The authors thank Mona Chambers, Geoffrey Hidalgo and Maurissa Two Two for their technical assistance. We also thank Dr. Earl White for amino acid analysis, Dr. Roger Simonds for analysis of pollen for pesticides, and Drs. Stanley S. Schneider, Vanessa Corby-Harris and Kirk Anderson for their reviews and discussion of earlier versions of the manuscript and many helpful suggestions.

1. AHB和EHB殖民地获得Beebread <ol><li>将花粉过滤器对蜜蜂的殖民地，并收集花粉。使用研磨咖啡研磨机花粉成细粉（类似于花粉花药棚）。 </li><li>建立每个AHB和EHB 5殖民地在一个封闭的飞行区（EFA），这样蜜蜂觅食，只能在规定的花粉。为了防止工人EHB和AHB殖民地之间漂移，分全民教育成独立的部分，使蜜蜂无法将它们之间的交叉。将个人EHB或AHB菌落3,500-4,000工蜂，蜡梳花蜜，蜂蜜，未成熟的育雏和空梳在全民教育的各个部分。 注意：当建立的殖民地不具备存储花粉。花粉中存储的速率可以增加不包括在所述的菌落一个铺设女王。 </li><li>通过放置一个托盘，在全民教育的各个部分花粉饲料地面花粉殖民地。每个托盘使foragin散布约60g花粉克蜜蜂可以收集它作为corbicular负载和花粉保存在他们的殖民地为beebread。继续每天提供新鲜花粉每个托盘上3周。 </li><li>参阅来自欧洲的菌落作为欧洲beebread（EBB），并从非洲化菌落作为非洲化beebread（ABB）至beebread。 </li></ol>在笼子里饲养2蜜蜂<ol><li>从单独的笼子里出现AHB和EHB殖民地密封工人育雏的环境室内放置帧定在32-34℃，相对湿度40％.. </li><li>当工人出现，大约24小时的时候，建立12有机玻璃笼生物测定（尺寸= 11.5 X 7.5 X16.5厘米3），并添加或者100新兴EHB或100新兴AHB工蜂每个笼子里。放置梳的截面与任意EBB或ABB的细胞在每笼一已知数量（每笼24-30个细胞）以产生下列治疗组合：AHB馈ABB，EHB馈ABB，AHB馈EBB和EHB馈EBB。 （4个处理; 6每次治疗笼;共24箱）。 </li><li>加水的小瓶和50％的蜂蜜和水的溶液体积为每个笼子制定。每天笔芯蜂蜜和水的小瓶11天的学习期。 </li></ol> 3.采样工人蜜蜂和Beebread和消费估计<ol><li>样品10新兴EHB和AHB工人将它们放置在鸡笼前。参阅这些作为第0天的蜜蜂，并让它们作为血淋巴蛋白浓度的基准。 </li><li>从每个网箱中取出10蜜蜂后，他们喂养EBB或ABB 4，7，和11天。 </li><li>放置在单个微量离心管活蜜蜂和冰袋设置。选择四个蜜蜂血淋巴蛋白浓度的分析子样本。 </li><li>在第一天，11取样蜜蜂后，计数梳细胞仍然含有beebread的数目。这是beebread消费的相对度量。 </li><li>从细胞中每个网箱中取出剩余beebread和存储组合通道根据笼吃离心管。保持beebread样品在-80℃下直到用于pH，可溶性蛋白浓度和氨基酸含量进行分析。 </li></ol> 4. pH值估计花粉和Beebread的<ol><li>取供给到在EFA蜜蜂花粉六个随机0.3克样品和溶解于300微升蒸馏水中。采用防水双结pH值矛与0.01的精度测量pH值。 </li><li>就拿beebread 0.3 g样品是留在每个网箱的11天喂养一段时间后。溶解在300微升蒸馏水和测量pH值的beebread所描述的花粉（4.1）。 </li></ol> 5.蛋白质分析<ol><li>采取花粉六个样品和EBB和ABB从每笼的样本。商店的样品在-20℃，直到用于可溶性蛋白浓度进行分析。 </li><li>混合20毫克或者花粉或beebread与1000微升的0.1M磷酸缓冲液（PBS）中。 </li><li> VORTEX持续10秒和离心机将混合物在571.2×g离心1分钟。 </li><li>除去上清液并将其放置在96孔平底的EIA / RIA聚苯乙烯板的孔中的10微升样品。复制每个样品的3口井中。 </li><li>从通过将20微升毛细管（已加热并拉至针尖点）插入邻近附着的翅膀的点胸廓的右侧部分从每笼收集蜜蜂绘制血淋巴。收集其他血淋巴，如果需要的话，通过将相同的管进入腹腔背甲之间的膜。 </li><li>加入1μl血淋巴至9微升0.1M的PBS中。储存在-20℃的血淋巴溶液直至分析可溶性蛋白。 </li><li>确定使用市售Bradford蛋白测定试剂盒总可溶性蛋白的浓度在花粉，beebread，和血淋巴样品。按照制造商的说明。 </li><li>建立的标准曲线来估计可溶性蛋白Concentration通过测定蛋白质吸光度与已知蛋白质浓度的牛血清白蛋白（BSA）的样品中。使用分光光度计测量蛋白质吸光度在595nm处。 </li></ol> 6.氨基酸分析<ol><li>从每一集落的梳细胞池个体样本来创建EBB和ABB的用于分析有代表性的样品。 </li><li>取50毫克，花粉或beebread样品称入自动进样器小瓶中，并加入1毫升蒸馏水的小瓶中，沿着用100μl的50毫微克/微升内标由-d 4中丙氨酸的溶液中，D 23 -lauric酸， 13 C 6葡萄糖和D 39 -arachidiac酸。 </li><li>盖上样品并超声5分钟。 </li><li>通过加入1毫升甲醇调节的HLB盒中，平衡加入1毫升蒸馏水，然后加入1毫升的beebread或花粉的样品。用1毫升5.0％甲醇/ H 2 O和埃卢洗仓德用1ml的80％的MeOH / H 2 O. </li><li>蒸发在氮气流中的样品至干燥。重构用50μl吡啶和100微升的N，O-双（三甲基硅基）三氟乙酰胺+三甲基氯硅烷（BSTFA + TMCS）的样品。 </li><li>帽和孵育所述样品在70℃下进行30分钟。 </li><li>使样品冷却，并转移到一个干净的自动进样器小瓶中。 </li><li>帽并将样品放入一个质量选择检测器接口到气相色谱仪来分析对挥发性化合物和有机酸的样品。分离糖和有机酸以下的TMS衍生使用一个柱（30米×0.25毫米内径）用1.0微米的膜厚度BSTFA + TMCS。 </li><li>设置列烘箱中于50℃2分钟，然后线性地增加温度至290℃以5℃/分钟。并保持7分钟。将GC注射器和GC / MS接口，以250℃，290℃，分别。 <ol><li>使用氦气作为载体在FLO为1.0毫升/分钟瓦特率。设置该MS源温度至230℃。 </li></ol></li><li>调整和每天用全氟三丁胺（PFTBA）校准质谱仪。使用在全扫描（35-700 AMU）正离子模式下的1微升注射PFTBA，以获得对所述在场数据和氨基酸浓度。 </li></ol>

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The authors have nothing to disclose.

使用上述的方法，我们发现，通过AHB所作的beebread被消耗在更大量由两个AHB和EHB。虽然EHB和AHB类似消费量的每种类型beebread的，AHB有较高的血淋巴中的蛋白质滴度。根据我们的方法的调查结果是相似的过去的报告，其中血淋巴蛋白水平在AHB比在EHB虽然都饲喂相同日粮26更高。通过测量EBB和ABB其中被消耗以不同的速率由两个EHB和AHB的消耗，它被确定在每个亚种血淋巴蛋白浓度无法通过增加食物消耗上升。似乎有一个高原在护士蜂年龄工人血淋巴蛋白浓度以及为高原设定点是AHB比EHB高。 有用于建立菌落beebread生产，这将优化花粉存储的速率几个重要的条件。首先，将共lonies需要开放育雏帧。没有开放的育雏饲料，工人不会收集花粉多。其次，殖民地必须queenless因此不需要额外的育雏产生。育雏饲养需要大量的花粉，只有多余的花粉被存储。在建立了全民教育的小菌落，会有一点花粉被存储为beebread如果育雏领域不断扩大使殖民地必须queenless。最后，对于要作出beebread，花粉必须收集作为corbicular负荷和存储在梳细胞。如果花粉被收集在花粉陷阱，它必须被研磨成细粉它呈现给蜜蜂，这样他们可以收集它作为corbicular载荷之前。 的方法来测量beebread的消耗产生定性而不是绝对的估计。这是数只消费时，细胞完全排空的蜂粮。总蜂粮消费的一个更准确的估计可能会被删除蜂BR获得EAD从细胞并使其成能之前和研究期间之后被测量肉饼。但是，我们要保持蜂粮的细胞，使蜜蜂可以在上面饲料，因为他们会在一个殖民地，也许继续在研究期间处理它。前和研究后的重量梳子部的差异不是作为消费的估计中使用，因为体重可能增加，因为蜜蜂把稀释蜂蜜在某些细胞饲喂给他们。 工人们可能还增加了一些稀释的蜂蜜蜜蜂面包。由于这些原因，含有前后馈送周期的蜂面包大致相等量的细胞进行计数，并产生一个质量测量。尽管如此，仍有在空的ABB细胞后第11天用EBB相比计数的数量的两种类型beebread之间的显着差异。 当确定存储花粉变得beebread可迪fficult因为蜜蜂不断增加花粉细胞。用于生产beebread的殖民地，建立开放式育雏的框架，以便将蜜蜂采集花粉。然而，菌落queenless所以有成立菌落后幼虫喂养只有约9天。在3周时间内的剩余时菌落呈全民教育，所收集的蜜蜂被存储并转换为beebread花粉。保持在梳子细胞所存储的花粉为它在笼子里饲养时对蜜蜂额外11天还可继续花粉的处理，beebread。花粉的蜜蜂面包的转换需要大约7天8。送入EHB和AHB的beebread有较低的pH值和降低蛋白质浓度的花粉美联储相比。转换beebread后改变花粉类似的调查结果已报告了其他7,10,27。我们的研究结果与以往不同的报告然而，在有浓度差异Øbeebread和花粉F之间的某些氨基酸。在蛋白质和氨基酸的浓度的变化可能是由于蛋白水解酶的活性，其中的源可能是蜜蜂本身或设立于beebread 7,8,28,29的微生物群落。 用于测量蛋白质浓度的方法是类似于先前描述以确定对非洲化饮食蛋白质和欧洲蜜蜂26的影响。作为该方法的延伸，我们能够估计可溶性蛋白在花粉和beebread。这些方法产生类似的结果之前的报道7,10,27。我们的研究结果提供了额外的证据表明，AHB更有效地吸收食物的蛋白质比EHB，并认为这可能是AHB的大部分地区的生态优势，其中已移居，并成为建立30-32的关键因素。

营养在蜂蜜蜂群的健康和活力，并在其设立的人口基础性作用。从食物中营养物质提供能量和必要的育雏饲养，体温调节的生化成分，觅食和免疫反应。对于蜜蜂的殖民地，营养生长所需的菌落群和维护他们的健康来自花蜜和花粉。花蜜提供碳水化合物和花粉耗材的剩余饮食要求，如蛋白质，脂质，维生素和矿物质1。 蜜蜂亚种可以根据营养菌落级参数，如工人长寿，育雏饲养和社会免疫力2-6的机制不同。这些差异可能与食物如何，特别是花粉由菌落处理并消化在个人。花粉被存储在梳细胞和通过微生物介导的乳酸发酵是化学变化<sup&gt; 7-10。发酵花粉被称为beebread。有可能是亚种间在他们从他们收集的花粉和存储获得营养物质的能力基因型差异，这可能会影响菌落生长和生存。食品加工和营养收购基因型效应已被记录在其他生物造成了一些个人同时消耗同样的食物11-14获得比别人更多的营养和热量。 在这里，我们描述了用于比较由蜜蜂亚种不同的制作成分和beebread的食用方法。方法测量也被描述所得血淋巴蛋白滴度工蜂。在beebread的营养成分先前的研究已经做完了欧洲蜜蜂（EHB）10,15,16。然而，可能会有在beebread由不同亚种蜂制成甚至当它们以相同的花粉的差异。 EHB和AHB进行比较，因为这些潜艇pecies有可能与食品加工和营养采集17独特行为和生理差异。其中最显着的区别在于AHB收集和消耗比EHB更多的花粉，似乎更容易转化为育雏18。 AHB殖民地有更高的蜂拥率比EHB和潜逃当食物资源变得有限19-23。潜逃是罕见的EHB。 AHB也有较高的代谢率比EHB 24。为EHB和AHB之间的菌落水平的差异的营养基础可能与花粉采集的速率，并以它的营养成分（ 例如 ，氨基酸和蛋白质）之后它被转换为beebread。 Beebread消耗和由此产生的蛋白质收购也可能在EHB和AHB之间的群体水平的差异的作用。使用开发的方法，EHB和AHB从相同花粉源所做beebread。该beebread然后反馈到EAC的蜜蜂ħ亚种和可能确定是否蜜蜂采集蛋白从beebread在独特的，以它们的亚种或往beebread源的方式进行。

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methods for comparing nutrients in beebread made by africanized and european honey bees and the effects on hemolymph protein titers

蜜蜂获得蜂巢的beebread花粉，他们收集和储存的营养物质。我们开发的方法来控制花粉源蜜蜂采集并转换通过将菌落在一个专门建造的封闭飞行区域beebread。方法被开发来分析花粉和beebread的蛋白质和氨基酸组成。我们还描述了如何在beebread的消耗量，测定和方法中使用的出苗后喂食beebread为4,7和11天后，以确定成年工蜂血淋巴蛋白滴度。方法被应用，以确定是否基因型影响花粉转化为beebread和蜜蜂消耗，并从中获得的蛋白质的速度。两个亚种（欧洲和非洲化蜜蜂;分别EHB和AHB）被提供有相同的花粉源。基于所开​​发的方法，beebread由两个亚种制成具有较低的蛋白质浓度和pH值下比花粉。在一般情况下，氨基酸CONcentrations在beebread由两种或EHB AHB取得相近，且发生在较高水平beebread比花粉。无论AHB和EHB消耗显著更由AHB比EHB的beebread的。虽然EHB和AHB类似消费量的每种类型beebread的，血淋巴蛋白浓度AHB均高于EHB高。在AHB和EHB之间的蛋白质收购的差异可能反映了相关的地理区域，其中每个亚种进化的环境适应性。这些差异可能有助于成功建立，因为对育雏饲养和菌落生长的影响AHB人口的新世界。

<html> Beebread贮存在-80℃下不到一个月的pH和蛋白质浓度被分析之前，而对于前氨基酸分析约4个月。 Beebread从在pH和蛋白质浓度（ 图1）的花粉不同。 beebread的pH比花粉下因为是蛋白质浓度。既EHB和AHB消耗更多的ABB比EBB（ 图2）。 可溶性蛋白在AHB的血淋巴水平显著高于EHB无论beebread它们所消耗的类型（ 图3）的。发生这些差异血淋巴中的蛋白质水平，即使EHB和AHB类似消费量的每种类型beebread的。蜜蜂在采样时的年龄显著影响可溶性蛋白的浓度在血淋巴。有一天-7或11，没有差异相比，蛋白质浓度显著低于一天4蜜蜂。 _content“&gt;的10个氨基酸，这对蜜蜂必不可少，但所有的组氨酸中的花粉进行检测。在大多数情况下，在beebread测定氨基酸的浓度比在花粉更高（ 图4）。例如，浓度亮氨酸和苏氨酸是在beebread高出约60％的花粉进行比较，缬氨酸浓度分别高出约25％。丙氨酸，天冬氨酸，谷氨酰胺和甲硫氨酸水平也均在beebread高于花粉。氨基酸浓度没有之间有很大的不同无论使用哪种EBB或花粉相比ABB和EBB除苯丙氨酸和半胱氨酸。苯丙氨酸水平高一倍左右的ABB。半胱氨酸浓度较低的EBB与ABB或花粉相比，色氨酸是存在于高浓度的唯一氨基酸花粉比EBB或脯氨酸的花粉和ABB的ABB。浓度比在EBB高。 图1：pH值（A）和可溶性蛋白质含量（B）中的花粉和欧洲（EHB）或非洲化（AHB）蜜蜂所做的beebread的比较。花粉的pH比beebread如通过方差分析确定显著更高（F 2,12 = 3725，P &lt;0.0001），接着是Tukeys W-多重比较检验。在花粉蛋白质浓度较beebread由EHB（EBB）或AHB（ABB）制成显著更高（F 2,27 = 16.49; P &lt;0.0001）。手段其次是在同一封信都没有显著的不同在0.05的水平。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig2highres.jpg" width="600" /> 图2：CEL的平均百分比LS含有beebread即完全由笼蜜蜂消耗在11天的间隔。该beebread是由使 ​​用相同的花粉源无论是欧洲（EHB）或非洲化（AHB）蜜蜂。装置从每个治疗5笼估计;那些具有相同的字母不显著的不同在0.05的水平由单向方差分析（F 3,16 = 7.3，P = 0.003）和杜克的读写测试来确定。该图已被修改为25。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig3highres.jpg" width="600" /> 图3：蛋白血淋巴欧洲（EHB）或非洲化的平均浓度（AHB）蜜蜂喂beebread 4，7，11天由欧洲（EBB）或非洲化（ABB）蜜蜂的重复测量分析。差异表明中4 TREA显著差异tment组（F = 3,20 19.7，P &lt;0.001）。在AHB美联储ABB可溶性蛋白的水平显著高于EHB美联储ABB（P = 0.008）或EBB（P = 0.018）。蜜蜂在采样时的年龄显著影响可溶性蛋白的浓度在血淋巴。有一天-7（P &lt;0.0001）或11（P = 0.001）相比，水平显著低于一天4蜜蜂。 7日和day11蜜蜂没有差异（P = 0.149）。这个数字已经被修改25。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52448/52448fig4highres.jpg" width="600" /> 图4：氨基酸浓度（每克花粉微克或beebread）的花粉，或从它的EBB 所作的beebread是beebread由欧洲蜜蜂和ABB是由非洲化蜜蜂。色氨酸，半胱氨酸，苯丙氨酸和脯氨酸分别绘制在预先清楚起见senting的金额。这个数字已经被修改25。

Watch this Scientific Journal Video about Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers at JoVE.com

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

methods-for-comparing-nutrients-beebread-made-africanized-european

Research

JoVE Journal

Biology

10.5K 次观看.  USDA-ARS. Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies. 

视频: Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

通过功能磁共振成像监测针灸对人脑的影响

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

科研

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

蜜蜂致死农药暴露对蜂群水平的影响

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...