We wish to acknowledge the partners in the Helmholtz-Alberta Initiative, the Helmholtz Association and the University of Alberta, for the support resulting from participation in this collaboration. Research funding is provided by the Helmholtz Association's Initiative and Networking Fund, the participating Helmholtz Centers and by the Government of Alberta through Alberta Environment's ecoTrust program.
Dr. Tanushree Ghosh is gratefully acknowledged for her critical inputs at a number of crucial stages.

注意:在下面描述的顺序执行的实验方案。细菌培养协议在第一节中讨论（也如图1所示 ）。第2节描述欲得使用外部添加剂的培养基中的协议。部分3描述了一种用于多模式显微镜的协议。所有的单个组分的权重可以使用分析天平进行测定。各溶液的体积可以使用量筒来测定。 注意:正确的生物安全协议必须遵守的第1 - 2咨询机构安全办公室了解详情。 1.细菌培养<ol><li> 文化细菌-琼脂平板培养基的制备 <ol><li>组装设备和成分如陪替氏培养皿，烧瓶，Tris碱，盐酸，琼脂，Millipore公司水，pH计etc.Sterilize使用前在121℃的所有容器高压灭菌。 </li><li>制备1升的0.1通过混合15.75克Tris-碱用1L微孔水的Tris-缓冲液的3M水溶液。为了降低原溶液（pH 10.4）的pH值增加2800微升盐酸（50％浓度）。连续查用pH计来设置的pH = 9。 </li><li>除以1升缓冲溶液分成两部分，如下所示: <ol><li>取800毫升此溶液。同样将其划分成各为400毫升双部分。溶解8克（NH 4）2 SO 4的一个溶液中，16克酵母提取物的其他解决方案。 </li><li>取剩余（200毫升）溶液，并再次将其分成的每次100ml两部分。混合2克（NH 4）2 SO 4为一个。添加4个克酵母提取物和4g琼脂到另一个。 </li></ol></li><li>在铝箔包装各自的瓶贴及胶带高压锅分开后的高压灭菌4解决方案。 注意:如果一个台式高压釜单元被使用时，体积应设定为500毫升（温度和压力下自动贩卖机ically指定为体积的函数）。 </li><li>从高压釜中取出来后，静置步1.3.1（下）设置两个400毫升解决方案。混合两个100ml的解决方案，有200毫升的溶液。将混合物倒入10 - 12培养皿中。 </li></ol></li><li> 文化细菌-琼脂平板样品制备 <ol><li>从冰箱（-80°C）中取出细菌股票，并允许它解冻。正确解冻后，将细菌股票和生物安全罩内的琼脂平板上。 </li><li>选择最小可用尺寸的微量（0.5 - 10微升是一个不错的选择）侵扰与Sporosarcina巴斯德股票一角。条纹的琼脂平板用枪头。将非振荡培养箱里面的条纹琼脂平板在31℃48小时。 </li><li> 48小时后，从孵化器中取出板，并目视检查单个菌落的存在。如果没有单个菌落，然后将其放置在叔他孵化另外24小时。 </li><li>直到检测单菌落重复上述过程。不要超过7天试用。 注:如果甚至一个星期后，然后可以得出结论的步骤没有正确地遵循和全过程必须从步骤1重复不会出现单菌落。 </li></ol></li><li> 文化细菌-最终样品制备 <ol><li>混合两种400毫升溶液（Tris缓冲液+（NH 4）2 SO 4和Tris缓冲液+酵母提取物（1.5.1）在一起，得到一个800 ml溶液。转移125毫升此溶液加入到烧瓶中。 </li><li>执行琼脂平板的表面上的可视检查，以确定与单个菌落的高浓度区。轻轻轻推，打破了枪头的殖民地之一。 </li><li>浸在同一枪头到125毫升烧瓶中，充分搅拌，以确保有足够数量的细胞的稳健乘法得到转移。放置烧瓶在振荡培养箱中于150rpm下，30℃下2 - 3天。经过2 - 3天，取出从孵化器的烧瓶中。 </li></ol></li><li> 文化细菌-最终的细胞计数 <ol><li>执行使用PBS以获得至少十元（10 -7）的稀释液，以确保可数单个菌落出现的非稀释的培养液连续稀释。借鉴琼脂平板一七平行等距线。 <ol><li>在培养皿中，足以突出的是由上可见的底面上画上粗线做到这一点。降3个非稀溶液的小滴成一个段。 1毫升未稀释的溶液添加到9毫升磷酸盐缓冲盐水（PBS）中以获得1:10稀释。 </li></ol></li><li>采取用吸管这个新稀释的溶液中的一小等分试样（〜0.1ml）中，并滴在下一段3更小滴。新近稀释溶液转移到一个新的烧瓶中，并进一步稀释至10倍（10倍）加入PBS。这使向下稀释至10 -2或1:100。 <ol><li>使用此1:在接下来的段100的解决方案。 1000一路:通过连续将其转让给新瓶和串联不断稀释十倍（10X）用PBS从10 -3或1获得越来越稀的样品重复这一点，他与新鲜稀释溶液的小体积处理下降到10 -7或1:10万成最后一段。 </li></ol></li><li>执行集落形成单位（CFU）平板计数孵育该板1之后，计数存在于琼脂平板上的细胞数量 - 在31℃2天。这使未稀释样本中的细菌计数的定量量度。 注:CFU值是基于系统对产生的菌落的营养培养基，温度和时间的假定每菌落是单独的，并通过一个单一的存活的微生物细胞创办在特定条件下的能力进行测定。 </li><li>密封培养皿具有自密封膜和储存以备将来使用冰箱剩余项目。 </li></ol></li></ol> 2.协议养分富集加速降水<ol><li>传输9传毫升制备的培养液（细胞+培养基）的成若干灭菌离心管，每次10毫升体积。 </li><li>准备在新鲜培养基下列浓度包含四个组件的外部富集的100毫升原液:尿素:2克/升，氯化铵1克/升，碳酸氢钠:212毫克/升，氯化钙:280毫克/升。使用分析的平衡仔细测量所有的成分，并与在烧杯中的高压釜新鲜培养基和地点（121℃，15psi的，15分钟）混合所有但尿素。 <ol><li>以下高压釜中，混合脲（2毫克）用1ml新鲜培养基的所需量和穿过装配有0.22微米的注射器过滤器，以完成浓缩过程的注射器。 </li><li>加1毫升添加剂，由9毫升准备好的培养液（细胞+培养基）的无菌离心管这种增菌培养基中（见步骤2.1）。 注:所有的这些组分中，尿素是在高温下可降解的，不能在高压灭菌器灭菌。因此，其它成分被高压灭菌后（121℃，15磅，15分钟），尿素最后通过0.22μm注射器式滤器加入。 </li></ol></li><li>涡流每个管彻底使用机械涡旋。放置富集液体（细胞+培养基+添加剂）在非振荡培养箱中于30℃。定期监测所有的单位降水开始。使用光学显微镜，一旦沉淀发作肉眼检测开始显微镜观察。实验开始后的36小时 - 这通常30之间。 </li></ol> 3.协议的多模式显微镜<ol><li>转移的流体的小体积（〜1ml）中，以高尊离子显微镜 - 腔玻璃罩系统执行与亮场模式初步意见。首先，先从最低放大倍率（4X）所有观测。其提供覆盖和关于析出物的分布，因此全局信息的广泛区域。 </li><li>选择特定区域与析出物显著卷和通过逐渐增大放大倍数放大（20X，40X，60X）。 注:由于这是不可能妥善解决从胞外聚合物物质（EPS）在明场细胞（由于非常接近的折射率），开启相衬模式必要时。在这种模式下，小区膜（作为一个不同的相位比体细胞）的仅有的轮廓是可见的，从而使视觉分化。 </li><li>最后，一​​些染色与商会选择性染带电部件的绿色，而着色红色死者组件活/死染色。参阅标准协议28 &lt;/ SUP&gt;。 </li><li>交换机上的荧光模式，以适当的红色和绿色区域之间区分。红（红色滤光器立方体540激发波长 - 580纳米，600 595 nm和屏障过滤器的分色镜 - 660 nm），而绿色（GFP长通绿色滤波器立方体440激发波长 - 480纳米，分色镜的510纳米）过滤器立方体505纳米和屏障过滤器是用来促进荧光显微镜照片。 </li><li>一旦所有的多模式数据已经被收集，选择最佳的图像和手动覆盖（个体明，相衬和荧光图像），以获得最佳的对比度和清晰度。 </li><li>在干涸的固体样品扫描电镜进行了解的晶体结构。这是一个行之有效的标准和程序。22执行XPS找出官能团（碳酸酯）的化学签名。这是一个行之有效的标准和程序以及23 </li></ol>

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The authors have nothing to disclose.

关键步骤:该手稿详细介绍了协议的培养S的一个可行的样本巴斯德 。一旦培养已准备好，必须适当地富集。这是在实验的成功是至关重要的关键步骤，因为未提供适当的化学环境导致要么沉淀或其完全缺乏的很长的时间尺度。S.巴氏是几个外部机构相当敏感，并且必须以高度小心和精确的进行培养，以确保生化鲁棒性和可重复性。浓缩的程度和剂量现在已知口授?...

Sporosarcina巴氏是革兰氏阳性细菌能够在高碱性环境（pH约10）1生存并且是细菌种类，可以成为一个所谓微生物学诱导方解石沉淀（MICP）2-4现象病原体之一。 MICP是一种方法，其中碳酸钙的沉淀是通过合适的环境的条件下某些微生物引起的。S.巴氏已假定近年来重要性由于其识别为在特定条件下诱导MICP的显著体积的可能剂。这种可能性来源于这样的事实即S.巴氏具有分泌丰富量的脲酶的独特能力。这种酶作为催化剂，促进尿素水分子的存在加速溶解（具有广泛和供应充足的天然存在的生化化合物）。通过反应的级联，该方法的最终LY导致带负电的碳酸根离子的生成。这些离子，又与正金属离子如钙，最终形成碳酸钙（方解石）的沉淀反应;因此标签MICP 5-9。 MICP的方法已经知道并研究了数十年10,11。在过去的几年中，MICP已经研究了一系列工程和环境应用，包括自下而上的绿化建设12，加强大型结构13,14和碳吸收和储存15,16的。 例如，Cunnigham 17等。人设计包含贝雷砂岩核心的高压中温流反应器。将反应器与细菌S.接种fridgidimarina和高压的超临界二氧化碳注入，生物量的孔隙体积内的大量积累的条件下超微电极观察到，这导致在透过性降低95％以上。 Jonkers和施兰根18研究在混凝土的自愈过程细菌的某些特殊菌株的效果。运入通过表面气孔进入孔隙网络外部的水有望激活休眠的细菌从而有助于结构强度通过MICP。托布勒19等。比较了S的尿素分解活性有利于大型MCIP条件下的地下水本土尿素分解缩影巴斯德发现S.巴斯德具有一致的能力，以提高方解石沉淀即使在土著社区缺乏事先脲酶活性。莫滕森20 et.al已经研究的外部因素，如土壤类型，氯化铵，盐度，氧浓度和MICP裂解细胞浓度的影响。他们论证了生物处理工艺非常稳健与RESP等在参数空间中的巨大差异证实了这一过程提供了一个合适的浓缩工艺，以加强细菌进行各种大型整治应用的健康。菲利普斯21等。人的实验设计与S.被注射后，研究在砂柱和砂岩核心的渗透性和强度的变化巴氏文化。他们发现，尽管渗透性下降了2 - 4倍，而断裂强度提高三倍。 S.巴氏及其在MICP的作用是积极的研究和有关化学沉淀机制若干问题的主题仍然没有完全理解。鉴于此，它拥有一套一致的标准化协议，以准确地文化S的适当丰富的股票是非常重要的巴斯德实现MICP。在这里，我们勾勒出一个严格的协议，以确保重复性和再现性。此马nuscript描述了详细的协议，培养S.巴斯德和适当丰富培养基，以诱使降雨。该过程通过各种显微镜技术如光学和扫描电子显微镜（SEM）和X射线光电子能谱（XPS）研究。手稿的重点是MICP的过程。程序，如:SEM和SIMS，是行之有效的标准协议，不单独介绍。

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microbiologically induced calcite precipitation mediated by sporosarcina pasteurii

被调查的特定的细菌在这里（S.巴氏 ）是其独特的能力，在合适的条件下，促使尿素（尿素水解）的水解通过脲酶分泌的自然发生的环境。尿素水解的过程中，通过化学反应的链，导致碳酸钙沉淀的形成。这被称为微生物学诱导方解石降水（MICP）。对于MICP适宜的培养协议这里详述。最后，在显微镜下的不同模式的可视化实验以了解沉淀过程的各个方面。像光学显微技术，扫描电子显微镜（SEM）和X射线光电子能谱（XPS）进行了用于化学表征终产物。此外，这些沉淀物会堵塞一个天然的多孔介质毛孔里面的能力，通过定性实验，其中海绵证明棒用来模仿细孔网络与一系列长度尺度。在含有细菌细胞培养液中浸渍的海绵棒变硬由于其孔从化学沉淀的连续方法得到的的堵塞。相比其中被压缩和施加的外部负载的作用下挤压的控制海绵条时，而硬化杆能够支持相同重量很少变形此硬化海绵棒表现出优异的强度。

S.巴斯德作为一个alkaliphile 24能够生存条件比较苛刻。当上述培养协议之后，和S.巴氏生长的腔室的内部，该细菌会导致碳酸钙的随时间（ 图2A）的沉淀。 图2（b）表示在培养基中的细菌细胞群体的相差光学显微镜图像。单个细胞能够清楚地区分，与芽孢杆菌类的细菌很明显的特征棒状的形状。小棕色箭头已用于特异性突出两...

Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

微生物诱导方解石沉淀通过介导<em&gt; Sporosarcina巴斯德</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

microbiologically-induced-calcite-precipitation-mediated-sporosarcina

Research

JoVE Journal

Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

21.6K 次观看.  University of Alberta. Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

视频: Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

通过电场诱导释放和测量（EFIRM）检测外体生物标志物

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...