我们对藏医学院“十四五”规划内涵建设项目（2022ZYYGH12）、藏医药大学教育部藏医与基础教育重点实验室2022年开放课题（ZYYJC-22-04）、宁夏市重点研发计划（2023BEG02012）的资金支持表示衷心感谢。 成都中医药大学杏林学者研究推广项目（XKTD2022013）。

将化合物溶液、蛋白质溶液和染料引入PBS溶液中。随后，使用数字加热振荡干浴对样品进行逐步加热，并通过测量复杂系统内荧光强度的变化来评估蛋白质的热稳定性。下面概述了详细的步骤， 图 1 说明了该协议的概述。
1. 溶液制备
<ol><li>通过将0.1mg伞形酮加入616.75μLDMSO溶液中来制备1mM伞形酮溶液（参见 材料表）。</li>
	<li>通过将 0.1 mg CD40 加入 500 μL ddH2O 溶液中来制备 200 μg/mL CD40 蛋白溶液（参见 材料表）。</li>
	<li>通过将 1 μL 5000x 橙色染料加入 9 μL 磷酸盐缓冲盐水 （PBS） 中来制备 500x 橙色染料溶液（参见 材料表）。 注意：在冰上执行上述操作，由于移液器的最小量程为 0.1 μL，因此最初将橙色蛋白染料稀释 10 倍以方便后续使用。</li>
</ol>2.染料性能检测
<ol><li>将 500x 橙色染料加入 PBS 溶液中并稀释以获得染料：PBS 比例为 1：500、1：1000、1：2000 和 1：4000。 注意：确保 DMSO 的最终浓度不超过 2% （v/v）。</li>
	<li>打开荧光酶标仪并预热10分钟。</li>
	<li>依次打开计算机和数据采集软件（见 材料表）。</li>
	<li>点击 仪器，选择对应的荧光微孔板型号，在数据采集软件中选择 确定 。 注意： 等待荧光微孔板完成预热并在面板上显示温度，然后再打开数据采集软件，以确保仪器与软件成功连接。</li>
	<li>单击 “采集设置 ”，然后在数据采集软件中选择“ 荧光 ”。</li>
	<li>在波长接口中编辑程序：Lm1 = 470 nm、570 nm，然后选择确定。 注：橙色染料可以用 300 nm 的紫外线或 470 nm 的可见光激发，发射可以在 570 nm 处测量。</li>
	<li>将不同浓度的橙色染料（1：500、1：1000、1：2000 和 1：4000）和 PBS 以 100 μL/孔的浓度加入 96 孔板中。 注意：在将溶液添加到PBS之前，将溶液溶解在DMSO中，并在制备过程中以2000× g 彻底涡旋，以确保由于染料的沉淀倾向而完全溶解。</li>
	<li>将96孔板放入仪器的检测台上，点击数据采集软件中的 读取 按钮，在室温下测量每种染料浓度的荧光13次，每次间隔2分钟。</li>
	<li>每次测定后单击“导出”按钮，选择“导出到 XML XLS TXT”，选择“所有板”，然后选择“输出格式的板”，最后单击“确定”以“XLS”格式将数据保存在文件夹中以供进一步分析。 注：这些步骤有助于确定连续测量对室温下橙色染料自发荧光的影响，并确定最佳染色浓度。</li>
	<li>打开数字加热摇动干浴，设置加热温度为35°C，加热时间为2分钟。</li>
	<li>在 1.5 mL 微量离心管中制备染料的最佳稀释比例，并将其置于数字加热振荡干浴中 2 分钟。</li>
	<li>将PBS溶液和在35°C加热的染色溶液以每孔100μL的体积加入96孔板中。</li>
	<li>将 96 孔板放入仪器的检测台，然后单击数据采集软件中的 读取 按钮。</li>
	<li>将加热温度设置为 40 °C，加热时间为 2 分钟。</li>
	<li>将 96 孔板中的染料溶液移回 1.5 mL 微量离心管中，并在仪器中加热 2 分钟。</li>
	<li>重复步骤2.12-2.15和步骤2.9的操作，分别测试染料溶液在35°C、40°C、45°C、50°C、55°C、60°C、65°C、70°C、75°C、80°C、85°C、90°C和95°C下的吸光度。 注：这些步骤有助于确定橙色染料在连续加热下自发荧光的稳定性，梯度为35°C至95°C。 至关重要的是，必须迅速工作，以避免温度快速下降或添加错误的液体，这可能导致实验错误。</li>
</ol>3. 检测蛋白质的温度熔融（Tm）
<ol><li>将 CD40 蛋白和橙色染料与 PBS 溶液混合，最终浓度分别为 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL 和 1：500。</li>
	<li>重复步骤 2.2-2.6 和步骤 2.10-2.16 中概述的操作，分别评估 CD40 蛋白溶液在 35 °C、40 °C、45 °C、50 °C、55 °C、60 °C、65 °C、70 °C、75 °C、80 °C、85 °C、90 °C 和 95 °C 的温度下的吸光度。</li>
	<li>重复步骤 2.9 保存和分析数据，然后使用数据分析软件计算 Tm 值（参见 材料表）。</li>
	<li>向 PBS 溶液中加入 CD40 蛋白、伞形酮和橙色染料，分别获得 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL、10 μM 和 1：500 的终浓度。 注意：在溶液制备过程中剧烈涡旋对于确保染料、CD40 蛋白和伞形酮在 PBS 中的均匀分布至关重要。</li>
	<li>重复步骤3.2-3.3的操作，分别在35°C，40°C，45°C，50°C，55°C，60°C，65°C，70°C，75°C，80°C，85°C，90°C和95°C的温度下测量CD40-伞形酮复合物溶液的吸光度。 注：执行这些步骤是为了确定 CD40 蛋白和 CD40-伞形酮复合物的 Tm 值，旨在确定与伞形酮的最佳 CD40 蛋白结合浓度。</li>
</ol>

使用荧光高灵敏度检测化合物-蛋白质结合

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作者没有什么可透露的。

DSF，也称为热位移测定或热荧光测定，是一种用于检测样品中蛋白质的热变性过程的技术，通过监测测试样品或染料的荧光信号在缓慢的程序化温度升高期间的变化。DSF 最初由 Pantoliano14 建立，是一种高通量方法。主要步骤包括在计算机控制的加热板上升高温度，使用长波长紫外线灯发射激发光，并通过电荷耦合器件相形成相机捕获荧光染料与蛋白质结合产生的荧光信号强度变?...

Vicatia thibetica H. Boissieu是伞形植物科的一种植物，在藏药中常用，是五种基本成分（Polygonatum sibiricum Delar. ex Redoute， Asparagus cochinchinensis （Lour.）Merr、 Vicatia thibetica H. Boissieu、 Oxybaphus himalaicus Edgew. 和 Gymnadenia conopsea （L.） R. Br.）1.主要分布在我国西南地区，如云南西北部、川西、西藏等地区，其干根作为当归1的局部替代品。这种根以其香味而闻名，经常被用作炖菜调味料，而被称为西藏芹菜的叶子则有助于制作美味的菜肴。因此， 紫 薇不仅作为一种独特的药用植物具有重要意义，而且作为西藏的食物来源也具有重要意义。
国内外研究表明，硫苜蓿具有补血和气（力量或能量）的特性，有利于调节月经。它用于解决由心悸和血虚引起的月经不规则痛经症状，具有抗氧化调节身体免疫力等药理作用2。Vicatia thibetica 的酒精提取物已证明能够恢复环磷酰胺诱导的免疫功能低下小鼠的体重和器官指数。此外，它还增加了血清中超氧化物歧化酶的活性，降低了丙二醛的含量，表明抗氧化能力有所提高，对脂质过氧化有平衡作用2,3。同时，它增强了血细胞和血红蛋白的数量，这不仅客观地反映了人体的造血功能，而且在免疫系统中起着至关重要的作用2,3。
Umbelliferone的特点是针状结晶和苦味，分子量小，易挥发，可以用蒸汽蒸馏。易升华，在水中溶解度低，在有机物中的溶解度高。作为 Vicatia thibetica 的主要化学成分之一，伞形酮在氢化可的松诱导的免疫抑制小鼠模型中对细胞免疫、体液免疫和非特异性免疫具有免疫调节作用 4,5。
细胞表面分子 CD40 是肿瘤坏死因子受体超家族的成员，在免疫细胞中广泛表达6.其同源配体 CD154，也称为 CD40L，是一种由活化的 T 淋巴细胞表达的 II 型跨膜蛋白。CD40 激活具有上调树突状细胞 （DC） 和单核细胞表面共刺激分子表达的能力。该过程促进主要组织相容性复合物 （MHC） 分子的抗原呈递功能，并进一步激活 CD8+ T 细胞7。
巨噬细胞在肿瘤微环境的形成和调控中起着至关重要的作用，CD40的激活可以增强巨噬细胞对肿瘤微环境的重塑8。CD40信号激活显著影响B细胞的增殖和活化。当 B 细胞被 CD40 激活时，可以作为有效的抗原呈递细胞发挥作用。它们呈递抗原，产生效应 T 细胞活性，从而有助于抗肿瘤作用9。此外，肿瘤细胞中的CD40激活可以诱导细胞凋亡并抑制肿瘤生长10。CD40 主要通过控制非受体酪氨酸蛋白激酶的活性来转导信号，包括 Lyn、Fyn、Syk 等。此外，它还具有刺激 Bcl-xL、Cdk4 和 Cdk6 蛋白、激活 Rel/NF-kB 转录因子以及磷酸化 CG-2 和 PI3K10 的能力。
差示扫描荧光 （DSF） 方法被广泛用于评估各种环境条件（如缓冲液组成、温度和小分子配体）对蛋白质结构热稳定性的影响。DSF 常用的染料是一种橙色、环境敏感的疏水染料。在正常情况下，蛋白质结构被折叠，在内部隐藏了其疏水部分。随着温度的升高，蛋白质的疏水区域变得更加暴露，导致天然蛋白质结构逐渐分解。染料选择性地与这种暴露的蛋白质部分结合，放大其荧光。然后通过监测荧光信号检测11的变化来计算Tm值。在一定程度上，Tm 值的变化可以衡量由于突变、缓冲液变化或配体结合导致的蛋白质稳定性变化。此外，它可以指示蛋白质折叠过程中的结构改变12。这种方法提供精确的数据、宽广的温度范围、高灵敏度和最小的蛋白质样品损失13。
在这项研究中，使用荧光酶标仪而不是荧光定量聚合酶链反应 （PCR） 仪器进行荧光测定。这种改进使得在没有荧光定量PCR仪器的实验室中进行DSF检测，使方法不那么复杂，减少了仪器设置所需的步骤，从而简化了实验过程。但是，这种方法存在某些缺点。虽然复杂性降低了，但过程变得更加繁琐。在不同温度下进行手动荧光检测是必要的，并且无法实现从系统中自动连续收集荧光。因此，本研究利用DSF技术探索CD40蛋白与伞形酮之间的相互作用，为藏医分子机制提供了新的见解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

研究不同分子之间的相互作用是理解疾病发病机制和筛选药物靶点的关键方面。Umbelliferone 是藏药 Vicatia thibetica 中的一种活性成分，具有未知机制的免疫调节作用。CD40蛋白是免疫反应的关键靶标。因此，本研究采用差示扫描荧光技术原理，利用荧光酶标记物分析CD40蛋白与伞形酮之间的相互作用。最初，通过实验验证了蛋白质荧光橙染料的稳定性，并确定了最佳稀释比例为1：500。随后观察到CD40蛋白的温度熔解（Tm）值随着浓度的增加而趋于降低。有趣的是，发现CD40蛋白与伞形酮之间的相互作用增强了CD40蛋白的热稳定性。这项研究代表了使用荧光微孔板和荧光染料检测小分子化合物和蛋白质结合潜力的首次尝试。该技术具有灵敏度和准确度高的特点，在蛋白质稳定性、蛋白质结构和蛋白质-配体相互作用方面取得了可观的进展，从而促进了进一步的研究和探索。

橙色染料在室温和高温下均在 Ex = 470 nm 和 Em = 570 nm 处始终表现出稳定的荧光激发。确定了1：500的最佳稀释比例（图2A，B）。当CD40蛋白浓度低于15μg/ mL时，Tm值的检测被证明是具有挑战性的（图3A，B）。然而，在浓度为15μg/ mL时，可检测到51.82°C的稳定Tm值，随着CD40蛋白浓度的增加，该值降至45.79°C（图3C，D</st...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

作者聚焦：简化 PCR 方法以提高可及性和效率

该协议描述了一种用于检测化合物和蛋白质分子之间结合的独特方法，具有最小的蛋白质样品损失和高数据准确性的优点。

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。

detection-cd40-protein-umbelliferone-interaction-via-differential

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

371 次观看.  University of Tibetan Medicine. 我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。

视频: 作者聚焦：简化 PCR 方法以提高可及性和效率

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

科研

医学

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer.

在检测之前乳房X光检查建筑失真<em&gt;通过</em面向模式的&gt;分析

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

降血脂药物对胆固醇颗粒形态的差异化作用

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. BMD is an important component of bone&#39;s nutritional status and in skeletal remains of both juveniles and adults, and it can provide information about bone quality. For adults remains, it can provide information on pathological conditions or when bone insufficiency may have occurred. In juveniles, it provides a useful metric to elucidate cases of fatal starvation or neglect, which are generally difficult to identify. This paper provides a protocol for the anatomical orientation and analysis of skeletal remains for scanning via dual-energy X-ray absorptiometry (DXA). Three case studies are presented to illustrate when DXA scans can be informative to the forensic practitioner. The first case study presents an individual with observed longitudinal fractures in the weight bearing bones and DXA is used to assess bone insufficiency. BMD is found to be normal suggesting another etiology for the fracture pattern present. The second case study employed DXA to investigate suspected chronic malnutrition. The BMD results are consistent with results from long bone lengths and suggest the juvenile had suffered from chronic malnutrition. The final case study provides an example where fatal starvation in a fourteen-month infant is suspected, which supports autopsy findings of fatal starvation. DXA scans showed low bone mineral density for chronological age and is substantiated by traditional assessments of infant health. However, when dealing with skeletal remains taphonomic alterations should be considered before applying this method.

Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes.

扫描骨骼遗骸的骨骼矿物密度在法医学背景下

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

使用扫描电子显微镜可视化膜褶皱形成

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

使用荧光辅助识别技术检测和去除牙齿颜色的复合树脂

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...