Bio-protocol最新文献

{"title":"Puromycin Proximity Ligation Assay (Puro-PLA) to Assess Local Translation in Axons From Human Neurons.","authors":"Raffaella De Pace, Juan S Bonifacino, Saikat Ghosh","doi":"10.21769/BioProtoc.5224","DOIUrl":"10.21769/BioProtoc.5224","url":null,"abstract":"<p><p>Local mRNA translation in axons is crucial for the maintenance of neuronal function and homeostasis, particularly in processes such as axon guidance and synaptic plasticity, due to the long distance from axon terminals to the soma. Recent studies have shown that RNA granules can hitchhike on the surface of motile lysosomal vesicles, facilitating their transport within the axon. Accordingly, disruption of lysosomal vesicle trafficking in the axon, achieved by knocking out the lysosome-kinesin adaptor BLOC-one-related complex (BORC), decreases the levels of a subset of mRNAs in the axon. This depletion impairs the local translation of mitochondrial and ribosomal proteins, leading to mitochondrial dysfunction and axonal degeneration. Various techniques have been developed to visualize translation in cells, including translating RNA imaging by coat protein knock-off (TRICK), SunTag, and metabolic labeling using the fluorescent non-canonical amino acid tagging (FUNCAT) systems. Here, we describe a sensitive technique to detect newly synthesized proteins at subcellular resolution, the puromycin proximity ligation assay (Puro-PLA). Puromycin, a tRNA analog, incorporates into nascent polypeptide chains and can be detected with an anti-puromycin antibody. Coupling this method with the proximity ligation assay (PLA) allows for precise visualization of newly synthesized target proteins. In this article, we describe a step-by-step protocol for performing Puro-PLA in human induced pluripotent stem cell (iPSC)-derived neuronal cultures (i3Neurons), offering a powerful tool to study local protein synthesis in the axon. This tool can also be applied to rodent neurons in primary culture, enabling the investigation of axonal protein synthesis across species and disease models. Key features • Establishment of quantitative local translation assay in axons of human iPSC-derived neurons. • Microscopy-based direct visualization of local translation events in neurons. • Puro-PLA is a sensitive method for detecting new protein synthesis occurring within minutes in neurons, enabling precise temporal analysis of translation dynamics.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5224"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896778/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627180","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":"Tissue-Specific Profiling of <i>O</i>-GlcNAcylated Proteins in <i>Drosophila</i> Using TurboID-<i>Cp</i>OGA^M.","authors":"Qin Lei, Haibin Yu, Fang Chen, Kai Yuan","doi":"10.21769/BioProtoc.5234","DOIUrl":"10.21769/BioProtoc.5234","url":null,"abstract":"<p><p>Protein <i>O-</i>GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, <i>O-</i>GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation, and protein homeostasis. Dysregulation of <i>O-</i>GlcNAc homeostasis has been implicated in a variety of diseases, including cardiovascular diseases, cancer, and neurodegenerative diseases. Studying <i>O-</i>GlcNAcylated proteins in different tissues is crucial to understanding the pathogenesis of these diseases. However, identifying phenotype-relevant candidate substrates in a tissue-specific manner remains unfeasible. We developed a novel tool for the analysis of <i>O-</i>GlcNAcylated proteins, combining a catalytically inactive <i>Cp</i>OGA mutant <i>Cp</i>OGA^CD and TurboID proximity labeling technology. This tool converts <i>O-</i>GlcNAc modifications into biotin labeling, enabling the enrichment and mass spectrometry (MS) identification of <i>O-</i>GlcNAcylated proteins in specific tissues. Meanwhile, TurboID-<i>Cp</i>OGA^DM, which carries two point mutations that inactivate both its catalytic and binding activities toward <i>O</i>-GlcNAc modification, was used as a control to differentiate <i>O-</i>GlcNAc-independent protein-protein interactions. We have successfully used TurboID-<i>Cp</i>OGA^CD/DM (TurboID-<i>Cp</i>OGA^M) to enrich <i>O</i>-GlcNAc proteins in <i>Drosophila</i> combining the UAS/Gal4 system. Our protocol provides a comprehensive workflow for tissue-specific enrichment of candidate <i>O-</i>GlcNAcylated substrates and offers a valuable tool for dissecting tissue-specific <i>O</i>-GlcNAcylation functions in <i>Drosophila</i>. Key features • Innovative approach to studying <i>O-</i>GlcNAcylation: Combines a catalytically inactive <i>Cp</i>OGA mutant (<i>Cp</i>OGA^CD), TurboID proximity labeling technology, and the UAS/Gal4 system for tissue-specific analysis. • Tissue-specific focus: Enables enrichment and mass spectrometry (MS) identification of <i>O-</i>GlcNAcylated proteins in specific tissues of <i>Drosophila</i>. • Biotin labeling conversion: Converts <i>O-</i>GlcNAc modifications into biotin tags, facilitating downstream enrichment and analysis. • Powerful tool for understanding the role of <i>O-</i>GlcNAcylation in cellular processes and its involvement in diseases such as cardiovascular diseases, cancer, and neurodegenerative disorders.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5234"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896768/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627184","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":"Monitoring Changes in Intracellular Chloride Levels Using the FRET-Based SuperClomeleon Sensor in Organotypic Hippocampal Slices.","authors":"Sam de Kater, Lotte J Herstel, Corette J Wierenga","doi":"10.21769/BioProtoc.5229","DOIUrl":"10.21769/BioProtoc.5229","url":null,"abstract":"<p><p>The reduction in intracellular neuronal chloride concentration is a crucial event during neurodevelopment that shifts GABAergic signaling from depolarizing to hyperpolarizing. Alterations in chloride homeostasis are implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Recent advancements in biosensor technology allow the simultaneous determination of intracellular chloride concentration of multiple neurons. Here, we describe an optimized protocol for the use of the ratiometric chloride sensor SuperClomeleon (SClm) in organotypic hippocampal slices. We record chloride levels as fluorescence responses of the SClm sensor using two-photon microscopy. We discuss how the SClm sensor can be effectively delivered to specific cell types using virus-mediated transduction and describe the calibration procedure to determine the chloride concentration from SClm sensor responses. Key features • Calibration and use of the ratiometric chloride sensor SuperClomeleon to measure intracellular chloride levels in mouse organotypic hippocampal slices. • SuperClomeleon sensor expression in pyramidal cells or GABAergic interneurons using adeno-associated viruses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5229"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896781/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627173","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":"Computational Cellular Mathematical Model Aids Understanding the cGAS-STING in NSCLC Pathogenicity.","authors":"Shweta Khandibharad, Pooja Gulhane, Shailza Singh","doi":"10.21769/BioProtoc.5223","DOIUrl":"10.21769/BioProtoc.5223","url":null,"abstract":"<p><p>Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. According to 2020 reports, globally, 2.2 million cases are reported every year, with the mortality number being as high as 1.8 million patients. To study NSCLC, systems biology offers mathematical modeling as a tool to understand complex pathways and provide insights into the identification of biomarkers and potential therapeutic targets, which aids precision therapy. Mathematical modeling, specifically ordinary differential equations (ODEs), is used to better understand the dynamics of cancer growth and immunological interactions in the tumor microenvironment. This study highlighted the dual role of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS/STING) pathway's classical involvement in regulating type 1 interferon (IFN I) and pro-inflammatory responses to promote tumor regression through senescence and apoptosis. Alternative signaling was induced by nuclear factor kappa B (NF-κB), mutated tumor protein p53 (p53), and programmed death-ligand1 (PD-L1), which lead to tumor growth. We identified key regulators in cancer progression by simulating the model and validating it with the following model estimation parameters: local sensitivity analysis, principal component analysis, rate of flow of metabolites, and model reduction. Integration of multiple signaling axes revealed that cGAS-STING, phosphoinositide 3-kinases (PI3K), and Ak strain transforming (AKT) may be potential targets that can be validated for cancer therapy. Key features • Procedures for the reconstruction of a robust and steady-state mathematical model with respective analysis in order to provide mechanistic insights. • The dynamic mathematical model allows an understanding of the multifaceted dual roles of cGAS-STING in NSCLC promotion and inhibition. • The inherent statistical tool in systems biology provides a novel immunotherapeutic target.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5223"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627079","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 Screening Host-Targeting Antivirals (HTAs) Using Human PBMCs and pDCs.","authors":"Zhao Xuan Low, Osamu Kanauchi, Sazaly AbuBakar, Vunjia Tiong, Pouya Hassandarvish","doi":"10.21769/BioProtoc.5230","DOIUrl":"10.21769/BioProtoc.5230","url":null,"abstract":"<p><p>This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs), HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection, especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs, known for their high production of humoral factors such as Type I interferons (IFNs), the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways, especially innate immunity, allows for species-independent antiviral activity, reducing the likelihood of viral escape mutations. Additionally, the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens, including emerging viruses, positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs, offering a novel avenue for HTA discovery. Key features • Optimized protocol for screening HTAs against dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV). • This protocol is ideal for screening soluble or intravenous-formulated compounds for evaluating their efficacy in experimental settings. • This protocol builds upon the method developed by Tsuji et al. [1] and extends its application to PBMCs and testing against DENV, CHIKV, and ZIKV.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5230"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627177","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":"Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector.","authors":"Luzi Yang, Congting Guo, Yueshen Sun, Yuxuan Guo","doi":"10.21769/BioProtoc.5235","DOIUrl":"10.21769/BioProtoc.5235","url":null,"abstract":"<p><p>The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver. Key features • This protocol describes a detailed procedure to construct and validate AAV-based cardiac-specific gene editing in mice. • MicroRNA-122 target sequences (miR122TS) in combination with a Tnnt2 promoter are used to enhance the cardiac specificity in genome editing. • Amplicon sequencing analysis is applied to precisely and sensitively quantify the genome editing efficiency and tissue specificity in mice.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5235"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896767/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627076","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":"Isolation and Biophysical Characterization of Extracellular Vesicles from Hairy Root Cultures.","authors":"Marisa Conte, Elisa Cappetta, Mariaevelina Alfieri, Michele Bifolco, Eleonora Boccia, Mariapia Vietri, Alfredo Ambrosone","doi":"10.21769/BioProtoc.5225","DOIUrl":"10.21769/BioProtoc.5225","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are membrane-bound, non-replicating particles released by virtually all types of cells. EVs concentrate and deliver a plethora of biomolecules driving very important biological functions, including intercellular communication not only between cells of the same organism but also across different kingdoms. Plant extracellular vesicles (PEVs) are a promising alternative to mammalian EVs in biomedical applications. Here, we present an optimized and reproducible protocol for isolating PEVs from the hairy root (HR) cultures of medicinal plants <i>Salvia dominica</i> and <i>S. sclarea.</i> Our methodological approach introduces a significant advancement in the standardization of HR-EVs purification processes from plant biotechnological platforms, paving the way for their broader application across various sectors, including agriculture, pharmaceuticals, and nutraceuticals. Key features • Hairy roots serve as a plant biotechnological platform for the production of bioactive compounds. • Plant extracellular vesicles (EVs) provide a safer alternative to mammalian EVs for biomedical applications. • This protocol represents a significant advancement in the standardization of plant EV purification.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5225"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896773/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627152","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":"Quantification of Neuromuscular Junctions in Zebrafish Cranial Muscles.","authors":"Ritika Ghosal, Johann K Eberhart","doi":"10.21769/BioProtoc.5219","DOIUrl":"10.21769/BioProtoc.5219","url":null,"abstract":"<p><p>Communication between motor neurons and muscles is established by specialized synaptic connections known as neuromuscular junctions (NMJs). Altered morphology or numbers of NMJs in the developing muscles can indicate a disease phenotype. The distribution and count of NMJs have been studied in the context of several developmental disorders in different model organisms, including zebrafish. While most of these studies involved manual counting of NMJs, a few of them employed image analysis software for automated quantification. However, these studies were primarily restricted to the trunk musculature of zebrafish. These trunk muscles have a simple and reiterated anatomy, but the cranial musculoskeletal system is much more complex. Here, we describe a stepwise protocol for the visualization and quantification of NMJs in the ventral cranial muscles of zebrafish larvae. We have used a combination of existing ImageJ plugins to develop this methodology, aiming for reproducibility and precision. The protocol allows us to analyze a specific set of cranial muscles by choosing an area of interest. Using background subtraction, pixel intensity thresholding, and watershed algorithm, the images are segmented. The binary images are then used for NMJ quantification using the Analyze Particles tool. This protocol is cost-effective because, unlike other licensed image analyzers, ImageJ is open-source and available free of cost. Key features • Immunostaining neuromuscular junctions in alcohol-exposed zebrafish. • Quantification of presynaptic and postsynaptic terminals in cranial muscles of zebrafish larvae. • Analysis of size and distribution of NMJs in cranial muscles of zebrafish larvae.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5219"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865821/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545200","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":"HPLC Analysis of tRNA-Derived Nucleosides.","authors":"Xingxing Chen, Fu Xu","doi":"10.21769/BioProtoc.5213","DOIUrl":"https://doi.org/10.21769/BioProtoc.5213","url":null,"abstract":"<p><p>Transfer RNAs (tRNAs), the essential adapter molecules in protein translation, undergo various post-transcriptional modifications. These modifications play critical roles in regulating tRNA folding, stability, and codon-anticodon interactions, depending on the modified position. Methods for detecting modified nucleosides in tRNAs include isotopic labeling combined with chromatography, antibody-based techniques, mass spectrometry, and high-throughput sequencing. Among these, high-performance liquid chromatography (HPLC) has been a cornerstone technique for analyzing modified nucleosides for decades. In this protocol, we provide a detailed, streamlined approach to purify and digest tRNAs from yeast cells and analyze the resulting nucleosides using HPLC. By assessing UV absorbance spectra and retention times, modified nucleosides can be reliably quantified with high accuracy. This method offers a simple, fast, and accessible alternative for studying tRNA modifications, especially when advanced technologies are unavailable. Key features • A streamlined protocol for purifying total tRNAs from yeast cells. • Adaptable for other RNA species and organisms, provided sufficient input material. • Enables the quantification of approximately 20 types of tRNA modifications. • Offers a cost-effective and rapid alternative for analyzing tRNA modifications by HPLC method.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5213"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545191","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":"Micrografting Technique of <i>Hevea brasiliensis</i> in Vitro Plantlets.","authors":"Florence Dessailly, Pascal Montoro, Sémi Melliti, Julie Leclercq","doi":"10.21769/BioProtoc.5215","DOIUrl":"https://doi.org/10.21769/BioProtoc.5215","url":null,"abstract":"<p><p>To prepare <i>Hevea brasiliensis</i> plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock-scion interaction studies. This article describes the implementation of an efficient micrografting technique from <i>Hevea</i> in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion. Key features • This protocol requires rejuvenated in vitro plantlets. • Requires between 40 and 60 days before scion bud break. • Sterile working conditions. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5215"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545194","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}