威尔弗雷德·哈根，德马蒂诺MICHELA，和Kourosh Honarmand埃布拉希米有益的讨论，我们非常感谢。这项工作是由代尔夫特理工大学的启动资金支持。

1。 DPS表达和纯化获得高纯度蛋白的DNA保护实验是必不可少的第一步。可以在4至5天的Dps蛋白的分离纯化。 <ol><li>变换的蛋白酶缺陷型菌株大肠杆菌大肠杆菌 （如BL21（DE3）pLysS中）已被克隆到其中的Dps的蛋白编码序列的pET载体（如pET17）。 </li><li>连胜转化细胞到的Luria肉汤（LB）琼脂平板，含适当的抗生素（如氨苄青霉素和氯霉素1.1中提供的例子）。过夜孵育板在37°C。 </li><li>从板的单菌落，接种到含有适当抗生素的LB培养基中30毫升。孵育过夜，在37°C，而在200-250转摇晃。 </li><li>物接种到2×1升含有适当抗生素的LB培养基中，用每次10毫升过夜培养。增长在37℃，一边摇晃，外径600 </分&gt;约为0.6。 （较高的OD增加蛋白质产量，生长的细胞没有明显影响最终产品的纯度。）诱导表达的通过加入IPTG浓度为0.3毫米，每秒伤害，并在37℃孵育3-4小时，而晃动。 </li><li>在6000×g离心15分钟，通过离心收获细胞。重悬细胞沉淀于7.5ml DEAE缓冲液A（50mM的HEPES-KOH，pH值7.5，100 mM氯化钠，0.1mM的EDTA）每L的诱导的细胞培养。样品在液氮中冷冻并储存在-80℃下，如果需要的话。然后，该过程可能会在水浴中解冻的样品在4℃下继续</li><li>蛋白酶抑制剂（Calbiochem公司的一套三蛋白酶抑制剂，例如，在每毫升的细胞悬浮液0.167微升）的混合物加入到细胞悬浮液，以防止过度表达的Dps退化。 </li><li>准备使用法国记者无细胞提取物。总理，法国媒体20 ml DEAE缓冲区中（如果采用连续流模型），然后disrup吨样品20 KPSI两次。离心溶胞产物在4℃下以30,000×g离心35分钟以澄清不溶颗粒，并保存上清液。然后将上清液用液氮冷冻并储存在-80℃下，如果需要的话。 </li><li>平衡30毫升DEAE琼脂糖凝胶CL-6B柱用DEAE缓冲液A，使用FPLC。运行缓冲液通过该柱在1-2毫升/分钟的最大压力为0.2MPa以上的背景。无细胞提取物加载到该列，开始收集流通过的OD 280信号一旦开始增加基线以上。用DEAE缓冲柱洗净A直到OD 280值返回到基线，同时不断收集流过。流过，应该包含大部分的DPS蛋白结合的DNA。用100％缓冲液B（50mM的HEPES-KOH，pH为7.5，1M NaCl的0.1mM的EDTA），清洗色谱柱。该洗脱液中包含的Dps-DNA复合物以及其他蛋白质，可以被丢弃。 </li><li>删除广告ditional污染通过硫酸铵沉淀蛋白质。确定汇集流过的体积。慢慢地（10-20分钟）过了一段加到干燥的小颗粒硫酸铵至62％饱和度（390毫克每毫升的溶液），在4℃，同时充分搅拌。搅拌该混合物20-30分钟后，加入硫酸铵的最后部分，以确保完全平衡。 DPS将仍然是可溶的，而杂蛋白溶液中沉淀出来。 </li><li>通过以20000×g离心30分钟除去沉淀的蛋白质在4℃下保存上清液。 </li><li>慢慢地添加一个额外的227毫克每毫升上清液硫酸铵达到94％饱和度，搅拌20-30分钟，以确保完全平衡。 Dps的析出物在这样高的盐的浓度，并且可以在4℃下以20,000 xg离心30分钟，离心收集将沉淀的Dps，可以被丢弃上清液。的颗粒可以保存在-80°C如果德IRED。 </li><li>重悬的DPS含有沉淀再悬浮缓冲（50毫米HEPES-KOH，pH值7.5，氯化钠150毫米，0.1毫米EDTA）。调整样本量为2.5毫升。 </li><li>缓冲液交换，以除去硫酸铵使用的PD-10凝胶过滤柱。 25毫升缓冲液，平衡凝胶床。 Dps的样品2.5ml的应用PD-10柱的顶部，并允许它浸泡英寸的流量通过丢弃。 3.5毫升缓冲液，洗脱，收集DPS。 </li><li>缓冲交换的样品在16000×g离心10分钟，在4℃以除去任何不溶性成分，用6-10毫升稀释缓冲液（50mM的HEPES-KOH，pH值7.5，0.1mM的EDTA）稀释，以确保盐的浓度足够低DPS绑定SP琼脂糖凝胶柱。尤其是浓缩的样品的Dps可能成为溶解后稀释到较低的盐缓冲液，就证明了混浊的液体，但也可以通过添加一个小型的AMOU resolubilizedNT 5 M氯化钠溶液（10-20毫米）。 </li><li>平衡30毫升的SP琼脂糖凝胶快速流动SP缓​​冲区A柱（50毫米HEPES-KOH，pH值7.5，氯化钠50毫米，0.1毫米EDTA）。运行缓冲液通过该柱为1.5-2毫升/分钟的压力限制为0.3MPa以上背景。将样品到列。与SP缓冲区A直到OD 280跟踪返回到基线进行清洗，并丢弃的流量通过。运行一个150毫升的从0到100％缓冲液B的线性梯度，同时收集2毫升馏分。 DPS将洗脱在锋利的峰值在约50％的B，虽然10％的变化是可能的。 </li><li>运行在15％SDS-PAGE凝胶上的洗脱级分，并通过测量OD 260 / OD 280的比例使用分光光度计检查DNA污染。凝胶应只显示一条带约19 kDa的，OD 260 / OD 280通常是0.7左右。游泳池最纯粹的DPS分数。使用离心式过滤器单元（如的Amicon超滤单元）与切断10K分子量集中的DPS和交换成一个存储缓冲区（50毫米HEPES-KOH，pH值7.5，氯化钠50毫米），。等分试样的纯化的Dps，并冷冻在液氮中储存于-80℃。 </li></ol> 2。 DNA保护实验实验包括对时间敏感的多个步骤，并应定时到第二，以获得可重复性的结果。涉及的许多成分和移液步骤，所以一个移液表（ 见表1）最好是跟踪的步骤和卷。为测定所需的总时间不超过3小时。 <ol><li>测量30毫升水放入密封的小瓶，用N 2冲洗的水10分钟（使用两个注射器针头，在流体中，它上面的一个）从溶液中除去氧气。添加硫酸亚铁0.0168克4·7H 2 O的水，获得了2毫米原液。密封的小瓶和冲洗5分钟，然后用氮气混合。解决方案必须清楚，如果检测到任何一丝黄色，准备一个新的铁溶液。 </li><li> 100毫米H 2 O 2溶液9.2MH 2 O 2（10.87微升到1毫升水）;保持在冰上，并通过包装在铝箔屏蔽光线。只可使用约1-2小时准备后，由于自发衰变。过氧化氢股票是同样的故障，这可能是部分通过适当的存储（在黑暗中，在4°C）改善。建议定期检查的股票浓度测量外径240。 </li><li>在室温水浴纯化DPS迅速解冻等分，并保持在冰上。离心沉淀物聚集在表顶部的picocentrifuge的在4000×g离心10秒。使用分光光度计（OD 280 = 1.547，显示其浓度为100μM的单体的Dps）离心分离后的浓度测量。 </li><li>使用高浓度的股票12X反应缓冲（1线性DNA稀释M MOPS-KOH，pH为7.0，1M NaCl的）的浓度为100毫微克/微升。使用线性DNA，使定量更容易，但也可以使用质粒DNA，以及。 </li><li>中加入1微升的PCR管的DNA的缓冲液混合物进入。添加足够的水加入到反应，使最终的反应体积的（减去SDS）将12微升。此量应在使用移液表预先计算。添加DPS一个的最终dodecamer浓度为3微米。在室温下孵育15分钟，以允许与DNA结合的Dps。 </li><li>加入足够的2mM的硫酸亚铁溶液，以达到所需的浓度。一个典型的实验中使用的浓度范围从0到1毫米硫酸亚铁。较高的浓度将导致更广泛的DNA降解，但也可能会导致形成的三价铁在反应混合物中析出。快速加入1微升100mM的H 2 O 2溶液（10mM的终浓度），5分钟，在室温下孵育，以允许芬顿介导的降解反应发生。 </li><li>加入0.8微升20％的SDS（十二烷基硫酸钠）。混合，在85°C孵育5分钟，扰乱的DPS-DNA复合物。 SDS的DPS-DNA复合物的稳定性，并防止不必要的凝胶的DNA的变化，提高了易用性量化。一个可选的步骤，是通过过氧化氢催化降解成无毒的产品，使反应停止。在这个协议中所描述的条件，这一步是没有必要的，得到广泛的DNA降解。 </li><li>在冰上孵育1分钟，加样染料，并加载在琼脂糖凝胶上。运行的凝胶，并使用溴化乙锭染色的DNA。电泳后的凝胶应被污染，而不是在电泳前，以防止干扰分布在凝胶中的溴化乙锭的SDS。 </li></ol>

<ol>
	<li>Almiron, M., Link, A. J., Furlong, D., Kolter, R. <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+DNA-binding+protein+with+regulatory+and+protective+roles+in+starved+Escherichia+coli.">A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli.</a> Genes Dev. 6, 2646-2654 (1992).</li><li>Chiancone, E., Ceci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+capacity+of+Dps+proteins+to+combat+bacterial+stress+conditions:+Detoxification+of+iron+and+hydrogen+peroxide+and+DNA+binding.">The multifaceted capacity of Dps proteins to combat bacterial stress conditions: Detoxification of iron and hydrogen peroxide and DNA binding.</a> Biochimica et biophysica acta. 1800 (8), 798-805 (2010).</li><li>Ali Azam, T., Iwata, A., Nishimura, A., Ueda, S., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase-dependent+variation+in+protein+composition+of+the+Escherichia+coli+nucleoid.">Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid.</a> J. Bacteriol. 181 (20), 6361-6370 (1999).</li><li>Grainger, D. C., Goldberg, M. D., Lee, D. J., Busby, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+repression+by+Fis+and+H-NS+at+the+Escherichia+coli+dps+promoter.">Selective repression by Fis and H-NS at the Escherichia coli dps promoter.</a> Molecular Microbiology. 68 (6), 1366-1377 (2008).</li><li>Martinez, A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+DNA+during+oxidative+stress+by+the+nonspecific+DNA-binding+protein+Dps.">Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.</a> J. Bacteriol. 179 (16), 5188-5194 (1997).</li><li>Nair, S., Finkel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dps+protects+cells+against+multiple+stresses+during+stationary+phase.">Dps protects cells against multiple stresses during stationary phase.</a> J. Bacteriol. 186 (13), 4192-4198 (2004).</li><li>Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R., Hogle, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+Dps,+a+ferritin+homolog+that+binds+and+protects+DNA.">The crystal structure of Dps, a ferritin homolog that binds and protects DNA.</a> Nat. Struct. Biol. 5 (4), 294-303 (1998).</li><li>Zhao, G., Ceci, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+hydrogen+peroxide+detoxification+properties+of+DNA-binding+protein+from+starved+cells.+A+ferritin-like+DNA-binding+protein+of+Escherichia+coli.">Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli.</a> J. Biol. Chem. 277 (31), 27689-27696 (2002).</li><li>Ceci, P., Cellai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+condensation+and+self-aggregation+of+Escherichia+coli+Dps+are+coupled+phenomena+related+to+the+properties+of+the+N-terminus.">DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.</a> Nucleic Acids Res. 32 (19), 5935-5944 (2004).</li><li>Ilari, A., Ceci, P., Ferrari, D., Rossi, G. L., Chiancone, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+incorporation+into+Escherichia+coli+Dps+gives+rise+to+a+ferritin-like+microcrystalline+core.">Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core.</a> J. Biol. Chem. 277 (40), 37619-37623 (2002).</li><li>Swift, J., Wehbi, W. A., 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=Design+of+functional+ferritin-like+proteins+with+hydrophobic+cavities.">Design of functional ferritin-like proteins with hydrophobic cavities.</a> Journal of the American Chemical Society. 128 (20), 6611-6619 (2006).</li><li>Ceci, P., Chiancone, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+iron+oxide+nanoparticles+in+Listeria+innocua+Dps+(DNA-binding+protein+from+starved+cells):+a+study+with+the+wild-type+protein+and+a+catalytic+centre+mutant.">Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.</a> Chemistry. 16 (2), 709-717 (2010).</li><li>Ceci, P., Ilari, A., Falvo, E., Chiancone, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dps+protein+of+Agrobacterium+tumefaciens+does+not+bind+to+DNA+but+protects+it+toward+oxidative+cleavage:+x-ray+crystal+structure,+iron+binding,+and+hydroxyl-radical+scavenging+properties.">The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure, iron binding, and hydroxyl-radical scavenging properties.</a> The Journal of Biological Chemistry. 278 (22), 20319-20326 (2003).</li><li>Su, M., Cavallo, S., Stefanini, S., Chiancone, E., Chasteen, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+so-called+Listeria+innocua+ferritin+is+a+Dps+protein.+Iron+incorporation,+detoxification,+and+DNA+protection+properties.">The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties.</a> Biochemistry. 44 (15), 5572-5578 (2005).</li><li>Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Koelreuteria+paniculata+Laxm.+on+oxidative+stress+and+hydrogen+peroxide-induced+DNA+damage.">Protective effects of Koelreuteria paniculata Laxm. on oxidative stress and hydrogen peroxide-induced DNA damage.</a> Phytopharmacology. 1 (5), 177-189 (2011).</li><li>Stinefelt, B., Leonard, S. S., Blemings, K. P., Shi, X., Klandorf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+scavenging,+DNA+protection,+and+inhibition+of+lipid+peroxidation+mediated+by+uric+acid.">Free radical scavenging, DNA protection, and inhibition of lipid peroxidation mediated by uric acid.</a> Annals of Clinical and Laboratory Science. 35 (1), 37-45 (2005).</li><li>Lopez-Gomollon, S., Sevilla, E., Bes, M. T., Peleato, M. L., Fillat, M. F. <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+role+of+Fur+proteins:+FurB+(All2473)+from+Anabaena+protects+DNA+and+increases+cell+survival+under+oxidative+stress.">New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress.</a> The Biochemical Journal. 418 (1), 201-207 (2009).</li><li>Ebrahimi, K. H., Hagedoorn, P. L., Hagen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+and+stimulation+of+formation+of+the+ferroxidase+center+and+the+iron+core+in+Pyrococcus+furiosus+ferritin.">Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.</a> Journal of Biological Inorganic Chemistry. 15 (8), 1243-1253 (2010).</li><li>Smith, F. E., Herbert, J., Gaudin, J., Hennessy, D. J., Reid, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+iron+determination+using+ferene+triazine.">Serum iron determination using ferene triazine.</a> Clinical Biochemistry. 17 (5), 306-310 (1984).</li></ol>

我们什么都没有透露。

在这篇文章中所描述的净化过程中的DPS是非常强大的。一直纯度高（&gt; 99％）;没有其他蛋白质出现在可见光波段的SDS-PAGE凝胶。尽管这样，似乎有部分批次纯化DPS核酸酶活性，证明了部分DNA的降解，当孵育浓度非常高的DPS。这可能表明在低浓度高活性的DNA酶的存在，我们无法通过净化除去。然而，这种DNA降解只观察到的浓度通常不用于在体外测定的 ，所以这通常不存在任何问题。 <...

需氧生物必须不断抗衡，可以破坏它们的DNA，以及其他重要的生物大分子的活性氧。对抗氧化损伤的毒性作用的一个有力的工具是从饥饿细胞的DNA结合蛋白（DPS）。自从被发现于1992年，从饥饿E.大肠杆菌培养1，DPS中已发现300余种细菌和古细菌2。 DPS在固定相的大规模上调使得它表达最高度的拟核相关蛋白的大肠杆菌饥饿条件下3，4的 大肠杆菌 。此外，已被证明的Dps保留在许多不同的应力，包括饥饿，铁浓度高，紫外线照射，热休克，氧化应激5，6细菌的生存能力和DNA完整性。 在结构上，DPS自联营成一个稳定的同源寡聚体复合物的12个单体，W一族组装成一个球形的空壳。 〜4.5 nm的范围内的内部腔体的溶剂可允许通过的小分子7的毛孔的外部访问，和，它可以螯合矿化的金属如铁8。 DPS的保护作用来源于几个生化活动，其中包括非特异性DNA结合，亚铁活动，和铁存储8。 对人体有益的生化活动的DPS，首先需要详细研究其净化。 Dps的净化是一个复杂的过程，Dps的必须不仅从其他蛋白质分离，但也从任何结合的DNA 7。我们已优化的纯化过程中使用许多常用的技术，包括两个离子交换柱和硫酸铵沉淀步骤。几个缓冲区的交流是必要的，高度集中的DPS可以在溶液中沉淀出来低盐条件。一旦DPS蛋白已被纯化，它可能是用于检测直接测量亚铁活性8，DNA-结合的化学计量学9，与铁结合的机制10。纯净DPS也有其他潜在的应用。稳定的中空球状结构的Dps被用作脚手架，用于存储在蛋白质内部空腔11的憎水颗粒，甚至作为一个反应室，以合成新颖的磁性纳米粒子12。 保护能力的DPS调解活性氧自由基造成的损害，可以清楚地直接证明使用DNA保护实验13，14。在这种体外过程中，产生自由基种，铁催化H 2 O 2通过Fenton化学降解。这些自由基直接损伤DNA存在于反应中，可以在高浓度下完全降解。两个关键的DPS活动都可能直接抵消的影响FENT在介导的自由基的产生。 Dps的催化铁的浓度降低通过矿化，在这个过程中消耗的可用过氧化氢。此外，DPS与DNA结合可能屏蔽它身体免受自由基的损害，并凝结成更小的体积与反应表面积少。这两个属性的组合，使得DNA为目的测量保护的Dps活动的适合的保护测定。 的DNA保护分析法是相当灵活的，可用于各种各样的应用程序之外的Dps表征。自由基损伤是一种常见的细胞形式的压力，和许多不同的蛋白质和化学品的使用，抵制它。测定的一般原则，使用作为自由基损伤的标记物的DNA的完整性，可以结合使用几乎任何产生自由基的反应剂或抵消。其中包括，该法已成功地用于确定抗氧化性能K的穿心莲提取物使用在食品行业15，表征尿酸对羟基损害调解16的影响，并获得新的见解毛皮转录调节蛋白17的功能。 尽管在发表的论文中检测的众多用途中，我们发现许多优化和故障排除步骤，这使得第一次不必要的艰苦的过程，许多研究者测定。在这篇文章中，我们提出的协议旨在消除这一屏障，为入境。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

氧化应激是一个不可避免的副产品的有氧生活。地面代谢分子氧气是必不可少的，但它也需要在许多破坏性反应于生物体的部分。通过Fenton化学降解细胞成分产生自由基相结合的有氧代谢和铁，这是生活的另一种重要的化合物，是足够的。 DNA降解无疑是最具破坏性的过程，涉及细胞内的自由基，DNA修复是远从琐碎。在这篇文章中提出的检测提供了一个定量的测量和可视化技术，分子和酶的作用自由基介导的DNA损伤。 DNA保护法是一种简单，快速和强大的工具， 在体外蛋白质或化学品的防护性能表征。它涉及曝光的DNA的损害性的氧化反应，加入不同浓度的化合物还。作为化合物浓度的函数的DNA损伤的减少或增加，然后可视化，使用凝胶电泳。在这篇文章中，我们展示了保护的DNA检测技术，通过测量DNA结合蛋白的保护性饥饿细胞（DPS）。 DPS是一个小型铁，动用300余种细菌，有力地打击环境压力。在这里，我们提出的的DPS净化协议和评估DNA保护DPS优化实验条件。

这里描述的Dps的纯化方法是高度可再现性。 Dps的纯化，根据所描述的协议，使用2升的大肠杆菌大肠杆菌文化为出发点，通常会产生2.5毫升含蛋白质浓度在5和12微米之间的DPS 12。较长的诱导时间（4小时）似乎减少这种可变性。蛋白质纯度为99％以上，始终SDS-PAGE凝胶（ 图1）可见一斑。 DNA污染的水平，一直是可以忽略不计，证明通过琼脂糖凝胶染色DNA和OD 260 / OD ...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

（DPS）从饥饿细胞的DNA结合蛋白起着至关重要的作用打击细菌应力的。本文讨论了净化<em&gt; E。大肠杆菌</em&gt; DPS和一个协议<em&gt;在体外</em&gt;试验示范DPS-介导的保护DNA降解的活性氧物种。

应用的<em在体外</em&gt;保护DNA检测，以可视化应力调解属性的DPS蛋白质

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

application-an-vitro-dna-protection-assay-to-visualize-stress

Research

JoVE Journal

Biology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

11.1K 次观看.  Delft University of Technology. （DPS）从饥饿细胞的DNA结合蛋白起着至关重要的作用打击细菌应力的。本文讨论了净化 E。大肠杆菌 DPS和一个协议在体外试验示范DPS-介导的保护DNA降解的活性氧物种。

视频: Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

科研

生物学

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

停流的动力学方法的应用研究的一种DNA修复蛋白的行动机制

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

彗星试验系统性氧化应激的间接测量

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...