Bio-protocol最新文献

{"title":"Protocol to Retrieve Unknown Flanking DNA Using Fork PCR for Genome Walking.","authors":"Hongjing Wu, Hao Pan, Haixing Li","doi":"10.21769/BioProtoc.5161","DOIUrl":"10.21769/BioProtoc.5161","url":null,"abstract":"<p><p>PCR-based genome walking is one of the prevalent techniques implemented to acquire unknown flanking genomic DNAs. The worth of genome walking includes but is not limited to cloning full-length genes, mining new genes, and discovering regulatory regions of genes. Therefore, this technique has advanced molecular biology and related fields. However, the PCR amplification specificity of this technique needs to be further improved. Here, a practical protocol based on fork PCR is proposed for genome walking. This PCR uses a fork primer set of three arbitrary primers to execute walking amplification task, where the primary fork primer mediates walking by partially annealing to an unknown flank, and the fork-like structure formed between the three primers participates in inhibiting non-target amplification. In primary fork PCR, the low-annealing temperature (25 °C) cycle allows the primary fork primer to anneal to many sites of the genome, synthesizing a cluster of single-stranded DNAs; the subsequent 65 °C cycle processes the target single-strand into double-strand via the site-specific primer; then, the remaining 65 °C cycles selectively enrich this target DNA. However, any non-target single-stranded DNA formed in the 25 °C cycle cannot be further processed in the following 65 °C cycles because it lacks an exact binding site for any primer. Secondary, or even tertiary nested fork PCR further selectively enriches the target DNA. The practicability of fork PCR was validated by walking three genes in <i>Levilactobacillus brevis</i> CD0817 and one gene in <i>Oryza sativa</i>. The results indicated that the proposed protocol can serve as a supplement to the existing genome walking protocols. Key features • This protocol builds upon the method developed by Pan et al. [1], which is applicable to genome-walking for any species. • The developed protocol is a random priming PCR-based genome-walking scheme. • Two rounds of nested fork PCR amplifications suffice to release a positive walking result.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5161"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769745/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054519","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}

{"title":"Primary Neuronal Culture and Transient Transfection.","authors":"Shun-Cheng Tseng, Peng-Tzu Chen, Eric Hwang","doi":"10.21769/BioProtoc.5169","DOIUrl":"10.21769/BioProtoc.5169","url":null,"abstract":"<p><p>Primary neuronal culture and transient transfection offer a pair of crucial tools for neuroscience research, providing a controlled environment to study the behavior, function, and interactions of neurons in vitro. These cultures can be used to investigate fundamental aspects of neuronal development and plasticity, as well as disease mechanisms. There are numerous methods of transient transfection, such as electroporation, calcium phosphate precipitation, or cationic lipid transfection. In this protocol, we used electroporation for neurons immediately before plating and cationic lipid transfection for neurons that have been cultured for a few days in vitro. In our experience, the transfection efficiency of electroporation can be as high as 30%, and cationic lipid transfection has an efficiency of 1%-2%. While cationic lipid transfection has much lower efficiency than electroporation, it does offer the advantage of a higher expression level. Therefore, these transfection methods are suitable for different stages of neurons and different expression requirements. Key features • Culture of primary neurons from the CNS. • Electroporation for freshly isolated neurons in suspension. • Cationic lipid transfection for adherent neurons.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5169"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054511","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}

{"title":"Mouse Model of Lipopolysaccharide (LPS)-Induced Pulpitis.","authors":"Lanting Shao, Baian Chen, Ying Zheng","doi":"10.21769/BioProtoc.5128","DOIUrl":"10.21769/BioProtoc.5128","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Pulpitis is an important and prevalent disease within the oral cavity. Thus, animal models are necessary tools for basic research focused on pulpitis. Researchers worldwide often use dogs and miniature pigs to construct animal models of pulpitis. However, gene editing in miniature pigs is difficult, the surgical modeling process is complex, and tooth demineralization time is lengthy. Although some researchers have attempted to establish a mouse model of pulpitis, most models have involved direct exposure of dental pulp. However, the causes of pulpitis vary considerably among individuals, hindering effective research. In this study, we established a mouse model of pulpitis by accessing the pulp cavity, exposing the pulp to lipopolysaccharide (LPS), and then filling the tooth. One day after surgery, we observed many necrotic tissues and extensive inflammatory exudate, including neutrophils, around the coronal cavity preparation. Additionally, we noted many more neutrophils and a small amount of chronic inflammatory cell infiltrates at the junction between inflamed and normal tissue. These findings indicated that our model can be used to explore the early stage of pulpitis. Ten days after surgery, we observed vacuolar degeneration in some fibroblasts and proliferation in others at the distal end of the inflamed tissue. We also noted dilation and congestion of the pulp blood vessels. Therefore, our model can also be used to explore the middle and later stages of pulpitis. Thirty days after surgery, we observed necrosis in the coronal pulp cavity and upper half of the root pulp, indicating that our model can also be used to explore the end stage of pulpitis. This model is easy to establish, shows pulpitis progression in the dental pulp, exhibits a clear inflammatory phenotype, and can be readily combined with gene editing techniques. Accordingly, it is suitable for basic research focused on pulpitis and has substantial practical value. Key features • Lipopolysaccharide (LPS) can induce pulpitis in mice. • The mouse model of LPS-induced pulpitis can be used in basic studies of pulpitis. • After 1 day, the mouse model of LPS-induced pulpitis can demonstrate the main phenotypes of early-stage pulpitis. • After 10 days, the mouse model of LPS-induced pulpitis can display the main phenotypes of middle and late stage pulpitis. Graphical overview Figure 1.Graphical overview of the C57BL/6 mouse model of lipopolysaccharide (LPS)-induced pulpitis.A. Weigh the mouse. B. Anesthetize the mouse. C. Secure the mouse to the surgical pad and expose its oral cavity. D. Open the pulp chamber of the right maxillary first molar. E. Rinse the medullary foramen with 0.9% NaCl solution. Apply a small cotton ball saturated with 1 mg/mL LPS to the medullary foramen for 5 min, then cover the medullary foramen with Esthet-X flow and irradiate the site. F. Perform tissue decalcification and paraffin embedding (sample collection, decalcification, dehydration, wax embedding,","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5128"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054509","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}

{"title":"An Optimized Protocol for Simultaneous Propagation of Patient-derived Organoids and Matching CAFs.","authors":"Jenny M Högström, Taru Muranen","doi":"10.21769/BioProtoc.5160","DOIUrl":"10.21769/BioProtoc.5160","url":null,"abstract":"<p><p>Recurrent hormone receptor-positive (HR+) breast cancer is a leading cause of cancer mortality in women. Recurrence and resistance to targeted therapies have been difficult to study due to the long clinical course of the disease, the complex nature of resistance, and the lack of clinically relevant model systems. Existing models are limited to a few HR+ cell lines, organoid models, and patient-derived xenograft models, all lacking components of the human tumor microenvironment. Furthermore, the low take rate and loss of estrogen receptor (ER) expression in patient-derived organoids (PDOs) has been challenging. Our protocol allows simultaneous isolation of PDOs and matching cancer-associated fibroblasts (CAFs) from primary and metastatic HR+ breast cancers. Importantly, our protocol has a higher take rate and enables long-term culturing of PDOs that retain ER expression. Our matching PDOs and CAFs will provide researchers with a new resource to study the influence of the tumor microenvironment on various aspects of cancer biology such as cell growth and drug resistance in HR+ breast cancer. Key features • Propagation of patient-derived organoids and matching cancer-associated fibroblasts from primary and metastatic hormone receptor (HR+) positive breast cancer. • Optimized media for long-term culturing of HR+ organoids from primary tumors and bone metastasis. • Co-culture model to assess the influence of the tumor stroma on breast cancer progression.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5160"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769750/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054541","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}

{"title":"Using DIMet for Differential Analysis of Labeled Metabolomics Data: A Step-by-step Guide Showcasing the Glioblastoma Metabolism.","authors":"Johanna Galvis, Joris Guyon, Thomas Daubon, Macha Nikolski","doi":"10.21769/BioProtoc.5168","DOIUrl":"10.21769/BioProtoc.5168","url":null,"abstract":"<p><p>Stable-isotope resolved metabolomics (SIRM) is a powerful approach for characterizing metabolic states in cells and organisms. By incorporating isotopes, such as ¹³C, into substrates, researchers can trace reaction rates across specific metabolic pathways. Integrating metabolomics data with gene expression profiles further enriches the analysis, as we demonstrated in our prior study on glioblastoma metabolic symbiosis. However, the bioinformatics tools for analyzing tracer metabolomics data have been limited. In this protocol, we encourage the researchers to use SIRM and transcriptomics data and to perform the downstream analysis using our software tool DIMet. Indeed, DIMet is the first comprehensive tool designed for the differential analysis of tracer metabolomics data, alongside its integration with transcriptomics data. DIMet facilitates the analysis of stable-isotope labeling and metabolic abundances, offering a streamlined approach to infer metabolic changes without requiring complex flux analysis. Its pathway-based \"metabologram\" visualizations effectively integrate metabolomics and transcriptomics data, offering a versatile platform capable of analyzing corrected tracer datasets across diverse systems, organisms, and isotopes. We provide detailed steps for sample preparation and data analysis using DIMet through its intuitive, web-based Galaxy interface. To showcase DIMet's capabilities, we analyzed <i>LDHA/B</i> knockout glioblastoma cell lines compared to controls. Accessible to all researchers through Galaxy, DIMet is free, user-friendly, and open source, making it a valuable resource for advancing metabolic research. Key features • Glioblastoma tumor spheroids in vitro replicate tumors' three-dimensional structure and natural nutrient, metabolite, and gas gradients, providing a more realistic model of tumor biology. • Joint analysis of tracer metabolomics and transcriptomics datasets provides deeper insights into the metabolic states of cells. • DIMet is a web-based tool for differential analysis and seamless integration of metabolomics and transcriptomics data, making it accessible and user-friendly. • DIMet enables researchers to infer metabolic changes, offering intuitive and visually appealing \"metabologram\" outputs, surpassing conventional visual representations commonly used in the field.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5168"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769753/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054534","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}

{"title":"Cochlear Organ Dissection, Immunostaining, and Confocal Imaging in Mice.","authors":"Chenyu Chen, Binjun Chen, Xiaoqing Qian, Haojie Sun, Xiao Fu, Dongdong Ren","doi":"10.21769/BioProtoc.5167","DOIUrl":"10.21769/BioProtoc.5167","url":null,"abstract":"<p><p>The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing. Key features • Protocol for the microdissection of the organ of Corti and suitable preparation for later immunostaining. • This technique involves the evaluation of mouse cochlea for planar-cell-polarity protein. • Quantitative and qualitative analysis of hair cell cilia in different dimensions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5167"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769749/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054556","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}

{"title":"Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate.","authors":"Mina O Seedhom, Devin Dersh, Jonathan W Yewdell","doi":"10.21769/BioProtoc.5165","DOIUrl":"10.21769/BioProtoc.5165","url":null,"abstract":"<p><p>Protein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not indicate ribosome translation status. Measurement of average transit rates entails in vitro pulse-chase radiolabeling of isolated cells or ribosome profiling after ribosome runoff, which is expensive and extremely demanding technically. Here, we detail protocols based on ribosome-mediated nascent chain puromycylation, harringtonine to stall initiating ribosomes while allowing ribosome elongation to continue normally, and cycloheximide to freeze translating ribosomes in place. Each compound is delivered intravenously to mice in the appropriate order, and after ex vivo cell fixation and permeabilization, translating ribosome numbers and transit rates are measured by flow cytometry using a directly conjugated puromycin-specific antibody. Key features • Measure relative numbers of translating ribosomes in mixed single-cell preparations. • Quantitate relative in vivo ribosome transit rates in mixed single-cell preparations. • Detect ribosome stalling in vivo.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 2","pages":"e5165"},"PeriodicalIF":1.0,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11769755/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143054558","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}

{"title":"A Highly Efficient System for Separating Glandular and Non-glandular Trichome of Cucumber Fruit for Transcriptomic and Metabolomic Analysis.","authors":"Lei Sun, Zhongxuan Feng, Fang Wang, Yu Qi, Menghang An, Lin Yang, Min Feng, Mingqi Wang, Huazhong Ren, Xingwang Liu","doi":"10.21769/BioProtoc.5154","DOIUrl":"10.21769/BioProtoc.5154","url":null,"abstract":"<p><p>Cucumber (<i>Cucumis sativus</i>) trichomes play a critical role in resisting external biological and abiotic stresses. Glandular trichomes are particularly significant as they serve as sites for the synthesis and secretion of secondary metabolites, while non-glandular trichomes are pivotal for determining the appearance quality of cucumbers. However, current methods for separating trichomes encounter challenges such as low efficiency and insufficient accuracy, limiting their applicability in multi-omics sequencing studies. This protocol introduces an efficient system designed for the precise separation of glandular and non-glandular trichomes from cucumber fruit. The process begins with the pre-cooling of sorbitol buffer or ethanol solution and the RNA-free treatment of laboratory supplies, followed by sterilization and pre-cooling. After filling glass bottles with pre-cooling buffer and glass beads, cucumber ovaries are then placed in the glass bottles and the trichome is harvested by bead-beating method. The separation process involves sequential filtration through various steel sieves and centrifugation to separate trichomes. The separated trichomes obtained from this method are well-suited for subsequent multi-omics sequencing analyses. This protocol achieved high precision in separating glandular and non-glandular trichomes, significantly enhancing the efficiency of separation and sample collection processes. This advancement not only addresses existing limitations but also facilitates comprehensive studies aimed at exploring the genetic and biochemical diversity present within cucumber trichomes, thereby opening avenues for broader agricultural and biological research applications. Key features • Use cucumber fruits on the day of flowering. • Pre-cooling and RNA-free treatment ensure supply quality and purity. • Efficiently separate glandular and non-glandular trichomes. • Trichome samples are suitable for multi-omics sequencing analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5154"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717712/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973755","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}

{"title":"In Vitro Assay to Examine Osteoclast Resorptive Activity Under Estrogen Withdrawal.","authors":"Cara Fiorino, Safia Omer, Nisha Gandhi, Rene E Harrison","doi":"10.21769/BioProtoc.5155","DOIUrl":"10.21769/BioProtoc.5155","url":null,"abstract":"<p><p>The bone is a highly dynamic organ that undergoes continuous remodeling through an intricate balance of bone formation and degradation. Hyperactivation of the bone-degrading cells, the osteoclasts (OCs), occurs in disease conditions and hormonal changes in females, resulting in osteoporosis, a disease characterized by altered microarchitecture of the bone tissue, and increased bone fragility. Thus, building robust assays to quantify OC resorptive activity to examine the molecular mechanisms underlying bone degradation is critical. Here, we establish an in vitro model to investigate the effect of estrogen withdrawal on OCs derived from the mouse macrophage RAW 264.7 cell line in a bone biomimetic microenvironment. This simple and robust model can also be adapted to examine the effect of drugs and genetic factors influencing OC resorptive activity in addition to being compatible with fluorescent imaging. Key features • A robust in vitro protocol that allows molecular and functional studies of mature osteoclasts in response to estrogen and its withdrawal. • Generation of inorganic bone-mimetic substrates for culturing and examining osteoclast resorptive behavior. • This quantitative image-based approach is compatible with brightfield and fluorescence microscopy to assess osteoclast resorptive activity.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5155"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717722/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973873","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}

{"title":"Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine.","authors":"Enrique Gamero-Estevez, Inga Viktoria Hensel, Michelle Steinhauer, Olivia Müllertz, Elizaveta Savochkina, Ibrahim Murathan Sektioglu, Bilgenaz Stoll, Shaghayegh Derakhshani, Sarah Devriese, Kyungbo Kim, Martin Resnik-Docampo","doi":"10.21769/BioProtoc.5157","DOIUrl":"10.21769/BioProtoc.5157","url":null,"abstract":"<p><p>The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures. Key features • Dissection and tissue analysis of whole human intestines. • Cryopreservation for biobank generation. • Optimized protocols for the isolation of epithelial and immune cells. • Autologous co-culture of organoids and lamina propria-derived immune cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 1","pages":"e5157"},"PeriodicalIF":1.0,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11717717/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142973877","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}