10SDG3500034和UTMB-NCB启动资金，SSM：这项工作是由美国心脏协会国家科学家开发资助。作者承认冷冻电镜和科学计算设施的西利结构生物学中心在UTMB（ <a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a> ），以及博士。史蒂夫Ludtke和Ed Egelman的帮助，二维和三维螺旋重建算法。

1，样品制备<ol><li>缓冲液交换的人FVIII-BDD 14和猪FVIII-BDD 15对HBS-钙缓冲液（20mM HEPES，150 mM氯化钠，5mM的氯化钙 ，pH值= 7.4）并浓缩至1.2毫克/毫升。保持蛋白溶液于-80℃。 </li><li>通过为1:4 w / w的比例在氯仿混合半乳糖（GC）和磷脂酰丝氨酸（PS）准备脂质纳米管（LNT）。蒸除氯仿在氩气下并溶解在HBS缓冲液中的脂质为1毫克/毫升。保持LNT的解决方案，在4°C。 </li></ol> 2，低温电子FVIII-LNT的显微镜<ol><li>冷冻电镜样品制备<ol><li>辉光放电300个网眼的Quantifoil R2 / 2的铜网（碳朝上）中的O 2和H 2气体的混合物，持续10秒，在50 W。 </li><li>混在HBS-钙缓冲液中的FVIII和LNT溶液以1:1 w / w的比率，并孵育15分钟，在室温下进行。 </li><li>申请下降了2.5微升的FVIII-LNT样品的亲水性电子显微镜电网中的Vitrobot Mark IV的加湿室（100％湿度）。 </li><li> blot和闪光灯冻结格（一个污点3.5秒，吸干力1）在液体C 2 H 6，冷却用液态氮来获得无定形冰。 </li><li>存储网格下液态氮（LN2）储物盒。 </li></ol></li><li>冷冻电镜数据收集 注：JEM2100-六硼化镧（2010年）配备一个TEMCON操作系统包括连接到所述电子显微镜，有窗读取真空系统，照明系统，HT，透镜电流等上的屏幕和两个计算机的面板：左边和右边，放置在柱的两侧。梁位移X，Y旋钮（SHIFT键Y，SIFT Y）和多功能（DEF / STIG）旋钮是在两个面板。在左边的面板照明（BRIGTHNESS）旋钮。在右侧面板有：放大倍率（MAG / CAM的长度）和焦点（FOCUS）旋钮和三个IMAGIN克（MAG1，MAG2，LOWMAG）和衍射（DIFF）模式的按钮。我们获得了我们的MAG1数据在α-2的最小剂量照射条件（MDS）所需的冷冻电镜数据采集到F6按键，右侧面板的最上面一排设置的F1。在这个协议中的通用设置用于：F1 - 升高/降低屏幕，F2 - 搜索模式，F3 - 对焦模式​​，F4 - 照片模式，F5 - MDS OFF / ON和F6 - 异型坯，用于从辐射屏蔽标本通过偏转光束损坏。 <ol><li>将冷冻支架插入冷冻站，并填写持有人及冷冻站与液氮的杜瓦。当温度达到-192℃，在支架末端开放的快门，取代先前冻结的冷冻电镜电网到指定地点，并与环夹固定。 </li><li>将冷冻支架插入电子显微镜。重新加注冷冻支架和防contaminator室用液氮的杜瓦。等待持有人30-60分钟稳定。按F6键上打开灯丝上。当灯丝处于饱和状态，按F6键关闭和打开快门的持有人查看网格。 </li><li>按低倍率/α-1（设定在200X MAG）和定位区域与薄冰上的网格。 </li><li>切换到MAG 1/alpha 2设置最小剂量（MDS）的模式，并获得超低温电磁数据在低剂量电子，而不破坏样品。 <ol><li>按F2键来设定搜索模式。以40,000×设定的放大倍率。放大梁BRIGTNESS旋钮，最小的电子剂量〜0.04É - / A 2·s以下。按DIFF切换到衍射模式。将相机长度为120厘米，MAG / CAM的长度计旋钮。在LOWMAG预先选定的区域中具有碳薄膜的孔的脂质纳米管内定位在网格区域。 </li><li>按F4键设置照片模式在40,000倍放大，并设置照明，亮度旋钮剂量为16-25É - / A 2·s以下。按标准对焦按钮将焦点设置条件调整Z轴高度用Z UP / DOWN按钮（右控制面板）的标本。 -1.5和-2.5微米之间设置的散焦。 </li><li>通过对应于在照片模式下进行成像的区域搜索模式数码相机显示屏上绘制的实时显示窗口的正方形排列搜索和照片模式。 </li><li>按F3键设置对焦模式为100,000倍放大。重点照明覆盖CCD芯片（〜4 - 5厘米半径）和离轴不照射在照片模式将被成像的区域。调整散焦〜-1.5和-2.5微米的调焦旋钮和正确使用DEF / STIG旋钮图像的散光。 </li></ol></li><li>选择FVIII-LNT由数码相机显示屏上获取实时图像的实时显示窗口进行成像的搜索模式。居中的FVIII-LNT中绘制的实时显示窗口上的正方形。 </li><li>通过切换到照片模式，并点击ACQUIR记录的CCD相机的数字图像在52,000 X有效放大倍率和0.5秒的曝光数码显微相机显示屏上的E键。图像采集条件设置，如光束空白（百叶窗）开，只有当图像在照片模式被获取。 </li><li>按Ctrl-F以获得快速傅立叶变换（FFT）检查获取的图像质量和散焦。 </li></ol></li></ol> 3，三维重建注：用于2D和3D分析图像分析软件：EMAN2和IHRSR都是免费的。 EMAN2可以从http://blake.bcm.edu/emanwiki/EMAN2/Install下载。 egelman@virginia.edu：该IHRSR软件可以从Egelman教授获得。最后IHRSR改进是在德州上运行高级计算中心集群：http://www.tacc.utexas.edu/在得克萨斯大学奥斯汀分校。在图1所示的三维重建算法包括两个主要步骤：首先选择一个同质组螺旋片（颗粒）与2D参考免费alignme在电子舱单2实施新台币（RFA）的算法，第二个实现基于在IHRSR注册成立的螺旋参数和反投影算法进行三维重建。第一步利用选择均匀的颗粒集三维重建与单粒子SPA（算法）为其EMAN2已专门开发和分布式开发的项目：http://blake.bcm.edu/emanwiki/EMAN2。这一步已经适应了冷冻电镜数据。第二个步骤是与IHRSR算法，它是专为与所述重组因子VIII的形式得到螺旋组件的类型设计的实现。该算法已经被广泛证明，通过科学文献12。 <ol><li>进行2D图像分析与EMAN2科学图像处理套件：http://blake.bcm.edu/eman2/doxygen_html 16选择均匀颗粒（螺旋段）套螺旋重建。 <ol><li>选择冷冻电镜显微照片与straigHT和组织良好的螺旋管与数码相机的软件和可视化工具。 </li><li>反转，标准化和筛选X射线像素的e2workflow.py图形用户界面，单粒子重建（SPR）选项：http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2projectmanager </li><li>在e2helixboxer.py GUI导入倒，归一化和X射线像素滤波后的图像。有90％的重叠使用选择的FVIII-LNT斜管和段在256×256像素（2.9 A /像素）：http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2helixboxer </li><li>从原来的显微照片评价散焦和运用对比传递函数（CTF）的校正（仅相位校正），从与在e2workflow.py注册成立的e2ctf.py选项相同的显微照片螺旋片：http://blake.bcm。 edu/emanwiki/EMAN2/Programs/e2ctf。 </li><li>生成初始粒子（螺旋段）设置在e2workflow.py图形用户界面 - SPR选项：http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2workflow。 </li><li>计算2D类平均与e2refine2d.py算法应用参考免费K-均值分类选择同质的数据集具有相同的直径LNT和螺旋有序度（ 图2），下面的脚本：&#39;e2refine2d.py - ITER = 8 - naliref = 5 - nbasisfp = 8 - 路径= r2d_001 - 输入= INPUT.hdf - NCLS = 51 - simcmp =点 - simalign = rotate_translate_flip - classaligncmp =点 - classraligncmp =相 - classiter = 2 - classkeep = 0.8 - classnormproc = normalize.edgemean - classaverager =均值 - normproj-classkeepsig&#39;，相应地改变NCLS值。 <ol><li>分类的初始数据在150类“NCLS = 151&#39;超过8次迭代&#39;classkeep = 0.8&#39;设置，这意味着少于80％的相似性的类平均粒子被排除在给定的类别。 http://blake.bcm.edu/emanwiki/EMAN2/Programs/e2refine2d </li><li>从班级的平均颗粒合并在e2display.py创建中间数据集显螺旋衍射。 </li><li>分类中间数据的50类“NCLS = 51&#39;设置为区分班级的顺序相同的直径和程度。 </li><li> http://blake.bcm.edu/emanwiki/EMAN2/Programs/emselector：从班级的秩序e2display.py创造三维重建的最终数据集相同的直径和融合度的颗粒。 </li></ol></li></ol></li><li>与迭代螺旋实时空间重构（IHRSR）算法进行螺旋重建，如Egelman 17，18所述。 <ol><li>估计上升，Δ狕从组合傅立叶FVIII-LNT螺旋（一）转变的最终数据集的颗粒。 </li><li>通过运行并行IHRSR改进的恒定Δ狕定义方位角ΔΦ（°），从5增加ΔΦ76;在5°增量60°。 </li><li>运行100个连续IHRSR周期每个最后的数据设置一个无特色缸作为初始体积和3.2.1中定义的初始螺旋参数Δ狕和ΔΦ。和3.2.2。 </li><li>检查最终的体积为从最终的数据集的二维分类类平均值和从最终IHRSR重建的突起之间的螺旋参数和对应的收敛性。 </li><li>强加对称性的最终三维重建，对应于在最终的非对称三维体积中观察到的不对称单元分配：4倍的人FVIII-LNT和5倍的猪FVIII-LNT。再跑100优化循环，以产生最终的对称三维重建。 </li><li> “e2proc2d.py &lt;infile&gt; &lt;outfil：首先把相应的数据在奇数和偶数设置计算两个卷傅立叶壳牌相关曲线e&gt;的 - 分裂= 2&#39;。然后在3.2.3中所述运行100 IHRSR连续改进。计算从奇数和偶数的数据集创建的三维体积的FSC：&#39;e2proc3d.py evenvolume.mrc fsc.txt - calcfsc = oddmap.mrc&#39;。 </li></ol></li><li>可视化和分类的FVIII-LNT卷在加州大学旧金山分校的嵌合体。 </li><li><ol><li>从步骤3.2.5打开最终卷。在加州大学旧金山分校的奇美拉，并在工具&gt;体数据&gt; VOLUME VIEWER选项的轮廓级别设置为0.005。 </li><li>在工具&gt;体数据&gt;分类地图，在段映射选项卡中选择卷，然后单击段段音量。 </li><li>对应于一个单元电池组片段通过选择用Ctrl + Shift并单击组。 </li><li>通过单元格颜色的部分和与行动&gt;色彩选项螺旋线来强调结构特点。 </li></ol></li></ol> 4。电子断层扫描<ol><li>负染的FVIII-LNT样品制备<ol> &lt;李&gt;准备FVIII-LNT样本作为冷冻电镜实验。 </li><li>辉光放电碳包覆的300网目铜网上（碳朝上）中的O 2和H 2气体，持续10秒，在50的混合物中W的用于冷冻电镜实验。 </li><li>申请一滴2.5微升的FVIII-LNT悬浮液与6纳米的胶体金粒子到电网，吸干多余的液体，并通过施加2分钟，5微升的1％乙酸双氧铀溶液负染色。吸干多余的液体和空气干燥的网格。 </li></ol></li><li>电子断层扫描数据采集<ol><li>转移网格成单一倾斜支架。 </li><li>传输在电子显微镜支架。 </li><li>与SerialEM软件19在2°增量的角度范围为-60°〜+60°，可拍摄具有CCD相机在52,000 X有效放大倍率自动采集系列倾斜，-6到-10微米的散焦和150电子剂量 - 170元素ctrons /每断层2·s以下。 </li></ol></li><li> FVIII-LNT的电子断层扫描重建 注：在重构4.2收购pFVIII-LNT和的hFVIII-LNT倾斜系列。与IMOD软件继教程ETomo选项：http://bio3d.colorado.edu/imod/doc/etomoTutorial.html <ol><li>使用一个终端窗口，打开3DMOD的断层。 http://bio3d.colorado.edu/imod/doc/3dmodguide.html。 </li><li> bin中的断层图像由4与IMOD BINVOL命令以减小X线断层图的大小。 </li><li>选择适当的角度旋转沿Y轴的脂质纳米管在使用IMOD ROTATEVOL命令的离散化层析X射线照片。 </li><li>旋转全断层与IMOD ROTATEVOL命令。 </li><li>裁剪沿Y轴与IMOD CLIP resize命令面向选定的脂质纳米管。 </li><li>打开裁剪子断层扫描与3DMOD。 </li><li>点击该断层图像沿想象的因子VIII分子的排列切片在Z轴方向和沿Y轴在子断层图像体。 </li></ol></li></ol>

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O., Mertens, K., Kemball-Cook, G., Holzenburg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+structure+of+membrane-bound+coagulation+factor+VIII:+modeling+of+the+factor+VIII+heterodimer+within+a+3-dimensional+density+map+derived+by+electron+crystallography.">3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography.</a> Blood. 99, 1215-1223 (2002).</li><li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+density+maps+with+UCSF+Chimera.">Visualizing density maps with UCSF Chimera.</a> Journal of structural biology. 157, 281-287 (2007).</li></ol>

作者宣称，他们有没有竞争经济利益，他们可以直接就任何发表在本手稿的程序进行联络。

在此工作的方法呈现高度同源性的蛋白质的2膜结合的组织之间进行区分：人和猪FVIII自组装于在人体中所遇到的条件下的脂质纳米管。 在描述的过程中，人类和猪FVIII成功举办螺旋状脂质纳米管，这是最关键的一步。下一个关键步骤是通过快速冷冻在接近液态氮的温度保持在样品中非晶薄的冰。保留在无定形冰和液氮温度下的样品保持水合的螺旋...

凝血因子VIII（FVIII）是由六个域2,332个氨基酸的糖蛋白大：A1-A2-B-A3-C1-C2 3。经凝血酶激活的FVIII作为内与膜结合的tenase复合物的辅因子以因子IXa。激活的FVIII（FVIIIa）来的FIXa中的膜-视方式结合增强FIXa的蛋白水解效率超过10 5倍，这是有效的凝血4是至关重要的。尽管重要作用的FVIII在凝固和tenase复合物的形成中起着，功能膜结合的FVIII结构尚未得到解决。 为了解决这个问题，单脂双层碳纳米管（LNT）富含磷脂酰丝氨酸（PS），能够结合的FVIII以高亲和性8,9和类似的活化的血小板表面已经开发了10。 FVIII的连续螺旋机构势必LNT已经被证明是effecti对于已经通过结构冷冻EM 5测定FVIII膜结合状态。官能化LNT是一个理想的系统通过冷冻电镜11，12，以研究蛋白质-蛋白质和螺旋组织膜相关蛋白的蛋白质-膜相互作用。冷冻电镜具有如下优点：与传统的结构的方法，如X-射线晶体学和核磁共振，作为试件被保存在最靠近生理环境（缓冲液，膜，pH值），无添加剂和同位素。在FVIII的情况下，使用此技术研究与膜结合的结构更是生理上相关的，因为在LNT类似密切的大小，形状和组成的活化的血小板，其中Tenase配合装配体内的伪足。 缺陷和FVIII的原因A型血友病，严重的出血性疾病影响1 5000男性的人口4,6的缺乏。在大多数电子ffective治疗血友病A是重组人FVIII（hFVIII的）终身管理。的重组FVIII血友病A治疗的显著并发症是抑制性抗体的发展到人的形式影响血友病A患者13约30％。在这种情况下，猪FVIII（pFVIII）浓缩物的情况下，如猪FVIII显示低交叉反应性抗人FVIII和形式的功能性复合体与人的FIXa 7的抑制性抗体。建立两个猪和人FVIII形式的膜结合组织是重要的，了解的FVIII辅助因子的功能和血止血的影响的结构基础。 在这项研究中，我们描述了脂质纳米技术，冷冻电镜和结构分析，旨在解决两个高度同源的FVIII形式的膜结合组织的结合。所提出的Cryo-EM数据和三维结构螺旋举办porci在带负电荷的LNT NE和人FVIII表明，所提出的纳米技术作为基础，FVIII的结构测定与膜结合的凝血因子和在生理环境中的膜复合物的潜力。

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	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;">JEM-2100</td>
			<td style="width:447px;">operated at 200 kV</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">626.DH</td>
			<td style="width:447px;">cooled to -175 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">with temperature controler unit</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan 4K x 4K CCD camera</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">US4000</td>
			<td style="width:447px;">4096 x 4096 pixel at 15 microns/pixel physical resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Solarus Model 950 plasma cleaner</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Vitrobot Mark IV</td>
			<td style="width:205px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">01821</td>
			<td style="width:447px;">plasma cleaned for 10 s on high power</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">Quantifoil R2/2 300 mesh</td>
			<td style="width:205px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Q225-CR2</td>
			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:239px;">Galactosyl ceramide</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dioleoyl-sn-glycero-phospho-L-serine</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">840035</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">EM software Spider</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://spider.wadsworth.org/spider_doc/spider/docs/spider.html">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

<html>低温电子显微镜（冷冻电镜）1是一款功能强大的方法在水合状态和膜环境2，调查的蛋白质和复合物的功能结构。 凝血因子VIII（FVIII） 图3是一个多结构域的血浆糖蛋白。缺陷或FVIII的缺乏是导致血友病A型 - 一个严重的出血性疾病。经蛋白水解活化，FVIII结合到丝氨酸蛋白酶因子IXa上的带负电荷的血小板膜，这是正常的凝血4是至关重要的。尽管举足轻重的作用FVIII凝血发挥，其膜结合态的结构信息是不完整的5。重组FVIII浓缩物是抗血友病A型的最有效的药物和市售的FVIII可以表示为人类或猪，既形成功能性复合物与人的因子IXa 6,7。 “&gt;在本研究中，我们提出低温电子显微镜（冷冻电镜）的组合，脂质纳米技术和结构分析应用到解决两个高度同源的FVIII形式与膜结合的结构：人和猪在我们实验室开发的方法以螺旋状组织两个功能重组FVIII形式对带负电荷的脂质纳米管（LNT）中说明。代表结果表明，我们的方法是足够灵敏定义序列中的两个高度同源（86％序列同一性之间的差异在螺旋机构）蛋白。为螺旋机构，冷冻电镜和电子断层扫描（ET）的数据采集的详细方案中给出的二维（2D）和三维（3D）结构分析应用于获得人和猪的三维重建FVIII-LNT进行了讨论。所提出的人类和猪FVIII-LNT结构表明了该方法的潜力度的计算E中的功能性，膜结合的凝血因子VIII在高分辨率的组织。

重组人类和猪FVIII被成功地组织上的螺旋形带负电荷的单一双层LNT，类似的活化的血小板表面。人类和猪FVIII-LNT的螺旋机构通过所收集的数字显微图是一致的（ 图2）。控制LNT和人类和猪FVIII-LNT螺旋管被选择和分割的e2helixboxer.py GUI和与e2workflow.py GUI，单一粒子的选择（ 表1）中创建的初始数据集。 膜结合人和猪FVIII-LNT的螺旋顺序从傅立叶?...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

我们提出了低温电子显微镜，脂质纳米技术和结构分析的组合应用，以解决两个高度同源的FVIII形式与膜结合的结构：人和猪。在我们研制的实验室，以螺旋状组织两个功能重组FVIII形式对带负电荷的脂质纳米管（LNT）的方法进行说明。

凝血因子VIII的脂质纳米管螺旋组织

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

helical-organization-blood-coagulation-factor-viii-on-lipid

Research

JoVE Journal

Bioengineering

12.2K 次观看.  University of Texas Medical Branch. 我们提出了低温电子显微镜，脂质纳米技术和结构分析的组合应用，以解决两个高度同源的FVIII形式与膜结合的结构：人和猪。在我们研制的实验室，以螺旋状组织两个功能重组FVIII形式对带负电荷的脂质纳米管（LNT）的方法进行说明。 

视频: Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

同时合成单壁碳纳米管和石墨在电弧等离子体磁增强

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

科研

生物工程

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in transcription of non-coding RNAs, specifically of microRNAs. Here, we propose a strategy to study the potential role of transcription factor in regulating transcription of microRNAs using publically available data, computational resources and high throughput data. We use the H3K4me3 epigenetic signature to identify microRNA promoters and chromatin immunoprecipitation (ChIP)-sequencing data from the ENCODE project to identify microRNA promoters that are enriched with transcription factor binding sites. By transfecting cells of interest with shRNA targeting a transcription factor of interest and subjecting the cells to microRNA array, we study the effect of this transcription factor on the microRNA transcriptome. As an illustrative example we use our study on the effect of STAT3 on the microRNA transcriptome of chronic lymphocytic leukemia (CLL) cells.

Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.

描述了微RNA转录的转录因子依赖调节

While the transcription regulation of protein coding genes was extensively studied, little is known on how transcription factors are involved in ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&amp;#946; on the Epithelial-to-Mesenchymal Transition

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...