我们感谢 UAntwerpen 的球体小组和 Ugent 的作物保护部对水样进行分析。该项目的支持是由 PF/10/007 研究基金的卓越中心的生态和社会进化动态提供的。afg 胶 (11Q0516N) 和 ESJT (FWO-SB151323) 以博士和 TP (12F0716N) 为博士后研究员, FWO 佛兰德 (全宗 Wetenschappelijk Onderzoek)。

这里描述的所有方法都得到了 KULeuven 伦理委员会的批准。
1. furzeri的孵化和一般维护
<ol><li>准备鱼培养基 (pH 值 7), 温度为14摄氏度, 并加入纯化型 II 水, 加标准化盐, 电导率为600µS/厘米 (24 摄氏度)。</li>
	<li>从已存储在标准条件13下的 GRZ 实验室线中选择鸡蛋。选择 DIII 阶段的鸡蛋 (即准备孵化), 可识别的金色眼睛9 , 并轻轻地把它们与软镊子对塑料 2 L 坦克 (不超过大约30鸡蛋每罐)。 注: 为了有足够数量的健康测试有机体, 孵化两次尽可能多的卵作为所需的鱼幼虫数量。</li>
	<li>添加1厘米的鱼培养基在12摄氏度, 让水温逐渐收敛到室温 (24 °c)9。鱼将孵化在前12小时。</li>
	<li>24小时后, 喂养幼仔一种浓缩剂量的新鲜孵化的Artemianauplii (有关食物频率和数量的详细信息, 请参阅 Polačik et 等) 的育种协议 20169), 并将水深提高到5厘米添加鱼培养基。</li>
	<li>36小时后, 喂养幼仔另一种浓缩剂量的新鲜孵化的卤虫幼体和添加鱼培养基, 增加水深到10厘米。</li>
	<li>在恒温条件下孵化鱼容器 (例如在孵化器、气候室或温水浴中) 在 14 h:10 h 灯以下: 黑暗政权。</li>
	<li>在实验开始之前, 复杂的鱼容器 (没有鱼) 与暴露化合物, 填充他们的最高浓度的接触介质, 并留在一夜之间, 以限制转移有毒物质的容器在实际实验。</li>
	<li>48小时孵化后, 选择健康的浮力幼虫开始接触实验。放弃所谓的肚皮滑块, 无法填满他们的游泳膀胱, 因此有一个受损的浮力 (持续下沉到底部)。</li>
</ol>2. 短期曝光协议
注: 研究人员应针对每项治疗至少20次复制 (20 条鱼在单独的罐子里)。除完全控制处理外, 如果用溶剂制备该化合物的溶液, 则应包括溶剂控制。溶剂控制应包含在最高接触浓度中溶剂浓度的溶剂量。
<ol><li>将实验容器 (0.5 升玻璃罐) 贴上标签, 用适当的曝光介质 (不同的毒物浓度) 填充。添加化合物以获得正确的浓度。</li>
	<li>将幼虫 (48 小时孵化后) 分别转移到容器 (每容器1条鱼进行单独监测)。 注意: 鱼类被单独暴露, 以尽量减少社会互动的潜在混淆效应, 例如对食物和侵略的竞争。然而, 鱼被允许视觉上的互动, 根据道德标准的实验室动物使用。</li>
	<li>随访2周以上的急性暴露。在这段时间里, 用卤虫幼体每天两次喂鱼ad 随意, 每星期7天/周。</li>
	<li>每隔一天刷新培养基以维持水质, 并尽量减少复合降解的潜在影响。监测关键水分变量 (溶解氧水平应超过 80%, 电导率应介于600和700µS/厘米之间, pH 值介于7.8 和8.2 之间, 硬度 (如碳酸钙3) 介于350和450毫克/升之间, 位于最佳饲养条件范围内。N. furzeri 9)。在刷新培养基之前和之后取水样品, 以确定实际的复合浓度。</li>
	<li>端点<ol>
<li>检查鱼的死亡率, 压力 (例如反常行为: 游泳颠倒) 或疾病每天 (上午, 晚上)。查阅谢德et的发布。(1999)12了解有关死亡率、压力或疾病的观察的详细信息。</li>
		<li>使用剂量-响应曲线 (丽思和 Streibig, 2005) 在不同的时间点计算 LC50值。使用 r v3.2.3 (r 开发核心团队, 2016) 中的刚果民主共和国包中的drm函数或类似的统计方法。</li>
	</ol>
</li>
</ol>3. 慢性暴露协议
注: 在实验开始时, 至少瞄准25条鱼/条件, 以尽量减少性别比例偏倚的几率, 并考虑自然原因导致的潜在背景死亡率 (i. e. 与年龄有关的死亡率)。
<ol><li>孵化 (见1节)</li>
	<li>第一阶段 (孵化后2天-孵化后16天)<ol>
<li>按照 2.1-2.4 所述的协议执行</li>
		<li>在实验的第二阶段, 培养基会在一周内降解 (没有茶点, 见下文)。将所需的介质在大型惰性容器中存放一周, 以允许化合物的类似降解。</li>
	</ol>
</li>
	<li>第二阶段 (孵化后16天)<ol>
<li>通过与化合物络合, 制备2升实验玻璃罐。用正确的曝光介质填充罐子, 并添加一个空气管通气罐子。在这些罐子里单独的把鱼放在实验的其余部分。允许视觉交互以适应道德标准。</li>
		<li>每星期刷新一次介质。将鱼与网转移到含有相同曝光介质的新罐子上。在每一周内每天抽取水样来监测化合物在每次浓度处理过程中的降解情况。如果测试多个压力源 (例如在不同温度下的化合物的毒性), 则计算每种处理的降解曲线。测量非生物参数 (pH 值, 温度, 溶解氧%, 电导率) 每周三次。</li>
		<li>从孵化后2天 (dph) 直到 23 dph, 每天喂鱼两次, 每周7天, ad 随意卤虫幼体。从 24 dph-37 dph, 补充ad libitumArtemia饮食与切碎的摇幼虫。从 38 dph, 每天喂鱼两次, 每周7天, ad 随意冷冻摇幼虫。</li>
	</ol>
</li>
	<li>端点<ol>
<li>每日检查鱼的死亡率, 压力或疾病12。</li>
		<li>为了确定生长, 每周测量身体大小 (9 dph-16 dph 21 dph-...) 通过把鱼转移到从他们的水库中填充培养基的培养皿中。采取 4-5 大小校准图片的鱼从上面 (在一个固定的高度) 和分析他们数字使用空间测量程序 (例如ImageJ)。 注: 对于成年鱼, 使用更高的培养皿, 以尽量减少处理压力, 保持所有的鱼淹没在整个测量过程中。</li>
		<li>对于男性成熟, 视觉检查鱼每天的着色从 15 dph 起。检查鳍的第一个迹象, 婚礼着色 (第二性特征)。使用第一天, 这是可见的作为一个代理的男性成熟时间。</li>
		<li>夫妇非奸鱼与男性相同的治疗组或非实验性男性每周三次从 30 dph 开始, 以确定女性成熟时间 (第一个卵子的一天)。为此, 请使用3.4.5 中描述的产卵协议。</li>
		<li>对于生育力, 夫妇成熟的女性与成熟男性3次/周从 30 dph 开始, 在他们的治疗使用交叉计划。<ol>
<li>为每对夫妇准备一个产卵池 (1 升), 使用暴露培养基从男性水族馆补充产卵基质 (细砂 < 500 µm)。</li>
			<li>将雄性和雌性转移到产卵池中, 让它们产卵两个小时。在此过程中尽量减少产卵容器周围的人类活动或干扰。</li>
			<li>然后, 将鱼轻轻地转移回原来的外壳容器, 不需要水的混合, 这将会在产卵基质中旋转卵。</li>
			<li>通过在500µm 网格上浇产卵基板来过滤卵。计数的鸡蛋和转移 (使用软镊子) 潮湿泥炭苔藓在培养皿中9,14。</li>
			<li>每天清除死卵。一个星期后, 密封的培养皿与密封膜, 并存储在一个温度控制孵化器在28摄氏度和14:10 小时的光: 黑暗循环, 立即发展到 DIII 阶段 (即准备孵化后约三周)。长期贮存时, 将卵存放在17摄氏度的恒定黑暗中, 卵子进入休眠阶段, 并保持多年存活。当从这些休眠卵子中招募鱼类进行实验时, 将休眠的卵转移到28°c 条件, 14:10 小时光: 黑暗周期约三周, 以允许发展到 DIII 阶段。</li>
		</ol>
</li>
		<li>测量成年鱼的临界热最大值 (CTmax) (用于性能15的度量值)。<ol>
<li>使用水浴, 以恒定的速度加热0.33 摄氏度/分钟, 并在其中的水不断循环。为每条鱼加1升水族馆。 注: 给定的空间限制在水浴, 有必要在几个系列工作。通过将 "系列" 作为随机因素进行统计分析时, 应考虑系列中条件之间的潜在差异。</li>
			<li>当水族馆的水达到实验饲养温度 (一般为28摄氏度) 时, 将鱼加入水族馆, 开始试验。使用数字温度计 (0.1 摄氏度) 监测 CTmax 浴1升水族馆中的温度每5分钟。</li>
			<li>当鱼无法保持 dorso 腹部直立位置或开始严重抽搐16,17时, 结束试验。测量 1 L 水族馆的温度, 这是临界最大温度。把鱼转回到实验房里恢复。</li>
		</ol>
</li>
		<li>在实验的最后一天测量鱼的重量 (0.1 毫克的准确度), 拍干并把它转移到一艘称量船上。注: 所有鱼类应在最后一次进食后四小时测量, 以规范肠道内的食物重量。</li>
		<li>用 0.1% Tricaine 弄死鱼。</li>
	</ol>
</li>
</ol>4. 跨代曝光协议
注: 要测量污染物对furzeri的跨代影响, 请遵循上文概述的第一代的慢性暴露协议。
<ol><li>每周两次, 检查生产的鸡蛋 (即第二代) 的发展, 储存在28°c 14:10h 光: 黑暗的周期条件通过检查培养皿的胚胎在 DIII 阶段 (见 Polačik et 等。20169)。当50多项父母治疗的复制被充分开发时, 在1.1 中根据该协议孵化他们。</li>
	<li>将健康的、浮力的鱼暴露在与父母鱼完全相同的设置和治疗中。</li>
</ol>

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作者没有什么可透露的。

这项工作描述了一种新的生物测定方法, 使用Nothobranchius furzeri(一种新兴的模型有机体) 来研究毒物和其他压力源的个体和组合的长期效应。提出的协议已成功地应用于测量物种对一系列有毒物质 (铜、镉、34-dichloroaniline 和毒死蜱) 的敏感性。由于它的快速生命周期, 这个脊椎动物模型允许评估致死和跨代的影响在四月内。使用这种鱼类作为毒性筛选模型的另一个主要优点是它生产抗旱蛋。这...

将一组物种的敏感度配置为战略性地选定的毒物被描述为欧洲范围立法 (注册、评估、授权和限制化学品) 的1 。急性或短期毒性试验主要用于这一目的, 因为它们给出了一个物种的敏感性的快速迹象。然而, 在它们的自然环境中, 生物体暴露在较长的时间内, 而完整的生命周期, 甚至几代可能会影响到2。此外, 受污染环境中的有机体通常会一次暴露于多个压力源, 这可能相互作用, 可能导致协同效应3。因此, 基于急性单源压力毒性试验计算的安全浓度可能低估了有毒物质在自然环境中的实际风险。因此, 最好还要研究欧洲委员会4、5和环保局 (联合致死) 所倡导的与环境有关的有毒物质的长期和几代效应。国家环境保护局)6,7。特别是在脊椎动物的研究中, 由于脊椎动物的寿命比无脊椎动物模型生物体长, 所以在从事慢性和几代人的暴露研究时, 劳动力、金钱和时间方面的成本很高。因此, 根据研究的问题, 选择最合适的鱼类模型有机体是可取的。此外, 还应提供广泛的脊椎动物种类, 以检验跨物种的反应的普遍性, 以便能够根据最敏感的物种来调整规章。目前, 有必要制定新的、有效的协议, 以短寿命周期为特征的脊椎动物模型物种, 以降低在脊椎动物身上进行慢性和多代暴露的成本7,8。
绿松石鳉鱼Nothobranchius furzeri 是一个有趣的鱼模型, 用于这种长期暴露实验, 因为它的成熟时间短和生命周期 (生成时间少于4周 9).这意味着与其他鱼类模型7相比, 可以在短时间内研究与生态相关的终点, 如成熟时间和繁殖力。此外, 这些鱼生产抗旱的休眠蛋, 在标准条件下储存数年后仍然可行, 从而消除了对连续养殖的需要9。在生态的研究中, 这也意味着复制鱼可以在完全相同的时刻孵化, 导致所有动物的时间同步, 甚至在不同时间生产的鸡蛋批次中。我们建议使用实验室 GRZ 应变进行暴露实验。这种菌株在实验室条件下表现良好, 是纯合的 (除了性染色体), 基因组的特征很好10,11。
在生态研究中, 选择合适的测试浓度范围是很重要的。为此, 可以使用几种互补方法。标称浓度范围可以基于相关物种的灵敏度, 如Nothobranchius guentheri12。或者, 该范围可以基于标准鱼模型的灵敏度, 如斑马鱼 (斑马斑马)2 , 对大多数毒物具有可比性的敏感性 (菲利普et 。(回顾))。结合上述两种选择, 应进行范围发现实验, 选择标称浓度范围。对于急性试验, 研究人员应瞄准100% 的死亡率, 中间死亡率和0% 的死亡后24小时接触毒物的浓度治疗。对于慢性试验, 最好在两周内进行范围发现实验, 以验证在这个参考期内, 测试浓度最高的条件下的幼虫死亡率是否不超过10%。
该议定书可以作为基线, 在furzeri上对水上污染物进行急性和长期接触, 检查压力源在个体和细胞层面的潜在影响。它还可用于执行多源压力研究, 以适应更高的生态相关性, 混合不同的有毒化合物或研究污染与其他自然压力源 (例如捕食) 之间的交互作用或人为压力源 (例如因气候变化而变暖)。

<table>
	<tbody>
		<tr>
			<td>purified water Type 1 (milli Q)</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sea Salt</td>
			<td>Instant Ocean</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2L plastic tank</td>
			<td>SAVIC</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>1L plastic tank (spawning)</td>
			<td>Avamoplast</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>nets</td>
			<td>Aqua bilzen</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>2L glass jars</td>
			<td>Sepac-Flacover</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>0,5L glass jars</td>
			<td>Sepac-Flacover</td>
			<td></td>
			<td>Always separate material for control and toxicity treatments</td>
		</tr>
		<tr>
			<td>Artemia eggs</td>
			<td>Ocean Nutrition</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chironomus</td>
			<td>Ocean Nutrition</td>
			<td></td>
			<td>frozen</td>
		</tr>
		<tr>
			<td>tricaine</td>
			<td>Sigma aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petri dishes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pipettes</td>
			<td>MLS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>FST</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;m mesh sieve</td>
			<td>/</td>
			<td></td>
			<td>self-made</td>
		</tr>
		<tr>
			<td>microcentrifuge tube (2ml)</td>
			<td>BRAND</td>
			<td></td>
			<td>To store fish in freezer</td>
		</tr>
		<tr>
			<td>glass vials</td>
			<td>Sigma aldrich</td>
			<td></td>
			<td>For water analysis</td>
		</tr>
		<tr>
			<td>weighing boat</td>
			<td>MLS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jiffy 7c pellets</td>
			<td>Jiffy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>Gilac</td>
			<td></td>
			<td>for Ctmax</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Air liquide</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>digital thermometer</td>
			<td>Testo AG</td>
			<td>testo 926</td>
			<td></td>
		</tr>
		<tr>
			<td>HETO therm heater</td>
			<td>Anker Schmitt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>calibrated balance</td>
			<td>Mettler-Toledo AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>platform for camera</td>
			<td>/</td>
			<td></td>
			<td>self-made</td>
		</tr>
		<tr>
			<td>Multiparameter kit</td>
			<td>HACH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80&#176;C)</td>
			<td>Panasonic Ultra low temperature freezer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fysio</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homogenisation buffer</td>
			<td>VWR</td>
			<td></td>
			<td>0.1&#160;M TRIS&#8211;HCl, pH 8.5, 15&#160;% polyvinyl pyrrolidone, 153&#160;&#181;M MgSO4&#160;and 0.2&#160;% Triton X-100</td>
		</tr>
		<tr>
			<td>chloroform:methanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glyceryl tripalmitate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>amyloglucosidase</td>
			<td>Sigma Aldrich</td>
			<td>A7420</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose assay reagent</td>
			<td>Sigma Aldrich</td>
			<td>G3293</td>
			<td></td>
		</tr>
		<tr>
			<td>Biorad protein dye</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtiter plate</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>384 microtiter plates</td>
			<td>Greiner Bio-one</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml glass tubes</td>
			<td>Fiers</td>
			<td></td>
			<td>For fat analysis</td>
		</tr>
		<tr>
			<td>2,5ml eppendorf tubes</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homogeniser</td>
			<td>Ultra-turrax TP 18/10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>photospectrometer</td>
			<td>Infinite M200 TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>heater for glass tubes</td>
			<td>Hach COD REACTOR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf Centrifuge 5415 R</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Bumako</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for acute and chronic ecotoxicity testing of the turquoise killifish nothobranchius furzeri

鳉鱼Nothobranchius furzeri 是生态领域新兴的模型有机体, 它在急性和慢性毒性试验中的适用性已经证明。总体而言, 该物种对有毒化合物的敏感性与其他模型物种的敏感程度相同。
这项工作描述了对furzeri的单一和联合应激作用的急性、慢性和几代生物鉴定的协议。由于它的成熟时间短和生命周期, 这脊椎动物模型允许研究终点, 如成熟时间和生育力在四月内。跨代全生命周期暴露试验可以在8月内进行。由于这一物种生产的鸡蛋具有抗旱性, 并且在多年内仍然可行, 所以不需要这种物种的现场养殖, 但在需要时可以招募个人。这些协议旨在测量生命历史特征 (死亡率、生长、繁殖力、体重) 和临界热最大值。

furzeri的严重暴露结果与不同的铜浓度 (如在2.5.2 中计算) 显示 cleardose 响应关系 (图 1)。随着毒物浓度的增加, 死亡率增加。LC50值随着时间的推移而减少, 这意味着随着浓度的降低, 50% 的复制死前会有更多的时间通过。有关furzeri对铜的急性和慢性暴露的详细结果, 以及与其他物种相比, 该物种的灵敏度比较, 我们指的是菲利普et</...

Watch this Scientific Journal Video about Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri at JoVE.com

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

在这项工作中, 我们描述了一种急性, 慢性和几代生物测定, 以研究单一和联合压力源对绿松石鳉鱼Nothobranchiusfurzeri 的影响。本协议旨在研究生命史特征 (死亡、生长、繁殖力、体重) 和临界热最大值。

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

protocol-for-acute-chronic-ecotoxicity-testing-turquoise-killifish

Research

JoVE Journal

Environment

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

7.4K 次观看.  University of Leuven. 在这项工作中, 我们描述了一种急性, 慢性和几代生物测定, 以研究单一和联合压力源对绿松石鳉鱼Nothobranchiusfurzeri 的影响。本协议旨在研究生命史特征 (死亡、生长、繁殖力、体重) 和临界热最大值。

视频: Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

一种用于收集和构建土芯协议测渗计

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

Protocol for Measuring the Thermal Properties of a Supercooled Synthetic Sand-water-gas-methane Hydrate Sample

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future energy resources and energy storage media. To develop a method for gas production from natural MH-bearing sediments and hydrate-based technologies, it is imperative to understand the thermal properties of gas hydrates.
The thermal properties' measurements of samples comprising sand, water, methane, and MH are difficult because the melting heat of MH may affect the measurements. To solve this problem, we performed thermal properties' measurements at supercooled conditions during MH formation. The measurement protocol, calculation method of the saturation change, and tips for thermal constants' analysis of the sample using transient plane source techniques are described here.
The effect of the formation heat of MH on measurement is very small because the gas hydrate formation rate is very slow. This measurement method can be applied to the thermal properties of the gas hydrate-water-guest gas system, which contains hydrogen, CO2, and ozone hydrates, because the characteristic low formation rate of gas hydrate is not unique to MH. The key point of this method is the low rate of phase transition of the target material. Hence, this method may be applied to other materials having low phase-transition rates.

We present a protocol for measuring the thermal properties of synthetic hydrate-bearing sediment samples comprising sand, water, methane, and methane hydrate.

协议测量过冷的合成砂水 - 气 - 甲烷水合物样品的热性能

Methane hydrates (MHs) are present in large amounts in the ocean floor and permafrost regions. Methane and hydrogen hydrates are being studied as future ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书 》

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

纳米材料在水介质中的分散: 对协议优化的研究

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking.
The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink&#39;s pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.

Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

用薄膜旋转圆盘电极法制备和测试燃料电池催化剂的研究

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

夏威夷咖啡莓科学监测协议: 全球咖啡农田模型

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

摘要

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此视频中的章节

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

人类血小板的LC-MS分析作为研究线粒体代谢的平台

一种用于收集和构建土芯协议测渗计

协议测量过冷的合成砂水 - 气 - 甲烷水合物样品的热性能

协议塑料微粒采样的海面和样品分析

生物毒理学方法与海洋细菌

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书》

纳米材料在水介质中的分散: 对协议优化的研究

用薄膜旋转圆盘电极法制备和测试燃料电池催化剂的研究

夏威夷咖啡莓科学监测协议: 全球咖啡农田模型

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

摘要

探索更多视频

此视频中的章节

优化负染:通过电子显微镜检查小和不对称蛋白结构的高通量协议

人类血小板的LC-MS分析作为研究线粒体代谢的平台

一种用于收集和构建土芯协议测渗计

协议测量过冷的合成砂水 - 气 - 甲烷水合物样品的热性能

协议塑料微粒采样的海面和样品分析

生物毒理学方法与海洋细菌

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书 》

纳米材料在水介质中的分散: 对协议优化的研究

用薄膜旋转圆盘电极法制备和测试燃料电池催化剂的研究

夏威夷咖啡莓科学监测协议: 全球咖啡农田模型

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书》