我们非常感谢詹尼Scimone与视频制作的优秀援助。

对感染艾滋病毒的血液样本的处理需要一些重要的建议。 所有血液标本必须在强大的防漏容器运送。采集标本时，必须小心，以避免污染容器的外观和附带任何手续的标本。 所有处理受感染的血液的人必须戴手套。手套必须改变和标本的处理完成后洗净双手。 艾滋病毒感染者的血液样品的处理，必须做到在II级生物危害柜罩。 应采用机械吹打艾滋病。 必须加以限制，其中有别无选择的情况下使用的针头或其他锐器（包括玻璃如移液器或毛细管）。 实验室表面必须与适当的化学消毒剂后溢出的血液净化，工作时活动结束。 必须净化污染的材料使用或重复使用前必须正确处置医疗废物的路线通过。 每一个可能具有传染性的血液或体液（即那些需要的普遍预防措施）的职业暴露事件应被视为医疗紧急干预必须迅速启动才能有效。 该协议要求5天完成。详细的时间表是在图1。 使用图2中所描述的方法，确定细菌种类。 1。样品采集<ol><li> 9毫升全血抽入EDTA的含管。 </li><li>于2000年在室温下10分钟的转速离心管获得等离子体。 </li><li>离子被收集在一个无菌2毫升的Eppendorf管。 </li></ol> 2。血浆样品中的DNA提取<ol><li>充分消毒罩，吸液管和实验所需的材料，以保证无菌。 </li><li>位置至少30分钟，在紫外线灯的所有材料。 </li><li>用消毒液擦拭手套。 </li><li>浸泡在乙醇一些纸巾和引擎盖下。每次小费是被丢弃擦拭湿纸巾吸管。 </li></ol> DNA是使用商业套件制造商的说明（易DNA试剂盒，Invitrogen公司，卡尔斯巴德CA，美国）提取。 <ol start="5"><li> 350μL血浆放置在无菌2毫升Eppendorf管中。 </li><li> 350μL超纯水过滤，作为阴性对照。 </li><li>添加10μL的溶菌酶（1mg/ml的）样品。 </li><li> 30分钟在37 ° C。 </li><li>加入500μL裂解液，轻轻混匀样品。 </li><li> 7分钟的孵育65 ° C。 </li><li>加入氯仿900μL的样品。 </li><li>涡大力直到样品均匀粘稠。 </li><li>添加200μL降水方案和涡大力。 </li><li>离心样品在室温下10分钟的转速在10500分开的阶段，并形成接口。 </li><li>上层水相转移到一个新的离心管中，含有1毫升乙醇100％。 </li><li>在4个样品离心10分钟的转速在10500 ° C。 </li><li>除去乙醇。 </li><li>加入乙醇70％1毫升。 </li><li>离心样品的最高速度为10分钟，在4 ° C。 </li><li>除去乙醇。应与pipettator删除残留的乙醇。 </li><li>在超纯水50μL重悬沉淀。 </li><li>用分光光度计读取DNA的浓度。 </li></ol> 3。 16S rRNA基因的PCR PCR扩增9如前所述。 <ol><li>扩增100μL反应10 10X PCR缓冲液，5μL25 mM的氯化镁，2毫米的总dNTPs 5μL，50微米底漆RW01（AACTGGAGGAAGGTGGGGAT）1μL，1μL50微米底漆DG74液组成的混合物的DNA （AGGAGGTGATCCAACCGCA），34μL的H20和0.5μLTaq DNA聚合酶（AmpliTaq金牌，应用生物系统，福斯特城，加利福尼亚，美国）。 </li><li>为了避免可能造成的污染细菌Taq聚合酶，PCR混合物中的过滤器传输。 </li><li>离心过滤器（单片机，Millipore公司，比尔里卡，马萨诸塞州，美国）为30分钟，在500 RCF在4 ° C。 </li><li>分装在PCR混合物，根据需要放大（正面和负面的PCR控制，样品和提取的水）样本数。 </li><li>使用以下热循环条件：94 ° C为10分钟，每次1分钟的40倍，94 ° C，55 ° C和72 ° C，并在72 ° C 10分钟</li><li>可视化的PCR产物在2％琼脂糖凝胶。使用100 bp的DNA阶梯。 PCR产物的大小约360基点（图3）。 </li></ol> 4。 PCR产物纯化只有PCR阳性标本应得到净化。净化是利用商业Kit中的下列制造商的说明（PureLink PCR扩增microkit，Invitrogen公司，卡尔斯巴德的CA，美国）。 <ol><li>新增4卷结合缓冲液，每1体积的PCR产物中含有异丙醇。 </li><li>传输与一列的结合缓冲液PCR产物。 </li><li>在室温下在10000 RCF离心1分钟。 </li><li>与洗涤缓冲液含有乙醇洗净列。 </li><li>在室温下在10000 RCF离心1分钟。 </li><li>以最大速度离心1分钟在室温下干燥的硅膜，并删除任何残留的洗涤缓冲液，用乙醇。 </li><li>加入10μL超纯水。 </li><li> 1分钟在室温下孵育。 </li><li>最大速度为2分钟，在室温下离心收集纯化的DNA。 </li></ol> 5。克隆溶原性肉汤（LB）板的制备<ol><li>溶于约800毫升的水混合的LB 25克。 </li><li>带来的最终体积至1 L。 </li><li>加入15克Bactoagar。 </li><li>高压灭菌器为20分钟。 </li><li>经过高压灭菌，大力摇动烧瓶中的解决方案混合熔化的琼脂。 </li><li>冷却到50 ° C的解决方案</li><li>添加氨苄青霉素（50微克/毫升）和漩涡，直到完全溶解。 </li><li>倒入深约3毫米的钢板。 </li><li>离开板在室温下。 </li><li>传播的X加仑（40mg/ml）和IPTG每个LB平板上，孵育（100毫米），在37 ° C，直到准备使用。 </li></ol>感受态细胞转化执行转换使用商业Kit中的下列制造商的说明（地形TA克隆试剂盒，Invitrogen公司，卡尔斯巴德的CA，美国）。 <ol><li>准备克隆反应的组合（1-4μL新鲜的PCR产物，1μL盐溶液，如有必要的载体和水1μL）。 </li><li>轻轻混合反应，并在室温下孵育5-10分钟。 </li><li>广场上冰的反应。 </li><li>新增2成一小瓶主管细胞克隆反应液，轻轻混匀。 </li><li>在冰上孵育30分钟。 </li><li>热休克的细胞在30秒42 ° C。 </li><li>立即转移到冰管。 </li><li>添加250μL介质。 </li><li>第tighly管和动摇管水平在37℃1小时。 </li><li> 50μL的蔓延，从每一个预热选择性板改造。 </li><li>过夜孵育在37 ° C。 </li></ol>潜伏期之后，白色和蓝色的菌落平板上发展。白色菌落呈阳性的PCR产物插入蓝色菌落PCR产物插入负。 6。测序分析测序反应<ol><li>混合使用这些试剂：2,5μLDNA，1μL引物（5pmol /μL）1,1μLBigDye（应用生物系统，福斯特城，加利福尼亚，美国）。 </li><li>热循环条件：96 ° C为1分钟10秒，55 ° C的25倍为4分15秒和60℃96℃。 </li></ol>柱纯化<ol><li>对于每一个反应，准备一个MicroSpin列（QIAGEN，米兰，意大利）。 </li><li>反相柱和旋涡混合树脂。 </li><li>柱底部对齐关闭，松开盖子¼圈，并将其放置在离心管列。 </li><li>离心1分钟3200转。 </li><li>列转移到一个干净的离心管。 </li><li>仔细吸管中心列到整个PCR测序反应。 </li><li>离心1分钟3200转。 </li><li>纯化的DNA eluite管（20μL超纯水）。 </li></ol>测序分析<ol><li>纯化样品装入96多孔板（20μL）。 </li><li>加载到音序器板。自动化DNA测序生成四色色谱图显示的测序运行的结果。 </li><li>作为对公共序列数据库的查询输入的核苷酸序列。在NCBI数据库和服务器上执行搜索，基本局部比对搜索工具（BLAST）。 </li><li>只考虑有98-100％的同源性的细菌。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2830/2830fig1.jpg" alt="图1" /> 图1。时间轴的过程。 <img src="/files/ftp_upload/2830/2830fig2.jpg" alt="图2" /> 图2整个细菌鉴定程序流程图。 <img src="/files/ftp_upload/2830/2830fig3.jpg" alt="图3" /> 图3显示广泛的16S rRNA基因的PCR产物2％琼脂糖凝胶。 1巷包含一个100bp的DNA梯状条带，2巷包含PCR阳性对照，泳道3显示负PCR控制。巷4显示了一个HIV阳性病人的样本，和5巷中含有水。 6巷SHOWS样品从一个健康的个体和阴性PCR反应; 7巷含有水。只有HIV阳性患者，显示了积极的PCR扩增。作为一个在提取步骤阴性对照用超纯水，表明没有发生污染。 <img src="/files/ftp_upload/2830/2830fig4.jpg" alt="图4" /> 图4。0.7％琼脂糖凝胶电泳显示质粒提取小量程序。 1巷包含1个碱基的DNA梯状条带，2巷包含蓝色的控制殖民地，车道从12月3日含有白色菌落。从蓝色菌落的质粒不包含的PCR产物插入。所有10个白色菌落中含有正确插入质粒。 <img src="/files/ftp_upload/2830/2830fig5.jpg" alt="图5" /> 图5显示了一个例子从一个艾滋病毒抗体阳性的个体血浆中的细菌测序分析。我们的结果表明，在HIV感染的微生物易位涉及到一个polimicrobic菌群，这是不是艾滋病毒阴性者中，建议在控制细菌移位肠道免疫功能的大量失败。

<ol>
	<li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat Rev Immunol. 10, 159-169 (2010).</li><li>Macpherson, A. J., Harris, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+commensal+intestinal+bacteria+and+the+immune+system.">Interactions between commensal intestinal bacteria and the immune system.</a> Nat Rev Immunol. 4, 478-485 (2004).</li><li>Kanauchi, O., Mitsuyama, K., Araki, Y., Andoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+intestinal+flora+in+the+treatment+of+inflammatory+bowel+disease.">Modification of intestinal flora in the treatment of inflammatory bowel disease.</a> Curr Pharm Des. 9, 333-346 (2003).</li><li>Brenchley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+a+cause+of+systemic+immune+activation+in+chronic+HIV+infection.">Microbial translocation is a cause of systemic immune activation in chronic HIV infection.</a> Nat Med. 12, 1365-1371 (2006).</li><li>Brenchley, J. M., Price, D. A., Douek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+disease:+fallout+from+a+mucosal+catastrophe.">HIV disease: fallout from a mucosal catastrophe.</a> Nat Immunol. 7, 235-239 (2006).</li><li>Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+levels+of+bacterial+DNA+correlate+with+immune+activation+and+the+magnitude+of+immune+restoration+in+persons+with+antiretroviral-treated+HIV+infection.">Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection.</a> J Infect Dis. 199, 1177-1185 (2009).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+translocation+is+associated+with+sustained+failure+in+CD4++T-cell+reconstitution+in+HIV-infected+patients+on+long-term+highly+active+antiretroviral+therapy.">Microbial translocation is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy.</a> AIDS. 22, 2035-2038 (2008).</li><li>Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Microbial+Translocation+and+Immune+Hyperactivation+in+Disease+Progression+of+HIV++Patients+with+Preserved+CD4+Count+in+the+Absence+of+ART.">Role of Microbial Translocation and Immune Hyperactivation in Disease Progression of HIV+ Patients with Preserved CD4 Count in the Absence of ART.</a> , (2010).</li><li>Greisen, K., Loeffelholz, M., Purohit, A., Leong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+primers+and+probes+for+the+16S+rRNA+gene+of+most+species+of+pathogenic+bacteria,+including+bacteria+found+in+cerebrospinal+fluid.">PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.</a> J Clin Microbiol. 32, 335-351 (1994).</li></ol>

<p class="jove_content"在此，我们显示的PCR /测序协议的特点艾滋病毒感染者外周血中的细菌易位。</p&gt;<p class="jove_content"&gt;最可靠的结果是使用EDTA含管收集血浆时获得。采血后，血浆应通过离心分离2 / 3小时内，以避免溶血和DNA片段退化。第一样本储存在-20 ° C，约5天，然后在-80 ° C应避免多个冻融循环，这可能会导致细菌的DNA损失。</p&gt;<p class="jove_content"&gt;血浆样品应解冻后12小时内使用。</p&gt;<p class="jove_content"&gt...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> Easty - DNA试剂盒</td><td> Invitrogen公司</td><td> K 180001 </td><td></td></tr><tr><td>氯仿</td><td> Sigma - Aldrich公司</td><td> C2432 </td><td></td></tr><tr><td>乙醇</td><td> Sigma - Aldrich公司</td><td> E7203 </td><td></td><tr/><tr><td>超纯娃儿</td><td> Invitrogen公司</td><td> 10977049 </td><td></td></tr><tr><td>溶菌酶</td><td> Fluka公司</td><td> 62970 </td><td> 10微克/毫升Distillated水</td></tr><tr><td> AmpliTaq金牌</td><td>应用生物系统</td><td> 26478701 </td><td></td></tr><tr><td>单片机100 </td><td> Millipore公司</td><td> 42413 </td><td></td></tr><tr><td> PCR引物</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td>琼脂糖</td><td> Eppendorf公司</td><td> C1343 </td><td></td></tr><tr><td> DNA梯状</td><td> Invitrogen公司</td><td> 15628019 </td><td></td></tr><tr><td> Purelink PCR扩增微套件</td><td> Invitrogen公司</td><td> K310050 </td><td></td></tr><tr><td> LB琼脂，粉末</td><td> Invitrogen公司</td><td> 22700025 </td><td></td></tr><tr><td> Bactoagar </td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td>氨苄青霉素</td><td> Invitrogen公司</td><td> 11593027 </td><td> 10毫克/毫升Distillated水</td></tr><tr><td> X - gal的</td><td> Invitrogen公司</td><td> 15520034 </td><td> 40mg/mL在DMF </td></tr><tr><td>经IPTG </td><td> Invitrogen公司</td><td> 15529019 </td><td> 100MM在Distillated水</td></tr><tr><td>地形助教克隆试剂盒</td><td> Invitrogen公司</td><td> K450002 </td><td></td></tr><tr><td> Purelink快速质粒小量制备试剂盒</td><td> Invitrogen公司</td><td> K210010 </td><td></td></tr><tr><td>大染料</td><td>应用生物系统</td><td> 4337455 </td><td></td></tr><tr><td> DyeEx 2.0自旋套件</td><td> Qiagen公司</td><td> 63204 </td><td></td></tr></tbody></table>

sequencing of bacterial microflora in peripheral blood: our experience with hiv-infected patients

生理健康的胃肠道是殖民地的影响共生微生物的体液免疫和细胞的粘膜免疫系统1,2的发展的种类繁多。 屏蔽的微生物是通过一个强大的黏膜屏障的免疫系统。感染和抗生素被称为改变正常的消化道障碍和居民的细菌组成，这可能会在可能的免疫异常导致3。 艾滋病毒会导致违反与胃肠黏膜免疫和全身血液循环进入细菌的生物制品，如内毒素和细菌的DNA片段，这有助于全身免疫激活4-7泄漏的渐进破坏屏障。在艾滋病毒/艾滋病的免疫病理及治疗 4,8微生物易位是牵连。 我们的目的是表征转位HIV感染患者的外周血中的细菌组成。为了追求我们的目标，我们成立了ribosomial panbacteric 16S基因测序分析PCR反应。 简单地说，艾滋病毒感染者和正常人全血使用。在这些患者中预计，鉴于目前健康的个人正常的肠道动态平衡没有菌群易位。静脉穿刺的全血和血浆分离收集，DNA是从血浆中提取和使用执行范围广泛的PCR反应ribosomial panbacteric 16S 基因 9 。执行下面的PCR产物纯化，克隆和测序分析。

Watch this Scientific Journal Video about Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients at JoVE.com

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

我们的实验将展示如何执行转位HIV阳性患者的外周血中的细菌种类的测序分析。

测序外周血中细菌菌群：我们的经验与艾滋病毒感染者

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

sequencing-bacterial-microflora-peripheral-blood-our-experience-with

Research

JoVE Journal

Medicine

12.2K 次观看.  San Paolo Hospital University of Milan, Italy. 我们的实验将展示如何执行转位HIV阳性患者的外周血中的细菌种类的测序分析。

视频: Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型：注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

科研

医学

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

在感染艾滋病毒的个体的测量弱者。体弱患者的识别是第一步改良与弱者拨回

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

在II-45同基因大鼠模型间皮瘤原位移植和外周免疫细胞监视

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

危重病人全血内毒素血症检测的内毒素活性测定

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Digital Handwriting Analysis of Characters in Chinese Patients with Mild Cognitive Impairment

An increasing amount of evidence shows that cognitive deficits and movement dysfunctions are not separated. Patients with mild cognitive impairment (MCI) can manifest fine motor disorders of the upper extremities. Handwriting is a complex and unique human activity involving both motor and cognitive coordination. Researchers from western countries have discovered that patients with MCI have abnormal handwriting features. However, no relevant studies have been conducted in the Chinese population. Owing to the cross-culture phenomenon of handwriting, the aim of this study is to find new handwriting tasks to demonstrate the differences in handwriting features between elderly patients with MCI and age-matched healthy individuals.

This study proposed a digital handwriting analysis of characters in individuals with mild cognitive impairment to find more information than is revealed by traditional pencil&#8211;paper handwriting analysis.

中国轻度认知障碍患者人物数字笔迹分析

An increasing amount of evidence shows that cognitive deficits and movement dysfunctions are not separated. Patients with mild cognitive impairment (MCI) ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

严重卵巢功能障碍患者的生育保护

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (rs = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (rs = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

用于测量外周血白细胞的床旁测试分析仪的评估

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At ...

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically easier, faster, becoming the gold standard for the surgical treatment of AF. However, the efficacy and safety of CMP-IV in situs inversus dextrocardia are largely unknown. This paper summarizes the CMP-IV procedure performed concomitantly with valvular surgery in patients with situs inversus dextrocardia at this institution.
From February 2016 to September 2020, three dextrocardia patients with persistent AF and valvular diseases were referred to this institution for valvular and CMP-IV surgery. CMP-IV was performed using either cryoablation with a nitrous oxide (N2O)-based cryoprobe or a bipolar radiofrequency clamp and bipolar radiofrequency pen. Mechanical valve replacement or mitral vavuloplasty was performed in another patient in addition to tricuspid annuloplasty. Transmurality of the ablated atrial tissues was evaluated by electron microscopy. Heart function was assessed by transthoracic echocardiography. Cardiac rhythm was monitored by 24 h Holter at 3, 6, 12, 18, 24, and 48 months follow-up. 
All the AF was successfully eliminated in the ablation procedure without recurrence or other complications during hospitalization. The mean bypass and crossclamp times were similar in all the patients. The postoperative ventilator support time, the duration of stay in the ICU, and postoperative residence time were also not significantly different among the patients. Transmural atrial necrosis was detected in the ablated atrial tissues. Sinus rhythm maintenance was achieved at 3, 6, 12, 18, 24, and 48 months follow-up in all the patients. All valve protheses switched freely; no tricuspid regurgitation was observed. The results of the present study demonstrate that the CMP-IV is safe and effective in eliminating AF in dextrocardia patients concomitant with valvular surgery.

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

Cox-Maze IV手术与瓣膜手术在位内与右旋心动过速中同时进行：中国的单中心经验

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The use of ablation technologies made the Cox-Maze IV procedure (CMP-IV) technically ...