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Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_17
Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx
{"title":"Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.","authors":"Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx","doi":"10.1007/978-1-0716-4430-0_17","DOIUrl":"https://doi.org/10.1007/978-1-0716-4430-0_17","url":null,"abstract":"<p><p>Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDXs) in immunodeficient NOD-scid-IL2rg<sup>null</sup> (NSG) mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"359-375"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657771","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
Utilization of Clinical Data and Evaluation of Biomarkers in the Investigation of Graft-Versus-Host Disease Outcomes.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_3
Serhat Çelik, Leylagül Kaynar
{"title":"Utilization of Clinical Data and Evaluation of Biomarkers in the Investigation of Graft-Versus-Host Disease Outcomes.","authors":"Serhat Çelik, Leylagül Kaynar","doi":"10.1007/978-1-0716-4430-0_3","DOIUrl":"https://doi.org/10.1007/978-1-0716-4430-0_3","url":null,"abstract":"<p><p>Graft-versus-host disease (GVHD) is one of the most important obstacles after allogeneic hematopoietic stem cell transplantation (allo-HCT). The mortality rate is around 50%, especially in severe GVHD. One of the most important clinical outcomes in GVHD is non-relapse mortality (NRM). NRM was defined as death without evidence of relapse or progression. Kaplan Meier, log-rank test, and Cox model are used in survival analysis methods. There are various biomarkers that assess clinical outcomes of GVHD. Damage-associated molecular patterns, pathogen-associated molecular patterns, microRNAs, markers of endothelial dysfunction, cytokines, and their receptors are used to predict the occurrence of GVHD and clinical outcomes in GVHD. Furthermore, the utilization of panels that assess many biomarkers has proven to be successful in predicting the clinical outcomes of GVHD, particularly NRM.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"71-83"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657784","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
Multiplexed Biomarker Detection Using DNA Payloads: Design, Assembly, and Analysis.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4394-5_16
Matthew Aquilina, Katherine E Dunn
{"title":"Multiplexed Biomarker Detection Using DNA Payloads: Design, Assembly, and Analysis.","authors":"Matthew Aquilina, Katherine E Dunn","doi":"10.1007/978-1-0716-4394-5_16","DOIUrl":"10.1007/978-1-0716-4394-5_16","url":null,"abstract":"<p><p>Most biomarker assays are typically designed to detect a single molecule type (DNA, RNA, proteins, etc.) in a single assay. This means that monitoring a diverse biomarker panel could quickly become a complex endeavor, requiring different techniques, labs, and expertise. In this chapter, we describe a method for multiplexed biomarker detection from a single sample using variable-length DNA payload chains as the output signal. Through the course of the assay, payloads are systematically disassembled in the presence of specific biomarkers. The resulting distinctly sized fragments then yield characteristic gel electrophoresis band patterns, which can be detected and quantified using image analysis algorithms. We detail the entire process for constructing DNA payloads and conducting a biomarker detection assay, including the sequence design, laboratory assembly, running the assay, and final image analysis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2901 ","pages":"203-226"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143772926","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
Rapid and High-Yielding Purification of DNA Self-Assembled Structures by Aqueous Two-Phase System. 利用水性两相系统快速高产地纯化 DNA 自组装结构
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4394-5_2
Marcos K Masukawa, Masahiro Takinoue
{"title":"Rapid and High-Yielding Purification of DNA Self-Assembled Structures by Aqueous Two-Phase System.","authors":"Marcos K Masukawa, Masahiro Takinoue","doi":"10.1007/978-1-0716-4394-5_2","DOIUrl":"10.1007/978-1-0716-4394-5_2","url":null,"abstract":"<p><p>Aqueous two-phase systems (ATPS) of dextran and polyethylene glycol (PEG) enable the purification of DNA structures such as DNA origami and DNA nanotubes in times as short as 10 min. This method, which has recovery yields >90% for a typical DNA origami, owes its efficiency to the highly selective partition of the DNA structures in the dextran phase of these emulsions. This purification method is carried out in conditions that promote the structural stability of these structures, making it particularly suitable for DNA nanotechnology. In this protocol, we will describe the materials and methods for purifying DNA origami and quantifying the purification yield by agarose electrophoresis and image analysis.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2901 ","pages":"13-25"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143772929","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
Trabecular Meshwork Regeneration for Glaucoma Treatment Using Stem Cell-Derived Trophic Factors. 利用干细胞衍生的营养因子再生小梁网治疗青光眼
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4087-6_4
Ajay Kumar, Enzhi Yang, Yiqin Du
{"title":"Trabecular Meshwork Regeneration for Glaucoma Treatment Using Stem Cell-Derived Trophic Factors.","authors":"Ajay Kumar, Enzhi Yang, Yiqin Du","doi":"10.1007/978-1-0716-4087-6_4","DOIUrl":"10.1007/978-1-0716-4087-6_4","url":null,"abstract":"<p><p>Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2848 ","pages":"59-71"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11971979/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140524","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
Using Real-Time Quaking-Induced Conversion (RT-QuIC) in Clinical Practice for Creutzfeldt-Jakob Disease Diagnostics.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4462-1_2
Tuane Cristine R G Vieira, Breno José Alencar Pires Barbosa
{"title":"Using Real-Time Quaking-Induced Conversion (RT-QuIC) in Clinical Practice for Creutzfeldt-Jakob Disease Diagnostics.","authors":"Tuane Cristine R G Vieira, Breno José Alencar Pires Barbosa","doi":"10.1007/978-1-0716-4462-1_2","DOIUrl":"https://doi.org/10.1007/978-1-0716-4462-1_2","url":null,"abstract":"<p><p>Prion diseases, a group of fatal neurodegenerative disorders, present significant diagnostic challenges due to their subtle clinical manifestations, which often mimic those of other treatable neurological conditions. This diagnostic ambiguity underscores the necessity for reliable and specific diagnostic tools. Real-Time Quaking-Induced Conversion (RT-QuIC) has emerged as a groundbreaking technique for diagnosing these diseases using cerebrospinal fluid (CSF) samples. This chapter outlines a comprehensive and standardized protocol for employing RT-QuIC to detect the pathological form of the prion protein in CSF, offering a powerful tool for clinical diagnosis. It provides step-by-step instructions for patient examinations, laboratory RT-QuIC analysis, and data interpretation. By adhering to these protocols, researchers and clinicians can achieve high sensitivity and specificity in diagnosing prion diseases, facilitating timely and accurate patient management.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2914 ","pages":"15-24"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143753409","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
Agrobacterium-Mediated Transformation of Prunus persica (Peach) Immature Cotyledons.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4450-8_18
Carolina Toro, Marion Barrera, Blanca Olmedo, Ricardo Vergara, Marisol Muñoz, Felipe Olivares, Humberto Prieto
{"title":"Agrobacterium-Mediated Transformation of Prunus persica (Peach) Immature Cotyledons.","authors":"Carolina Toro, Marion Barrera, Blanca Olmedo, Ricardo Vergara, Marisol Muñoz, Felipe Olivares, Humberto Prieto","doi":"10.1007/978-1-0716-4450-8_18","DOIUrl":"https://doi.org/10.1007/978-1-0716-4450-8_18","url":null,"abstract":"<p><p>Genetic transformation in Prunus persica genotypes could add single horticultural traits in existing cultivars without modifying their commercial characteristics. Despite this opportunity, the final setting of transformation protocols in the species endures two major limiting factors preventing the development of new varieties: (a) explants are recalcitrant in regenerating adventitious transformed shoots and (b) have a limited regeneration capability, usually extended to just a few genotypes (i.e., cultivar dependence). This protocol illustrates a procedure that has allowed the establishment of transformation methodologies in several varieties of the species and is presented here for Red Top and Elegant Lady; this methodology could help extend technical baselines for successful transformation procedures in other, potentially more recalcitrant, and newly developed cultivars or genus members.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2911 ","pages":"189-198"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730752","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
Genetic Transformation of Lettuce (Lactuca sativa) Using Agrobacterium tumefaciens.
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4450-8_9
Damaris Godinez-Vidal, Javier Narváez-Vásquez, Martha L Orozco-Cárdenas
{"title":"Genetic Transformation of Lettuce (Lactuca sativa) Using Agrobacterium tumefaciens.","authors":"Damaris Godinez-Vidal, Javier Narváez-Vásquez, Martha L Orozco-Cárdenas","doi":"10.1007/978-1-0716-4450-8_9","DOIUrl":"https://doi.org/10.1007/978-1-0716-4450-8_9","url":null,"abstract":"<p><p>Lettuce (Lactuca sativa L.) plays a pivotal role in global agriculture and food supply chains as a widely cultivated commercial crop. This chapter presents a detailed protocol for transforming lettuce using Agrobacterium tumefaciens. The procedure includes a two-day pre-culture period of cotyledon explants, followed by a 20-min inoculation with Agrobacterium without explant wounding, and a two-day co-culture on the same medium. Afterward, the selection and regeneration phases under kanamycin selection led to the establishment of transgenic plants in the greenhouse. The entire process from the initial explant infection to the greenhouse cultivation of transgenic plants spans roughly 120 days, achieving an average transformation efficiency of 45%. This protocol offers a reliable method for the genetic modification of lettuce, for use in research or crop improvement.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2911 ","pages":"83-95"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730754","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
An Evolutionary Framework Exploiting Virologs and Their Host Origins to Inform Poxvirus Protein Functions. 利用病毒学及其宿主起源为痘病毒蛋白功能提供信息的进化框架。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4160-6_17
Dustin C Hancks
{"title":"An Evolutionary Framework Exploiting Virologs and Their Host Origins to Inform Poxvirus Protein Functions.","authors":"Dustin C Hancks","doi":"10.1007/978-1-0716-4160-6_17","DOIUrl":"10.1007/978-1-0716-4160-6_17","url":null,"abstract":"<p><p>Poxviruses represent evolutionary successful infectious agents. As a family, poxviruses can infect a wide variety of species including humans, fish, and insects. While many other viruses are species-specific, an individual poxvirus species is often capable of infecting diverse hosts and cell types. For example, the prototypical poxvirus, vaccinia, is well known to infect numerous human cell types but can also infect cells from divergent hosts like frog neurons. Notably, poxvirus infections result in both detrimental human and animal diseases. The most infamous disease linked to a poxvirus is smallpox caused by variola virus. Poxviruses are large double-stranded DNA viruses, which uniquely replicate in the cytoplasm of cells. The model poxvirus genome encodes ~200 nonoverlapping protein-coding open reading frames (ORFs). Poxvirus gene products impact various biological processes like the production of virus particles, the host range of infectivity, and disease pathogenesis. In addition, poxviruses and their gene products have biomedical application with several species commonly engineered for use as vaccines and oncolytic virotherapy. Nevertheless, we still have an incomplete understanding of the functions associated with many poxvirus genes. In this chapter, we outline evolutionary insights that can complement ongoing studies of poxvirus gene functions and biology, which may serve to elucidate new molecular activities linked to this biomedically relevant class of viruses.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2860 ","pages":"257-272"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142770398","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
Bioinformatics for the Structural Genomics of Poxviruses. 痘病毒结构基因组学的生物信息学。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4160-6_5
Paolo Ribeca
{"title":"Bioinformatics for the Structural Genomics of Poxviruses.","authors":"Paolo Ribeca","doi":"10.1007/978-1-0716-4160-6_5","DOIUrl":"10.1007/978-1-0716-4160-6_5","url":null,"abstract":"<p><p>Poxviruses are large, complex viruses, and their host species are widespread across the tree of life. As a result, the bioinformatics analysis of their genomes can be complex. Here we show how a few helpful tools and strategies can be used to inform the analysis, leading to a better understanding of the structural properties of poxvirus genomes and to a more accurate quality control of, or comparison between, assembled sequences.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2860 ","pages":"65-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142770400","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
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