We thank Dr&#8217;s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.

潜在的小分子封端基团的1计算识别注意：原则上，各种各样的对接或药效搜索方法可用于预测潜在封盖基。计算研究中REPLACE的主要目的是确定的小分子，其保留的被取代的氨基酸的功能和相互作用。 <ol><li>在LigandFit对接协议14的验证</li></ol>注意：在以前的研究中，对接方法（LigandFit 15，在分子建模程序套件的模块，发现工作室3.0）进行验证，以确保该算法足以重现已知NCAPS的结合方式，并表明，该结果获得的未知化合物的预测14。 <ol style="margin-left: 40px;"><li>通过以下步骤（1.2 - 1.5），识别LigandFit最佳条件为：PLP1恩ERGY网格姿势代，最小化产生的姿势和PLP1打分函数姿势的分析。 </li><li>为了限制所得到的构象，并在封端的肽适当地定位的配体形成共价键，设置Trp217和Gln254的吲哚氮与酰胺氮原子分别作为相互作用滤波器氢键。 </li><li>设计和码头的片段替代品库到CDK2 /细胞周期蛋白受体（2UUE）。优先潜力封盖组​​进行综合和基于PLP1得分，以互动的过滤器和互补的CBG互动的商业采购。是所需LigandFit对接的各个步骤如下。 </li></ol><ol start="2"><li>的受体结合位点14制备 <ol><li>导入CDK2 /细胞周期蛋白晶体结构（PDB ID：2UUE）到软件中的一个可视化窗口。这个结构包含先前标识FLIP 5-甲基-1-苯基-1H-1，2,4-三唑-3-甲酰（3,5- DCPT）RLIF结合于细胞周期蛋白槽（图3）。 </li><li>删除或守在原地的残留RLIF（C端）。当被保持在肽序列中，使用肽的N末端氨基作为相互作用滤波器的配位体。从“受体 - 配体相互作用”工具，创建一个围绕的N帽3,5- DCPT来自显示/隐藏部位球的球体。球体被创建以限定在配体 - 受体相互作用允许发生期间结合所述结合位点的活性区。 </li><li>定义的结合位点进一步从“找到站点作为选择的配体的量”，并从蛋白质删除配体3,5- DCPT。执行此步骤来定义创建的范围内具有约束力的体积。 </li><li>设置残基Trp217和Gln254作为相互作用滤波器氢键的吲哚氮与酰胺氮原子。设置的过滤器相互作用，使得只有那些INTE姿势RACT与集残留的原子将在对接过程中，因此保留在停靠姿势重要的相互作用进行过滤。 </li></ol></li><li>配14的制备<ol><li>设计的100潜在封盖基根据标准的库包括分子量小于500时，存在羧酸酯基的结扎用截短的肽和夹杂物的范德华相互作用适当疏水性基团（取代的苯基）在二次口袋作为表 1所示。 </li><li>选择杂环用于模仿肽氢键相互作用与Trp217和取代基包括更换HAKRRLIF的碱性侧链的离子配对相互作用。停靠的配位体作为相应醛分子，以使羰基氧可以与Gln254的酰胺氮原子的母体肽（表1）相似的肽主链相互作用。 </li><li>此前DOC王，最大限度地减少设计的配体低能量构象，并使用“准备配体”协议，在适当的电离状态的准备。绘制和中的ChemDraw .sdf文件格式的电子表格之前导出这些潜在的N帽被导入到软件中。 注意：准备配位体协议用于最小化所有的配体停靠到它们的最低能量状态和电离指控被施加到适用原子。默认参数用于此步骤。 </li></ol></li><li>对接的配体进入细胞周期蛋白A槽14 <ol><li>选择从受体 - 配体相互作用协议的软件的集合的LigandFit例行程序。使用受体和配体准备作为输入此协议。 </li><li>这些试验选择PLP1作为能量网格，指定构成是能量最小化，并生成姿势为10的数目，保持所有其他参数为默认值。 注：LigandFit是对接方法，它可以识别并产生高的互补性和潜力的亲和姿势与使用被结合蒙特卡洛构象搜索算法基于形状进行比较滤波器的蛋白质的活性位点。使用对接程序的能量网格是力场用于产生配体 - 受体相互作用和潜在姿势。 PLP1是特异性优先氢键相互作用和分段线性电位，其中所述PLP原子类型分配每个非氢原子的配体或非氢原子受体。 </li></ol></li><li>结果的分析<ol><li>在第一个实例，显示由降PLP1得分值带来的可视化程序，然后排序。使用计分函数如PLP1来估计根据一个候选配体对接的配体的结合亲和力姿势几何和非共价相互作用与靶受体结构。 注：在这里，PLP1打分函数被发现与G香港专业教育学院最好的结果，因为它包括的氢键的估计。 </li><li>目测分析打进姿势为1的顶部25％）superimposability与已知封端基团（具有相对均方偏差（RMSD）值小于2的），2）满足相互作用的过滤器和3）视觉的互补与细胞周期蛋白槽。它接受一个正确的停靠姿势（相较于实验结构）具有&lt;2的RMSD值。 </li><li>检查姿势为视觉的互补性，其被定义为具有的方式，是与已知的结构 - 活性关系一致的结合口袋的有效填充。相互作用滤波器被设置为包括需要已知可用于结合和/或需要在用于酰胺键形成正确的几何定位的电位加帽基临界分子间接触原子约束。潜在的N-端封基团使氢键与interactio停靠姿势三个例子N个滤波器示于图4。 </li></ol></li></ol> 2.合成和潜在的N -capping组表征<ol start="1"><li>合成的N-封端基团。 </li><li>对于合成，使用所有的商业原料，溶剂和试剂而获得。通过常规有机合成化学（图5 12,13的例子）合成潜在的N-封端基团。 </li><li>用于监测反应进行硅胶薄层层析（TLC）。 <ol><li>溶解起始原料和反应混合物（1毫克）中的流动相，发现它们对使用细管的TLC板上。 </li><li>将TLC板中含有流动相（乙酸乙酯和己烷以35:65的比例）的腔室。一旦流动相达到90％的薄层板上，捞出晾干板。 </li><li>使用UV光来检测起始材料和反应混合物作为可见的斑点。卡尔culate所有部位中的R F（距离由点和流动相行进的比）。 注意：该反应完成时，没有光斑见于起始材料中的R F。 </li></ol></li><li>在反应完成后通过放置在分液漏斗和洗涤用含水酸或碱溶液适当工作了反应混合物中。收集有机溶剂，在旋转蒸发器中蒸发，并在真空下干燥所得到的粗产物。 </li><li>溶解500毫克粗产物在3-5毫升合适的溶剂，并加入到一个1克的二氧化硅或RP18 samplet和下空气干燥。放置含粗产物在二氧化硅/ RP SNAP闪光灯墨盒samplet和使用自动高性能快速色谱法（根据制造商的协议）采用SNAP 100个克柱和梯度运行的6％乙酸乙酯开始纯化粗物质：94 ％己烷至50％乙酸乙酯和50％己烷以上15个柱体积。 </li><li>通过蒸发所有溶剂至干干燥使用旋转蒸发器收集在溶剂从快速色谱纯化产物，并进一步干燥该产物在真空下除去所有的残留溶剂。通过NMR，MS和分析型HPLC进行纯化产物的表征。 <ol><li> 1 H NMR和13 C-NMR，重约10毫克的纯化样品，将其溶解在适当的氘代溶剂中，并将内容物转移到干燥NMR管，用于记录NMR谱2,14。 </li><li>记录1 H NMR和 13 C NMR谱与高场核磁共振仪（最小300兆赫）。收购质谱使用QTOF（串联四-1飞行时间质谱仪），电喷雾离子化（ESI）或VG 70S（双聚焦扇形磁场质谱仪，EI）2,14。 </li><li>通过HPLC用二极管阵列检测器分析产品的纯度和配备在C18（2）100，250×4.6毫米，5微米柱进行分析。使用的梯度运行从100％水（0.1％三氟乙酸）开始至60％乙腈（0.1％三氟乙酸）在30分钟内并保持梯度为4分钟。提取色谱图在226和254纳米2,14。 注：评价化合物的分析纯度分别&gt; 95％。 </li></ol></li></ol> 3.固相合成的翻转2代<ol start="1"><li>正封顶翻转合成<ol><li>装配通过使用以下步骤的标准固相合成方法的N-封端的肽化合物。 <ol><li>在2ml激活5当量的C-末端氨基酸（的Fmoc-Phe的生产）的，在O3 -Benzotriazole- N，N，N&#39;，N&#39;四甲基脲六氟磷酸盐（HBTU，221.93毫克）4.4当量DMF 5分钟。装载的Fmoc-PHE HBTU混合到使用6当量二异丙基胺的溜冰场树脂（DIPEA，103.13毫克）处理1小时，在室温。 </li><li>使用20％哌啶在3ml DMF中的10分钟的C-末端残基除去Fmoc保护基。夫妇随后的氨基酸（ 如 ，FMOC，异亮氨酸，FMOC亮氨酸，FMOC-精氨酸- PMC）一步一步来。在每一步，一对5当量使用6当量DIPEA（103.13毫克）和HBTU（221.93毫克）在2毫升DMF中4.4当量的1小时在室温下一个氨基酸。 </li><li>适用的洗涤周期（5×10毫升DMF + 5×10ml DCM中的）上述氨基酸偶联和Fmoc去保护步骤后。肽装配后，夫妇N-二封盖使用6当量DIPEA（103.13毫克）和HBTU（221.93毫克）的4.4当量的群体在2ml DMF中进行1小时，在室温。 </li><li>在肽组装完成后，治疗用2ml的裂解混合物的反应混合物（90：5：5的TFA / H 2 O / TIPS）O / N以除去侧链保护基团，并从树脂裂开翻转。用冷乙醚沉淀和我磨碎所得到的产物˚F必要的浓缩在旋转蒸发器。 </li></ol></li></ol></li><li>的N-封顶-C皑皑翻转的合成<ol><li>称重并溶解N-末端封端的和受保护的精氨酸-亮氨酸肽（16.1毫克，0.02毫摩尔）在二氯甲烷中，并添加[4-（3-氯苯氧基）吡啶-2-基]甲胺（C-帽）沿带 O -苯并三唑-N，N，N&#39;，N&#39;四甲基脲六氟磷酸盐（HBTU; 16.7毫克，0.02毫摩尔）和N，N-二异丙基 （DIPEA; 6.5毫克，0.02毫摩尔）。 </li><li>搅拌并监测在RT反应直至TLC / HPLC显示起始材料的完全消耗。反应完成后，浓缩，用乙酸乙酯和水之间在旋转蒸发器然后分区粗反应混合物。 </li><li>分离水和有机层;用1N NaOH，1N HCl和盐水洗涤洗涤有机层。与约1克硫酸钠干燥有机层并浓缩产物在旋转蒸发器中。菲娜LLY治疗产品O / N与裂解混合物（95：2.5：2.5三氟乙酸/ H 2 O / TIPS）进行脱保护。 </li><li>用冷的二乙醚，并在必要的浓缩在旋转蒸发至完全干燥以除去所有的溶剂磨碎所得到的产物。进一步在真空下干燥产物以除去所有的残留溶剂。 </li></ol></li><li>纯化和翻转的特性<ol><li>使用半制备反相HPLC纯化法粗翻转。冻干纯化的翻转和用质谱2,14描述他们的分子质量。 </li><li>通过HPLC用二极管阵列检测器确定所述翻转的分析纯度和配备有C18（2）100，250×4.6毫米，5微米柱。使用从95％的H 2 O（0.1％三氟乙酸）/ 5％乙腈（0.1％三氟乙酸）到35％ 的 H 2 O（0.1％三氟乙酸）/ 65％乙腈（0.1％trifluoroace开始梯度法抽动酸）在30分钟内并保持梯度为4分钟。提取色谱在226和254纳米。 </li></ol></li></ol> 4.荧光偏振结合分析的竞争性结合2,14的测定<ol start="1"><li>请在黑色384孔（138微升）使用以下步骤板的测定法。 </li><li>稀释的样品中，示踪肽（4纳米），和激酶复合物（CDK2 /细胞周期蛋白A - 18微克/毫升，CDK4 /细胞周期蛋白D - 37微克/毫升），以所需浓度在测定缓冲液（25mM HEPES pH为7，10毫氯化钠，0.01％的Nonidet P-40，1mM的二硫苏糖醇（DTT）。 </li><li>向384孔板的各孔中，添加：5微升CDK4D1或CDK2CA（0.3微克/孔纯化重组人激酶复合物），5微升化合物溶液，5微升30nM的示踪肽（荧光素AHX-亲缬氨酸的-Lys精氨酸基-Arg-Leu-（3ClPhe）-Gly或荧光素AHX-PRO-VAL-赖氨酸 - 精氨酸 - 精氨酸 - 亮氨酸 - 苯丙氨酸 - 甘氨酸示踪肽）。使用每个DMSO（6％），buffe的5微升R，示踪和5μlDMSO（6％），激酶复合物，示踪物作为对照孔</li><li>在加入所有组分后，离心平板在500rpm下1分钟（41.16 XG）在RT，然后孵育该板与搅拌在室温45分钟。使用多模板读取器和检测器装有485毫微米/ 535毫微米的激发/发射滤​​光片和分色镜读取各孔在板的荧光强度。 </li><li>计算相对平均偏振（熔点）使用该方程表示下面的所有测试样品。确定IC 50值用对数回归通过关联相对MPS和测试浓度。 </li></ol><img alt="式（1）" src="/files/ftp_upload/52441/52441eq1.jpg" />

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M., Jiang, X., Oldfield, T., Waldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LigandFit:+a+novel+method+for+the+shape-directed+rapid+docking+of+ligands+to+protein+active+sites.">LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites.</a> J Mol Graph Model. 21, 289-307 (2003).</li><li>Mendoza, N., 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=Selective+cyclin-dependent+kinase+2/cyclin+A+antagonists+that+differ+from+ATP+site+inhibitors+block+tumor+growth.">Selective cyclin-dependent kinase 2/cyclin A antagonists that differ from ATP site inhibitors block tumor growth.</a> Cancer Res. 63, 1020-1024 (2003).</li><li>Denizot, F., Lang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cell+growth+and+survival.+Modifications+to+the+tetrazolium+dye+procedure+giving+improved+sensitivity+and+reliability.">Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.</a> J Immunol Methods. 89, 271-277 (1986).</li><li>Zheleva, D. I., 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=Highly+potent+p21(WAF1)-derived+peptide+inhibitors+of+CDK-mediated+pRb+phosphorylation:+delineation+and+structural+insight+into+their+interactions+with+cyclin+A.">Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.</a> The journal of peptide research : official journal of the American Peptide Society. 60, 257-270 (2002).</li><li>Ivanov, A. A., Khuri, K. F. R., Fu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein-protein+interactions+as+an+anticancer+strategy.">Targeting protein-protein interactions as an anticancer strategy.</a> Trends in Pharmacological Sciences. 34, (2013).</li><li>Thayer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+peptides.">Improving peptides.</a> Chemical and Engineering News. 89, 13-20 (2011).</li><li>Yin, H., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+targeting+protein-protein+interactions+with+synthetic+agents.">Strategies for targeting protein-protein interactions with synthetic agents.</a> Angew Chem Int Ed Engl. 44, 4130-4163 (2005).</li><li>Jubb, H., Higueruelo, A. P., Winter, A., Blundell, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+and+drug+discovery+for+protein-protein+interactions.">Structural biology and drug discovery for protein-protein interactions.</a> Trends Pharmacol Sci. 33, 241-248 (2012).</li><li>Cannon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceutics+and+drug+delivery+aspects+of+heme+and+porphyrin+therapy.">Pharmaceutics and drug delivery aspects of heme and porphyrin therapy.</a> J Pharm Sci. 82, 435-446 (1993).</li></ol>

The authors declare that they have no competing financial interests.

Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI&#8217;s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and...

在这篇文章中，应用REPLACE（更换使用计算富集部分配体的替代品）策略，以蛋白质-蛋白质相互作用的肽酶抑制剂转换成多种药物相关的分子为例进行说明1-3。虽然生产者价格指数代表了丰富，但开发不足潜在的药物靶点的来源，现有方法在很大程度上不足以让这些普及。当前策略包括基于片段设计4，高通量筛选5和6提供了进步装订肽，但是这些在许多情况下是无效的。其结果是，更多的进展和更有效的方法是必需的。 REPLACE已激酶抑制剂具有改善的类药性的属性和有潜力的进一步发展为抗肿瘤治疗剂的开发已充分验证。这种策略是例示的电池的非ATP酶抑制剂的开发周期CDK的并涉及如下：1）获得关于HAKRRLIF / RRLIF与细胞周期蛋白结合槽相互作用三维结构信息; 2）确定的重要结合决定肽相互作用;含有一种或多种结合决定簇的肽的N-末端的3）截断; 4）计算确定可能的小分子的替代品（局部的配位体的替代品，聚乳酸类）的肽和其中的截断部分保留母体肽的关键相互作用; 5）合成或PLA中的商业采购预测与先前所删除的肽残基所占据的子站点贪婪地结合; 6）的合成通过使用固相合成截短的肽最好PLA中的结扎翻转的; 7）检测结合在体外翻转或功能测定（在CDK /细胞周期蛋白上下文荧光偏振），随后在细胞生存力测定法进一步表征。更换的战略中一个示意图y为如图 1所示。在这篇文章中，REPLACE策略的迭代被讨论，并应用到CDK2 /细胞周期蛋白A进行详细说明。激酶被认为是直接或间接地解除管制在大多数肿瘤中，因此被视为适当的抗癌药物靶 7。 CDK的要求协会细胞周期的完全激活，随后磷酸化参与细胞周期调控8关键蛋白。激酶的两个主要群体是控制细胞周期检查站同型[G1 / S（CDK4 /细胞周期蛋白D，CDK6 /细胞周期蛋白D和CDK4 / cyclin E的），S期（CDK2 /细胞周期蛋白A）和G2 / M（CDK1 /细胞周期蛋白B）]和RNA聚合酶通过磷酸化调节（CDK7 /细胞周期蛋白H，CDK8 /周期蛋白C，CDK9 /周期蛋白T）。当E2F1转录因子形成与DP蛋白质然后与DNA结合，并启动基因转录的复合S期进展中的关键步骤发生。 CDK2 /细胞周期蛋白A需要中和E2F1转录通过磷酸化活性，从而导致释放E2F1-DP的复杂和其随后的退化。 CDK2 /细胞周期蛋白A的抑制被认为是维持E2F1在其DNA绑定状态，导致持续激活。的E2F-1的活性得到的水平将超过诱导的p53依赖的细胞凋亡，因此暗示治疗策略所需的阈值。由于E2F-1的失调p53和pRb的通路，高含量经常发生在肿瘤细胞中和抑制CDK2 /细胞周期蛋白A的应导致选择性凋亡的肿瘤和可以被认为是一个有效的癌症靶7。 临床研究CDK抑制剂靶向高度保守的ATP结合位点，导致跨越大于500蛋白激酶在人激酶组之间的反应性和潜在地引起副作用和毒性9。另一种方法是非ATP竞争抑制通过在CBG靶向性底招聘存在于细胞周期正调控亚基并因此显着，并从遥远的ATP结合位点10,11。所述CBG主要是存在于细胞周期蛋白A，细胞周期蛋白D和cyclin E疏水槽并已被证明为识别在基片和肿瘤抑制发现的共有序列。作为一种分离的肽，细胞周期蛋白结合基序（CBM）的结合到CBG，并已显示出抑制的细胞周期的CDKs激酶活性。煤层气已经被优化到八肽（HAKRRLIF，CDK2 /细胞周期蛋白A的IC 50 0.07±0.02μM，CDK4 /细胞周期蛋白D，IC 50 0.88±0.34μM），进而截断为代表的分子量与毒品之间的良好折衷五肽肖像和效力（RRLIF，CDK2 /细胞周期蛋白A的IC 50 1.01±0.17μM， CDK4 /细胞周期蛋白D，IC 50 25.12±2.97μM）12,13。该CBGs由一个大的初级和二级小疏水口袋它是由一个酸化桥接的C区（包括Glu220，Glu224和Asp283）。 HAKRRLIF的关键结合决定簇包括ALA2与Lys3，精氨酸4和Arg5的二次疏水口袋，离子配对和氢键与酸性区域和高度Leu6和Phe8的互补性与亲脂性主要部位的相互作用。此外，大量的氢键被从肽骨架贡献而Ile7充当间隔物残基允许与主口袋最佳的接触。装订模式和HAKRRLIF与CBG的相互作用如图2。 靶向CBM / CBG蛋白-蛋白相互作用将抑制CDK2 /细胞周期蛋白A，CDK2 /细胞周期蛋白E与CDK4 /细胞周期蛋白D的激酶活性，这一点应引起E2F1癌细胞介导的凋亡，而不影响正常细胞7。虽然煤层气衍生肽是细胞周期CDK的有效抑制剂，这是不可能的，他们将作为药物有用，因为它们的代谢不稳定和普遍缺乏的细胞通透性。为此，我们已经为这些有效的肽酶抑制剂转化为更多的类似药物的化合物的抗肿瘤治疗通过CDK2 /细胞周期蛋白利用放松管制E2F1一个抑制进一步的发展应用替换策略。下面的协议，总结了已更换的周期槽的应用程序已经完成的工作。在第一个实例，类药性封端基团替代HAKRRLIF的N末端四肽进行鉴定。此外改善这些群体进行了调查，一个额外的验证研究更换。从这些研究中有代表性的结果也列。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Computational Chemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accelyrs Discovery studio 3.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dell Optiplex Workstations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Organic Chemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6mm, 5&#956;m column (Phenomenox Luna) for purity determination, 1H NMR and 13C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flourescence Polarization Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>384 micro well plates, ,&#160; Micro pipets,&#160;</td>
			<td>Grenier Bio-one</td>
			<td>110256602</td>
			<td></td>
		</tr>
		<tr>
			<td>CDK4D1 and CDK2CA (well purified recombinant human kinase complex)</td>
			<td>BPS Bio Sciences</td>
			<td>40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)</td>
			<td></td>
		</tr>
		<tr>
			<td>assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1mM dithiothretiol (DTT),</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 nM HEPES</td>
			<td>CALBIOCHEM</td>
			<td>375368</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>127838</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>US Biological</td>
			<td>N3500</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DTX880 multimode detector&#160; fitted with 485 nm/535 nm excitation/emission filters and a dichroic mirror suitable for fluorescein</td>
			<td>Beckman Coulter, Brea, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates, &#160;&#160;</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NU serum, DMEM media, trypsin, PEN/STRIP,&#160; MTT reagent,</td>
			<td colspan="2">Fisher, Life technology, Alfa Aesar</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Heamocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-70c freezer</td>
			<td>Revco (Ultima II)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo electron corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>5804 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator 4-8C</td>
			<td>Isotemp Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DTX880 multimode detector fitted with 595nm filter.</td>
			<td>Beckman Coulter, Brea, CA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors

REPLACE是发展到更有效地针对蛋白质 - 蛋白质相互作用（质子泵抑制剂）一个独特的策略。其目的是通过对抑制剂的识别这样的结合位点并提供改进的方法代表了大多数潜在的药物靶点，扩大可用药物靶点的空间。本文的主要目的是提供替换策略，涉及的计算和合成化学方法的使用和应用的方法概述。取而代之的是通过其应用到非ATP竞争性细胞周期蛋白依赖性激酶（CDK）抑制剂作为抗肿瘤治疗学的发展例举。激酶经常放松管制在癌症，因此被认为是药物开发的重要目标。在S期CDK2 /细胞周期蛋白A的抑制已报道通过E2F1途径促进癌细胞中p53的独立的方式选择性凋亡。靶向蛋白质 - 蛋白质相互作用的细胞周期蛋白结合蛋白摹槽（CBG）是一种方法，这将使细胞周期在转录激酶特异性抑制。该CBG是由CDK底物和肿瘤抑制蛋白的共识序列中，识别被称为细胞周期蛋白结合基序（CBM）。煤层气先前已优化，从P21WAF（HAKRRIF），然后八肽进一步截短为五肽保持足够的活性（RRLIF）。在一般不细胞渗透性肽，是代谢不稳定，因此，策略REPLACE（通过计算富集代用偏配体替代品）已被以产生更多的类药性的抑制剂的应用。策略开头片段结扎抑制肽（翻转），其选择性地抑制细胞周期CDK /细胞周期蛋白复合物的设计。通过迭代地更换HAKRRLIF / RRLIF的残基片段像小分子（封端基团），从N末端（NCAPS）开始生成翻转，随后更换上C末端。这些化合物开始的非ATP竞争性CDK抑制剂作为抗肿瘤疗法的产生点。

HAKRRLIF与细胞周期蛋白槽的相互作用示于图2。表示键结合决定的肽残基包括ALA2，ARG4，Leu6和Phe8与其他残基提供较小的贡献12,13,18。在这种情况下，研究替换策略已被用于为了找到残留HAKRRLIF的N末端四肽片段的替代品，主要是模仿ALA2和ARG4的相互作用。潜在NCAP片段（ 表1）库是基于标准构建。每个分子停靠到由N个封端基的截断其晶体结构与细胞周期蛋白A（PDB ID：2U...

Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

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11.2K 次观看.  University of South Carolina. We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

视频: Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

SC1（Pluripotin）使用，以支持在没有LIF的自我更新MESC

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

科研

生物学

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

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 ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

在 NAFLD/NASH 的鼠模型中应用超声波和剪切波弹性成像

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...