Methods and Protocols最新文献_第8页

Quantitative Image Analysis of Axonal Morphology in In Vivo Model 体内模型轴突形态的定量图像分析

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-12-01 DOI: 10.3390/mps6060116

Laurie Nemoz-Billet, Jacques Brocard, Florence Ruggiero, Sandrine Bretaud

Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.

量化轴突分支对于理解神经回路功能、发育和再生过程以及疾病机制至关重要。调节轴突树突形成模式和调节神经元回路的因素在各种脑连接障碍中的含义进行了研究。缺乏一种可靠的、用户友好的方法使得轴突形态的定量分析变得困难。具体来说，可视化和量化复杂轴突树突的方法在实际应用中具有挑战性。我们的研究旨在开发一种强大而简单的量化方法，使用imagej2d分析，并将其与Imaris可视化和3D图像分析进行比较。我们使用斑马鱼荧光转基因系对发育中的运动神经元轴突进行体内成像，充分反映了轴突网络的复杂性。我们在ImageJ上开发的新方法既简单又快速，可以获得新的信息，例如沿轴突轴的侧枝分布。这项研究描述了一步一步的程序，可以很容易地应用于各种生物体和体外系统。我们的研究为进一步探索神经回路提供了基础，以获得对神经疾病和潜在治疗干预的新见解。

{"title":"Quantitative Image Analysis of Axonal Morphology in In Vivo Model","authors":"Laurie Nemoz-Billet, Jacques Brocard, Florence Ruggiero, Sandrine Bretaud","doi":"10.3390/mps6060116","DOIUrl":"https://doi.org/10.3390/mps6060116","url":null,"abstract":"Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138610525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Protocol Article: A Cross-Sectional Evaluation of Children’s Feet and Lower Extremities 协议条款：儿童足部和下肢横断面评估

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-12-01 DOI: 10.3390/mps6060115

Christian Wong, Christina Ystrøm Bjerge, Ales Jurca, M. M. Petersen, Soren Boedtker, Andreas Balslev-Clausen, Steen Harsted

Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases.

背景:儿童下肢和足部的健康是护理人员和医疗保健专业人员(如医生、学校护士和足科医生)关注的焦点领域。我们的研究旨在调查丹麦儿童下肢和足部的一般健康状况，以确定可能是疼痛和足部疾病评估的先决条件的人体测量参数，以及它们是否与疼痛强度和位置、三维足部尺寸和足压力测绘、鞋子尺寸、体育活动的类型和强度、生活质量和足部健康有关。目的是，我们将能够识别疼痛的参数预移位，从而为儿童的人体测量条件相关的体育活动提供建议，作为疼痛的潜在预防措施。这种分析将在群体层面上按社会经济地位分层，这种观点将能够提供预防疼痛的建议。方法:本研究是对随机选择的丹麦小学一年级、五年级和九年级的1000名儿童进行横断面检查。我们将对下肢和足部进行临床检查，检查错位、畸形和疾病，以及旋转状态和活动范围。此外，我们将评估他们的疼痛程度、体育活动、三维足部尺寸、足底压力、鞋类和足部健康和生活质量的患者相关结果测量(PROMs)。结果:我们的目标是提供儿童下肢和足部的人体测量学概述。对儿童健康正常物质的基本了解将在群体水平上分析其与疼痛程度、体育活动和社会经济地位的关系。这可能会让我们了解影响儿童下肢和足部疾病的因素。总之，对丹麦小学儿童的下肢和足部进行检查是确定自我保健和鞋履方面需要改进的领域，绘制儿童足病相关护理需求的图，并为家长提供关于鞋履预防行动以及与疼痛有关的体育活动的选择和强度的建议。结论:本研究的宗旨是通过长期随访来评估群体水平、足部状况和体育活动的长期社会经济过程，使用与患者相关的结果测量来评估生活质量和其他生活方式因素，如情感功能、社会功能和互动、学校功能。这可能会改善儿童的生活质量，预防未来的疾病。

{"title":"Protocol Article: A Cross-Sectional Evaluation of Children’s Feet and Lower Extremities","authors":"Christian Wong, Christina Ystrøm Bjerge, Ales Jurca, M. M. Petersen, Soren Boedtker, Andreas Balslev-Clausen, Steen Harsted","doi":"10.3390/mps6060115","DOIUrl":"https://doi.org/10.3390/mps6060115","url":null,"abstract":"Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138621553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Validity and Reliability of POM-Checker for Measuring Shoulder Range of Motion in Healthy Participants: A Pilot Single-Center Comparative Study. POM-Checker 用于测量健康参与者肩关节活动范围的有效性和可靠性：单中心试点对比研究

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-27 DOI: 10.3390/mps6060114

Hongmin Chu, Weonjin Kim, Seongsu Joo, Eunsik Park, Yeong Won Kim, Cheol-Hyun Kim, Sangkwan Lee

Background: The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400).

Methods: This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included.

Results: The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility.

Conclusion: The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint.

研究背景本研究旨在比较基于 Kinect 的无标记 ROM 评估设备（POM-Checker）和三维运动捕捉分析系统（BTS SMART DX-400）的肩关节运动测量结果：这是一项单次临床试验，旨在评估POM-Checker的有效性和可靠性。主要结果是评估在同一组参与者中两种测量设备之间的等效性，目的是评估 POM-Checker 与黄金标准设备（三维运动分析系统）相比的有效性。由于这是一项试验性研究，共有六名参与者参加：采用类内相关系数（ICC）和相应的 95% 置信区间（CI）来评估测量结果的可重复性。在分析的 18 个动作中，有 16 个动作的 ICC 值大于 0.75，表明重现性极佳：结果表明，POM-检查器在测量肩关节活动范围方面是可靠和有效的。

{"title":"Validity and Reliability of POM-Checker for Measuring Shoulder Range of Motion in Healthy Participants: A Pilot Single-Center Comparative Study.","authors":"Hongmin Chu, Weonjin Kim, Seongsu Joo, Eunsik Park, Yeong Won Kim, Cheol-Hyun Kim, Sangkwan Lee","doi":"10.3390/mps6060114","DOIUrl":"10.3390/mps6060114","url":null,"abstract":"Background: The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400).Methods: This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included.Results: The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility.Conclusion: The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745328/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138830499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables. 巨型籼稻栽培品种 MTU1010 高效转化和再生的多功能方案：通过激素变量进行优化。

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-23 DOI: 10.3390/mps6060113

Pragya Yadav, V V Santosh Kumar, Jyoti Priya, Shashank Kumar Yadav, Shivani Nagar, Meenu Singh, Viswanathan Chinnusamy

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

水稻是最重要的粮食作物之一，可以满足世界大多数人口的日常热量和饮食需求。然而，水稻的产量受到各种生物和非生物属性的严重限制，对全球粮食安全造成了严重威胁。要利用功能基因组学和基因组编辑技术生成性状增强的基因型，就必须制定高效的遗传转化和再生方案。籼稻栽培品种的抗逆性以及高效、通用的遗传转化和再生方案的匮乏仍然是一个制约因素。在本研究中，我们优化了 MTU1010（一种超大型籼稻品种）的组织培养方法。我们对不同植物生长调节剂对胚性生殖茧诱导效率的影响进行了组合分析，结果表明，在 MSB5 培养基中添加 2.5 mg/L 2-4D 和 0.25 mg/L 6-BAP，胚性生殖茧诱导率最高，达到 92%。在生长培养基中添加 1 毫克/升酮素以及 2.5 毫克/升 BAP 和 0.5 毫克/升 NAA，可使转化胼胝体的再生效率提高 50%。此外，我们的研究结果表明，用 150 µM 乙酰丁香酮预激活农杆菌培养 30 分钟可显著提高胼胝体的转化效率。此外，在再悬浮和共培养过程中将盐浓度降到一半，可使转化效率提高 33%。因此，本研究开发的方案将有助于对籼稻栽培品种进行基因组编辑和基因工程，以进行功能基因组学研究和作物改良。

{"title":"A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables.","authors":"Pragya Yadav, V V Santosh Kumar, Jyoti Priya, Shashank Kumar Yadav, Shivani Nagar, Meenu Singh, Viswanathan Chinnusamy","doi":"10.3390/mps6060113","DOIUrl":"10.3390/mps6060113","url":null,"abstract":"Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10745540/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138830497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma. 一个简单，快速，有效的肝素酶方案，使核酸研究从冷冻肝素化血浆。

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-20 DOI: 10.3390/mps6060112

Rownock Afruza, Nicole Minerva, Justin B Lack, Moumita Chakraborty, James A Haddad, Rabab O Ali, Christopher Koh, Elliot B Levy, Ohad Etzion, Theo Heller

Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at -80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.

无细胞rna (cfRNAs)是一种很有前途的非侵入性生物标志物，如果与代谢组学结合起来，它将具有更大的潜力。血浆是cfrna的最佳来源，但通常来源于多种抗凝血剂。从肝素中获得的血浆适合于代谢组学，但难以用于基于qpcr的下游分析。在本研究中，我们的目标是开发一种简单、高效、经济的肝素酶方案，然后对提取的人血浆cfrna进行文库制备和测序，并在-80°C的肝素中保存数年。从美国国立卫生研究院(NIH)临床中心的慢性HCV感染患者(NCT02400216)的CPT™钠肝素管中采集血液。血浆cfrna用肝素酶I处理，用于文库制备和下一代测序(NGS)。肝素酶处理保持了RNA的完整性，即使以7 ng的cfRNAs作为起始材料，也可以成功地为所有研究对象制备文库。从Pavian R包v1.2.0中导出的分类报告没有人为读取。在微生物读数中脊索动物的丰度表明通过肝素酶I处理没有增加实验误差。我们报告了一种新的实用的肝素酶治疗方法，用于收集并在肝素钠中冷冻数年的人血浆。这是利用肝素血浆进行NGS和下游转录组学研究的有效证明，然后可以将其与来自相同样品的代谢组学相结合，最大限度地提高效率并减少抽血。

{"title":"A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma.","authors":"Rownock Afruza, Nicole Minerva, Justin B Lack, Moumita Chakraborty, James A Haddad, Rabab O Ali, Christopher Koh, Elliot B Levy, Ohad Etzion, Theo Heller","doi":"10.3390/mps6060112","DOIUrl":"10.3390/mps6060112","url":null,"abstract":"Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at -80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":2.4,"publicationDate":"2023-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10660533/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138176753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Methods and Protocols-Aims and Scope Update. 方法和协议-目标和范围更新。

IF 2.4 Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-17 DOI: 10.3390/mps6060111

Fernando Albericio, Philip Hublitz

As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...].

正如我们的读者所知，《方法与协议》是一本多学科同行评议的科学杂志，为生命科学、化学和生物医学科学领域的新方法的发表提供了一个论坛，这些领域与其他相关科学领域(如物理学、地球科学和环境研究)的交叉。

引用次数: 0

Protocol for Facile Synthesis of Fmoc-N-Me-AA-OH Using 2-CTC Resin as Temporary and Reusable Protecting Group 用2-CTC树脂作为临时可重复使用的保护基团快速合成Fmoc-N-Me-AA-OH的方案

Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-13 DOI: 10.3390/mps6060110

Tanya Román, Gerardo Acosta, Constanza Cárdenas, Beatriz G. de la Torre, Fanny Guzmán, Fernando Albericio

One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity.

提高多肽体内生物利用度和半衰期的一种方法是对多肽序列中的一个或多个氨基酸进行n -甲基化。然而，市售的fmoc - n - me - aa - oh数量有限，而且往往价格昂贵。本研究采用2-氯三酰氯(2-CTC)树脂作为羧酸策略的临时保护基，建立了固相合成Fmoc-N-Me-AA-OH的方法。比较了在Biron - Kessler法中采用硫酸二甲酯或碘化甲酯的两种烷基化步骤。在这项工作中，我们测试了两种氨基酸:Fmoc- thr (tBu)-OH和Fmoc-βAla-OH。第一个是α氨基酸，非常受阻，胺基直接受到羧基电子效应的影响，而在Fmoc-βAla-OH中，亚甲基的存在由于中间的碳原子而减弱了这种影响。两种方法合成的氨基酸Fmoc-N-Me- thr (tBu)-OH和Fmoc-N-Me-β - ala -OH收率高，纯度高。

引用次数: 0

Bio-SELEX: A Strategy for Biomarkers Isolation Directly from Biological Samples Bio-SELEX:一种直接从生物样品中分离生物标志物的方法

Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-11 DOI: 10.3390/mps6060109

Juan David Ospina-Villa, Valentina Restrepo-Cano, Miryan Margot Sánchez-Jiménez

Bio-SELEX is a revolutionary method for the discovery of novel biomarkers within biological samples, offering profound insights into diagnosing both infectious and non-infectious diseases. This innovative strategy involves three crucial steps: Traditional SELEX, Pull Down, and mass spectrometry. Firstly, Traditional SELEX involves the systematic selection of specific nucleic acid sequences (aptamers) that bind to the target molecules of interest. These aptamers are generated through iterative rounds of selection, amplification, and enrichment, ultimately yielding highly selective ligands. Secondly, the Pull-Down phase employs these aptamers to capture and isolate the target biomarkers from complex biological samples. This step ensures the specificity of the selected aptamers in binding to their intended targets. Lastly, mass spectrometry is utilized to identify and quantify the captured biomarkers, providing precise information about their presence and concentration in the sample. These quantitative data are invaluable in disease diagnosis and monitoring. Bio-SELEX’s significance lies in its ability to discover biomarkers for a wide range of diseases, spanning infectious and non-infectious conditions. This approach holds great promise for early disease detection, personalized medicine, and the development of targeted therapies. By harnessing the power of aptamers and mass spectrometry, Bio-SELEX advances our understanding of disease biology and opens new avenues for improved healthcare.

Bio-SELEX是一种革命性的方法，用于在生物样品中发现新的生物标志物，为诊断传染性和非传染性疾病提供了深刻的见解。这一创新策略包括三个关键步骤:传统SELEX，下拉和质谱。首先，传统的SELEX涉及系统地选择与感兴趣的靶分子结合的特定核酸序列(适体)。这些适体是通过反复的选择、扩增和富集产生的，最终产生高度选择性的配体。其次，Pull-Down阶段使用这些适体从复杂的生物样品中捕获和分离目标生物标志物。这一步骤确保了所选适配体与预期靶标结合的特异性。最后，质谱法用于识别和量化捕获的生物标志物，提供关于它们在样品中的存在和浓度的精确信息。这些定量数据在疾病诊断和监测中具有不可估量的价值。Bio-SELEX的重要意义在于它能够发现各种疾病的生物标志物，包括传染性和非传染性疾病。这种方法对早期疾病检测、个性化医疗和靶向治疗的发展有着巨大的希望。通过利用适体和质谱的力量，Bio-SELEX推进了我们对疾病生物学的理解，并为改善医疗保健开辟了新的途径。

{"title":"Bio-SELEX: A Strategy for Biomarkers Isolation Directly from Biological Samples","authors":"Juan David Ospina-Villa, Valentina Restrepo-Cano, Miryan Margot Sánchez-Jiménez","doi":"10.3390/mps6060109","DOIUrl":"https://doi.org/10.3390/mps6060109","url":null,"abstract":"Bio-SELEX is a revolutionary method for the discovery of novel biomarkers within biological samples, offering profound insights into diagnosing both infectious and non-infectious diseases. This innovative strategy involves three crucial steps: Traditional SELEX, Pull Down, and mass spectrometry. Firstly, Traditional SELEX involves the systematic selection of specific nucleic acid sequences (aptamers) that bind to the target molecules of interest. These aptamers are generated through iterative rounds of selection, amplification, and enrichment, ultimately yielding highly selective ligands. Secondly, the Pull-Down phase employs these aptamers to capture and isolate the target biomarkers from complex biological samples. This step ensures the specificity of the selected aptamers in binding to their intended targets. Lastly, mass spectrometry is utilized to identify and quantify the captured biomarkers, providing precise information about their presence and concentration in the sample. These quantitative data are invaluable in disease diagnosis and monitoring. Bio-SELEX’s significance lies in its ability to discover biomarkers for a wide range of diseases, spanning infectious and non-infectious conditions. This approach holds great promise for early disease detection, personalized medicine, and the development of targeted therapies. By harnessing the power of aptamers and mass spectrometry, Bio-SELEX advances our understanding of disease biology and opens new avenues for improved healthcare.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135086480","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparison of Light-Sheet Fluorescence Microscopy and Fast-Confocal Microscopy for Three-Dimensional Imaging of Cleared Mouse Brain 光片荧光显微镜与快速共聚焦显微镜用于清除小鼠脑三维成像的比较

Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-10 DOI: 10.3390/mps6060108

Youngjae Ryu, Yoonju Kim, Sang-Joon Park, Sung Rae Kim, Hyung-Jun Kim, Chang Man Ha

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.

全脑成像通过破译组织结构、神经元回路和单个神经元追踪来理解大脑功能是很重要的。因此，已经开发了许多清除方法来获取全脑图像或三维厚组织图像。然而，成像全脑体积有几个限制，包括较长的图像采集时间，大量的数据和较长的图像后处理。基于这些限制，许多研究人员不确定哪种光学显微镜最适合成像厚组织。在这里，我们比较了快速共聚焦显微镜与光片荧光显微镜进行全脑三维成像，它们可以最快地获得图像。为了比较两种显微镜对大体积成像的影响，我们对整个小鼠大脑进行了组织清理，改变了光片荧光显微镜的样品室和低倍率物镜，并改进了样品架。我们发现，与使用4倍物镜的快速共聚焦显微镜相比，使用2.5倍物镜的光片荧光显微镜具有节省时间、大容量图像采集和高z分辨率等优点。因此，我们认为光片荧光显微镜适用于全脑成像和获得高分辨率的三维图像。

{"title":"Comparison of Light-Sheet Fluorescence Microscopy and Fast-Confocal Microscopy for Three-Dimensional Imaging of Cleared Mouse Brain","authors":"Youngjae Ryu, Yoonju Kim, Sang-Joon Park, Sung Rae Kim, Hyung-Jun Kim, Chang Man Ha","doi":"10.3390/mps6060108","DOIUrl":"https://doi.org/10.3390/mps6060108","url":null,"abstract":"Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135186412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of a Quantitative PCR Method for Detecting Enterococcus faecalis Cytolysin in Human Stool Samples 人粪便中粪肠球菌溶细胞素定量PCR检测方法的建立

Q2 Biochemistry, Genetics and Molecular Biology

Methods and Protocols

Pub Date : 2023-11-08 DOI: 10.3390/mps6060107

Noemí Cabré, Yongqiang Yang, Yanhan Wang, Bernd Schnabl

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials.

酒精相关性肝病(ALD)是一个主要的全球健康问题，是全球发病率和死亡率的重要因素。在ALD亚型中，酒精相关性肝炎是一个严重而紧迫的医学挑战，具有高短期死亡率。尽管进行了广泛的研究，但目前酒精相关性肝炎的治疗方法疗效有限，需要新的干预措施。最近的研究强调了肠道微生物群在ALD发病中的关键作用，特别是粪肠球菌(E. faecalis)及其溶细胞素外毒素。本研究提出了一种标准化的实时定量聚合酶链反应(RT-qPCR)方法，用于检测和定量酒精相关性肝炎患者粪便样本中的细胞溶酶。诊断分析允许细胞溶素阳性粪肠球菌与疾病严重程度以及死亡率之间的关联分析。该检测方法的开发是为了规范细胞溶血素阳性患者的鉴定，这些患者可以被选择用于临床试验。

引用次数: 0