这项工作得到加拿大卫生研究所的支持, MOP-42546 和 MOP-119514 的运作赠款。

1. 氨基酸溶液的制备 <ol> <li> 为每种氨基酸准备和标记两个50毫升圆锥离心管 (分别用于两个和 #34; 低和 #34; 和 #34; 高 #34; 浓度). </li>
	<li> 准备一个大2升瓶, 含纯净水 (18.2 米和 #937; #183; cm 在25和 #176; C). </li>
	<li> 计算每种氨基酸 (克) 达到所需浓度, 并使用刮刀将适当数量的氨基酸称量到各自的50毫升离心管中. 
	注: 对于 #34; 高 #34; 两种带负电荷的氨基酸的浓度, 用 PBS 代替水。由于水的溶解度低, 我们不能增加其浓度, 而使用 PBS 代替水有助于维持低 pH 值。此外, 每种氨基酸溶液的浓度计算都是通过40毫升的最终体积得到的。所有氨基酸浓度在 表 3 中概述。一定要清洗在氨基酸之间的刮刀, 以避免污染。我们建议用水冲洗, 然后用70% 乙醇擦拭. </li>
	<li> 使用血清吸管将40毫升纯净水 (或 PBS) 加入每50毫升的试管中。帽管和涡旋或剧烈摇动直至溶解。水浴超声 (室温, 130 W, 40 赫) 也可用于协助溶解度过程. 
	注: 以下氨基酸溶液对光敏感, 应覆盖铝箔: 色氨酸、苯丙氨酸和酪氨酸 (包括芳香环状结构) 和半胱氨酸 (活性 SH 组). </li>
</ol> 2。准备 SAP-AA 解决方案 <ol> <li> 为自肽准备20毫升闪烁小瓶。对于一个给定的自肽, 准备一瓶每个准备的氨基酸溶液 (每个组合将在一个单独的小瓶). </li>
	<li> 使用高性能的分析平衡 (可读性降低到0.1 毫克或更低), 重约1和 #177; 0.2 毫克的肽到每个小瓶的底部。盖后称量, 并记录的确切重量的肽的上限. </li>
	<li> 将适当数量的氨基酸溶液 (在1节中制备) 放入每一个含有肽的小瓶中, 以便达到所需的自肽浓度 (0.1 毫克/毫升, 用于长肽, 长度为16氨基酸, 或0.2 毫克/毫升短肽, 长度为8氨基酸). </li>
	<li> 几种在室温下为10分钟的水浴 sonicator (130 W, 40 赫), 确保瓶子内的溶液完全浸入水浴中. </li>
</ol> 3。药物-二甲基亚砜或药物乙醇库存溶液的制备 <ol> <li> 将1毫克的药物 (在本例中为姜黄素) 与100% 的二甲基亚砜结合, 另1毫克与100% 乙醇一起创建两个股票解决方案. 
	注: 我们增加了200和 #181; l 二甲基亚砜和400和 #181; l 乙醇, 分别为5毫克/毫升和2.5 毫克/毫升, 由于每个溶剂中的溶解度不同, 而使其成为两甲基苯甲酸二甲砜-姜黄素和乙醇-姜黄素的股票;然而, 重要的是要注意, 股票的浓度应调整取决于疏水性药物的利益。诸如药物溶解度和有效生物浓度等因素在确定这一价值时很重要。同时, 请记住, 当与 SAP-AA 解决方案结合使用时, 该股票将分别被稀释100倍和50倍的二甲基亚砜和乙醇制剂 (见第4节)。根据所需配方的数量和 #8211, 最好准备大量的存货; 在这种情况下, 将使用超过1毫克的药物。库存可储存在-20 和 #176;使用前解冻冰和涡旋. </li>
	<li> 漩涡瓶十五年代完全溶解药物. </li>
</ol> 4。药物制剂的制备 <ol> <li> 为每个配方准备清晰的、1.5 毫升的离心管。一定要用预定的自肽、氨基酸 (和浓度) 和溶剂标签管. </li>
	<li> 添加10和 #181; 药物-二甲基亚砜库存, 或20和 #181; l 药物乙醇库存到适当的离心管. </li>
	<li> 添加990和 #181; SAP-AA 酸解决方案的适当标记离心管含有药物-二甲基亚砜的股票, 和980和 #181; 我对那些含有药物乙醇的股票。这产生1毫升药物配方与1% 二甲基亚砜或2% 乙醇. 
	注: 根据该协议, 所有姜黄素制剂的最终浓度为0.5 毫克/毫升。同样, 当使用其他疏水化合物和/或开始与不同的存货集中 (参见步骤 3.1) </li> <li> 漩涡强烈三十年代和允许公式化休息为 30 min. </li> </ol> 时, 这将有所不同。
 5. 溶解度测试 <ol> <li> 在休息期过后, 漩涡又一次剧烈地三十年代. </li>
	<li> 离心 14220 x g 的配方1分钟. </li>
	<li> 分析离心管底部的降水 (通过可视化). </li>
</ol>

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作者没有什么可透露的。

在制定过程中, 有许多关键步骤和问题需要考虑。首先, 当我们使用不同的组件和浓度时, 整个协议中的多个涡旋步骤确保所有浓度都是一致和正确的。一些高浓度的疏水性氨基酸溶液在涡流后仍可能无法完全溶解, 在这种情况下, 它们可以用手使劲摇动, 以协助过程。同样, sap-AA 解决方案必须经过步骤2.4 中概述的超声步骤, 因为结构调整结构自然倾向于聚合, 而超声将帮助分割 sap 集群, 从而产生更?...

sap 是一类生物材料广泛研究的3D 支架在再生医学1,2,3,4。然而, 最近, 由于其独特的生物学特性5、6、7、8, 它们被作为治疗药物的载体而被利用。sap 自然组装成稳定的纳米结构9, 从而提供了一种药物的封装和保护手段。sap 是亲, 由一个特定的疏水性和亲水性氨基酸重复的模式, 驱动他们的自组装9,10 , 并允许他们作为一个介乎疏水性和亲水性环境.因此, 对于疏水性药物的临床交付--由于在水环境中由于缺乏溶解度而在人体内具有极低的生物利用度和吸收率11,12 -sap 是希望作为交付车辆.此外, 他们的序列模式还意味着, sap 可以合理设计和工程, 以最大限度地与任何特定药物或化合物 (即, 基于功能基团) 的兼容性, 并进一步协助溶解度。
在许多体外和体内设置13、14、15、16中, sap 已被应用为有效的药物运载工具。它们也显示出极大的安全性和生物相容性。然而, 由于低渗透的 SAP 药物制剂, 它们不能用于静脉注射, 在临床设置13。考虑到这种限制, 我们最近制定了一项战略, 将 sap 与氨基酸解决方案结合起来, 以减少毒性 co-solvents 的使用, 并增加处方渗透, 从而提高临床相关性。我们选择使用氨基酸, 因为它们是结构化结构的基石, 已经被临床接受, 并与 sap 结合, 它们增加了疏水性药物的溶解度, 同时减少了需要的17,18的数量。
我们已经审查了 SAP-AA 组合作为一个通用平台的疏水性药物溶解度和后续交付通过创建一个多步骤筛选管道, 并将其应用于 Src 抑制剂, PP2, 作为一个模型疏水性化合物。在这个过程中, 我们研究了改变配方的成分-最终测试6种不同的结构, 所有20种氨基酸在2不同浓度 (低和高; 低根据现有临床应用的浓度, 和高浓度是 2x, 3x, 或5x 的临床浓度的基础上, 每个氨基酸在水中的最大溶解度), 和2不同的 co-solvents-和选择的组合, 溶解 PP2 进一步分析。这种药物制剂在细胞培养中被证明是一种有效的药物载体, 同时也在使用气管和静脉的管理的体内模型。同样, 我们的工作涉及的多功能性的 SAP-AA 组合在磷多, 结构不同的疏水性化合物在水环境中;具体来说, 药物 rottlerin 和姜黄素18。本文概述了液-AA 的配方方法和姜黄素溶解度分析为例, 在我们的筛选管道的主要步骤。该协议提供了一个简单的, 可重现的方式来筛选最佳的 SAP-AA 组合, 它可以溶解任何给定的疏水性化合物。

<table style="border-collapse:collapse;width:1034pt" width="1379">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-I</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20" style="width:510pt" width="680">Sequence: AEAKAEAKAEAKAEAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAKAKAEAEAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-IV</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAEAEAKAKAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EFK8-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: FEFEFKFK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">A6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AAAAAAKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">P6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: PPPPPPPKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Alanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7469-100G</td>
			<td class="xl20">L-Alanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Isoleucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">I7403-100G</td>
			<td class="xl20">L-Isoleucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Leucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">L8912-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Leucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Methionine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">M5308-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Methionine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Proline</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5607-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Proline</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Valine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">V0513-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Valine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Phenylalanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5482-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Phenylalanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tryptophan</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8941-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Tryptophan</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tyrosine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8566-100G</td>
			<td class="xl20">L-Tyrosine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glycine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8790-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glycine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Asparagine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A4159-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Asparagine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8540-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Serine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Serine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Threonine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8441-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Threonine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Histidine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">H6034-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Histidine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Lysine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl17" style="width:101pt" width="135">L5501-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Lysine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Arginine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A8094-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Arginine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Aspartic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Aspartic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8415-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Cysteine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">C7352-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Cysteine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Dimethyl Sulfoxide</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">D4540-500ML</td>
			<td class="xl22">DMSO</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Ethanol</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">277649-100ML</td>
			<td class="xl22">Anhydrous</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Curcumin</td>
			<td class="xl21" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl22">08511-10MG</td>
			<td class="xl22">Hydrophobic drug, curcumin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Rottlerin</td>
			<td class="xl21" style="width:106pt" width="141">EMD Millipore</td>
			<td class="xl22">557370-10MG</td>
			<td class="xl22">Hydrophobic drug, rottlerin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">PP2</td>
			<td class="xl21" style="width:106pt" width="141">Enzo&#160;</td>
			<td class="xl22">BML-EI297-0001</td>
			<td class="xl21" style="width:510pt" width="680">Hydrophobic drug, PP2</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Scintillation Vials</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl21" style="width:101pt" width="135">2650-66022-081</td>
			<td class="xl22">Borosilicate Glass, with Screw Cap, 20 mL. Vials for weighing peptide.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Falcon 50 mL Conical Centrifugation Tubes</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl23" style="width:101pt" width="135">352070</td>
			<td class="xl22">Polypropylene, Sterile, 50 mL. For amino acid solutions.</td>
		</tr>
	</tbody>
</table>

solubility of hydrophobic compounds in aqueous solution using combinations of self-assembling peptide and amino acid

自组装多肽 (sap) 是有前途的车辆提供疏水疗法的临床应用;它们的亲特性使它们能够在人体的水环境中溶解疏水化合物。然而, 自肽解决方案有较差的血液相容性 (例如, 低渗透), 阻碍了他们的临床应用通过静脉注射管理。我们最近开发了一个通用的疏水药物输送平台, 它将 sap 与氨基酸溶液 (液-AA) 结合起来, 提高药物的溶解度, 增加配方渗透, 达到临床使用的要求。在三种不同的疏水性化合物--PP2、rottlerin 和姜黄素--的背景下, 对这种配方进行了彻底的测试, 以证明其通用性。此外, 我们研究了改变配方成分的影响, 分析了6种不同的结构, 20 种天然氨基酸在低和高浓度, 和两个不同的 co-solvents 二甲基亚砜和乙醇。我们的策略被证明是有效的, 以优化一个给定的疏水性药物的成分, 和治疗功能的配方抑制剂, PP2, 被观察到的体外和在体内。这篇手稿概述了我们的通用配方方法使用 SAP-AA 组合疏水性化合物, 并分析溶解度作为第一步, 以潜在使用这些配方在更多的功能性研究。我们包括有代表性的溶解度结果的配方疏水性化合物, 姜黄素, 并讨论如何作为一个平台, 未来的生物研究和疾病模型的方法。

对于疏水性药物, 姜黄素, 我们生产的配方使用所有20种天然存在的氨基酸在低浓度, 结合只有一个 SAP, EAK16-II, 作为一个原理。我们还测试了使用二甲基亚砜和乙醇作为 co-solvents 的配方。总的, 这产生了40姜黄素配方, 每个含有不同的成分。重要的是要注意, 在我们以前的研究使用 Src 抑制剂, PP2, 我们包括更多的选择的 SAP (共 6) 和氨基酸浓度 (临床, 以及较高的浓度), 这产生?...

Watch this Scientific Journal Video about Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid at JoVE.com

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

本议定书描述了一种临床适用的方法, 溶解疏水化合物在水环境中使用自肽和氨基酸溶液的组合。我们的方法解决疏水疗法的一个主要局限, 缺乏安全, 有效的手段溶解和交付方法入临床设置。

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

solubility-hydrophobic-compounds-aqueous-solution-using-combinations

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Organic Chemistry

Medicine

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

16.6K 次观看.  University Health Network. 本议定书描述了一种临床适用的方法, 溶解疏水化合物在水环境中使用自肽和氨基酸溶液的组合。我们的方法解决疏水疗法的一个主要局限, 缺乏安全, 有效的手段溶解和交付方法入临床设置。

视频: Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

使用的非肥胖型糖尿病小鼠胃排空的评估[<sup&gt; 13</sup&gt; C-辛酸呼气试验

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

科研

医学

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

酸化浸渍法评价全肾肾素数

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

柚皮素溶液的制备

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

评估培养骨细胞和分离骨干中的氨基酸消耗

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

使用相干拉曼散射成像对皮肤内的药物化合物进行可视化和定量

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

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使用的非肥胖型糖尿病小鼠胃排空的评估[