我们感谢 Martina Gáliková 博士为我们提供 果蝇 菌株。我们感谢Michelle Crozatier-Borde和Marc Haenlin团队对我们的研究提供反馈，并帮助我们改进我们的模型。我们要感谢Napo Pharmaceuticals Company为我们提供药物Crofelemer。作者还感谢客座编辑Hugues Petitjean博士为我们提供了发表该协议的机会。这项研究由国家研究署（ANR）在ANR-22-CE03-0001-01项目下资助。

1.制备植物提取物
<ol><li>从一棵成年树上收集 Psidium guajava L.叶子30 ，并按以下方法处理：将叶子在40°C的烤箱中干燥6天，然后风干6天，然后在40°C的烤箱中再次干燥4天，最后通过在研磨机或咖啡研磨机中研磨干燥的叶子来制备叶粉。</li>
	<li>将100g干燥粉末浸泡在1L96%乙醇中24小时，使用摇床连续搅拌。再次将植物残留物置于相同的过程中，并在40°C下使用真空旋转蒸发器在减压（175mbar）下将所得滤液蒸发至干燥。</li>
	<li>将植物提取物溶解在乙醇中以获得所需的浓度。通过测试一系列浓度来确定最佳浓度（使用此处描述的方案）。<ol><li>对于植物提取物，测试浓度范围为 100 μg/mL、1 mg/mL、10 mg/mL、100 mg/mL，并使用不影响苍蝇存活率的浓度范围。对于纯化合物，测试以下浓度范围：0.05、0.5、5 和 50 mM12。</li>
	</ol></li>
</ol>2. 配制食品培养基
<ol><li>测量 100 mL 蒸馏水，然后用 4 g 糖和 0.8 g 琼脂倒入烧杯中（见 材料表）。加热至100°C（同时搅拌）并保持10分钟。</li>
	<li>将温度降低至80°C，在搅拌的同时加入7.4克面粉和2.8克酵母。加热至少20分钟，仍然搅拌并控制温度，温度应在80°C左右。</li>
	<li>加入moldex-丙酸溶液（1 mL moldex 和 0.3 g 丙酸混合均匀）。等待温度降至50°C左右，加入植物提取物溶液（1mg / mL）和0.5g溴酚蓝粉。 注意：有关要测试的其他浓度的更多详细信息，请参阅第 1.3.1 节。</li>
	<li>将食物倒入培养皿中，并在培养皿装满时停止（图1A）。让培养皿冷却至室温（约3小时），然后盖上盖子，并储存在4°C的冰箱中。 注意：培养皿应在冰箱中存放不超过 2 周，以避免水分蒸发。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65877/65877fig01.jpg"> 图1：粪便沉积试验的实验过程演示。 （A）显示装满食物培养基的培养皿的图像。确保培养皿中有足够的食物，这样就不会有缝隙困住苍蝇并阻止它们移动。但是，不要在培养皿中装满食物，以便均匀地覆盖表面。（B）协议中描述的刮刀图像。（C）协议中描述的粪便沉积测试的图像。 <a href="https://www.jove.com/files/ftp_upload/65877/65877fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3.准备苍蝇
<ol><li>准备 CO2 罐、带针的 CO2 吹气枪、飞垫、画笔和显微镜。飞行站为飞垫和吹气枪提供 CO2 。苍蝇垫用于分拣苍蝇，而 CO2 吹气枪用于麻醉小瓶、瓶子和培养皿中的苍蝇。</li>
	<li>通过选择含有蛹（至少10个）的小瓶来标准化苍蝇的年龄，并使用以下方法从管中丢弃成蝇。一个接一个地将小瓶倒置，将针头插入棉塞和小瓶侧壁之间。用CO2 吹气枪麻醉成年苍蝇，直到所有苍蝇都在棉塞上睡着（麻醉它们需要几秒钟，松开吹气枪后麻醉作用将持续几秒钟）。打开装有70%乙醇的玻璃瓶上方的小瓶，将苍蝇放入其中。用棉塞关闭小瓶，并将其保存在25°C和60%湿度的培养箱中。将培养箱的光照周期设置为12小时光照/12小时黑暗。</li>
	<li>孵育后（最多8小时），在显微镜下将它们分类为处女雌性和雄性，并通过将它们翻过来并观察它们的生殖器来飞垫。<ol><li>与男性生殖器相比，女性生殖器是苍白的，男性生殖器呈红色。雄性也可以通过前腿上存在的深色刚毛（称为性梳）来识别。将苍蝇分成两个新鲜管（一个用于雄性，一个用于雌性），并在25°C下孵育6-8天。 注：在25°C下，雌性在孵化后保持处女状态约8小时。</li>
	</ol></li>
</ol>4.粪便沉积试验
<ol><li>在培养皿上贴上相应的菌株、性别和药物标签，以避免培养皿之间混淆。将培养皿堆叠在一起。</li>
	<li>获取装有染色食物的培养皿，并将它们倒置在吸墨纸上以吸收多余的液体。使用刮刀（图1B），将食物切成12等份，然后用刮刀将一片放入空的培养皿中。 注意：根据重复次数，每个培养皿最多可以增加要切割的切片数量，最多 20 个。每个切片的大小应相同。</li>
	<li>用 CO2 麻醉苍蝇，直到所有苍蝇都在棉塞上睡着了。在每个培养皿中转移六只健康的苍蝇（图1C），并立即盖上盖子，然后将它们放入培养箱中（25°C，60%湿度，12小时光照/ 12小时黑暗）。</li>
	<li>为确保苍蝇在实验过程中不会从培养皿中逸出，请用胶带固定培养皿的顶部和底部盖子。对于每个测试组，准备六个重复的培养皿（至少）。</li>
	<li>让苍蝇饲养24小时后，使用CO2 麻醉它们，将苍蝇转移到装有70%乙醇的容器中，并处理任何剩余的食物。</li>
	<li>保留培养皿并继续执行步骤5。</li>
</ol>5. 培养皿的定量
<ol><li>在计算机上设置一个文件夹并重命名它，包括实验菌株名称、苍蝇的性别和使用的药物类型。在此文件夹中，创建名为“原始”、“剪切”和“分析”的子文件夹。</li>
	<li>使用光学分辨率为 6,400 像素/英寸 （ppi） 的高分辨率扫描仪扫描培养皿。分别扫描每个培养皿的顶盖和底盖，将它们分别放置在扫描仪区域的中间。</li>
	<li>打开计算机上安装的应用程序。计算机屏幕上将打开一个欢迎窗口，其中包含所有常规设置（图 2A）。 注意： 请勿修改高级设置。此步骤仅对拥有与 材料表中建议的相同扫描仪的用户有效。如果使用其他软件，请参阅指南。<ol><li>在应用程序中命名培养皿，包括序列号、顶部或底部盖、性别和所用食物类型。选择在步骤 5.1 中设置的原始子文件夹。</li>
		<li>预览培养皿。单击窗口底部的“ 预览 ”，等待几秒钟让扫描仪进行预扫描，然后计算机屏幕上会出现一个窗口。移动屏幕上显示的正方形以包围培养皿。</li>
		<li>单击计算机屏幕上窗口右下角的 扫描 ，扫描会自动将扫描内容另存为所选文件夹中的图像。</li>
	</ol></li>
	<li>使用应用程序（例如，开源斐济应用程序）裁剪图像，以便不将人工制品和食物残渣视为沉积物。 注意：以下步骤仅对拥有斐济应用程序的用户有效。如果使用其他软件，请参阅指南。<ol><li>打开 Fiji 应用程序，等待几秒钟，直到屏幕上出现工具栏。</li>
		<li>将要裁剪的图像拖动到工具栏上。在工具栏中选择第三个图标多边形选择，通过单击屏幕在饼图周围标记角度来裁剪照片中不需要的部分（彩色饼图）（图 2B-1,2）。 注意：在图像中描绘裁剪区域时，所选帧必须端到端无缝连接。</li>
		<li>单击栏左上角的“编辑”，然后单击“填充/清除外部”（图 2B-3）。</li>
		<li>要保存照片，请单击栏左上角的 “文件 ”，然后单击 “另存为”，最后单击 “Tiff”。选择在步骤 5.1 中设置的 子文件夹 Cut 。 注意：保存裁剪后的图像时，请确保文件名中没有特殊字符（例如 ！、&、$、#、_,-,...）或字符过多。</li>
	</ol></li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65877/65877fig02.jpg"> 图2：分析粪便沉积物测试数据过程中的关键步骤。 （A） 显示扫描应用程序设置信息的屏幕截图。（B） 使用斐济应用程序裁剪的图像。确保没有人工制品和食物残渣被视为沉积物。（C） 显示打开 Excel_merge-v4 应用程序时的屏幕截图。 <a href="https://www.jove.com/files/ftp_upload/65877/65877fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
6. 使用粪便开源软件的终极阅读器进行粪便沉积物识别
注：粪便软件终极阅读器的介绍和使用可以在 补充文件1中找到。
<ol><li>首先，打开 T.U.R.D. 软件（补充图 1）。创建一个新实验并为文档命名，然后将其保存在步骤 5.1 中设置的“分析”子文件夹中。</li>
	<li>单击 “Plates ”，然后单击 “Add plates”。选择要处理的培养皿。将出现一个新窗口，其中包含所选板的名称和新参数。设置块大小、偏移量、最小大小和最大大小（补充图 2）。</li>
	<li>要验证检测到的粪便沉积物是否正确，请单击“ 板”，然后单击“ 检查所选板”，然后单击“ 图形”，然后单击“ 查看注释图像 ”（补充图3）。放大并查看计数。如果只有少数矿床不应包含在分析中，请取消选择要排除的矿床（补充图4）。</li>
	<li>对于要处理的每个新映像，请从步骤 6.3 重新启动。</li>
	<li>使用 T.U.R.D. 软件分析板后，通过单击“否”来修改苍蝇数量 。苍蝇 （补充图5）。</li>
	<li>通过单击 “板”>“编辑组”>“添加”来修改组名称，然后在 “组 列”上选择组名称。</li>
	<li>通过单击 “分析>描述性统计量”>选择组 （补充图 6）分别导出不同的仿行数据。将所有电子表格文件 （.csv） 保存在同一文件夹中。</li>
	<li>为了将所有文件（在步骤6.7中获得）收集到一个唯一的电子表格中，打开应用程序Excel_merge-v4（补充编码文件1），等到出现以下句子选择文件夹的路径（包含.csv文件）：，然后粘贴上面的文件夹地址。例如，路径可以是 C：\Experiment\Fecal deposit test\，然后在键盘上单击 Enter 两次（图 2C）。之后，将在同一文件夹中创建一个新的电子表格文件。新的电子表格文件包括不同工作表中的所有导出文件。</li>
	<li>在上一个电子表格文件中，添加另一个工作表以收集所有仿行的每个参数的平均值（使用 VLOOKUP 函数处理数据）。 附表1中给出了一个例子。</li>
	<li>分析 p 值。</li>
</ol>

果蝇 用于药物发现的粪便沉积物分析

<ol>
	<li>Cheng, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+system.">Gastrointestinal system.</a> WIREs Sys Bio Med. 2 (1), 65-79 (2010).</li><li>Greenwood-Van Meerveld, B., Johnson, A. C., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+physiology+and+function.">Gastrointestinal physiology and function.</a> Handb Exp Pharmacol. 239, 1-16 (2017).</li><li>Doyle, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinicopathologic+study+of+24+cases+of+systemic+mastocytosis+involving+the+gastrointestinal+tract+and+assessment+of+mucosal+mast+cell+density+in+irritable+bowel+syndrome+and+asymptomatic+patients.">A clinicopathologic study of 24 cases of systemic mastocytosis involving the gastrointestinal tract and assessment of mucosal mast cell density in irritable bowel syndrome and asymptomatic patients.</a> Am J Surg Pathol. 38 (6), 832-843 (2014).</li><li>Levine, G. A., Walson, J. L., Atlas, H. E., Lamberti, L. M., Pavlinac, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+pediatric+diarrhea+in+low-resource+settings.">Defining pediatric diarrhea in low-resource settings.</a> J Pediatric Infect Dis Soc. 6 (3), 289-293 (2017).</li><li>Abraham, B., Sellin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+diarrhea.">Drug-induced diarrhea.</a> Curr Gastroenterol Rep. 9 (5), 365-372 (2007).</li><li>Badry, A. H. H., Jameel, A. Y., Mero, W. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+microorganisms+associated+with+arrhea+in+infants+and+children+in+Duhok+Province,+Kurdistan+Region+/+Iraq.">Pathogenic microorganisms associated with arrhea in infants and children in Duhok Province, Kurdistan Region / Iraq.</a> Sci J Uni Zakho. 2 (2), 266-275 (2014).</li><li>Manetu, W. M., M'masi, S., Recha, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrhea+disease+among+children+under+5+years+of+age:+A+global+systematic+review.">Diarrhea disease among children under 5 years of age: A global systematic review.</a> Open J Epidemiol. 11 (3), 207-221 (2021).</li><li>Fu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+animals+promote+antibiotic+resistance+gene+dissemination+in+water+via+conjugation:+Role+of+different+regions+within+the+zebra+fish+intestinal+tract,+and+impact+on+fish+intestinal+microbiota.">Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract, and impact on fish intestinal microbiota.</a> Mol Ecol. 26 (19), 5318-5333 (2017).</li><li>Zhang, Q., Widmer, G., Tzipori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pig+model+of+the+human+gastrointestinal+tract.">A pig model of the human gastrointestinal tract.</a> Gut Microbes. 4 (3), 193-200 (2013).</li><li>Cox, C. R., Gilmore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+microbial+colonization+of+Drosophila+melanogaster+and+its+use+as+a+model+of+Enterococcus+faecalis+pathogenesis.">Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis.</a> Infect Immun. 75 (4), 1565-1576 (2007).</li><li>Jasper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+physiology+and+pathology+of+aging+in+the+intestine+of+Drosophila+melanogaster.">Exploring the physiology and pathology of aging in the intestine of Drosophila melanogaster.</a> Invertebr Reprod Dev. 59, 51-58 (2015).</li><li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacol Rev. 63 (2), 411-436 (2011).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+digestive+tract+of+Drosophila+melanogaster.">Anatomy and physiology of the digestive tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Jennings, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila-+a+versatile+model+in+biology+&amp;+medicine.">Drosophila- a versatile model in biology &amp; medicine.</a> Materials Today. 14 (5), 190-195 (2011).</li><li>Kumar, V. S., Navaratnam, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neem+(Azadirachta+indica):+Prehistory+to+contemporary+medicinal+uses+to+humankind.">Neem (Azadirachta indica): Prehistory to contemporary medicinal uses to humankind.</a> Asian Pac J Trop Biomed. 3 (7), 505-514 (2013).</li><li>Shrestha, P., Adhikari, S., Lamichhane, B., Shrestha, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+screening+of+the+medicinal+plants+of+Nepal.">Phytochemical screening of the medicinal plants of Nepal.</a> J Environ Sci Tech Food Tech. 1 (6), 11-17 (2015).</li><li>Perveen, S., Al-Taweel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacognosy:+Medicinal+Plants.">Pharmacognosy: Medicinal Plants.</a> IntechOpen. , (2019).</li><li>Noumi, E., Yomi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicinal+plants+used+for+intestinal+diseases+in+Mbalmayo+Region,+Central+Province,+Cameroon.">Medicinal plants used for intestinal diseases in Mbalmayo Region, Central Province, Cameroon.</a> Fitoterapia. 72 (3), 246-254 (2001).</li><li>Njoku, V. O., Obi, C., Onyema, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+constituents+of+some+selected+medicinal+plants.">Phytochemical constituents of some selected medicinal plants.</a> African J Biotechnol. 10 (66), (2011).</li><li>Rokaya, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+uses+of+medicinal+plants+in+gastrointestinal+disorders+in.">Traditional uses of medicinal plants in gastrointestinal disorders in.</a> Nepal. J Ethnopharmacol. 158, 221-229 (2014).</li><li>Birdi, T., Krishnan, G. G., Kataria, S., Gholkar, M., Daswani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+open+label+efficacy+clinical+trial+of+oral+guava+leaf+decoction+in+patients+with+acute+infectious+diarrhoea).">A randomized open label efficacy clinical trial of oral guava leaf decoction in patients with acute infectious diarrhoea).</a> J Ayurveda Integr Med. 11 (2), 163-172 (2020).</li><li>van Vuuren, S. F., Nkwanyana, M. N., de Wet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+evaluation+of+plants+used+for+the+treatment+of+diarrhoea+in+a+rural+community+in+northern+Maputaland,+KwaZulu-Natal,+South+Africa.">Antimicrobial evaluation of plants used for the treatment of diarrhoea in a rural community in northern Maputaland, KwaZulu-Natal, South Africa.</a> BMC Complement Altern Med. 15, 53 (2015).</li><li>Firenzuoli, F., Gori, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbal+medicine+today:+Clinical+and+research+issues.">Herbal medicine today: Clinical and research issues.</a> Evid Based Complement Alternat Med. 4, 37-40 (2007).</li><li>Rawat, P., Singh, P. K., Kumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+based+traditional+anti-diarrheal+medicinal+plants+and+their+phytocompounds.">Evidence based traditional anti-diarrheal medicinal plants and their phytocompounds.</a> Biomed Pharmacother. 96, 1453-1464 (2017).</li><li>Palombo, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemicals+from+traditional+medicinal+plants+used+in+the+treatment+of+diarrhoea:+modes+of+action+and+effects+on+intestinal+function.">Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: modes of action and effects on intestinal function.</a> Phytother Res. 20 (9), 717-724 (2006).</li><li>Koyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nutrient-responsive+hormonal+circuit+mediates+an+inter-tissue+program+regulating+metabolic+homeostasis+in+adult+Drosophila.">A nutrient-responsive hormonal circuit mediates an inter-tissue program regulating metabolic homeostasis in adult Drosophila.</a> Nat Commun. 12 (1), 5178 (2021).</li><li>Wayland, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spotting+the+differences:+Probing+host/microbiota+interactions+with+a+dedicated+software+tool+for+the+analysis+of+faecal+outputs+in+Drosophila.">Spotting the differences: Probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila.</a> J Insect Physiol. 69, 126-135 (2014).</li><li>Cognigni, P., Bailey, A. P., Miguel-Aliaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neurons+and+systemic+signals+couple+nutritional+and+reproductive+status+with+intestinal+homeostasis.">Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis.</a> Cell Metab. 13 (1), 92-104 (2011).</li><li>G&#225;likov&#225;, M., Dircksen, H., N&#228;ssel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thirsty+fly:+Ion+transport+peptide+(ITP)+is+a+novel+endocrine+regulator+of+water+homeostasis+in+Drosophila.">The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila.</a> PLoS Genet. 14 (8), 1007618 (2018).</li><li>Chassagne, F., Quave, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+extraction,+and+in+vitro+antibacterial+evaluation+of+plants+used+in+traditional+medicine.">Collection, extraction, and in vitro antibacterial evaluation of plants used in traditional medicine.</a> Methods Mol Biol. 2296, 19-41 (2021).</li><li>Patel, T. S., Crutchley, R. D., Tucker, A. M., Cottreau, J., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crofelemer+for+the+treatment+of+chronic+diarrhea+in+patients+living+with+HIV/AIDS.">Crofelemer for the treatment of chronic diarrhea in patients living with HIV/AIDS.</a> HIVAIDS. 5, 153-162 (2013).</li><li>Cottreau, J., Tucker, A., Crutchley, R., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crofelemer+for+the+treatment+of+secretory+diarrhea.">Crofelemer for the treatment of secretory diarrhea.</a> Expert Rev Gastroenterol Hepatol. 6 (1), 17-23 (2012).</li><li>Wu, P. E., Juurlink, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loperamide+cardiac+toxicity:+Pathophysiology,+presentation,+and+management.">Loperamide cardiac toxicity: Pathophysiology, presentation, and management.</a> Can J Cardiol. 38 (9), 1378-1383 (2022).</li><li>Benguettat, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DH31/CGRP+enteroendocrine+peptide+triggers+intestinal+contractions+favoring+the+elimination+of+opportunistic+bacteria.">The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria.</a> PLoS Pathog. 14 (9), 1007279 (2018).</li><li>Thakare, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intake+estimation+and+longitudinal+tracking+of+solid-food+consumption+(DIETS)+in+Drosophila.">Direct intake estimation and longitudinal tracking of solid-food consumption (DIETS) in Drosophila.</a> bioRxiv. , 543033 (2023).</li><li>Miller, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+an+emerging+translational+model+of+human+nephrolithiasis.">Drosophila melanogaster as an emerging translational model of human nephrolithiasis.</a> J Urol. 190 (5), 1648-1656 (2013).</li><li>Zierer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fecal+metabolome+as+a+functional+readout+of+the+gut+microbiome.">The fecal metabolome as a functional readout of the gut microbiome.</a> Nat Genet. 50 (6), 790-795 (2018).</li></ol>

作者没有什么可透露的。

由于黑腹果蝇与人类之间的基因相似性，黑腹果蝇已被广泛接受为各种生物过程的模型36.使用 D. melanogaster 作为研究肠道的模型很普遍，并且 T.U.R.D. 的应用已被用于估计粪便沉积的数量、面积和数量。然而，表型检测方法尚未用于评估果蝇的腹泻。因此，该协议引入了一种新方法，通过检测粪便沉积物来粗略评估腹泻的存在。
粪便沉积是肠道功能和健康的重要指标37.在此背景下，提出了一种在含药培养基上饲养 黑腹果蝇 的方法，以研究粪便沉积物的各种参数。通过监测沉积物的数量，可以确定排便的频率并评估药物是否对肠道运输有任何影响。可以测量沉积物的总面积来评估粪便的浓度和稀释度，这是决定肠道整体健康的重要因素。此外，总积分光密度 （IOD） 可用于检测沉积物中存在的粪便物质总量。该协议提供了一种有效的方法来筛选和评估影响肠道的药物以及植物提取物。当 黑腹果蝇 被用作模式生物时，可以评估潜在药物的疗效，这有助于加速药物发现过程。通过将这种方法应用于植物提取物，研究人员可以帮助验证它们作为止泻剂的用途。
使用该方案研究黑腹果蝇中的粪便沉积物时，需要考虑几个关键步骤。首先，必须计算在培养基中达到所需药物浓度所需的质量。此外，在将药物添加到培养基中时，确保良好的制备条件很重要，因为高温会降解药物并影响其效力。其次，雌性苍蝇的选择在该协议中很重要。重要的是使用处女雌性苍蝇，以避免处女和交配雌性之间的粪便输出量差异。例如，处女雌性产生的斑点比交配雌性更圆，交配雌性往往比处女雌性排泄更多的粪便物质27,28。因此，建议在封闭8小时之前收集苍蝇，以确保所有收集的雌性都是处女。此外，被测试的苍蝇应该强壮健康，因为它们的健康会影响食物摄入量和粪便输出量。例如，翅膀形状异常的苍蝇可能难以获得食物。最后，要成功使用 T.U.R.D.，块大小（像素）和偏移设置至关重要。由于图像的光对比度不同，可能需要尝试不同的设置，以尽可能最好地识别粪便沉积物。
尽管所提出的方法有效，但存在一些局限性。一是培养基中药物浓度的准确性。当培养基在制备过程中被加热时，一些水可能会蒸发，从而影响药物的浓度。另一个限制是培养皿的扫描。培养皿的某些部分（即边缘）未被扫描，这可能导致对总粪便沉积物的错误计算。此外，苍蝇不会在培养皿的顶部和底部盖上产生相同数量的粪便沉积物。由于它们往往会在底盖上产生更多的沉积物，因此顶盖和底盖之间的分析标准偏差可能很高，这可能会影响结果的准确性。
使用该协议，研究人员可以研究 黑腹果蝇的腹泻。通过对含药培养基进行改性，该方法可用于筛选止泻植物，为药物发现提供了一种新的方法。几个世纪以来，传统医学和天然产品一直被用来治疗不同的疾病，包括胃肠道疾病。通过使用该协议来评估植物提取物对粪便沉积物的功效，可以确定肠道疾病的潜在新疗法，并可以提供将其用作止泻药的科学原理。这种方法可以为药物发现和民族药理学领域做出有价值的贡献。

胃肠道 （GI） 也称为消化道，负责营养物质的消化和吸收以及未消化产物的排泄1.胃肠道容易受到一系列疾病的影响，这些疾病会引起不适、疼痛和日常生活中断。胃肠道疾病包括腹痛和不适、腹胀、胃灼热、消化不良或消化不良、恶心、呕吐、腹泻和便秘2.腹泻是胃肠道疾病3 最常见的症状，它被定义为在 24 小时内至少有 3 次稀便和水样便的疾病4。腹泻由多种病原体引起，包括细菌、病毒、寄生虫、真菌，也可能由药物引起 5,6。在世界范围内，腹泻仍然是 5 岁以下儿童死亡的第二大原因7.虽然腹泻可以自行消退，但如果持续超过几天，也可能表明存在更严重的潜在疾病。
为了研究肠道，研究人员转向小鼠、大鼠和猪等动物模型 8,9。然而，使用这些动物可能既昂贵又耗时，因为它们需要专门的设施和道德考虑。最近的研究表明，黑腹果蝇可以作为研究胃肠道的模型，并研究一些机制，例如维持再生稳态、免疫衰老的发展、上皮屏障功能的丧失和代谢稳态的下降10,11。D. melanogaster，被称为果蝇，与人类具有高度的遗传同源性;大约 75% 的人类疾病基因被认为在苍蝇12 中具有功能同源物。它们还有一个简单的消化系统，包括前肠、中肠和后肠13。黑腹果蝇易于在实验室培养，可以以不同的方式进行基因改造14.因此，使用黑腹果蝇进行体内测试是一种强大的工具，使研究人员能够在受控环境中研究复杂的生物过程。
根据世界卫生组织 （WHO） 的数据，生活在发展中国家的约 80% 的人使用传统医学来满足其基本卫生需求15.药用植物的大量使用可以解释为它们易于获得、价格低廉且副作用小16.草药疗法中使用的主要植物部分包括叶子、树皮、根、种子17，而主要制备方法是输液、煎煮和浸渍18。这些草药含有植物化学物质，如生物碱、萜类、类黄酮、类固醇、单宁和碳水化合物19，对人体有治疗作用。人们使用各种药用植物来治疗胃肠道疾病，如腹泻、胃痛和痢疾20.例如，番石榴是世界上最常用的治疗腹泻的植物之一。各种药理学和临床测试已经表明其安全性，这使其成为研究21,22 的良好止泻候选药物。然而，草药的主要局限性是缺乏效率和安全性评估，以及缺乏关于所用植物提取物成分的明确和完整的信息23。为了验证草药的有效性和安全性，需要一种涉及实验和临床验证的系统方法，并且该方法应得到来自体内和体外研究的足够数据的支持。
为了评估传统疗法在治疗腹泻方面的疗效，近几十年来，小鼠和大鼠的使用一直占主导地位24,25。由于前面提到的主要优点，即易用性、价格实惠、可复制、保守的果蝇和哺乳动物之间的吸收和消化功能，我们建议使用黑腹果蝇作为模型来评估植物的止泻活性。黑腹果蝇的腹泻表型可以表征为几个特征，包括粪便沉积物的丰度增加、沉积物大小更大、颜色较浅（浓度较低）和粪便物质较高26。这种表型可以使用各种参数进行量化：粪便沉积物数量、沉积物总面积、平均亮度和总综合光密度 （IOD）。总IOD定义为沉积物的总染料含量，即排泄的粪便总量27。以前，已经开发了一种分析黑腹果蝇粪便沉积物的测定法 27,28。在该测定中，粪便的最终读取器（T.U.R.D.）被用作粪便分析工具，可以检查粪便沉积物的数量，大小和轻度，从而监测果蝇的肠道生理学。然而，这种方法从未用于评估果蝇的腹泻表型。离子转运肽 （ITP） 基因是口渴和排泄的重要内分泌调节剂，将水稳态与黑腹果蝇的摄食相结合。在最近的一项研究中，研究表明，食物在整个胃肠道中的运输速度和排便事件的频率因 ITP 过表达而降低，而 ITP 敲除则增加。本研究的作者将后一种表型描述为腹泻29.
在该协议中，通过使用ITPi菌株作为腹泻模型，采用粪便沉积测定的修改版本来评估抗腹泻剂（即番石榴叶提取物）对黑腹果蝇胃肠道的影响。该方法的总体目标是：1）提供一种简单可靠的方法来评估药物和植物提取物的止泻作用，以及2）通过应用生物活性引导的方法，允许发现负责植物提取物中止泻作用的生物活性化合物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical &#38; Food medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma Aldrich</td>
			<td>A7002</td>
			<td>5 Kg bucket</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma Aldrich</td>
			<td>34725-61-6</td>
			<td>B5525-25G</td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>Nature et Cie</td>
			<td>*910007</td>
			<td>25 Kg bag</td>
		</tr>
		<tr>
			<td>Crofelemer</td>
			<td>Napo pharmaceuticals</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Loperamide</td>
			<td>Sigma Aldrich</td>
			<td>L4762</td>
			<td>5 grams</td>
		</tr>
		<tr>
			<td>Moldex</td>
			<td>VWR</td>
			<td>1.06757.5000</td>
			<td>5 Kg bag</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Dutscher</td>
			<td>409553-CER</td>
			<td>1 Liter bottle</td>
		</tr>
		<tr>
			<td>Sugar</td>
			<td>Pomona EpiSaveurs</td>
			<td>52705</td>
			<td>1 Kg bag</td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>Dutscher</td>
			<td>789195</td>
			<td>10 Kg bag</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>DWK LIFE SCIENCE</td>
			<td>-</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Centrifugation tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td>Eppendorf tubes&#160;&#160;5.0 mL</td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Erlen Meyer flask</td>
			<td>-</td>
			<td>-</td>
			<td>500 mL (for extraction)</td>
		</tr>
		<tr>
			<td>Filter paper grade</td>
			<td>Whatman</td>
			<td>-</td>
			<td>3 mm chr.</td>
		</tr>
		<tr>
			<td>Flowbuddy socle</td>
			<td>Genesis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flugs Narrow Plastic vials</td>
			<td>Genesis</td>
			<td>49-102</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flystuff Blow gun</td>
			<td>Genesis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flystuff Ultimate Flypad</td>
			<td>Genesis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flystuff Foot pedal</td>
			<td>Genesis</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumostar</td>
			<td>11295-51</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>-</td>
			<td>-</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Inox spatula</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Eppendorf</td>
			<td>4924000088</td>
			<td>Eppendorf Reference&#160;2</td>
		</tr>
		<tr>
			<td>Micropipette tip</td>
			<td>Eppendorf</td>
			<td>30000919</td>
			<td>epT.I.P.S.&#160;Standard</td>
		</tr>
		<tr>
			<td>Narrow Drosophila vials</td>
			<td>Genesis</td>
			<td>32-120</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>628162</td>
			<td>Size: 60 x 15mm</td>
		</tr>
		<tr>
			<td>Round-bottom flask</td>
			<td>-</td>
			<td>-</td>
			<td>500 mL (for evaporation)</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Avantor</td>
			<td>620-0916</td>
			<td></td>
		</tr>
		<tr>
			<td>Whisk</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chiller</td>
			<td>HUBER</td>
			<td>Minichiller</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating bath</td>
			<td>B&#220;CHI</td>
			<td>B-490</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>BIOBLOCK SCIENTIFIC</td>
			<td>-</td>
			<td>Magnetic stirrer hot plate</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>-</td>
			<td>HPP110eco</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>B&#220;CHI</td>
			<td>R-200</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>Epson</td>
			<td>V850 pro</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmund B&#252;hle</td>
			<td>KS 10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Stereomicroscope binocular</td>
			<td>Zeiss</td>
			<td>Stemi 305</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>VACUUBRAND</td>
			<td>PC500 series</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Sigma Aldrich</td>
			<td>CLS6776-1EA</td>
			<td>Corning&#160;LSE&#160;vortex mixers</td>
		</tr>
		<tr>
			<td>Weighing scale</td>
			<td>OHAUS Scout</td>
			<td>SKX622</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

assessment of the effect of antidiarrheal drugs and plant extracts on drosophila melanogaster

为了研究人类胃肠道生理学，生物医学科学家依赖于模式生物的使用。尽管许多研究人员使用小鼠作为模型来研究肠道功能，但只有少数报告关注黑腹果蝇（D. melanogaster）。与小鼠相比，果蝇具有许多优点，例如生命周期短、成本效益高且维护简单，并且没有伦理问题。此外，哺乳动物的胃肠道生理学、解剖学和信号通路在黑腹果蝇中高度保守。植物提取物传统上用于治疗腹泻和便秘。例如，番石榴（P. guajava）是热带地区最著名的止泻药之一。然而，没有研究评估止泻药和泻药以及植物提取物对黑腹果蝇的作用，并且与哺乳动物相比，果蝇是否可能发生类似的作用（例如，在止泻药的情况下，粪便沉积物更小、更集中、更丰富）是否可能发生。在这项研究中，番石榴在呈现腹泻表型的黑腹果蝇菌株中证明了由番石榴诱导的止泻作用。使用添加染料的食物监测苍蝇产生的粪便样本。该协议概述了用于用药物制备食物的方法，评估以这些食物制剂为食的苍蝇的粪便沉积物，并解释获得的数据。

这里介绍的研究表明，可以通过使用粪便沉积测定来实现黑 腹果蝇 腹泻的测量。表型（腹泻与否）之间的显着差异可以通过分析各种参数来确定，包括粪便沉积物的数量、沉积物的总面积、沉积物的平均面积、平均亮度和总综合光密度 （IOD），IOD 是沉积物中存在的染料总量的量度，代表排泄的总粪便物质含量27。
果蝇中的 ITP 基因敲低可诱导腹泻表型，其特征是排便频率增加，使其成为研究腹泻的合适模型29。在该实验的背景下，使用 ITPi 菌株（w1118;daughterless-GeneSwitch，UAS-ITPi /（CyO））并在标准培养基上饲养。鉴于这种植物在热带地区广泛用于治疗腹泻，因此选择 番石榴 叶提取物作为止泻干预措施。Crofelemer是一种止泻药，被美国食品和药物管理局（FDA）批准用于缓解接受抗逆转录病毒治疗的成年HIV/AIDS患者的非感染性腹泻症状31。Crofelemer 是从 Croton lechleri Müll.Arg 的乳胶中提取的。茎皮32.洛哌丁胺是一种合成药物，在世界范围内用于治疗腹泻33.Crofelemer 和洛哌丁胺均被用作潜在的阳性对照。
假设是，与用正常食物喂养的果蝇相比，用番石榴提取物、Crofelemer 和洛哌丁胺喂养苍蝇会减少腹泻表型。为了验证这一假设，通过比较喂食正常食物的果蝇与喂食番石榴提取物（1 g/100 mL）、Crofelemer （1 g/100 mL） 和洛哌丁胺 （10 mM） 的果蝇之间的几个参数，对黑腹果蝇粪便沉积物进行了测量。对于实验设置，使用6-7天大的处女雌性或雄性。每个培养皿包含六只苍蝇，并进行了六次重复。将苍蝇饲养24 h，然后对每组进行分析。使用学生的 t 检验来比较测试组之间的显着差异。结果表明，与番石榴提取物（1 g/100 mL）组相比，正常食物组的粪便沉积物数量（图3A）、沉积物总面积（图3B）和总IOD（图3C）在处女雌性和雄性中都表现出显着更高的值。不幸的是，洛哌丁胺在两性中都没有显示出任何效果（但已经证明它在黑腹果蝇中起到解痉剂的作用）34，而克罗菲勒默只对雌性有影响。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65877/65877fig03.jpg"> 图 3：ITPi 应变分析。在4种条件下分析ITPi菌株：以正常食物为食，补充1 g/100 mL番石榴提取物的食物，1 g/100 mL Crofelemer和10 mM洛哌丁胺。数据以雌性和雄性每种情况的平均±SD表示（培养皿两侧的六次重复）。使用比较两组的学生 t 检验进行统计分析。p 值如下所示： *： p < 0.05;**： p < 0.01;： p < 0.001， ****： p < 0.0001.（A） 比较了喂食补充有 1 g/100 mL Crofelemer、10 mM 洛哌丁胺、1 g/100 mL 番石榴提取物的食物的果蝇和喂食正常食物的果蝇的粪便沉积量。此外，还分析了处女雌性和雄性之间粪便沉积物数量的差异。在两组中，喂食正常食物的果蝇的粪便沉积数量明显高于喂食 1 g/100 mL 番石榴提取物的果蝇。（B） 比较了喂食正常食物的果蝇和喂食补充有 1 g/100 mL 番石榴提取物、1 g/100 mL Crofelemer 和 10 mM 洛哌丁胺的食物的果蝇的粪便沉积总面积。在雄性和雌性中，喂食正常食物的果蝇的粪便沉积总面积明显高于喂食 1 g/100 mL 番石榴提取物的果蝇。（C） 分析了以正常食物为食的果蝇与以补充 1 g/100 mL 番石榴提取物、1 g/100 mL Crofelemer 和 10 mM 洛哌丁胺为食的食物喂养的果蝇之间 ITPi 菌株总 IOD 的差异。在雄性和雌性中，喂食正常食物的果蝇的总IOD显著高于喂食1 g/100 mL番石榴提取物的果蝇。缩写：F = 女性;M = 男性;Crofe = Crofelemer;Lope = 洛哌丁胺;也没有食物=正常食物;P. gua ext = 番石榴提取物。<a href="https://www.jove.com/files/ftp_upload/65877/65877fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
为了证明在 番石榴 提取物组中观察到的排泄减少是由于提取物的抑制作用而不是食物消耗减少，我们进行了固体食物消耗量的直接摄入量估计和跟踪 （DIETS） 方法35。结果显示，除了雄性洛哌丁胺导致果蝇摄入的食物少于正常人外，药物供应组和未使用药物组之间的食物消耗没有显着差异（图4）。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65877/65877fig04.jpg"> 图4：喂养试验。 喂养试验测量了果蝇的固体食物消耗量。用四种不同的培养基喂养果蝇：1 g/100 mL 番石榴 提取物、1 g/100 mL Crofelemer、10 mM 洛哌丁胺和普通食物。每组由 20 只苍蝇组成，有 5 次重复。数据以女性和男性每种情况的平均±标准差表示。使用比较两组的学生 t 检验进行统计分析。 p 值如下所示： *： p < 0.05;**： p < 0.01;： p < 0.001， ****： p < 0.0001. <a href="https://www.jove.com/files/ftp_upload/65877/65877fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
粪便沉积物和饲养试验结果表明， 番石榴 提取物是治疗果蝇腹泻的可靠药用植物。
补充图1：T.U.R.D.打开窗口。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%201.zip" target="_blank">请点击这里下载此文件。</a>
补充图 2：T.UR.D. 窗口，设置有待调整。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%202.zip" target="_blank">请点击这里下载此文件。</a>
补充图 3：带有注释图像的 T.U.R.D. 窗口。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%203.zip" target="_blank">请点击这里下载此文件。</a>
补充图 4：T.U.R.D. 窗口显示已处理图像中检测到的每个点。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%204.zip" target="_blank">请点击这里下载此文件。</a>
补充图 5：T.U.R.D. 窗口显示每个处理后的图像。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%205.zip" target="_blank">请点击这里下载此文件。</a>
补充图 6：T.U.R.D. 窗口，显示导出每个组数据的过程。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Figure%206.zip" target="_blank">请点击这里下载此文件。</a>
补充文件1：使用T.U.R.D.软件的快速指南。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20File%201_RE.zip" target="_blank">请点击这里下载此文件。</a>
补充表 1：准备分析的最终电子表格示例。<a href="https://www.jove.com/files/ftp_upload/65877/Supplementary%20Table%201_RE.zip" target="_blank">请点击这里下载此文件。</a>
补充编码文件 1：合并电子表格的应用程序。 <a href="https://www.jove.com/files/ftp_upload/65877/Supplemental%20Coding%20File.zip" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Advancing Antidiarrheal Research with the Drosophila Model at JoVE.com

作者聚焦：利用果蝇模型推进止泻药研究 

在这里，描述了一种用药物和植物提取物喂养 果蝇 的方法，并通过分析果蝇粪便沉积物来评估它们对胃肠道的影响。药物治疗的苍蝇可以用作进一步研究的模型。

止泻药和植物提取物对黑腹果蝇效果的评估

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

我们的研究重点是对传统疗法的科学验证，包括药用植物。我们正试图更好地了解这些药物的药理作用，特别是止泻和抗菌作用。为了测试药用植物的止泻作用，通常采用小鼠模型，但该过程耗时且昂贵，并且需要大量的植物材料进行测试。通过使用黑腹果蝇作为模型，我们的目标是通过减少实验时间和使用的植物材料数量来改善新型止泻药的发现过程。我们还旨在通过使用简单有效的模型为在传统医学中使用止泻植物提供科学依据。我们的研究结果将帮助科学家对植物提取物进行生物引导分离，从而发现负责抗腹泻活性的化合物。

assessment-effect-antidiarrheal-drugs-plant-extracts-on-drosophila

Universidad San Sebastian

Research

JoVE Journal

Biology

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

1.3K 次观看.  UMR 152 PharmaDev, Université Paul Sabatier, Institut de Recherche pour le Développement. 我们的研究重点是对传统疗法的科学验证，包括药用植物。我们正试图更好地了解这些药物的药理作用，特别是止泻和抗菌作用。为了测试药用植物的止泻作用，通常采用小鼠模型，但该过程耗时且昂贵，并且需要大量的植物材料进行测试。通过使用黑腹果蝇作为模型，我们的目标是通过减少实验时间和使用的植物材料数量来改善新型止泻药的发现过程。我们还旨在通?...

视频: 作者聚焦：利用果蝇模型推进止泻药研究 

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

过程评价絮凝剂的效率的分散粒子的从植物提取物的去除

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

果蝇的口腔细菌感染和脱落

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...