作者感谢佛兰德研究基金会对玛尔塔卡拉塔余德 (FWO 博士后 fellowship-12N2815N) 的财政支持。艾玛-加西亚萨纳布里亚是一个博士后研究员支持的富兰德创新和企业家精神 (Agentschap 与客厅里 Innovatie 门 Wetenschap créative, 内陆水运)。

1. 菌株和文化条件
注: 综合口腔群落由口腔微生物群中常见的菌株组成3。
<ol><li>从美国类型文化收集 (ATCC) 获得以下菌株: Aggregatibacter actinomycetemcomitans (ATCC 43718),梭杆菌 nucleatum (ATCC 10953),牙龈菌(ATCC33277),普氏菌中间体(ATCC 25611), 变形链球菌 (ATCC 25175),链球菌远缘(ATCC 33478),放线菌对内氏(ATCC 51655), 仙人掌链球菌 (ATCC 49818),放线菌放线菌(ATCC 15987) 和链球菌缓症(ATCC 49456)。</li>
	<li>获得Veillonella parvula从莱布尼茨学院 DSMZ-德国收集的微生物和细胞培养 (DSM 2007),链球菌血统从 BCCM/LMG 细菌收集 (LMG 14657) 和使用链球菌唾液菌株 TOVE-R9, 口腔链球菌 10。</li>
</ol>2. 开发多种群社区代表口腔微生物群
注: 生成合成群落, 模拟口腔细菌对体外肠道上皮的潜在黏附能力。在血液琼脂板上生长细菌补充与5微克/毫升血红, 1 微克/毫升甲萘醌, 5% 无菌 defibrinated 马血, 如埃尔南德斯-加西亚萨纳布里亚等。(2017)11. 简言之:
<ol><li>将100毫克的血红溶解在2毫升的1米氢氧化钠中, 加入100毫升的蒸馏蒸压水。储存在一个黑暗的容器里。在添加到培养基前, 通过0.22 µm 过滤器进行消毒。</li>
	<li>在20毫升96% 乙醇中溶解100毫克的甲萘醌。在添加到培养基前, 通过0.22 µm 过滤器进行消毒。</li>
	<li>根据制造商的说明和高压釜, 准备1升的血液琼脂培养基 (见材料表)。在添加马血和补充剂之前, 让冷却下来。混合并倒入盘子。</li>
	<li>准备改良的脑心输液 (BHI) 汤, 如前所报告的12。在热处理后加入5微克/毫升的血红和1微克/毫升甲萘醌。</li>
	<li>在亨盖特管中分配9毫升的培养基, 并与橡胶塞子和铝盖紧密结合。用 N2/CO2 (90%/10%) 冲洗管子。</li>
	<li>用注射器检索1毫升厌氧培养基, 并将其放在1.5 毫升的离心管中。选取每个细菌种类的单一菌落, 在培养基中并用重悬, 然后转移回厌氧改性 BHI。孵化48小时在37摄氏度, 除了S. 唾液, 必须孵化24小时。</li>
	<li>
如果需要, 在组装合成群落之前确认文化的纯度。简言之:
	<ol>
<li>从液体培养中提取 DNA, 如加西亚萨纳布里亚等. (2010)13和放大 16S rRNA 基因使用引物 27F (AGAGTTTGATYMTGGCTCAG) 和 1492R (TACGGYTACCTTGTTACGACT) 和以下节目: 5 分钟在95°c, 30 个周期95°c 为1分钟, 55 °c 为1分钟和72°c 为1.5 分钟, 然后最后的延长步5分钟, 在72摄氏度。</li>
		<li>按照制造商的说明, 用商业套件 (见材料表) 净化 PCR 产品。</li>
		<li>在 10 ul 总容积中, 执行纯度为0.5 的染料 (见材料表)、3.2 pmol M13f 底漆 (CGCCAGGGTTTTCCCAGTCACGAC)、2.0 ul 5x 序缓冲器和 20 ng 模板的纯化产品的排序反应, 使用商业测序系统用套件 (参见材料表)。 注意: 我们在商业上执行了这个程序。</li>
		<li>在所有序列 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) 上执行一次爆破搜索, 以确定最接近已知的分类, 并使用 RDP 分类器在线工具 (http://rdp.cme.msu.edu/) 和新浪与原始应变序列进行比较。对齐服务 (https://www.arb-silva.de/aligner/)。</li>
	</ol>
</li>
	<li>用流式细胞仪和 SYBR 绿/碘碘染色等方法在孵化末期测量细胞数, 加西亚萨纳布里亚等。(2017)11. 利用改良的 BHI 将培养物稀释为 105细胞毫升-1。</li>
	<li>加入1毫升的缓症到75毫升的厌氧 BHI 和孵化直到固定相 (6 小时)。以下, 收集0.75 毫升的每稀释应变和混合在厌氧条件下。</li>
	<li>一旦所有的文化混合, 采取1毫升的缩影, 并添加到75毫升的 BHI。孵化合成社区在 10% CO2, 10% H2, 80% N2为 48 H 在37°c 和 200 rpm (参见材料表)。</li>
	<li>测量细胞密度如2.8。在向细胞模型展示社区之前。使用斯洛姆卡等表1中的引物和退火温度, 可对群落组成进行定量实时 PCR 评价。(2017)10. 此外, 使用 16S rRNA 基因扩增子测序来提供有关最初成员13、14的信息。对于两种身份确认协议, 使用 cDNA 作为模板, 表明细菌积极转录蛋白, 并使用 DNA 作为模板, 以显示存在/没有的菌株。</li>
</ol>3. 一般细胞培养做法
注: 对于细胞培养的一般方面, 作者指的是硕士和史黛西 (2007)15。获得从欧洲收集的经鉴定的细胞培养 (Caco-2 ECACC 86010202 和 HT29-MTX-E12 ECACC 12040401, 英国公共卫生) 使用的细胞线。除非另有规定, 细胞培养试剂可以购买 (见材料表)。
<ol><li>
Caco-2 和 HT29-MTX 细胞系的维护与传代 注: Caco-2 和 HT29-MTX 细胞通常生长在25厘米2的组织培养烧瓶中, 含有补充的 DMEM 培养基 (表 1)。当60–70% 汇合到达时, 细胞 trypsinized。<ol>
<li>从烧瓶中取出细胞培养基。清洗两次与5毫升 PBS 没有 Ca ++/毫克+ +。</li>
		<li>加入2毫升的0.5 毫克/升溶液的胰蛋白酶和0.22 克/l-乙烯二胺四乙酸四酸 (EDTA)。通过轻轻地移动烧瓶来分发胰蛋白酶溶液。去掉1.5 毫升的胰蛋白酶溶液, 在37摄氏度孵化出10分钟的烧瓶。</li>
		<li>在显微镜下检查细胞是否与瓶子表面分离。如果需要, 孵育额外的5分钟。</li>
		<li>添加5毫升的补充细胞培养培养基, 以使胰蛋白酶钝化。吸管向上和向下, 以获得一个均匀的单细胞悬浮。</li>
		<li>立即, 采取50µL 整除细胞悬浮和混合与无菌0.4% 台盼蓝溶液在 1:1 (v/v) 比例为 Caco-2 细胞或1:5 伏/五为 HT29-MTX。混合吹打和负载到一个计数室 (见材料表)。</li>
		<li>在显微镜下计数可行的细胞 (白色明亮的细胞) (见制造商的说明)。</li>
		<li>种子在一个新的烧瓶密度为 4 x 105细胞/cm2和 1 x 104细胞/厘米2, 分别为 Caco-2 和 HT29-MTX。 注: (1) Caco-2 细胞分化并形成紧密连接, 一旦到达融合。在 trypsinization 之前避免过度融合是非常重要的。trypsinization 和/或细胞团簇后, 烧瓶细胞过度生长会导致细胞脱离。(2) HT29-MTX 细胞为粘液生成细胞, 代谢活性高16。这些特征可能导致细胞培养基的快速酸化, 特别是当细胞处于高密度时。HEPES 缓冲可以添加到细胞培养基 (表 1) 中, 以保持 pH 值在7和7.2 之间。如果 pH 值下降, 培养基可以在融合小于50% 时进行交换。但是, 如果融合 > 50%, 细胞培养必须分裂。</li>
	</ol>
</li>
</ol>4. 模拟肠道宿主-微生物界面的 Multicompartment 细胞模型的组装
注: 完成双腔井共培养 (直径24毫米, 孔径0.4 微米; 见材料表)。
<ol><li>添加1.5 毫升完整的细胞培养基到顶端的隔间和2毫升到基部。预孵化20–30分钟, 以获得均匀分布的细胞悬浮时, 播种。</li>
	<li>胰蛋白酶处理 Caco-2 和 HT29-MTX 的细胞培养, 如第3节所述。trypsinization 程序必须准确地遵循, 以实现两个细胞系的均匀分布, 在单个细胞悬浮, 没有团簇。</li>
	<li>按3.1.8 中所述计算可行细胞数。</li>
	<li>将细胞密度调整为 6.5 x 104细胞/cm2 , Caco-2/HT29-MTX 细胞的90/10 比例。</li>
	<li>融汇细胞悬浮液, 并将相应体积的 Caco-2 和 HT29-MTX 细胞添加到预填充的无菌容器中, 并辅以细胞培养培养基。将预孵化的介质从腔室的顶端侧取出。</li>
	<li>用吹打轻轻地混合细胞悬浮液, 并将1.5 毫升转移到腔体插入物的顶端隔间。播种过程需要恒定的均匀化细胞悬浮, 因为细胞倾向于沉淀物。根据用户的经验和工作速度, 在配药1–3井后必须混合细胞悬浮液。</li>
	<li>移动板块轻轻向后和向前, 然后从右向左向右 (5–10倍), 以获得均匀扩散的细胞在井中。转移到孵化器和重复的板块移动。</li>
	<li>保持 Caco-2/HT29-MTX 细胞的共培养15天, 刷新顶端和基底隔间每2天与补充 DMEM。在此之后, 细胞培养培养基可以改变为补充 DMEM 没有抗生素/防霉溶液。</li>
	<li>保持系统, 直到20–21天播种后, 每2天刷新培养基。</li>
	<li>
使用电阻系统 (见材料表), 按照制造商的指示, 测量上皮屏障的完整性。
	<ol>
<li>在开始测量之前, 确保志愿者设备全部充电 (> 24 小时)。</li>
		<li>从电源上拔下志愿者设备并盖上塑料蒸压袋。在袋子里打一个洞, 介绍电极并插入仪表上的输入端口。喷洒70% 乙醇/水 (v/v), 然后将志愿者设备引入流柜。</li>
		<li>要对电极进行消毒, 请在70% 乙醇/水 (v/v) 溶液中浸泡电极提示15分钟, 使空气干燥十五年代. 在预热细胞培养基中冲洗电极。</li>
		<li>将细胞从孵化器中取出, 并允许细胞在流动柜内进入室温。</li>
		<li>请确保仪表与充电器断开。将模式开关设置为欧姆, 然后打开电源开关。</li>
		<li>通过将电极浸泡在插入物中, 用较长的尖端从井中浸出, 测量细胞电阻。将电极保持在90°角到板插入。</li>
	</ol>
</li>
</ol>5.体外胃肠道转运条件下的细菌存活
注: 制备模拟消化液后, Minekus等协议。(2014)17、下面描述的修改。用超纯水制备所有的稀释。过滤-通过0.22 µm 过滤器消毒所有解决方案, 并在无菌条件下执行消化过程。
<ol><li>
口腔消化:
	<ol>
<li>在50毫升不育管中放置3毫升细菌群落, 与3毫升模拟唾液液混合, 含 (在毫摩尔 L-1): 氯化钾, 15.1;2PO4, 3.7;NaHCO3, 6.8;氯化镁2(H2O)6, 0.5, (NH4) CO3, 0.06;HCl, 1.1;CaCl2(H2O)2, 0.75。添加75毫升的α淀粉酶从人类唾液类型 IX A 和2克/升黏蛋白从猪胃 ii. 型。</li>
		<li>孵育混合物2分钟, 在37摄氏度和 100 rpm。收集2毫升的 DNA 提取和流式细胞仪定量 (S1) 样本。</li>
	</ol>
</li>
	<li>
胃消化:
	<ol>
<li>添加4毫升模拟胃液 (SGF), 含 (在毫摩尔 L-1): 氯化钾, 6.9;2PO4, 0.9;NaHCO3, 25;氯化钠, 47.2;氯化镁2(H2O)6, 0.1, (NH4) CO3, 0.5;HCl, 15.6;CaCl2(H2O)2, 0.075。此外, SGF 中含有2克/升的猪胃 ii. 型粘蛋白。</li>
		<li>如果需要, 测量 ph 值并调整为 ph 值3和1米 HCl。孵育在37°c 和100转每分钟。收集2毫升样品 (S2)。</li>
	</ol>
</li>
	<li>
小肠消化:
	<ol>
<li>添加4毫升模拟肠道液 (SIF), 含 (在毫摩尔 L-1): 氯化钾, 6.8;2PO4, 0.8;NaHCO3, 85;氯化钠, 38.4;氯化镁2(H2O)6, 0.33;HCl, 8.4;CaCl2(H2O)2, 0.3;消化管。</li>
		<li>如果需要, 调整到 pH 值7与1米氢氧化钠。孵育在37°c 和100转每分钟。收集2毫升样品 (S3)。</li>
	</ol>
</li>
</ol>6.体外胃肠道转运条件下的细菌定植能力
<ol><li>离心2毫升小肠消化, 10 分钟在 2650 x g。</li>
	<li>除去上清, 并将细菌颗粒与5毫升补充 DMEM, 无抗生素和防霉。</li>
	<li>使用流式细胞仪 (参见材料表) 染色并量化完整/损坏的单元格, 如加西亚萨纳布里亚等所述。(2017)11。</li>
	<li>在没有抗生素和防霉的情况下, 通过稀释补充 DMEM, 调节细胞密度为 105活细胞/毫升。</li>
	<li>去除 transwell 系统的顶端介质, 加入1.5 毫升的细菌悬浮液。共同养殖系统为 2 h 在一般细胞培养情况下 (37 °c, 95% 湿气, 5% CO2)。 注: 此时间可扩展到 48 h, 具体取决于所需的实验协议。共同培养可以维持在不同的孵化器, 而不是常规的细胞维护, 以避免污染。</li>
	<li>在孵化后测量志愿者值, 以评估4.11 中所描述的上皮屏障的完整性。</li>
	<li>完成潜伏期后, 取出顶端培养基, 收集0.5 毫升作进一步分析 (S4)。在制造商的指导下, 亚组的培养基可以用来评估乳酸脱氢酶 (LDH) 测定的细胞活力 (见材料表)。</li>
	<li>在 HBSS (NAC 缓冲器) 中加入0.5 毫升10毫米 n-乙酰半胱氨酸, 溶解 HT29-MTX 产生的粘液, 并恢复附着的细菌。孵育板材在37°c 为 1 h, 在鼓动下 (135 rpm, 看见材料表)。</li>
	<li>恢复离心管 (S5) 中的 NAC-HBSS, 并用1毫升 DPBS 清洗两次细胞。</li>
	<li>添加0.5 毫升0.5% 海卫 X-100 (瓦特/v) 在单层膜上, 通过吹打扰乱细胞, 恢复液体和漩涡1分钟. 速度对于避免用洗涤剂破坏细菌细胞是至关重要的。</li>
	<li>将细胞离心5分钟, 在 2650 x g处分离出上清 (S6) 和颗粒 (S7)。</li>
</ol>7. 样品分析
注: 分析收集的样品 (S1-S7), 以评估细菌的生存能力和黏附能力的肠道细胞, 经过模拟胃肠消化。
<ol><li>细菌生存能力: 通过样品 S1-S5 两次通过0.45 µm 和量化细菌细胞使用活/死细胞染色和流式细胞术, 如步骤 2.811所示。</li>
	<li>
粘附电位: 为 S1-S6 和条纹10µL 在血琼脂和改良 BHI 板上准备连续稀释 (-1 到-8)。在厌氧条件下孵育37摄氏度, 24–48的 S7。
	<ol>
<li>粘附细菌的分类鉴定:<ol>
<li>选择一个文化循环的个体殖民地, 并并用重悬在10µL 的核酸酶无水。收集4µL 的悬浮和使用它作为一个模板, PCR 扩增的 16S rRNA 基因使用引物和条件在2.7.1 描述。</li>
			<li>按照2.7.3 中的协议执行排序, 并使用2.7.4 中包含的过程验证序列。</li>
		</ol>
</li>
	</ol>
</li>
	<li>做进一步的下游分析, 如总 DNA 提取, qPCR 量化和/或 16S rRNA 扩增子测序, RNA 提取, 和 transcriptomic 分析的期望。 注: 在取样后立即进行流式细胞仪定量。作为对膜透细胞的一种控制, 使用了70摄氏度的样品进行3分钟的接触。利用流式细胞仪测量无细菌细胞培养模型中的消化液或样品, 对背景噪声进行了评价。每样样品均用三份计量。</li>
</ol>

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作者没有什么可透露的

口服微生物菌群是人类健康的一个关键因素, 最近有几个作者20、21发表了报告。先前的研究表明, 摄取含有大量细菌的唾液会影响小肠的微生物生态系统, 这是免疫启动的主要部位之一。静态上消化道消化模型与由肠道上皮和粘液分泌细胞代表的宿主界面的结合, 有助于解开微生物成分对宿主的影响。
体外模型是研究的基本工...

人与细菌同居, 是存在与人细胞1的同一个数字。因此, 对人类微生物群的全面了解是至关重要的。口腔是一个独特的环境, 因为它被分成几个较小的栖息地, 从而包含了大量的细菌和生物膜在这些不同的地方。作为一个开放的生态系统, 一些物种在口中可能是短暂的访客。然而, 某些微生物在出生后不久就会殖民, 形成组织的生物膜2。这些在牙龈缝隙、龈下缝隙、舌头、粘膜表面和牙科修复体和填充物3中的牙齿表面被发现。细菌也可以作为絮凝体和浮游细胞存在于牙管腔内, 或者与坏死的牙髓组织混合或悬浮在液相中。
宿主细胞与居民微生物群之间有积极、连续的交叉谈话4。细菌在物种内部和之间进行交流, 只有很小一部分自然殖民者能够坚持组织, 而其他细菌则依附于这些主要殖民者。例如, 微生物之间的细胞结合是将次级殖民者纳入口腔生物膜的关键, 并建立相互作用的微生物细胞的复杂网络4。唾液样品中大约70% 的细菌聚集物由牙龈sp、普氏菌链球菌、 Veillonella sp 和不明Bacteroidetes组成。nucleatum是龈下生物膜中的中间体殖民者, 与晚期殖民者菌、denticola和Tannerella 连翘有牵连, 这与牙周炎5有关。此外,缓症链球菌还占据粘膜和牙科栖息地, 而血统和仙人掌则更倾向于将牙齿殖民3。因此, S. 血统是存在于下门牙和犬齿, 而放线菌对内氏已发现在上部 anteriors6。
此外, 土著微生物群在维护人类健康方面起到了2的作用。居民微生物参与免疫教育和防止病原体扩张。这种殖民抵抗的发生是因为当地的细菌可以更好地适应于附着在表面上, 并且更有效地代谢可利用的养分促进生长。虽然益生菌菌株在胃肠道中存活并保持活跃, 但仍未充分描述从胃肠道上部位置吞下的本土细菌的持久性。因此, 我们接受了一个人工群落, 代表口腔生态系统, 模拟胃肠道转运条件。用类似肠道上皮的 multicompartment 模型评估细菌细胞的生存能力。目前肠道模拟器提供了适当的重现性分析的腔内微生物群落7。然而, 细菌黏附力和寄主微生物相互作用是分开解决的, 因为结合细胞系与微生物群落是挑战8。相反, 我们提出了一个框架, 提供了潜在的机械解释的成功的殖民事件报告的肠道接口。事实上, 这个模型可以与静态肠道模型联合使用, 以评估微生物群落对宿主表面信号的影响。

<table>
	<tbody>
		<tr>
			<td>STRAINS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregatibacter actinomycetemcomitans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 43718</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium nucleatum </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10953</td>
			<td></td>
		</tr>
		<tr>
			<td>Porphyromonas gingivalis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33277</td>
			<td></td>
		</tr>
		<tr>
			<td>Prevotella intermedia </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25611</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mutans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25175</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sobrinus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33478</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomyces viscosus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15987</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus salivarius&#160; TOVE-R</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mitis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49456</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sanguinis </td>
			<td>BCCM/LMG Bacteria Collection</td>
			<td>LMG 14657</td>
			<td></td>
		</tr>
		<tr>
			<td>Veillonella parvula </td>
			<td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures</td>
			<td>DSM 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus gordonii </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49818</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>86010202</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29-MTX cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>12040401</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS AND CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) broth</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar 2</td>
			<td>Oxoid</td>
			<td>CM0055</td>
			<td>Blood Agar medium</td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M9429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>H9039</td>
			<td></td>
		</tr>
		<tr>
			<td>5% sterile defibrinated horse blood</td>
			<td>E&#38;O Laboratories Ltd,</td>
			<td>P030</td>
			<td></td>
		</tr>
		<tr>
			<td>InnuPREP PCRpure Kit</td>
			<td>Analytik Jena</td>
			<td>845-KS-5010250</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>Big Dye</td>
			<td>Applied Biosystems</td>
			<td>4337454</td>
			<td>Dye for sequencing</td>
		</tr>
		<tr>
			<td>ABI Prism BigDye Terminator v3.1 cycle sequencing kit</td>
			<td>Applied Biosystems</td>
			<td>4337456</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 culture flasks uncoated, cell-culture treated, vented, sterile</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 
			0.4%, liquid, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture media, with GlutaMAX and Pyruvate</td>
			<td>Life technologies</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3450-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach Type II&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactivated fetal bovine serum</td>
			<td>Greiner Bio One</td>
			<td>758093</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100 for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium, magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce LDH Cytotoxicity Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>88953</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning HTS Transwell-24 well, pore size 0.4 &#181;m</td>
			<td>Corning Costar Corp</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Serva Electrophoresis</td>
			<td>28539010</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber improved</td>
			<td>Carl Roth</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>653118</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>MoBio</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Flush system</td>
			<td>Custom made</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>InnOva 4080 Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1007</td>
			<td>Shaker for 2.10</td>
		</tr>
		<tr>
			<td>Memmert CO2 incubator</td>
			<td>Memmert GmbH &#38; Co.</td>
			<td>ICO150med</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS (Electrical Resistance System)</td>
			<td>EMD Millipore, Merck KGaA</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Milli-Q academic, ultra pure water system</td>
			<td>Millipore, Merck KGaA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker (ROCKER 3D basic)</td>
			<td>IKA</td>
			<td>4000000</td>
			<td>Shaker for 6.10</td>
		</tr>
	</tbody>
</table>

assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface

宿主和微生物群之间的相互作用已被长期承认和广泛描述。口腔与胃肠道的其他部分相似, 因为居民的微生物群发生, 防止外源细菌的定植。事实上, 在口腔中发现了600多种细菌, 单个个体在任何时候都可能携带100左右。口腔细菌具有在口腔生态系统中坚持各种利基的能力, 从而在居民微生物群落中得到整合, 有利于生长和生存。然而, 在吞咽过程中细菌进入肠道的流动已经被提出来扰乱肠道微生物群的平衡。事实上, 口服菌转移了回肠菌群中的细菌组成。我们利用合成社区作为自然口腔生态系统的简化表示, 以阐明在模拟胃肠道条件下口腔细菌的存活和生存能力。选择十四种, 经体外唾液、胃和肠道消化过程, 并提出一个 multicompartment 细胞模型, 包含 Caco-2 和 HT29-MTX 细胞模拟肠道黏膜上皮。该模型有助于解开吞噬细菌对肝肠循环细胞的影响。使用合成群落可以进行可控性和重现。因此, 这种方法可以适应评估病原体的生存能力和随后的炎症相关变化, 益生菌混合物的殖民化容量, 最终, 潜在的细菌对 presystemic 循环的影响。

该协议导致了一个模型的生成, 适合于阐明口腔细菌在模拟胃肠道转运条件下的生存和存活。从单个菌株的完整细胞计数约 108细胞 mL-1之前, 合成社区的创建, 而多种群的缩影包含90% 的可行细胞在社区的建立 (图 1A和1B)。根据活/死定量, 细菌的生存能力减少后, 每消化步骤 (图 2)。这可能?...

Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

采用综合口服群落、体外胃肠消化和小肠上皮模型相结合的新方法对肠道宿主-微生物相互作用进行评价。我们提出了一种方法, 可以适应评估细胞入侵的病原体和多物种生物膜, 甚至测试益生菌制剂的生存性。

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

assessing-viability-synthetic-bacterial-consortium-on-vitro-gut-host

Research

JoVE Journal

Biochemistry

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

11.5K 次观看.  Ghent University. 采用综合口服群落、体外胃肠消化和小肠上皮模型相结合的新方法对肠道宿主-微生物相互作用进行评价。我们提出了一种方法, 可以适应评估细胞入侵的病原体和多物种生物膜, 甚至测试益生菌制剂的生存性。

视频: Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

体外F-肌动蛋白在早期体的聚合

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

使用支撑浮块在透射电子显微镜中制备样品支撑膜

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...