Bio-protocol最新文献

{"title":"A Protocol to Purify Human Mediator Complex From Freestyle 293-F Cells.","authors":"Hui-Chi Tang, Kuang-Lei Tsai, Ti-Chun Chao","doi":"10.21769/BioProtoc.5185","DOIUrl":"10.21769/BioProtoc.5185","url":null,"abstract":"<p><p>The Mediator, a multi-subunit protein complex in all eukaryotes, comprises the core mediator (cMED) and the CDK8 kinase module (CKM). As a molecular bridge between transcription factors (TFs) and RNA polymerase II (Pol II), the Mediator plays a critical role in regulating Pol II-dependent transcription. Considering its large size and complex composition, conducting in vitro studies on the Mediator complex is challenging, especially when isolating the intact and homogeneous complex from human cells. Here, we present a method to purify the intact CKM-cMED complex from FreeStyle 293-F cells (293-F cells), which offers advantages for performing large-scale protein purification. To isolate the CKM-bound cMED without the presence of Pol II, FLAG-tagged CDK8, a subunit of the CKM complex, was expressed in 293-F cells for purification, as CKM and Pol II are mutually exclusive in their interaction with cMED. The complex is isolated from nuclear extracts through immunoaffinity purification and further purified by glycerol gradient to enhance its homogeneity. This protocol provides a time- and cost-efficient way to purify the endogenous Mediator complex for structural- and functional-based studies. Key features • This protocol describes a method for purifying the endogenous Mediator complex, free of Pol II, from 293-F cells. • Does not require the use of crosslinkers, offering advantages for structural and functional studies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5185"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545175","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":"Leveraging Circular Polymerization and Extension Cloning (CPEC) Method for Construction of CRISPR Screening Libraries.","authors":"Bengisu Dayanc, Sude Eris, Serif Senturk","doi":"10.21769/BioProtoc.5183","DOIUrl":"10.21769/BioProtoc.5183","url":null,"abstract":"<p><p>Recent advancements in high-throughput functional genomics have substantially enhanced our comprehension of the genetic and molecular dimensions of cancer, facilitating the identification of novel therapeutic targets. One of the key methodological innovations in this field is the CRISPR screening strategy, which has proven efficacy in elucidating essential gene functions and pathway alterations critical to cancer cell survival and fitness. The construction of custom CRISPR libraries permits the integration of tailored single-guide RNAs (gRNAs), offering greater flexibility as well as specificity in comparison to the commercially available libraries, and enables more refined secondary screening strategies to attenuate the selection of false positive potential gene candidates. Among various molecular cloning techniques, circular polymerase extension cloning (CPEC) has emerged as a highly efficient and cost-effective approach. CPEC utilizes polymerase overlap extension to assemble overlapping DNA fragments into circular plasmids, eliminating the need for restriction digestion and ligation and thus streamlining the creation of both single and multi-fragment constructs. In this protocol, we present the application of the CPEC method to construct the EpiTransNuc knockout gRNA library, specifically designed to target epigenetic regulators, transcription factors, and nuclear proteins. The custom library, assembled using the lentiGuide-Puro backbone, comprises 40,820 gRNAs, with 10 gRNAs per gene, along with 100 non-targeting control gRNAs. Importantly, the CPEC method can be tailored to meet the specific requirements of other custom gRNA libraries, offering flexibility for diverse research applications. Key features • Involves PCR-based linearization of the backbone with designed primer sets. • Facilitates flexibility in gRNA composition and number in library construction. • Skips conventional cloning techniques such as restriction digestion and ligation. Graphical overview Schematic representation of the circular polymerase extension cloning (CPEC) procedure.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5183"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545192","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":"Purification of Native Acetyl CoA Carboxylase From Mammalian Cells.","authors":"Yaxue Sun, Jiachen Li, Lianmei Zhao, Hongtao Zhu","doi":"10.21769/BioProtoc.5221","DOIUrl":"https://doi.org/10.21769/BioProtoc.5221","url":null,"abstract":"<p><p>Fatty acid (FA) biosynthesis is a crucial cellular process that converts nutrients into metabolic intermediates necessary for membrane biosynthesis, energy storage, and the production of signaling molecules. Acetyl-CoA carboxylase (ACACA) plays a pivotal catalytic role in both fatty acid synthesis and oxidation. This cytosolic enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which represents the first and rate-limiting step in de novo fatty acid biosynthesis. In this study, we developed a rapid and effective purification scheme for separating human ACACA without any exogenous affinity tags, providing researchers with a novel method to obtain human ACACA in its native form. Key features • Detailed protocol for the purification of native ACACA. • ACACA is biotinylated in mammalian cells. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5221"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865823/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545199","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":"Development and Application of MLB Human Astrovirus Reverse Genetics Clones and Replicons.","authors":"Hashim Ali, David Noyvert, Valeria Lulla","doi":"10.21769/BioProtoc.5201","DOIUrl":"10.21769/BioProtoc.5201","url":null,"abstract":"<p><p>Human astroviruses pose a significant public health threat, especially to children, the elderly, and immunocompromised individuals. Nevertheless, these viruses remain largely understudied, with no approved antivirals or vaccines. This protocol focuses on leveraging reverse genetics (RG) and replicon systems to unravel the biology of MLB genotypes, a key group of neurotropic astroviruses. Using reverse genetics and replicon systems, we identified critical genetic deletions linked to viral attenuation and neurotropism, pushing forward vaccine development. We also uncovered novel replication mechanisms involving ER membrane interactions, opening doors to new antiviral targets. Reverse genetics and replicon systems are essential for advancing our understanding of astrovirus biology, identifying virulence factors, and developing effective treatments and vaccines to combat their growing public health impact. Key features • Provides a basic understanding of the molecular biology of MLB astroviruses, aiding in addressing open questions related to virus evolution, replication, and pathogenesis. • Facilitates the development of novel therapeutics and vaccines. • Enables rapid testing of antiviral drugs against MLB astroviruses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5201"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545178","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":"Novel Workflows for Separate Isolation of Pathogen RNA or DNA from Wastewater: Detection by Innovative and Conventional qPCR.","authors":"Kristina M Babler, Helena M Solo-Gabriele, Mark E Sharkey, Ayaaz Amirali","doi":"10.21769/BioProtoc.5189","DOIUrl":"10.21769/BioProtoc.5189","url":null,"abstract":"<p><p>Wastewater-based surveillance (WBS) can provide a wealth of information regarding the health status of communities from measurements of nucleic acids found in wastewater. Processing workflows for WBS typically include sample collection, a primary concentration step, and lysis of the microbes to release nucleic acids, followed by nucleic acid purification and molecular-based quantification. This manuscript provides workflows from beginning to end with an emphasis on filtration-based concentration approaches coupled with specific lysis and nucleic acid extraction processes. Here, two WBS processing approaches are presented, one focusing on RNA-specific pathogens and the other focused on DNA-specific pathogens found within wastewater: 1) The RNA-specific approach, employed for analyzing RNA viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) couples electronegative filtration of wastewater with the placement of the filter within a lysis buffer followed by direct RNA extraction. 2) The DNA-specific approach, employed for analyzing DNA pathogens like <i>Candida auris</i>, uses size selection membranes during filtration, subsequently followed by a lysis buffer, bead-beating, and DNA extraction. Separate workflows for RNA versus DNA isolations have the advantage of improving the detection of the target pathogen. A novel aspect of the RNA-specific workflow is the direct extraction of nucleic acids from filter lysates, which shows enhanced recoveries, whereas the DNA-specific approach requires bead beating prior to extraction. Novelty is also provided in a new qPCR approach called Volcano 2nd Generation (V2G), which uses a polymerase capable of using RNA as a template, bypassing the reverse transcriptase step normally required for qPCR. Key features • Membrane filtration approaches for concentrating suspended solids from wastewater. After concentration, workflows are optimized for separate recovery of RNA and DNA. • Unique polymerase utilized to perform qPCR analysis, foregoing reverse transcription, for RNA. • Sample products for use with other molecular techniques (e.g., sequencing) as workflow approaches generate high-quality, concentrated nucleic acid extracts with minimal inhibitors. • Validated through COVID-19 surveillance where >1,000 samples of wastewater and >3,000 filter concentrates produced from these samples have been created and analyzed, with published results. <b>This complete protocol was used in:</b> J Biomol Tech (2023), DOI: 10.7171/3fc1f5fe.dfa8d906.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5189"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865829/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545195","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":"Streamlined Quantification of Microglial Morphology in Mouse Brains Using 3D Immunofluorescence Analysis.","authors":"Maria Helena de Donato, Armin Kouchaeknejad, Andreu de Donato, Gunter Van Der Walt, Emma Puighermanal","doi":"10.21769/BioProtoc.5218","DOIUrl":"10.21769/BioProtoc.5218","url":null,"abstract":"<p><p>Microglial cells are crucial patrolling immune cells in the brain and pivotal contributors to neuroinflammation during pathogenic or degenerative stress. Microglia exhibit a heterogeneous \"dendrite-like\" dense morphology that is subject to change depending on inflammatory status. Understanding the association between microglial morphology, reactivity, and neuropathology is key to informing treatment design in diverse neurodegenerative conditions from inherited encephalopathies to traumatic brain injuries. However, existing protocols for microglial morphology analyses lack standardization and are too complex and time-consuming for widescale adoption. Here, we describe a customized pipeline to quantitatively assess intricate microglial architecture in three dimensions under various conditions. This user-friendly workflow, comprising standard immunofluorescence staining, built-in functions of standard microscopy image analysis software, and custom Python scripts for data analysis, allows the measurement of important morphological parameters such as soma and dendrite volumes and branching levels for users of all skill levels. Overall, this protocol aims to simplify the quantification of the continuum of microglial pathogenic morphologies in biological and pharmacological studies, toward standardization of microglial morphometrics and improved inter-study comparability. Key features • Comparison of 3D microglial architecture between physiological and pathological conditions. • Quantitative assessment of critical microglial morphological features, including soma volume, dendrite volume, branch level, and filament length. • Simplified, semi-automated data export and analysis through simple Python scripts. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5218"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11873448/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143543939","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":"Quantifying Bacterial Chemotaxis in Controlled and Stationary Chemical Gradients with a Microfluidic Device.","authors":"Adam Gargasson, Carine Douarche, Peter Mergaert, Harold Auradou","doi":"10.21769/BioProtoc.5184","DOIUrl":"10.21769/BioProtoc.5184","url":null,"abstract":"<p><p>Chemotaxis refers to the ability of organisms to detect chemical gradients and bias their motion accordingly. Quantifying this bias is critical for many applications and requires a device that can generate and maintain a constant concentration field over a long period allowing for the observation of bacterial responses. In 2010, a method was introduced that combines microfluidics and hydrogel to facilitate the diffusion of chemical species and to set a linear gradient in a bacterial suspension in the absence of liquid flow. The device consists of three closely parallel channels, with the two outermost channels containing chemical species at varying concentrations, forming a uniform, stationary, and controlled gradient between them. Bacteria positioned in the central channel respond to this gradient by accumulating toward the high chemoattractant concentrations. Video-imaging of bacteria in fluorescent microscopy followed by trajectory analysis provide access to the key diffusive and chemotactic parameters of motility for the studied bacterial species. This technique offers a significant advantage over other microfluidic techniques as it enables observations in a stationary gradient. Here, we outline a modified and improved protocol that allows for the renewal of the bacterial population, modification of the chemical environment, and the performance of new measurements using the same chip. To demonstrate its efficacy, the protocol was used to measure the response of a strain of <i>Escherichia coli</i> to gradients of α-methyl-aspartate across the entire response range of the bacteria and for different gradients. Key features • The protocol is based on a previously proposed system [1] that we improved for higher throughput. • Setup allowing a rapid quantification of motility and chemotaxis responses. • Seventeen hours were required from the start of an <i>E. coli</i> culture to the measurements to obtain the chemotactic velocity under various chemical conditions. Graphical overview Schematic illustration of the three-channels chip architecture and its use with bacteria.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5184"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11911661/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143652583","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":"<i>Campylobacter jejuni</i> Biofilm Assessment by NanoLuc Luciferase Assay.","authors":"Tjaša Čukajne, Petra Štravs, Orhan Sahin, Qijing Zhang, Aleš Berlec, Anja Klančnik","doi":"10.21769/BioProtoc.5192","DOIUrl":"10.21769/BioProtoc.5192","url":null,"abstract":"<p><p><i>Campylobacter jejuni</i>, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into <i>C. jejuni</i> biofilm dynamics that could indirectly help in the development of improved food safety measures. Key features • Quantification of <i>C. jejuni</i> using NanoLuc luciferase. • The assay is linear in the range of 1.9 × 10⁷ to 1.5 × 10⁸ CFU/mL. • Following biofilm growth, less than 1 h is required for detection. • The assay requires genetically modified bacterial strains.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5192"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865840/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545172","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":"Locomotor Activity Monitoring in Mice to Study the Phase Shift of Circadian Rhythms Using ClockLab (Actimetrics).","authors":"Andrea Brenna, Jürgen A Ripperger, Urs Albrecht","doi":"10.21769/BioProtoc.5187","DOIUrl":"https://doi.org/10.21769/BioProtoc.5187","url":null,"abstract":"<p><p>The circadian clock regulates biochemical and physiological processes to anticipate changes in light, temperature, and food availability over 24 h. Natural or artificial changes in white/blue lighting exposure (e.g., seasonal changes, jet lag, or shift work) can advance or delay the clock phase to synchronize physiology with the new environmental conditions. These changes can be monitored through behavioral experiments in circadian research based on the analysis of locomotor activity by measuring wheel-running revolutions. The protocol includes measuring the internal period length in constant darkness and administering nocturnal light pulses to mice kept either in light/dark conditions (LD 12:12, Aschoff-type II protocol) or continuous darkness (DD, Aschoff-type I). Here, we describe a step-by-step guide for researchers to analyze the mouse circadian clock using wheel-running experiments and ClockLab (Actimetrics) to quantify data. Key features • This protocol builds upon the method developed by Jud et al. [1], optimized for digital analysis using the ClockLab software. • Step-by-step tutorial on measuring period length, analyzing periodograms, assessing general activity, and determining phase shifts (Aschoff Type I and II).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5187"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865828/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545193","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 PDMS-based Microfluidic Chip Assembly for Time-Resolved Cryo-EM (TRCEM) Sample Preparation.","authors":"Xiangsong Feng, Joachim Frank","doi":"10.21769/BioProtoc.5193","DOIUrl":"10.21769/BioProtoc.5193","url":null,"abstract":"<p><p>Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here, we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO₂-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid. As we have demonstrated in recent experiments, this setup is capable of addressing problems of severe sample adsorption and ineffective mixing of fluids and leads to highly reproducible results in applications to the study of translation. As an example, we used our TRCEM sample preparation method to investigate the molecular mechanism of ribosome recycling mediated by High frequency of lysogenization X (HflX), which demonstrated the efficacy of the TRCEM device and its capability to yield biologically significant, reproducible information. This protocol has the promise to provide structural and kinetic information on pre-equilibrium intermediates in the 10-1,000 ms time range in applications to many other biological systems. Key features • Design and fabrication of high-performance splitting-and-recombination-based micromixer and planar microsprayer. • Protocol for SiO₂ coating on the PDMS surface and fabrication of the microfluidic chip assembly. • Preparation of time-resolved cryo-EM sample in the time range of 10-1,000 ms. • Data collection on EM grid covered with droplets from the microsprayer.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5193"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11877145/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143560380","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}