Methods in molecular biology最新文献

{"title":"Transient Transfection and T Cell Activation in the Assessment of Endoplasmic Reticulum Aminopeptidase 1 and 2 Peptide Trimming Function.","authors":"Emma Reeves, Edward James","doi":"10.1007/978-1-0716-5092-9_18","DOIUrl":"10.1007/978-1-0716-5092-9_18","url":null,"abstract":"<p><p>Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play a critical role in antigen processing by trimming N-terminally extended peptides within the ER, thereby shaping the repertoire of peptides presented by Major Histocompatibility Complex Class I (MHC I) molecules. Polymorphic variants in these enzymes give rise to functionally distinct allotypes, influencing peptide trimming efficiency and specificity, modulating CD8+ T cell responses in both health and disease. This chapter outlines a cellular model system for assessing ERAP1 and ERAP2 peptide trimming activity, using peptide-specific T cell activation as a surrogate readout. By employing transient transfection and co-culture with either T cell hybridoma (B3Z) or cytotoxic T lymphocytes (CTL), this approach enables the evaluation of peptide processing efficiency of ERAP1/2 based on the presentation and recognition of optimally generated MHC I-restricted epitopes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3007 ","pages":"275-289"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889828","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":"Analysis of PD-L1 Transcriptional Activity by Chromatin Immunoprecipitation.","authors":"Suprataptha U Reddy, Riddhi Mehta, Ivana Vancurova","doi":"10.1007/978-1-0716-4901-5_19","DOIUrl":"10.1007/978-1-0716-4901-5_19","url":null,"abstract":"<p><p>Immune checkpoint PD-L1 was originally identified as a cell surface transmembrane protein, but recent studies have demonstrated its presence also in the nucleus and suggested its role in transcriptional regulation. Interleukin-8 (IL-8, CXCL8) is a pro-angiogenic chemokine that promotes cancer progression. We have recently shown that in ovarian cancer (OC) cells, IFNγ induces nuclear accumulation of PD-L1, which is then recruited to IL-8 promoter, resulting in increased transcription of IL-8. Since the increased expression of IL-8 induces proliferation and invasion in OC cells, understanding the role of PD-L1 in transcriptional regulation of IL-8 is important for increasing the effectiveness of PD-L1 targeting immunotherapies. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) followed by real time PCR to quantitatively measure PD-L1 recruitment to IL-8 promoter. The main points of the protocol are the ChIP analysis of PD-L1 recruitment to IL-8 promoter using antibody that specifically recognizes endogenous PD-L1, and quantitative real time PCR using primers for human IL-8 promoter spanning the transcription start site (TSS) and a region ~500 bp upstream of the TSS. Our results show that IFNγ significantly increases PD-L1 recruitment to TSS of IL-8 promoter and this recruitment is even higher to the ~500 bp upstream region containing multiple transcription factors binding sites, suggesting that PD-L1 associates with IL-8 promoter by binding to other transcription factors.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"217-229"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889349","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":"In Silico Approaches in Predictive Genetic Toxicology.","authors":"Meetali Sinha, Tanya Jamal, Alok Dhawan, R Parthasarathi","doi":"10.1007/978-1-0716-4976-3_26","DOIUrl":"10.1007/978-1-0716-4976-3_26","url":null,"abstract":"<p><p>In silico toxicology methods facilitate clear interpretation of the observed experimental genetic toxicity trends and provide faster, more economical, and animal-free tools for the benefit of environmental and human health in conjunction with other relevant in vitro and in vivo methods. Regulatory agencies are emphasizing the use of computational approaches to predict genetic toxicity to reduce the use of animals in toxicity testing of chemicals. Combination of in vivo (existing data), in vitro, high-throughput, and content screening data together with computational predictions might improve the predictive confidence in the genotoxicity assessment. This is an immensely difficult task, and twenty-first-century toxicology will not become animal-free overnight but is likely to use relevant and reliable computational approaches that will evolve and adapt to best use these scientific and technological advances. Expert-based, statistical QSAR models, and read-across are the most commonly used computational methodologies, and each predictive model adheres to the OECD QSAR validation principle along with an expert review system to conclude. The key steps involved in the in silico genotoxicity prediction are identification of the problem, data collection, generation of chemical descriptors, construction of the Q/SAR model, internal/external validation of the model, statistical evaluation of the models, and optimization of modeling parameters to further enhance SAR performance, updating a validated model with new chemical sets and finally reporting and documentation of the complete study. This chapter describes various in silico toxicology approaches and standardized protocols for conducting genetic toxicity predictions of chemicals and also highlights various parameters for the validation of the prediction results obtained from QSAR models.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2986 ","pages":"565-586"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582326","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":"Application of the Comet Assay in Advanced In Vitro Models.","authors":"Elise Rundén-Pran, Naouale El Yamani, Sivakumar Murugadoss, Tanima SenGupta, Eleonora Marta Longhin, Ann-Karin Hardie Olsen, Tatiana Honza, Alexandra Misci Hudecova, Erin McFadden, Solveig Brochmann, Xiaoxiong Ma, Maria Dusinska","doi":"10.1007/978-1-0716-4976-3_16","DOIUrl":"10.1007/978-1-0716-4976-3_16","url":null,"abstract":"<p><p>The comet assay (single-cell gel electrophoresis) is a simple, cost-efficient, robust, reliable, and user-friendly method for measuring DNA damage. The in vitro comet assay can be applied in advanced in vitro mini-organ and mini-tissue models. Higher-throughput formats of the assay, such as 48/96 mini-gels on GelBond^® film, and the 12-mini-gel slide format, in combination with automated scoring, make the comet assay a valuable screening method for the genotoxic potential of chemicals. In compliance with the 3Rs to reduce, refine, and replace animal experiments, the development of new approach methods (NAMs) is an important part of the paradigm shift in toxicology toward Next Generation Risk Assessment (NGRA), based on non-animal hazard identification and characterization of chemicals. In the case of advanced cell models, the cells are often grown in three-dimensional (3D) culture, and multiple cell types representative of different organs can be co-cultivated. We demonstrate the applicability of the comet assay with commonly applied advanced models from liver, lung, breast, gut, skin, and brain. We describe both the in vitro standard alkaline version of the comet assay (ACA) which measures DNA strand breaks, and the enzyme-linked modification (ELCA) that allows detection of specific base alterations by applying lesion-specific endonucleases (e.g., formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) for oxidized purines and pyrimidines, respectively). We consider basic methodological issues, experimental design including treatment conditions, and the importance of including cytotoxicity testing-all of which could have an impact on and/or give biased results. Protocols are provided for both the standard 2-gel and 12-gel slide formats. Further, we address critical points that need to be taken into consideration when assessing genotoxicity. The adaptation of the comet assay to advanced models such as 3D cell cultures, co-cultures, and air-liquid interface (ALI) exposure systems marks a significant advance in genotoxicity testing. These models offer more biologically relevant contexts for measuring DNA damage and repair, leading to better risk assessment and the development of safer chemicals, such as pharmaceuticals, cosmetics, food additives, as well as influencing environmental policies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2986 ","pages":"337-383"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582237","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":"Multicolor Laser Scanning Confocal Immunofluorescence Microscopy of DNA Damage Response Biomarkers.","authors":"Julian Laubenthal, Bart Krist, Alok Dhawan, Diana Anderson, Michał R Gdula","doi":"10.1007/978-1-0716-4976-3_14","DOIUrl":"10.1007/978-1-0716-4976-3_14","url":null,"abstract":"<p><p>DNA damage through endogenous and environmental toxicants is a constant threat to both a human's ability to pass on intact genetic information to its offspring as well as in somatic cells for its own survival. To counter these threats posed by DNA damage, cells have evolved a series of highly choreographed mechanisms, collectively defined as the DNA-damage response (DDR), to sense DNA lesions, signal their presence, and mediate their repair. Thus, regular DDR signaling cascades are vital to prevent the initiation and progression of many human diseases including cancer. Consequently, quantitative assessment of DNA damage and response became an important biomarker for assessment of human health and disease risk in biomonitoring studies. However, most quantitative DNA damage biomarker techniques require dissolution of the nuclear architecture and hence loss of spatial information. Laser scanning confocal immunofluorescence microscopy (LSCIM) of three-dimensionally preserved nuclei can be, quantitative and maintain the spatial information. Here we describe the experimental protocols to quantify individual key events of the DDR cascade in three-dimensionally preserved nuclei by LSCIM with high resolution, using the simultaneous detection of Rad50 as well as phosphorylated H2AX and ATM and in somatic and germ cells as an example.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2986 ","pages":"297-311"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145582392","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":"Interaction Between an Individual Cohesin Complex and DNA Revealed by Optical Tweezers and Single-Molecule Microscopy.","authors":"Martina Richeldi, Maxim Molodtsov","doi":"10.1007/978-1-0716-5005-9_2","DOIUrl":"10.1007/978-1-0716-5005-9_2","url":null,"abstract":"<p><p>Establishment of cohesion between sister chromatids in mitosis is one of the fundamental principles used by cells to ensure accuracy of the chromosome segregation. Cohesion allows spindle microtubules to generate tension across sister chromatids that acts as a signal that correct spindle-chromosome connections have been made. Cohesion is established by cohesin-a ring-shaped protein complex that is thought to link sister chromatids by physically entrapping their DNA. However, molecular mechanisms underlying cohesin interaction with DNA and its ability to form DNA-DNA interactions are not well understood. Here, we describe a protocol to reconstitute this process at the single-molecule level and investigate how single cohesin protein complexes establish DNA-DNA interactions and resist mechanical forces exerted on DNA.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2991 ","pages":"21-36"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329558","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":"Development of Electric Field Stimulation (EFS)-Mediated Skeletal Muscle Training with iPSCs.","authors":"Tomoya Uchimura, Hidetoshi Sakurai","doi":"10.1007/978-1-0716-4738-7_13","DOIUrl":"10.1007/978-1-0716-4738-7_13","url":null,"abstract":"<p><p>Functional analysis of skeletal muscle in vitro requires maturation and stimuli to induce excitation-contraction coupling as well as a quantitative method to analyze contraction. Electric field stimulation (EFS) has been traditionally used as a method to excite skeletal muscle in vitro and in vivo. A combination of human-induced pluripotent stem cell (hiPS) technology and EFS will lead to recapitulation of functional phenotypes of skeletal muscle diseases. Here, we describe the combinational technology using hiPS technology and EFS to induce the maturation of skeletal muscle and analyze contractile performance in vitro.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2963 ","pages":"185-194"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145199974","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":"DG9-Conjugated Morpholino-Based Exon 51-Skipping Therapy for Duchenne Muscular Dystrophy.","authors":"Md Nur Ahad Shah, Laura Edellein Sutanto, Toshifumi Yokota","doi":"10.1007/978-1-0716-4738-7_1","DOIUrl":"10.1007/978-1-0716-4738-7_1","url":null,"abstract":"<p><p>Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene that leads to the loss of the dystrophin protein. Exon-skipping therapy with phosphorodiamidate morpholino oligomers (PMOs) has been shown to restore the disrupted open-reading frame to produce a shortened yet functional dystrophin. The applications of this treatment, however, are limited in terms of its efficiency. A potential solution to this problem is to conjugate the PMOs to cell-penetrating peptides. DG9 is a novel peptide that has exhibited promising results in terms of its ability to enhance the cellular uptake of PMOs to increase exon-skipping efficiency in both the skeletal and heart muscles. In this chapter, we describe the systemic injection of DG9-conjugated PMOs to induce the skipping of exon 51 in an exon 52-deleted mouse model, hDMDdel52;mdx, which is a humanized dystrophic mouse model that mimics human DMD patients by having a human DMD gene integrated into them with a disrupted reading frame. As a result, this genetic makeup makes the model useful for testing the safety and efficacy of DG9-PMO as a potential treatment for DMD. We also describe the methodology to assess the efficacy and safety of the DG9-PMO treatment, including systematic DG9-PMO injections, RT-PCR, western blotting, histology, and functional tests.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2963 ","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145199987","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":"Evaluation for the Efficacy of Exon-Skipping Therapy Using In Vivo Assessment of Skeletal Muscle Function and Fragility.","authors":"Yuki Ashida, Katsura Minegishi, Norio Motohashi, Yoshitsugu Aoki, Takashi Yamada","doi":"10.1007/978-1-0716-4738-7_6","DOIUrl":"10.1007/978-1-0716-4738-7_6","url":null,"abstract":"<p><p>Splice-switching oligonucleotide (SSO) therapy is expected to be a promising treatment for muscular diseases such as Duchenne muscular dystrophy (DMD). Preclinical studies using animal models of muscle diseases are essential for the development of SSO therapy. In addition to the histological and biochemical analyses, an in vivo muscle torque measurement using supramaximal electrical stimulation has become a standard protocol for the evaluation of drug efficacy. Nevertheless, in order to accurately measure the torque production in animal models, there are some critical points that should be brought to attention. In this chapter, we describe detailed methods for in vivo muscle torque measurement in animal models for DMD, i.e., mdx52 mouse and canine X-linked muscular dystrophy in Japan (CXMD_J), that set the standard for reproducibility in this field.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2963 ","pages":"91-104"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200043","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":"Utilization of Induced Pluripotent Stem Cell-Derived Neurons to Investigate the Splice-Modification Efficacy of Splice-Switching Drug Candidates.","authors":"Yukiko Ueta, Hiroaki Ohara, Tomonari Awaya, Masatoshi Hagiwara","doi":"10.1007/978-1-0716-4738-7_12","DOIUrl":"10.1007/978-1-0716-4738-7_12","url":null,"abstract":"<p><p>Modification of RNA splicing represents a promising therapeutic strategy for treating genetic diseases. Several splice-switching drugs have already been approved, notably for spinal muscular atrophy and specific genotypes of Duchenne muscular dystrophy. While peripheral blood mononuclear cells and fibroblasts are commonly used to evaluate the efficacy of these drug candidates in patient-derived samples, such approaches may lead to misleading conclusions due to the cell type-specific nature of RNA splicing.Induced pluripotent stem cells (iPSCs), which can be differentiated into various somatic cell types, offer a valuable tool to overcome this limitation. Once efficient differentiation protocols are established, iPSCs can give rise to neurons, retinal cells, cardiomyocytes, and other hard-to-obtain cell types. In this chapter, we describe a protocol for evaluating the splice-modification efficacy of drug candidates in iPSC-derived neurons, using the removal of a poison exon in SCN1A gene, associated with Dravet syndrome as a model.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2963 ","pages":"173-184"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200158","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}