{"title":"Precision Cell-Cell Assembly Through Light-Mediated DNA Interactions.","authors":"Katelyn Mathis, Brian Meckes","doi":"10.1007/978-1-0716-4402-7_11","DOIUrl":"https://doi.org/10.1007/978-1-0716-4402-7_11","url":null,"abstract":"<p><p>Complex interactions between diverse cell populations influence processes ranging from cell division to programmed cell death. Replicating this complexity with reproducibility remains challenging. We introduce an innovative approach that combines DNA-mediated interactions with photolithography to achieve controlled cell-cell interactions. By coating cells with DNA sequences responsive to light, we enable meticulous spatial organization through light activation, facilitating rapid and programmable construction of intricate cell structures. This method not only addresses the challenges of orchestrating cell arrangements in vitro, particularly in three-dimensional settings but also offers a new avenue for the on-demand creation of complex cellular architectures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"173-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542675","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}
{"title":"Surface Functionalization of Elastomers with Biopolymers.","authors":"Emilie Morin, Elana Muzzy, Andrea S Carlini","doi":"10.1007/978-1-0716-4402-7_13","DOIUrl":"https://doi.org/10.1007/978-1-0716-4402-7_13","url":null,"abstract":"<p><p>Biopolymer coatings on elastomeric surfaces have significant impact for advancements in biomedicine as they combine flexible devices with complex biological functionality. Biopolymers offer increased ability for antimicrobial coatings, sensing of relevant biological markers, and controlled drug delivery. The methodologies available to conjugate these important biopolymers to flexible elastomeric substrates are vast and rapidly evolving. This chapter aims to compile methodologies across the application space of biopolymer conjugation to elastomers. We present a guide to the field and methods ranging from surface activation and functionalization, grafting-to and grafting-from of biopolymers, and characterization of the resulting substrates.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"197-227"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542676","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}
Ana Flores, Laura Fernández-Sánchez, Oksana Kutsyr, Alberto Yáñez, María Luisa Gil, Daniel Gozalbo, Victoria Maneu, Pedro Lax
{"title":"Nonhematopoietic Stem Cell Identification and Sorting in Adult Retina.","authors":"Ana Flores, Laura Fernández-Sánchez, Oksana Kutsyr, Alberto Yáñez, María Luisa Gil, Daniel Gozalbo, Victoria Maneu, Pedro Lax","doi":"10.1007/978-1-0716-4386-0_5","DOIUrl":"https://doi.org/10.1007/978-1-0716-4386-0_5","url":null,"abstract":"<p><p>Flow cytometry enables the detection and characterization of specific cell populations, and proper identification of particular cell types can be assessed by immunohistochemistry. Here, we describe the identification and sorting of nonhematopoietic stem cells expressing stem cell antigen-1 from adult murine retinas using flow cytometry and immunohistochemistry strategies. These sorted cells can be further used for regenerative purposes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2899 ","pages":"59-65"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605437","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}
Ane Rodríguez-Bodero, Paolo Bonifazi, Jan Tønnesen, Juan Manuel Encinas-Pérez
{"title":"Picrotoxin-Induced Epileptogenic Hippocampal Organotypic Slice Cultures (hOTCs).","authors":"Ane Rodríguez-Bodero, Paolo Bonifazi, Jan Tønnesen, Juan Manuel Encinas-Pérez","doi":"10.1007/978-1-0716-4386-0_23","DOIUrl":"https://doi.org/10.1007/978-1-0716-4386-0_23","url":null,"abstract":"<p><p>Cultured organotypic hippocampal slices (hOTCs) have become increasingly popular as a model for studying brain function. This model offers significant advantages over traditional in vitro methods, as they allow the examination of mid to long-term manipulations while preserving the structure of the dentate gyrus (DG) in the hippocampus. In this chapter, we focus on a protocol based on hOTCs of mouse entorhinal cortex and hippocampus, which by integrating techniques such as retroviral injections, immunohistochemistry, and microscopy imaging, physiological or pathological processes can be easily investigated.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2899 ","pages":"367-388"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605499","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}
{"title":"Agroinoculation of Plants with Geminiviruses.","authors":"Anelise F Orílio","doi":"10.1007/978-1-0716-4454-6_5","DOIUrl":"https://doi.org/10.1007/978-1-0716-4454-6_5","url":null,"abstract":"<p><p>Agroinoculation refers to the introduction of genetic information from plant viruses into leaves or other organs of the host plant through Agrobacterium tumefaciens. Agroinoculation of geminiviruses requires the insertion of tandem copies of the genome between the T-DNA borders of an Agrobacterium binary vector. The technique is applicable to most plant species and is limited only by the host range of the virus and of the A. tumefaciens strain. Herbaceous plants can be easily agroinoculated using a needleless syringe. The successful agroinoculation of plants with begomoviruses involves the preparation of A. tumefaciens competent cells, the transformation of A. tumefaciens with the cloned tandem copy of the viral genome, and the agroinoculation procedure itself. This chapter describes a straightforward protocol for the agroinoculation of a cloned infectious begomovirus into plants.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2912 ","pages":"29-34"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597341","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}
Christiane Eliza Motta Duarte, João Paulo Batista Machado, Bianca Gouveia-Mageste, Fredy Davi Albuquerque Silva, Elizabeth Pacheco Batista Fontes
{"title":"Subcellular Localization of Geminivirus Proteins by Laser Scanning Confocal Microscopy.","authors":"Christiane Eliza Motta Duarte, João Paulo Batista Machado, Bianca Gouveia-Mageste, Fredy Davi Albuquerque Silva, Elizabeth Pacheco Batista Fontes","doi":"10.1007/978-1-0716-4454-6_18","DOIUrl":"https://doi.org/10.1007/978-1-0716-4454-6_18","url":null,"abstract":"<p><p>In eukaryotic cells, the subcellular localization of proteins is inherently linked to their function. Since viruses rely on the host cellular machinery to complete their life cycle, viral proteins are expected to employ the host transport machinery to reach various compartments. Several factors, including the multifunctional nature of viral proteins, the stage of virus infection, and interactions with both viral and host proteins, influence the final destination of viral proteins. For instance, NSP (nuclear shuttle protein) from bipartite begomoviruses and CP (coat protein) from monopartite begomoviruses typically exhibit nuclear localization, yet their subcellular distribution can vary depending on coexpression partners and stage of infection. Virtually all viral proteins display dynamic subcellular distribution patterns that change under their specific functions at different stages of the virus life cycle. Thus, identifying the subcellular distribution of viral proteins is essential for comprehending their multiple roles during infection. This chapter outlines a protocol for efficiently determining the subcellular localization of viral proteins during infection or when expressed with protein partners. The protocol essentially consists of three steps: (i) cloning the viral protein and protein partners fused to fluorescent tags, (ii) transiently expressing the tagged proteins in N. benthamiana leaves, and (iii) determining the subcellular localization of the tagged proteins using confocal microscopy.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2912 ","pages":"205-226"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597373","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}
{"title":"Investigating the Pathology-Related Astroglial Plasticity in the Human Cerebral Cortex.","authors":"Swetlana Sirko, Patrizia Della Vecchia","doi":"10.1007/978-1-0716-4366-2_11","DOIUrl":"https://doi.org/10.1007/978-1-0716-4366-2_11","url":null,"abstract":"<p><p>Evaluation of astrocyte proliferation in human brain pathology plays a key role in the diagnostic and prognostic assessment of diseases, especially in cases of benign or malignant brain neoplasms. However, the proliferative potential of nonneoplastic astrocytes within the affected human brain parenchyma has not been well defined. Given the beneficial functions of proliferating reactive astrocytes for brain repair in experimental models of stroke and trauma, investigating the context-specific potential of human astrocytes to proliferate will be a major step forward in exploiting their roles in the genesis, progression, and outcome of neurological diseases in patients. Here, we describe a step-by-step protocol for immunofluorescent staining and neurosphere-forming assay tailored to uncover the astrocyte proliferation and neural stem cell properties in samples of human brain tissue obtained during neurosurgical resections or in a small brain biopsy. This protocol allows for reliable assessment and evaluation of adoptive astrocyte plasticity in the context with various neuropathological conditions in the human brain.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2896 ","pages":"147-164"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670325","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}
{"title":"Retro-Orbital Delivery of AAVs for CNS Wide Astrocyte Targeting.","authors":"Domenico Natale, Matthew Holt","doi":"10.1007/978-1-0716-4366-2_2","DOIUrl":"https://doi.org/10.1007/978-1-0716-4366-2_2","url":null,"abstract":"<p><p>Viral vector-mediated astrocyte targeting in live mice is a popular and valuable method to investigate astrocyte function in the context of intact neural circuits and complex brain physiology. Targeted genetic manipulation and functional investigation of this cell population can be accomplished by utilizing cell type-specific promoters to drive adeno-associated virus (AAV)-mediated transgene expression specifically in astrocytes. Here, we provide a comprehensive protocol for non-invasive retro-orbital (RO) administration of blood-brain barrier (BBB)-crossing AAVs in neonatal and adult mice, such as AAV-PHP.B, AAV-PHP.eB, and AAV.CAP-B22, which results in central nervous system (CNS)-wide transduction. Key procedures outlined include the preparation of AAV solutions for injection, a modified two-handed injection technique for precise and consistent RO injections, and a training strategy to practice mock RO injections using non-toxic dyes. This protocol serves as a valuable resource for researchers interested in exploring the roles of astrocytes in brain functions and neurological disorders.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2896 ","pages":"13-31"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670390","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}
{"title":"Drug Repositioning Based on Cerebrospinal Fluid Proteomes Using Connectivity Map Framework.","authors":"Adriana Cortés, Silvia Romero-Murillo, Elena Anaya-Cubero, Paz Cartas-Cejudo, Leire Extramiana, Mercedes Lachén-Montes, Joaquín Fernández-Irigoyen, Enrique Santamaría","doi":"10.1007/978-1-0716-4462-1_22","DOIUrl":"https://doi.org/10.1007/978-1-0716-4462-1_22","url":null,"abstract":"<p><p>Selecting a fluid near an affected organ can improve the likelihood of identifying a biomarker panel from pathological tissue. Cerebrospinal fluid (CSF), in close contact with the brain, is a valuable source of biomarkers for neurological disorders due to the inaccessibility of brain tissue. Moreover, the altered CSF proteome identified in neurological diseases can facilitate the repurposing of drugs already used for other therapeutic purposes. In this context, Connectivity Map (CMap) is a valuable tool as it provides information on compounds and gene modifications that can be utilized to reverse specific pathological signatures. Analyzing CSF differential proteomics through the CMap framework offers an efficient and cost-effective approach to identifying potential novel therapies for neurodegenerative diseases.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2914 ","pages":"323-332"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753483","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}
Jessica Elliott, Rachel Koldej, Amit Khot, David Ritchie
{"title":"Graft-Versus-Host Disease Mouse Models: A Clinical-Translational Perspective.","authors":"Jessica Elliott, Rachel Koldej, Amit Khot, David Ritchie","doi":"10.1007/978-1-0716-4430-0_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-4430-0_1","url":null,"abstract":"<p><p>A variety of graft-versus-host disease (GVHD) models have been developed in mice for the purpose of allowing laboratory investigation of the pathobiology, prevention, and treatment of GVHD in humans. While such models are crucial in advancing our knowledge in this field, there are some key limitations that need to be considered when translating laboratory discoveries into the clinical context. This chapter will discuss current clinical practices in transplantation and GVHD and the relative strengths and weaknesses of mouse models that attempt to replicate these states.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"1-56"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657764","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}
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