作者要感谢他们的感激之情美国陆军环境质量6.1基础研究发展计划，外科研究的美国陆军研究所和NSRDEC的持续产品改进计划，并远征大本营TECD为这项工作提供资金。我们感谢亚当Driks（Loyola大学医学中心），在图6B中所示的显微照片。

1，便携式化学消毒（PCS） <ol><li>设备。在PCS是一个创新的设备为便携式，能独立使用点的医用灭菌。就此而言，商业鹈鹕硬质塑料手提箱被饰以特殊设计特点，以适应灭菌（ 图1）。 </li><li>设备设计。 a）一个广口的反应容器接收到的干化学试剂和水; B）安装在墙上的情况下两个止回阀缓解压力1磅; c）一种过滤入口阀允许空气被泵在冲洗腔室; D）循环管分配空气通过室灭菌后;五）一次性干式洗涤器（活性炭）栽种在出口止回阀装置去除残留物，并确保用户和环境的健康和安全;和f）在该个案的基础高跷容纳手术器械托盘或其它穿孔托盘，并在最大化气流潮红。 </li><li>操作。将只在一个水平面上，并打开盖子。将手术托盘含清洁，非无菌器械和包裹在里面的情况高跷蓝色釜纸。混合干粉和水（ 例如 ，93克亚氯酸钠，63个亚硫酸钠，加入25克硫酸氢钠抗坏血酸，和300毫升水-其他排列是可能的），在广口反应器启动化学反应，然后关闭并锁定盖子。下2分钟后，将反应产生丰富的二氧化氯的消毒剂，热量和湿度。在25分钟内，连接一个电池操作或手动力空气泵的入口阀和流动空气进入腔室为约。 5分钟（ 图2）。 </li><li>周期结束与再利用。打开机箱，取出无菌器械的手术托盘。处理反应器（水和良性的化学盐）的。可用于与手术器械的另一个纸盘立即再次使用PCS和一套新的干粉和水。 </li><li>验证灭菌与生物指标。将市售B / T含孢子浸在任G的纸张（10〜5个/单位）确定生物指标嗜热脂肪 （用于湿热）或B。 atrophaeus（用于环氧乙烷气体灭菌），用于消毒周期的情况下的内部。在二氧化氯灭菌周期完成后，删除并激活指标，然后孵化指标24-48小时，以验证灭菌。 </li><li>验证与灭菌孢子悬浮液。放置G的水悬浮液嗜热脂肪芽孢（〜10 5 CFU /毫升）的只为暴露于二氧化氯消毒周期内。恢复G。嗜热脂肪芽孢暴露于抗生素测定培养基中的二氧化氯处理，用1％可溶性淀粉8（无恢复指示不育）。检查的汤治疗refractility相衬显微镜ð孢子（孢子二氧化氯灭活保留相亮字14）。 </li><li>验证硬表面的消毒。接种带有G。嗜热脂肪芽孢水悬浮液（〜10 5）由玻璃或金属制成的坚硬的，无孔的表面。将材料接种到只为与二氧化氯消毒周期治疗。采样处理的表面与市售Difco公司HY-拭子检查，并获得无增长证实不育。 </li></ol> 2“D-FENS” <ol><li>设备。 “D-FENS”是一个可折叠的手持瓶装有一个手动喷淋触发装置。柔性塑料瓶具有裆部底部充满时，站立，并且耐化学腐蚀的塑料能提供多种重新使用每喷雾器（ 图3）。 </li><li>生成水CLO 2 S辨率。 D-FENS使用的干重1-10克总数量剂，以产生高达800毫升50 - 500ppm的二氧化氯溶液（ 例如 4.7克的亚氯酸钠和1.6克的亚硫酸钠，和1.3克抗坏血酸钠，以置换可能的）。使用“动力学控制，”一种新型的2 -步混合方法，其包括：我前浓度 -溶解在小体积的所有试剂，以及ii反应后的稀释 -稀释该溶液至其最终的工作体积，以生成含水氯氧化钛溶液在喷雾瓶在2-9分钟。该消毒液被喷成细雾或喷雾消毒或净化目的的表面。在D-FENS二氧化氯溶液至少8小时轮班和任何剩余的溶液可以清空向下下沉或地面排水在变速清除生物膜的末端保持稳定。 </li><li>微生物学验证 - 接种多孔表面。准备贝尔德 - 帕克琼脂（BPA）的含蛋黄培养皿碲酸盐（EYT）和酵母提取物（YE），接种琼脂表面用0.1毫升〜10 6 CFU / ml的悬浮液对金黄色葡萄球菌 3株鸡尾酒（ 金黄色葡萄球菌 A-100能产生肠毒素A， 金黄色葡萄球菌 ATCC 14458，产生肠毒素B，和金黄色葡萄球菌 993，产生肠毒素D）7。S.金黄色葡萄球菌被选定为目标生物体，因为它产生鲜明的显黑色的菌落，如果没有失活。 </li><li>微生物检验 - 测试多孔表面。使用D-FENS喷雾瓶含有二氧化氯液剂，喷洒消毒液在多孔琼脂表面。使用一致的，稳定的力量来分配每个喷雾触发脉冲的解决方案大致相等的体积。旋转板连续脉冲之间90º到应用琼脂表面均匀覆盖。您也可以使用这种技术用玻璃曲棍球棒和应用光压来传播绿泥石NE氧化钛溶液在琼脂表面进行机械磨损（相当于擦拭或洗涤-参见图4）。 </li><li>微生物学验证 - 硬表面。接种灭菌定制的不锈钢（304型）的优惠券（4“×4”，总表面积10.16厘米2）用0.2ml体积的细菌细胞的含水悬浮液（ 如 ， 金黄色葡萄球菌，大肠杆菌，或李斯特菌 ）和接种扩散均匀地券面。风干券30分钟，在室温下，在层流罩。拿起接种券用无菌镊子和浸泡在20ml含二氧化氯在100ml抹胸袋试品溶液。后的0.5〜5分钟的接触时间，淬灭消毒液，加入少量的亚硫酸钠固体，然后在一个抹胸咀嚼溶液2分钟。除去袋，并在层流罩撤回上清液并连续稀释solutioÑ​​到预先制作的琼脂平板上，然后培养24小时列举幸存者。 </li></ol> 3，只为新鲜水果和蔬菜使用定点接种的方法，其中高水平的病原体被点样到番茄楔子的外表面的减少只处理以杀灭有害食源性致病菌（ 大肠杆菌和单增李斯特菌 ）在新鲜的农产品的能力进行了测试。 <ol><li>接种。接种25克样品番茄楔子与任何10 5 CFU / g的L。外表面单核细胞增生 OSY-8578或106 CFU / g的E。杆菌 ATCC 11229，然后空气干燥在无菌生物罩15分钟。 </li><li> PCS的治疗。接种物干燥后，将番茄楔子（戴无菌手套）的二氧化氯浓度和暴露时间不同条件下的PCS和测试。在某些情况下，将孢子生物指标G的一个嗜热脂肪ND B。 atrophaeus在PCS内陪番茄楔子和验证的杀菌处理（ 图5）。 </li><li>微生物采油。后在PCS治疗，将番茄楔子在一个抹胸袋用50毫升含水磷酸盐缓冲液（pH 7）中，然后咀嚼2分钟用抹胸搅拌机。 </li><li>枚举。分配所述塑炼溶液作为连续10倍稀释到琼脂平板胰蛋白酶大豆琼脂酵母提取物（TSAYE）和营养琼脂（NA）为L。 monocyotgenes和E。大肠杆菌 ，分别和传播与玻璃曲棍球棒，盖，板孵育在T = 35℃24-48小时。用菌落计数器，以确认微生物灭活列举的幸存者。 </li><li>钝化多酚氧化酶（“褐变”）酵素。将未接种苹果切片在PCS内独立的培养皿中，并暴露于二氧化碳（ 图5）氯。治疗后，取出培养皿和揭露日Ë苹果切片在周围环境中。肉眼观察表明没有褐变为长达1周后处理。 </li></ol> 4，“D-FEND ALL” <ol><li>生成水CLO 2 S辨率。 D-FEND全部采用少量干粉（绿泥石和SAMIA）在水中产生0.5-3.0分钟二氧化氯溶液。例如，混合0.8-3.3克试剂在15-1,200毫升的水溶液，以产生二氧化氯溶液。 </li><li>微生物学验证 - 纺织品。接种纺织品样品的无菌条带的炭疽杆菌斯特恩或解淀粉芽孢杆菌的孢子水悬浮液，并让纺织带风干在层流通风橱。拾取带用无菌镊子和浸泡在20毫升的二氧化氯溶液在100ml抹胸袋。在10分钟，淬火过程中无需加入少量亚硫酸钠固体影响孢子，然后磨烂纺织带和解决方案用于在胸衣2分钟。除去袋，并在层流罩串联稀溶液涂布预先制作的琼脂板上，然后孵育24小时，并列举验证去污。 </li></ol>

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我们没有进一步披露。

这个基础研发已通过合作与学术界，其他政府机构和业内人士认为，导致新的商业化，环保（“绿色”）技术提出了新的研究和技术方向。二氧化氯是经20年来的美国国家卫生基金会更安全，更快捷，更环保的消毒比传统治疗方法的第一种方法。该PCS，D-沼泽，和D-FEND ALL原型被验证为在实验室环境实验室规模的原型，和结果的范围赋予这些系统具有通用性的巨大广度来灵活适应个别用户的需求。?...

在PCS技术所得款项在没有商业设备以前存在和产生的消毒剂二氧化氯（CLO 2），有一个证明有能力杀死植物病原菌对新鲜农产品3,6,9-13,15或净化的细菌孢子。6， 14,15,17的PCS已验证实验室专门以实现杀菌对嗜热脂肪土芽孢杆菌属 （GS）的孢子（见相关参考8）和孢子生物指标G的活文化和嗜热脂肪芽孢杆菌atrophaeus（BA）6,15,16。在PCS也已适应了不太严格的条件下工作，以确保食品安全失活的植物病原体李斯特菌和大肠杆菌的新鲜农产品，如整个西红柿，并延长保质期的鲜切产品，例如，由INACtivating在苹果切片6,15的多酚氧化酶褐变。以产生二氧化氯，在PCS采用新颖的化学效应通过在近中性pH值的氧化还原反应，前进，从而消除了使用酸和运输的固有困难，存储，处理，并在远前方军事布置酸性废物部署1,2,4,17。除了军队，只也可用于国土安全/国防;在自然灾害（超级风暴桑迪，海啸，卡特里娜飓风）变为不可接入电源，饮用水，以及清除废物;现场由紧急第一反应;而在社区医院或在停电（停电和棕色奏）的学校。 在D isinfectant -喷涂机适用于F洪灾和EN vironmentally友好S anitation（D-FENS）也使用化学效应（3化学成分）和一个2步混合法（ <em&gt;我。预浓缩，然后为ii。反应后稀释），生成水二氧化氯，主要是在一个可折叠的喷雾瓶用于净化的军队物资，食品处理设备和现场给料设备在陆军野战厨房和卫生中心和海军表面厨房，医疗单位，淋浴和厕所随地大量部署 ​​的人员共同存在于接近5,6。验证测试表明，D-FENS消除致病菌为金黄色葡萄球菌 ，一种常见的食源性致病菌，对多孔表面14。 “D-FEND ALL”（D isinfectant为EN vironmentally友好Ðecontamination，所有用途）提供了一个更简单的（2化学成分），使用更方便（1步混合）替代具有无与伦比的多功能性生产水性二氧化氯净化细菌纺织品孢子，用于表面消毒，以促进卫生设施tation和卫生，以及提高水的质量和安全，对要求快速生产大量使用少量起始原料的应用在新型灰回收利用技术，旨在生产清洁，饮用水的稀二氧化氯的应用解决方案，特别是优势远征基地营2。 多种机制存在按照联邦技术转移法案，以促进联邦技术转移到非联邦实体，以此来鼓励技术为国家的重大利益的发展和商业化。因此，他们的许多军事和民用用途新兴的潜力，PCS，D-沼泽，和D-FEND所有的技术已经通过专利许可协议和商业评估许可证和专利转让给业界的商业化。 D-FENS（的缓，控释版本，称为＆＃8220，D-FENS精简版“）是技术转移到商业行业纳入包装材料，延长新鲜浆果的保质期，和PCS也已技转给学术界和其他政府机构的对比测试与其他技术，对于与新鲜农产品商品食品安全的研究，并加强本科科学教育。在PCS和化学的技术转移导致批准用于生物罩灭菌用改进的时间，成本和环保相对于传统的甲醛处理的商业产品。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chlorite</td>
			<td>Sigma-Aldrich</td>
			<td>244155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>239312</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D-16</td>
			<td></td>
		</tr>
		<tr>
			<td>BT Sure biological indicator (steam)</td>
			<td>Thermo Fisher Sci</td>
			<td>AY759X3</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Test (EtO)</td>
			<td>SGM Biotech Inc</td>
			<td>EZG/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Hy-check</td>
			<td>Becton-Dickinson/ Difco</td>
			<td>290002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Agar</td>
			<td>Difco</td>
			<td>213000</td>
			<td></td>
		</tr>
		<tr>
			<td>Baird-Parker Agar</td>
			<td>Difco</td>
			<td>276840</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Yolk-Tellurite</td>
			<td>Difco</td>
			<td>277910</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Yeast extract</td>
			<td>Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Difco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Tryptone</td>
			<td>Difco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Soluble starch</td>
			<td>Difco</td>
			<td>0178-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Lemco Beef Extract</td>
			<td>Oxoid</td>
			<td>L29</td>
			<td></td>
		</tr>
		<tr>
			<td>Masticator - Classic</td>
			<td>IUL Instruments</td>
			<td>Cat. No. 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher bags</td>
			<td>Seward</td>
			<td>Stomacher &#8216;400&#8217; bags</td>
			<td></td>
		</tr>
	</tbody>
</table>

the portable chemical sterilizer (pcs), d-fens, and d-fend all: novel chlorine dioxide decontamination technologies for the military

有一个说陆军需要一种便携式现场，非蒸汽灭菌器技术，可用于正向手术团队，牙科公司，兽医服务保障分队，战斗支援医院和地区医疗实验室手术器械消毒和灭菌病理标本前处理在手术室，急诊区，重症监护病房。小说“清洁和绿色”二氧化氯技术的以下合奏是通用，灵活，以适应和满足了一些去污6,15关键军事需求的。具体来说，在P ortableçhemical S terilizer（PCS）的发明，以应付紧急战场需求和关闭的关键能力差距的能源独立，轻巧便携，快速机动和坚固耐用的高强度推进部署3。由于在手术sterilizat一个革命性的技术突破使用点的离子技术，只为一个现代的现场高压灭菌器，它依赖于现场，在意志的产生二氧化氯代替蒸汽。两（2）只单位消毒4手术托盘中1小时，这是（因为它的繁琐的大小，尺寸庞大，重量和绰号“贝莎”在部署）的吞吐量相当于一个大型高压蒸汽。不过，只经营使用较少的100％电量（0与9千瓦）和98％的水少（10对640盎司），显著由95％降低体重（20主场迎战450磅，一个4人电梯）和由96％立方体（2.1对比60.2英尺3），并实际上消除了在维修和维护可靠的操作，提升和输送，以及所需的蒸汽高压灭菌器的电功率的向前部署了困难的挑战。

在易于操作只被设计在30分钟的治疗细菌灭活孢子悬液或细菌孢子生物指标涉及控制生产二氧化氯的独特的化学效应，实现无菌。具体来说，微生物验证研究证实，该只由含有灭活或者G的孢子（10 5孢子/ ml）生物指标达到不育嗜热脂肪或B。 atrophaeus，这是为了通过蒸汽高压灭菌器（湿热），或环氧乙烷气体，以分别指示灭菌。该电脑还灭菌G的悬浮液嗜热脂肪<...

Watch this Scientific Journal Video about The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military at JoVE.com

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

在P ortableçhemical S terilizer（PCS）是一种革命性的，能独立，几乎无水的杀菌技术，军队医疗单位。在PCS产生二氧化氯的干燥试剂与水现场混合，在意志，并在点的使用（POU）在一个塑料手提箱。在D isinfectant -喷涂机适用于F洪灾和EN vironmentally友好S anitation（D-FENS）和D isinfectant为EN vironmentally友好Ðecontamination，多用途 （D-FEND ALL）的水溶液产生二氧化氯在一个可折叠的喷雾瓶和其他潜在的实施方式。这些多功能去污技术杀死微生物在无数不同两用应用军用和民用消费者的青睐。

便携式化学消毒（PCS），D-沼泽，和D-FEND ALL：新型二氧化氯去污技术用于军事

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

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Research

JoVE Journal

Bioengineering

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

14.4K 次观看.  United States Army-Natick Soldier RD&E Center, Warfighter Directorate. 在P ortableçhemical S terilizer（PCS）是一种革命性的，能独立，几乎无水的杀菌技术，军队医疗单位。在PCS产生二氧化氯的干燥试剂与水现场混合，在意志，并在点的使用（POU）在一个塑料手提箱。在D isinfectant -喷涂机适用于F洪灾和EN vironmentally友好S anitation（D-FENS）和D isinfectant为EN vironmentally友好Ðecontamination，多用途

视频: The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

科研

生物工程

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.