本研究由厦门市科学技术局（3502Z20227197）和福建省科学技术局（No.2019J01070，No.2021Y0027）资助。

该研究是按照华侨大学医学伦理委员会的机构指南进行的。经患者知情同意，在福建医科大学附属泉州市第一医院手术切除并采集脑血凝块。
1.血凝块预处理
<ol><li>将凝块放在干净的培养皿上，用镊子加入 5 mL 生理盐水，轻轻摇晃培养皿，然后用移液管除去生理盐水。 注意: 重复冲洗三次。</li>
	<li>用剪刀将凝块切成小块（小于 5 mm x 5 mm x 5 mm）。加入 5 mL 生理盐水，轻轻摇晃培养皿，然后用移液管除去生理盐水。 注意: 重复冲洗三次。确保在冲洗过程中没有凝块被吸入。</li>
	<li>将凝块转移到另一个干净的 U 型板上。 注意:可以在此处暂停方案（将板储存在2-8°C）并稍后重新启动。</li>
</ol>2. 溶栓
<ol><li>用生理盐水将sFE浓度调节至2000U / mL来制备sFE工作溶液。 注:sFE是根据Tang Lab15的既定方案制备的。</li>
	<li>将 300 μL sFE 工作溶液加入预处理的凝块中。</li>
	<li>执行第一轮降级。<ol><li>将sFE和凝块的混合物在37°C孵育0.5小时。 注:用生理盐水处理的印迹设置为阴性对照。不要旋转样品在摇床上。</li>
		<li>轻轻混合样品。用干净的移液管将液体部分转移到另一个干净的管中。 注意:剩余的凝块用于另一轮降解。</li>
		<li>将液体部分在4°C下以200× g 离心5分钟。</li>
		<li>用移液管将上清液转移到另一个干净的管中，以获得回收的sFE溶液。</li>
		<li>将细胞沉淀物与 50 μL 生理盐水轻轻混合。 注:将细胞混合物储存在2-8°C以备后用。</li>
	</ol></li>
	<li>执行后续降级。<ol><li>用回收的sFE溶液（步骤2.3.4）在37°C下降解剩余的凝块（步骤2.3.2）0.5小时。 注意:其余步骤与第一轮降级相同。降解过程完成，直到整个凝块降解。</li>
	</ol></li>
	<li>执行单元格收集。<ol><li>通过收集每轮降解的细胞混合物来制备血细胞样品。</li>
	</ol></li>
</ol>3. 赖特染色
<ol><li>进行细胞涂片。<ol><li>将细胞调节至 1 x 106 个细胞/mL 的密度。</li>
		<li>将 5 μL 细胞加入 poly-L 赖氨酸包被的载玻片中，然后使用盖玻片涂抹它们。</li>
		<li>在室温下蒸发液体。</li>
	</ol></li>
	<li>进行染色。<ol><li>向细胞涂片中轻轻加入 200 μL Wright 染料（参见 材料表），加入 600 μL Wright 染料 B 混合均匀。 在室温下染色10分钟。</li>
		<li>用清水轻轻冲洗染料。 注意:染色的细胞涂片可以在室温下储存以备后用。</li>
	</ol></li>
	<li>进行显微成像。<ol><li>使用光学显微镜检查染色细胞（参见 材料表）。</li>
	</ol></li>
</ol>4.血常规检查
<ol><li>将细胞调节至 1 x 106 个细胞/mL 的密度。</li>
	<li>根据制造商的说明，使用自体血液分析仪分析细胞成分，例如血小板，红细胞，白细胞，淋巴细胞，中性粒细胞，单核细胞，嗜酸性粒细胞，嗜碱性粒细胞和未成熟粒细胞（参见 材料表）。</li>
</ol>

一种基于纤溶酶的研究脑血栓细胞成分的方法

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作者没有要披露的利益冲突。

sFE 是一种纤溶剂，可直接有效地降解纤维蛋白12,16。本研究采用sFE技术降解脑血凝块的交联纤维蛋白，释放血栓内封闭的细胞，并定性和定量分析血栓的细胞成分。显微镜数据和血常规检查表明，封闭的细胞从血凝块中释放出来。此外，sFE处理后释放细胞的细胞类型和结构没有受到显着影响。因此，首次创造了一种独特、简单、有效的方法来分析脑血?...

脑血管疾病是威胁人体健康的三大疾病之一，其中缺血性脑血管疾病占80%以上。脑血栓形成和脑静脉血栓形成是当今最受关注的缺血性脑血管疾病，主要由脑血栓引起1,2。如果治疗不当，将有很高的残疾率和死亡率，出院后复发率很高3.
最近，越来越多的研究表明，脑血栓的细胞成分与脑血栓形成的诊断、治疗和预后密切相关4,5,6。因此，血栓组成数据的可用性，尤其是细胞成分的数据，对于临床诊断和治疗非常重要。不幸的是，目前可用的方法无法定量和定性地全面分析血凝块成分。例如，基于 Martius Scarlett Blue 的原位染色只能研究凝块7 某些切片的红/白细胞。基于免疫组织化学 （IHC） 的原位染色只能使用其抗体研究凝块某些切片的有限血液成分8。基于显微图像的方法仅关注凝块9的特定结构。此外，所有这些方法都费力且耗时10.迄今为止，尚未报道定量和定性研究脑血栓细胞成分的程序。人们普遍认为，交联的纤维蛋白将血细胞紧紧包裹在凝块中11.因此，交联纤维蛋白的特异性降解和完整细胞的释放对于细胞成分的准确分析至关重要。
先前的工作是从 Sipunculus nudus （sFE） 中分离出一种纤维蛋白溶解酶，它可以特异性地快速降解纤维蛋白12。在此，提出了一种基于sFE独特活性的脑血栓细胞成分分析方法。该方案首先利用sFE降解凝块的纤维蛋白，然后通过Wright染色和常规血液检查分析细胞成分13,14。根据这种方法，可以对脑血栓的细胞成分进行定量和定性研究。这种简单有效的方案可用于其他血凝块的细胞成分分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agglutination Reaction Plate</td>
			<td>ROTEST</td>
			<td>RTB-4003</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Hematology Analyzer</td>
			<td>SYSMEX</td>
			<td>XNB2</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Vertical Pressure Steam Sterilizer&#160;</td>
			<td>SANYO</td>
			<td>MLS-3750</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>AIRTECH</td>
			<td>BLB-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dish (100 mm)</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
		<tr>
			<td>DHG Series Heating and Drying Oven&#160;</td>
			<td>SENXIN</td>
			<td>DGG-9140AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td>SLGP033NK</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Refrigerated Centrifuge&#160;</td>
			<td>Cence</td>
			<td>H1650-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>01-30253-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CISEN</td>
			<td>H37022337</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE E100</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (1 mL )</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>MARTOR</td>
			<td>23111</td>
			<td></td>
		</tr>
		<tr>
			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td>VORTEX KB3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Hystic</td>
			<td>HKQS-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright Staining Solution</td>
			<td>Beyotime</td>
			<td>C0135-500ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive approach to analyze the cell components of cerebral blood clots

脑血栓形成是脑动脉或静脉中的血凝块，是最常见的脑梗塞类型。脑血栓细胞成分的研究对诊断、治疗和预后具有重要意义。然而，目前研究凝块细胞成分的方法主要基于 原位 染色，由于细胞被紧密包裹在凝块中，因此不适合对细胞成分进行综合研究。先前的研究已成功从 Sipunculus nudus中分离出一种纤维蛋白溶解酶（sFE），该酶可以直接降解交联的纤维蛋白，释放细胞成分。本研究建立了一种基于sFE的综合方法，用于研究脑血栓的细胞成分。该方案包括凝块溶解、细胞释放、细胞染色和常规血液检查。根据这种方法，可以对细胞成分进行定量和定性研究。显示了使用该方法的实验的代表性结果。

在降解过程的初始阶段，发现血凝块呈红色致密结构，工作溶液为无色。孵育30 min后，工作溶液变为浅红色，表明交叉的血细胞被释放到工作溶液中。当孵育时间延长至5 h时，大多数凝块溶解，工作溶液变为浅红色。相反，即使在孵育10小时后，生理盐水组（NC）也没有显着变化（图1）。这些结果表明，该方案成功释放了包裹在凝块中的血细胞。
Wright染...

Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

作者聚焦:一种脑血栓综合细胞成分分析的新方法 

本研究描述了一种通过溶栓、细胞染色和常规血液检查对脑血凝块进行细胞成分分析的快速有效方法。

一种分析脑血凝块细胞成分的综合方法

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

我们的研究主要集中在脑血栓形成上。许多研究表明，脑血凝块细胞成分与脑血栓形成的诊断、治疗和预后相关。在这项研究中，我们建立了一种分析脑血栓细胞成分的新方法。分析脑血凝块细胞成分的这一挑战是从血凝块中释放出整个血轴。这是因为血凝块泄漏了纤维性皮疹，即血栓中的血细胞。靶向启动凝块细胞成分的方法最好在 C2 站立下进行，这不适合细胞成分的全面启动。相反，我们的方法能够对脑斜率细胞成分进行定量和定性启发。我们的方法利用纤维蛋白溶解酶来延缓血栓中的纤维形成，释放完整的细胞。从细胞成分分析中获得的数据将有助于在分子水平上研究脑血栓形成的机制。我们的团队将继续专注于纤维蛋白溶解和凝血系统的研究。

a-comprehensive-approach-to-analyze-cell-components-cerebral-blood

Research

JoVE Journal

Biochemistry

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

506 次观看.  Quanzhou First Hospital Affiliated to Fujian Medical University. 我们的研究主要集中在脑血栓形成上。许多研究表明，脑血凝块细胞成分与脑血栓形成的诊断、治疗和预后相关。在这项研究中，我们建立了一种分析脑血栓细胞成分的新方法。分析脑血凝块细胞成分的这一挑战是从血凝块中释放出整个血轴。这是因为血凝块泄漏了纤维性皮疹，即血栓中的血细胞。靶向启动凝块细胞成分的方法最好在 C2 站立下进行，这不适合细胞成分...

视频: 作者聚焦:一种脑血栓综合细胞成分分析的新方法 

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

体外荧光显微镜基于动感检测的肌素特异性适应

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...