发布求助
文献互助 智能选刊 最新文献

Methods in molecular biology最新文献

筛选
英文 中文
Imaging Mitochondrial Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Neurons. 人类诱导多能干细胞衍生神经元线粒体轴突运输成像
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4264-1_10
Carla Lopes
{"title":"Imaging Mitochondrial Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Neurons.","authors":"Carla Lopes","doi":"10.1007/978-1-0716-4264-1_10","DOIUrl":"10.1007/978-1-0716-4264-1_10","url":null,"abstract":"<p><p>Neuronal mitochondria are essential organelles to maintain synaptic activity due to the high calcium buffering capacity and ATP production. In neurons, mitochondria transport occurs along the microtubules mediated by motor proteins, kinesins and dynein, to drive mitochondria toward the synapses. Disruption of axonal transport is an early pathogenic event in neurodegenerative disorders and growing evidence supports that it may precede neurodegeneration. Here, we describe a method to label mitochondria with fluorescent proteins to monitor their movement along the axons in hiPSC-derived medium spiny neuron-like cells. We also included a detailed protocol for differentiation of hiPSC that produces electrophysiologically mature GABAergic striatal neurons with low amount of glial population.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2878 ","pages":"201-209"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639384","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
Metabolic Phenotyping of Synaptic Mitochondria Using MitoPlates™ and Synaptoneurosomes. 利用 MitoPlates™ 和突触线粒体对突触线粒体进行代谢表型。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4264-1_4
Aleksandra Stawikowska, Magdalena Dziembowska, Bozena Kuzniewska
{"title":"Metabolic Phenotyping of Synaptic Mitochondria Using MitoPlates™ and Synaptoneurosomes.","authors":"Aleksandra Stawikowska, Magdalena Dziembowska, Bozena Kuzniewska","doi":"10.1007/978-1-0716-4264-1_4","DOIUrl":"10.1007/978-1-0716-4264-1_4","url":null,"abstract":"<p><p>Mitochondrial functional assays using MitoPlates™ S-1 allow us to characterize mitochondria in terms of substrate metabolism. MitoPlates™ are 96-well microplates pre-coated with a diverse set of substrates. The electron flow from NADH and FADH<sub>2</sub> producing mitochondrial substrates is measured based on the reduction of redox dye, that acts as a terminal electron acceptor. Here, we describe the application of MitoPlates™ to characterize the metabolism of synaptic mitochondria enclosed in isolated pre- and postsynaptic terminals (synaptoneurosomes).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2878 ","pages":"67-74"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639390","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
Molecular Dynamics Simulations of the SNARE Complex Interacting with Synaptotagmin, Complexin, and Lipid Bilayers. SNARE复合物与Synaptotagmin， Complexin和脂质双层相互作用的分子动力学模拟。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_1
Maria Bykhovskaia
{"title":"Molecular Dynamics Simulations of the SNARE Complex Interacting with Synaptotagmin, Complexin, and Lipid Bilayers.","authors":"Maria Bykhovskaia","doi":"10.1007/978-1-0716-4314-3_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_1","url":null,"abstract":"<p><p>Molecular dynamics (MD) simulations enable in silico investigation of the dynamic behavior of proteins and protein complexes. Here, we describe MD simulations of the SNARE bundle forming the complex with the neuronal proteins Synaptotagmin-1 (Syt1) and Complexin (Cpx). Syt1 is the synaptic vesicle (SV) protein that serves as the neuronal calcium sensor and triggers synaptic fusion upon calcium binding, and this process is promoted and accelerated by Cpx. The fusion depends on the Syt1 interactions with the SNARE-Cpx complex and with the lipid bilayer of the presynaptic membrane (PM). The MD simulations of the PM-Syt1-SNARE-Cpx-SV molecular system described here enabled us to investigate how this protein-lipid complex promotes the merging of SV and PM, triggering synaptic fusion.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"3-16"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979167","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
Immunophenotyping of Hematopoietic Cells in the Spleen in Hypothyroid Mice. 甲状腺功能减退小鼠脾脏造血细胞的免疫分型
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4252-8_7
Diego Rodríguez Muñoz
{"title":"Immunophenotyping of Hematopoietic Cells in the Spleen in Hypothyroid Mice.","authors":"Diego Rodríguez Muñoz","doi":"10.1007/978-1-0716-4252-8_7","DOIUrl":"10.1007/978-1-0716-4252-8_7","url":null,"abstract":"<p><p>Hypothyroidism, which is characterized by insufficient production of thyroid hormones, and malaria, a mosquito-borne infectious disease caused by Plasmodium parasites, are significant global health challenges. Studying how these two conditions interact could provide valuable insights into their complex relationship and potential treatment options.To induce hypothyroidism in the research, scientists used drugs to block the production of thyroid hormones. Then, they infected mice with Plasmodium berghei ANKA to mimic cerebral malaria infection. The spleen is essential in the body's immune response to malaria. It is involved in both innate and adaptive immunity, iron recycling, and the removal of old red blood cells or damaged cells infected with Plasmodium. Monitoring disease progression in male mice is crucial for early detection, and techniques like flow cytometry can help identify specific immune system populations within the spleen that are relevant to the research.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2876 ","pages":"105-115"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695694","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
A Sensitive Radioimmunoassay for T3 and T4 Determinations in Plasma and Tissues. 用于测定血浆和组织中 T3 和 T4 的灵敏放射免疫测定法
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4252-8_3
Ana Montero-Pedrazuela, Carmen Grijota-Martínez, Ana Guadaño-Ferraz, Soledad Bárez-López
{"title":"A Sensitive Radioimmunoassay for T3 and T4 Determinations in Plasma and Tissues.","authors":"Ana Montero-Pedrazuela, Carmen Grijota-Martínez, Ana Guadaño-Ferraz, Soledad Bárez-López","doi":"10.1007/978-1-0716-4252-8_3","DOIUrl":"10.1007/978-1-0716-4252-8_3","url":null,"abstract":"<p><p>This chapter details protocols for determining plasma thyroid hormone (TH) levels and tissue TH content by competitive radioimmunoassays (RIAs). These protocols include: an initial test of the chromatographic performance, isotopic labeling to produce high activity <sup>125</sup>I-T3 and <sup>125</sup>I-T4, free iodide estimation of the labeled products, purification of tracers from iodide by paper electrophoresis, extraction of THs from plasma and tissue samples, and the RIA procedures. The RIA involves the competition between radioactive labeled and unlabeled hormones for specific antibody binding, and due to its high sensitivity is capable of detecting a minimum of 2.5 pg of T4 and 0.4 pg of T3.Drs. María Jesús Obregon and Gabriella Morreale de Escobar improved these protocols in the 1970s, enhancing sensitivity and accuracy. Their improvements enabled the detection of TH content in tissues, providing crucial insights into maternal THs role in fetal development and the importance of iodine intake during gestation. Their research also facilitated the early detection of congenital hypothyroidism, preventing neurological impairments in newborns. Internationally and in Spain, their contributions are widely acknowledged, leading to substantial public health impacts, including the implementation of nationwide neonatal screening programs. Despite progress, thyroid diseases remain prevalent, underscoring the need for continued research into thyroid physiology and associated disorders, employing highly sensitive techniques like the RIA outlined herein that we continue using in our research.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2876 ","pages":"37-59"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695677","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
Olive Fruit Phenolic Profiling Using High Resolution-Magic Angle Spinning (HR-MAS) Solid-State NMR Spectroscopy. 利用高分辨率-魔角旋转（HR-MAS）固态核磁共振光谱分析橄榄果酚类物质。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_16
Εfstathia Manolopoulou, Apostolos Spyros
{"title":"Olive Fruit Phenolic Profiling Using High Resolution-Magic Angle Spinning (HR-MAS) Solid-State NMR Spectroscopy.","authors":"Εfstathia Manolopoulou, Apostolos Spyros","doi":"10.1007/978-1-0716-4334-1_16","DOIUrl":"https://doi.org/10.1007/978-1-0716-4334-1_16","url":null,"abstract":"<p><p>High Resolution-Magic Angle Spinning (HR-MAS) solid-state NMR spectroscopy is finding increasing application in the analysis of solid foods, bypassing the need for complicated solvent extraction procedures. In the present protocol, we report a simple analytical approach based on HR-MAS NMR spectroscopy for the phenolic profiling of olive fruits, flesh, or skin. This approach allows the facile characterization of phenolic compounds in olive fruits cultivated for extra-virgin olive oil production as a function of maturation and variety, in addition to processing technology for table olives.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2891 ","pages":"277-284"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984036","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
Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria. 细菌中合成小 RNA 的表达质粒的金门克隆。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_17
Sophie Dittmar, Bork A Berghoff
{"title":"Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.","authors":"Sophie Dittmar, Bork A Berghoff","doi":"10.1007/978-1-0716-4220-7_17","DOIUrl":"10.1007/978-1-0716-4220-7_17","url":null,"abstract":"<p><p>Bacterial small RNAs (sRNAs) are well known for their ability to modulate gene expression at the post-transcriptional level. Their rather simple and modular organization provides the user with defined building blocks for synthetic biology approaches. In this chapter, we introduce a plasmid series for Escherichia coli and describe protocols for fast and efficient construction of synthetic sRNA expression plasmids based on Golden Gate assembly. In addition, we present the G-GArden tool, which assists with the design of oligodeoxynucleotides and overhangs for scarless assembly strategies. We propose that the presented procedures are suitable for many applications in different bacteria, which are related to E. coli and beyond.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2850 ","pages":"307-328"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372288","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
Monitoring Accessible Cholesterol Levels in Immune Cells. 监测免疫细胞中可达胆固醇水平。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4318-1_7
Duo H Ma, Neal M Alto, Arun Radhakrishnan
{"title":"Monitoring Accessible Cholesterol Levels in Immune Cells.","authors":"Duo H Ma, Neal M Alto, Arun Radhakrishnan","doi":"10.1007/978-1-0716-4318-1_7","DOIUrl":"10.1007/978-1-0716-4318-1_7","url":null,"abstract":"<p><p>Cholesterol is a critical lipid that is present at high concentrations in the plasma membranes of animal cells. Most of the membrane cholesterol is sequestered by other membrane lipids and the transmembrane domains of proteins. Cholesterol in excess of such sequestration forms a pool that is referred to as \"accessible cholesterol.\" This pool of cholesterol plays a crucial role in maintaining lipid homeostasis and in controlling cell growth. The accessible cholesterol pool can also be exploited by bacteria and viruses to promote infection and host immune responses rapidly lower levels of this pool to confer protection. We had previously developed a bacterial toxin sensor called ALOD4 to monitor and quantify accessible cholesterol in cultured cells. Here, we report the characterization of a modified version of ALOD4 that is specialized to detect and monitor accessible cholesterol levels in primary immune cells by flow cytometry analysis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2888 ","pages":"83-99"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142854802","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
SNARE-Mediated Membrane Fusion Probed Using a Synthetic Organelle in the Living Bacterium. 利用合成细胞器在活细菌中探测snare介导的膜融合。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_19
Christian Vannier, Thierry Galli
{"title":"SNARE-Mediated Membrane Fusion Probed Using a Synthetic Organelle in the Living Bacterium.","authors":"Christian Vannier, Thierry Galli","doi":"10.1007/978-1-0716-4314-3_19","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_19","url":null,"abstract":"<p><p>Studies on the mechanisms and regulation of functional assemblies of SNARE proteins mediating membrane fusion essentially make use of recombinant proteins and artificial phospholipid bilayers. We have developed an easy-to-use in vivo system reconstituting membrane fusion in living bacteria. It relies on the formation of caveolin-dependent intracytoplasmic cisternae followed by the controlled synthesis of members of the synaptic SNARE machinery. Only when a SNARE complex is formed with its intact components does the docking and subsequent fusion occur between the cisternae and the plasma membrane that is accompanied by the disappearance of the former. The phenotypic response of the bacterial cell to fusion events is a remarkable increase in cell body length due to an expansion of the plasma membrane. Therefore, such an easy-to-observe phenotype makes this system amenable to structure-function studies of SNAREs. We describe here the specific ways to produce caveolin and the SNARE proteins from compatible plasmids upon bacterial transformation and to obtain the elongated cell phenotype. We also provide protocols to carry out the preparation of cell culture samples suitable for biochemical and light microscopy analysis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"263-280"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979102","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
Assays of Platelet SNARE-actin Interactions. 血小板snare -肌动蛋白相互作用的测定。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_9
Kamil Woronowicz, Robert Flaumenhaft
{"title":"Assays of Platelet SNARE-actin Interactions.","authors":"Kamil Woronowicz, Robert Flaumenhaft","doi":"10.1007/978-1-0716-4314-3_9","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_9","url":null,"abstract":"<p><p>The actin cytoskeleton serves an important, but poorly characterized, role in controlling granule exocytosis. The dynamic nature of actin remodeling allows it to act both as a barrier to prevent indiscriminate granule release and as a facilitator of membrane fusion. In its capacity to promote exocytosis, filamentous actin binds to components of the exocytotic machinery through actin binding proteins, but also through direct interactions with SNAREs. The platelet is an excellent cellular model to evaluate SNARE-actin interactions because of the marked reorganization of its actin cytoskeleton that occurs with activation and because of its abundance of secretory granules. This chapter will describe methods to evaluate SNARE-actin interactions in platelets using isolated platelet actin cytoskeleton, granule-enriched membrane fractions in a cell-free secretory system, and purified actin and recombinant SNAREs.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"135-147"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979134","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
首页 上一页 下一页 尾页
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整，为了账户安全，请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
请完成安全验证×
相关产品
×
本文献相关产品
联系我们：info@booksci.cn Book学术提供免费学术资源搜索服务，方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号：481959085
Book学术官方微信