放在注射器的尖端，并在上面加入 1 克硫酸镁。将无菌注射器过滤器固定在注射器的尖端，并将滤液收集在无菌管中。</li>
</ol>

铜 绿假单胞 菌分离株铁载体产量的评估

<ol>
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K., Verma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+microplate+method+for+rapid+and+efficient+estimation+of+siderophore+produced+by+bacteria.">Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria.</a> 3 Biotech. 7 (381), 1-9 (2017).</li><li>Frac, M., Gryta, A., Oszust, K., Kotowicz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+microplate+method+(biolog+mt2)+for+detection+of+fusarium+fungicides+resistance/sensitivity.">Fast and accurate microplate method (biolog mt2) for detection of fusarium fungicides resistance/sensitivity.</a> Frontiers in Microbiology. 7 (4), 1-16 (2016).</li><li>Cezard, C., Farvacques, N., Sonnet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biology+of+pyoverdines,+pseudomonas+primary+siderophores.">Chemistry and biology of pyoverdines, pseudomonas primary siderophores.</a> Current Medicinal Chemistry. 22 (2), 165-186 (2015).</li><li>Braud, A., Hoegy, F., Jezequel, K., Lebeau, T., Schalk, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+metal+specificity+of+the+pseudomonas+aeruginosa+pyoverdine-iron+uptake+pathway.">New insights into the metal specificity of the pseudomonas aeruginosa pyoverdine-iron uptake pathway.</a> Environmental Microbiology. 11 (5), 1079-1091 (2009).</li><li>Brandel, J., 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+siderophore+of+pseudomonas+aeruginosa:+Physicochemical+characterization+of+the+iron(iii),+copper+(ii)+and+zinc+(ii)+complexes.">a siderophore of pseudomonas aeruginosa: Physicochemical characterization of the iron(iii), copper (ii) and zinc (ii) complexes.</a> Dalton Transactions. 41 (9), 2820-2834 (2012).</li><li>Hoegy, F., Mislin, G. L. A., Schalk, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+methods+and+protocols.">Pseudomonas methods and protocols.</a> Methods in Molecular Biology. 1149, (2014).</li><li>Cunrath, O., 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=The+pathogen+pseudomonas+aeruginosa+optimizes+the+production+of+the+siderophore+pyochelin+upon+environmental+challenges.">The pathogen pseudomonas aeruginosa optimizes the production of the siderophore pyochelin upon environmental challenges.</a> Metallomics. 12 (12), 2108-2120 (2020).</li><li>Ji, A. J., 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+novel+and+sensitive+LC/MS/MS+method+for+quantification+of+pyochelin+in+human+sputum+samples+from+cystic+fibrosis+patients.">A novel and sensitive LC/MS/MS method for quantification of pyochelin in human sputum samples from cystic fibrosis patients.</a> Biomarkers &amp; Applications. 4 (1), 135 (2019).</li><li>Visaggio, D., 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+highly+sensitive+luminescent+biosensor+for+the+microvolumetric+detection+of+the+pseudomonas+aeruginosa+siderophore+pyochelin.">A highly sensitive luminescent biosensor for the microvolumetric detection of the pseudomonas aeruginosa siderophore pyochelin.</a> ACS Sensors. 6 (9), 3273-3283 (2021).</li><li>Miethke, M., Marahiel, M. 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作者没有什么可透露的。

该方案使研究人员能够从细菌游离上清液中定量铜绿假单胞菌的总铁载体和两种不同的铁载体，即pyoverdine和pyochelin。在CAS琼脂平板测定中，CAS染料和Fe3+离子形成络合物。当细菌产生铁载体时，它们会淬灭 CAS-Fe3+ 复合物中的 Fe3+ 离子，导致细菌生长周围的颜色发生变化。这种变化导致细菌生长周围出现明显的橙色光晕14,15...

生物体需要铁来执行各种重要功能，例如电子传输和 DNA 复制1。 铜绿假单胞菌是一种革兰氏阴性机会性病原体，已知具有多种毒力因子在宿主中建立感染，其中一种机制是铁载体形成2。在缺铁条件下， 铜绿假单 胞菌会释放出称为铁载体的特殊分子，这些分子会从周围环境中淬灭铁。铁载体在细胞外螯合铁，产生的铁载体复合物被主动转运回细胞3。
已知铜绿假单胞菌产生两种铁载体，pyoverdine 和 pyochelin。已知 Pyoverdine 具有较高的铁螯合亲和力 （1：1），而已知 pyochelin 具有较低的铁螯合亲和力 （2：1）4。脓蘑菇素也被称为次级铁载体，因为它具有较低的铁螯合亲和力5。铜绿假单胞菌 6 中铁载体的产生和调节受到群体感应 （QS） 系统的主动控制。
除铁淬火外，铁载体还参与调节毒力因子，并在生物膜形成中发挥积极作用7。铁载体还发挥着其他关键作用，包括参与细胞信号传导、防御氧化应激和促进微生物群落之间的相互作用8.铁载体通常根据它们螯合铁的特定官能团进行分类。该分类中的三种主要双酸配体是儿茶酚酸盐、羟肟酸盐和α-羟基羧酸盐3。Pyoverdines 是荧光假单胞菌属物种的标志，例如铜绿假单胞菌和荧光假单胞菌 5。它们由与含有 6-12 个氨基酸的寡肽偶联的混合绿色荧光发色团组成。几种非核糖体肽合成酶 （NRP） 参与其合成9.参与吡啶产生和调控的四个基因是 pvdL、pvdI、pvdJ 和 pvdD10。Pyoverdine 还负责哺乳动物的感染和毒力11.铜绿假单胞菌在中度限铁条件下产生脓蘑菇素，而吡脓草定在严格的限铁环境中产生12。参与 pyochelin 生产的两种操纵子是 pchDCBA 和 pchEFGHI13。值得注意的是，在碧蓝蛋白存在下，脓脓苷（儿茶酚酸酯）诱导氧化损伤和炎症，并产生羟基自由基，其对宿主组织有害11。
与微生物测定相比，蓝铝磺酸铬 （CAS） 测定因其全面、高灵敏度和更大的便利性而被广泛采用，微生物测定虽然灵敏，但可能过于特异性14.CAS测定可以在琼脂表面或溶液中进行。它依赖于当三价铁离子从其强烈的蓝色络合物转变为橙色时发生的颜色变化。CAS 比色法定量了 Fe-CAS 表面活性剂三元络合物中铁的消耗。这种特殊的络合物由金属、有机染料和表面活性剂组成，呈蓝色，并在 630 nm 处表现出吸收峰。
本报告提出了一种定性检测铁载体产生的方法，其中可以检测琼脂平板上铁载体的产生。还提供了一种定量估计微量滴定板中总铁载体产生的方法，以及来自 铜绿假单胞菌的两种铁载体pyoverdine和脓脓明的检测和定量分析方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar Agar, Type I</td>
			<td>HIMEDIA</td>
			<td>GRM666</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Hydroxyquinoline</td>
			<td>Loba Chemie</td>
			<td>4151</td>
			<td></td>
		</tr>
		<tr>
			<td>Casamino Acid</td>
			<td>SRL Chemicals</td>
			<td>68806</td>
			<td></td>
		</tr>
		<tr>
			<td>Cetyltrimethyl Ammonium Bromide (CTAB)</td>
			<td>HIMEDIA</td>
			<td>RM4867-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>1070242521</td>
			<td></td>
		</tr>
		<tr>
			<td>Chrome azurol sulfonate</td>
			<td>HIMEDIA</td>
			<td>RM336-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Merck</td>
			<td>100241</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose monohydrate</td>
			<td>Merck</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Merck</td>
			<td>107020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferric chloride hexahydrate</td>
			<td>HIMEDIA</td>
			<td>GRM6353</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Flasks</td>
			<td>Borosil</td>
			<td>5100021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Test-tubes</td>
			<td>Borosil</td>
			<td>9820U05</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SDFCL</td>
			<td>20125</td>
			<td></td>
		</tr>
		<tr>
			<td>King&#39;s medium B base</td>
			<td>HIMEDIA</td>
			<td>M1544-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Minimal Medium Salts</td>
			<td>HIMEDIA</td>
			<td>G013-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulphate&#160;</td>
			<td>Qualigens</td>
			<td>10034</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiskanGO UV Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone Type I, Bacteriological</td>
			<td>HIMEDIA</td>
			<td>RM667-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES free acid</td>
			<td>MP Biomedicals</td>
			<td>190257</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Merck</td>
			<td>1048731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>HIMEDIA</td>
			<td>RM005-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Shimadzu UV-Vis Spectrophotometer</td>
			<td>Shimadzu</td>
			<td>2072310058</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma Laborzentrifuge</td>
			<td>Sigma-Aldrich</td>
			<td>3-18K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Qualigens</td>
			<td>15915</td>
			<td></td>
		</tr>
	</tbody>
</table>

qualitative and quantitative analysis of siderophore production from pseudomonas aeruginosa

铜绿假单胞 菌（铜绿假单胞菌）以其产生多种毒力因子以在宿主中建立感染而闻名。其中一种机制是通过铁载体的产生来清除铁。 铜绿假单 胞菌产生两种不同的铁载体：铁螯合亲和力较低的脓脓苷和铁螯合亲和力较高的脓苷。本报告表明，吡啶可以直接从细菌上清液中定量，而脓皮单在定量前需要从上清液中提取。
定性分析铁载体产生的主要方法是蓝铝磺酸铬 （CAS） 琼脂平板测定。在该测定中，Fe3+-染料复合物中释放的 CAS 染料导致颜色从蓝色变为橙色，表明铁载体的产生。为了定量总铁载体，将细菌上清液与微量滴定板中的CAS染料等比例混合，然后在630nm处进行分光光度分析。通过将Pyoverdine与50 mM Tris-HCl等比例混合，直接从细菌上清液中定量，然后进行分光光度法分析。380 nm处的峰证实了pyoverdine的存在。至于Pyochelin，不可能从细菌上清液中直接定量，因此必须首先提取。随后的分光光度分析显示存在 pyochelin，峰值为 313 nm。

在对临床分离株的铁载体进行定量定量之前，对铁载体的产生进行了定性筛选，以确保铁载体的产生。通过在CAS琼脂平板上划线细菌来观察来自临床分离株的铁载体的定性检测。本研究选择了三种临床分离株，即MR1、TL7、J3以及PAO1（参考菌株）。所有三种临床分离株和 PAO1 都显示出铁载体产生的阳性结果，其中蓝色琼脂表面细菌生长周围的透明橙色光晕表明铁载体产生阳?...

Watch this Scientific Journal Video about Quantifying Siderophores and Pyochelin for Infection Control at JoVE.com

作者聚焦：定量铁载体和绿茶素用于感染控制 

该方案提供来自 铜绿假单胞菌的总铁载体，pyoverdine和脓霉素的定性和定量分析。

铜绿假单胞菌产生铁载体的定性和定量分析

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

铁载体与铁形成复合物，并在铁限制条件下将其转运到细胞中。铁载体是有效的毒力因子，有助于在宿主中建立严重感染，并且已知在调节铜绿假单胞菌的群体感应中发挥作用。我们的研究旨在定量估计假单胞菌中铁载体的产生。铁载体是细胞外分子，因此使用游离上清液定量总铁载体以及吡喃蕊素和脓粒素。由于不能检测到小体积的脓蘑菇素，因此从大量无细胞上清液中提取。最近，LCMS和生物传感器已被开发用于定量化脓皮脂素。LCMS对于小型实验室来说是一种昂贵的设置，而生物传感器只能定量化pyochelin。这种简单且图文并茂的方案使用最少的实验室试剂和仪器，并允许使用更短的时间从无细胞上清液中估计铁载体。

qualitative-quantitative-analysis-siderophore-production-from

Research

JoVE Journal

Biology

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

3.7K 次观看.  The Maharaja Sayajirao University of Baroda. 铁载体与铁形成复合物，并在铁限制条件下将其转运到细胞中。铁载体是有效的毒力因子，有助于在宿主中建立严重感染，并且已知在调节铜绿假单胞菌的群体感应中发挥作用。我们的研究旨在定量估计假单胞菌中铁载体的产生。铁载体是细胞外分子，因此使用游离上清液定量总铁载体以及吡喃蕊素和脓粒素。由于不能检测到小体积的脓蘑菇素，因此从大量无细胞上清?...

视频: 作者聚焦：定量铁载体和绿茶素用于感染控制 

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

科研

生物学

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

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 ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

细胞扩散过程中细胞边缘动力学的定量分析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

硬骨模型日本青鳉鱼（Oryzias latipes）中的精子收集和计算机辅助精子分析

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...