Bio-protocol最新文献

{"title":"DepStep: An Efficient One-Step rRNA Depletion Workflow for RNA Sequencing in Non-model Organisms.","authors":"Suleman M Qasim, Peter L Sarin","doi":"10.21769/BioProtoc.5764","DOIUrl":"10.21769/BioProtoc.5764","url":null,"abstract":"<p><p>RNA sequencing (RNA-seq) has revolutionized transcriptomics, ribosome footprinting, and polysome profiling, providing a wealth of data. Many RNA-based omics typically remove ribosomal RNA (rRNA) or select for messenger RNA (mRNA) prior to sequencing, thereby enriching reads that map to the translationally active part of the transcriptome. Prokaryotic mRNA lacks the 3' polyadenylated tail, which excludes the use of poly(A)-based selection methods. While commercial rRNA depletion products exist for prokaryotes, their proprietary nature and potential inefficiency with non-model organisms are factors that may limit broad-scale application. To mitigate this issue, we designed DepStep, a consolidated workflow for one-step rRNA depletion using species-specific biotinylated antisense probes for selective hybridization and removal of the target rRNA molecules. As a proof-of-concept, RNA-seq libraries of the psychrophilic gram-negative bacterium <i>Shewanella glacialimarina</i> TZS-4_T were prepared using both DepStep and a commercial rRNA depletion kit for gram-negative bacteria, to which DepStep was benchmarked. DepStep compares favorably to the commercial depletion kit; it removes >98.6% of the rRNA content in the sample, resulting in sequencing libraries where the coding DNA sequence (CDS) reads account for >80% of the total read count. Importantly, DepStep's cost-per-sample is three times lower than the commercial kit, establishing DepStep as a simple yet cost-effective alternative to commercial solutions. Key features • DepStep is a comprehensive workflow for one-step rRNA depletion in non-model species. • Guidelines for design and implementation of specific biotinylated antisense probes. • Compares favorably in benchmarking to commercial products. • Alternative for budget-conscious labs with 3× lower cost-per-sample.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5764"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461757/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148724647","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 Streamlined and Time-Saving Approach to Generate HLA-DR15 MHC Class II Tetramers via In Vivo Biotinylation.","authors":"Xue-Yao Zhao, Heng-Hui Li, Hong-Yan Ma, Bin Yang, Ru-Yi Qian, Xiang Zhang, Ling-Jie Luo, Yan-Wei Wu, Liang Chen","doi":"10.21769/BioProtoc.5755","DOIUrl":"10.21769/BioProtoc.5755","url":null,"abstract":"<p><p>This protocol describes an optimized strategy for the efficient generation of peptide-loaded major histocompatibility complex (MHC) class II (pMHC) tetramers, which are essential tools for detecting and characterizing antigen-specific T cells in immunological research. Traditional methods require separate expression of MHC proteins followed by in vitro biotinylation-a multi-step process that is time-consuming and prone to protein loss. Here, we present an integrated approach based on co-expression of MHC monomers and BirA biotin ligase in Expi293F T cells, enabling site-specific biotinylation in vivo during protein synthesis. At the same time, the incorporation of a thrombin-cleavable class II-associated invariant chain peptide (CLIP) peptide into the MHC construct allows flexible loading of any antigenic peptide of interest without the need for re-cloning or re-expression of the MHC molecule. Pre-biotinylated MHC molecules are subsequently purified, loaded with antigenic peptides, and assembled into fluorescent tetramers via streptavidin conjugation. This streamlined workflow significantly reduces handling steps, improves protein yield, and enhances reproducibility. The resulting tetramers are suitable for sensitive detection and isolation of antigen-specific T cells by flow cytometry, supporting applications in T-cell immunogenicity studies, vaccine development, and autoimmune disease research. Key features • One-step in vivo biotinylation. • Eliminates purchased BirA enzyme, saves ~16 h, and reduces protein loss. • CLIP-based peptide exchange without re-cloning. • Produces antigen-specific tetramers validated by flow cytometry.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5755"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403119/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611162","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":"Preparation and Characterization of Neutrophil Membrane-Fused Mitochondria (nMITO).","authors":"Qing Zhang, Yuqin Yue, Xing Zhou","doi":"10.21769/BioProtoc.5744","DOIUrl":"10.21769/BioProtoc.5744","url":null,"abstract":"<p><p>Mitochondrial transplantation is an emerging strategy for cellular repair, yet its efficiency is often limited by poor targeting and environmental instability. This protocol details the fabrication and comprehensive characterization of neutrophil membrane-fused mitochondria (nMITO), a hybrid organelle platform designed to combine the metabolic vigor of natural mitochondria with the targeting and anti-inflammatory properties of neutrophil membranes. We describe an optimized workflow for mouse heart mitochondrial isolation, lipopolysaccharide (LPS)-activated neutrophil membrane (NEM) extraction, and the subsequent sonication-mediated fusion process. Characterization techniques include dynamic light scattering (DLS) for size and zeta potential, transmission electron microscopy (TEM) for ultrastructural integrity, and bioenergetic assays [ATP synthesis and tetramethylrhodamine methyl ester (TMRM)-based membrane potential] to ensure functional preservation. Key features • The protocol provides a methodology for the isolation of neutrophil membranes from mouse bone marrow and mitochondria from the heart. • The protocol provides a methodology for the fabrication of neutrophil membrane-fused mitochondria (nMITO).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5744"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403121/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611111","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 Measuring Drug-Target Engagement in Mouse Colorectal Cancer Organoids Using NanoBRET Assay.","authors":"Hammed A Badmos, Colin Steele, Ross Cagan","doi":"10.21769/BioProtoc.5752","DOIUrl":"10.21769/BioProtoc.5752","url":null,"abstract":"<p><p>Organoids as a drug discovery platform represent an emerging field that continues to refine its tools. NanoBRET (bioluminescence resonance energy transfer) has emerged as a proximity-based and highly sensitive assay to measure protein-protein and protein-ligand interactions. NanoBRET assays were developed and are currently used for 2D cell line experiments. Here, we present the development of the first organoid-compatible Nanoluciferase (Nluc) for 3D model systems. We utilise the Nluc for NanoBRET assays to test drug-target engagement. We describe steps for seeding, transfecting, and replating of mouse colorectal cancer organoids. In addition, we provide detailed procedures for the NanoBRET assay. Various lines of evidence have shown significant difference in drug response between 2D human cell lines and 3D model systems, including patient-derived organoids. Our protocol provides a template for measuring this difference in the context of drug-target engagement. Key features • Development of organoid-compatible Nluc vector. • Lentiviral delivery and antibiotic selection of MEK1-Nluc organoids. • NanoBRET assay for the real-time measurement of drug-target engagement in organoids. • Quantitative measurement of competitive drug binding in living 3D organoids. • Impact of drug combinations on drug-target engagement in organoids.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5752"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403122/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611149","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":"Optimized Field Collection and Gut Dissection Workflows for Microbiome Studies of the Citrus Root Weevil, <i>Diaprepes abbreviatus</i>.","authors":"Paola G Figueroa-Pratts, Tasha M Santiago-Rodriguez, Imilce A Rodriguez-Fernandez","doi":"10.21769/BioProtoc.5761","DOIUrl":"10.21769/BioProtoc.5761","url":null,"abstract":"<p><p>Careful dissection of insect gut tissues is essential for microbiome studies to ensure accurate characterization of internal microbial communities and preservation of DNA integrity. Because insect-associated microbiomes are highly sensitive to contamination, effective removal of external microbes prior to dissection is critical to minimize bias in downstream analyses. While ethanol- and bleach-based surface sterilization methods are commonly used, standardized workflows integrating field collection, sterilization, and dissection remain limited. Here, we present a step-by-step protocol for the field collection, surface sterilization, and dissection of gut tissues from the agricultural pest <i>Diaprepes abbreviatus</i> (Coleoptera: Curculionidae), optimized for genomic DNA extraction and microbiome analyses. Using wild-caught specimens, this workflow incorporates a rigorous surface sterilization and dissection strategy that minimizes external contamination while preserving biologically relevant microbial signatures and DNA integrity for downstream microbiome analyses. The protocol provides a standardized framework for insect gut microbiome studies and can be broadly adapted to other wild-caught insect species requiring careful collection, disinfection, and sterile dissection prior to molecular analysis. The protocol integrates field collection and laboratory processing steps into a streamlined workflow that minimizes contamination while preserving tissue integrity for downstream applications. Key features • Designed for wild-caught <i>Diaprepes abbreviatus</i> collected directly from agricultural host trees, this protocol can also be adapted for other insect species. • Integrates field collection, surface sterilization, and sterile gut dissection into a single workflow to minimize contamination. • Sequential ethanol and diluted bleach treatment effectively removes external microbes prior to dissection. • Enables isolation of intact gut tissues suitable for high-quality DNA extraction and downstream microbiome sequencing.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5761"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403129/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611139","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":"Measuring PINK1 Activity in Single Cells Using a PINK1 Kinase Activity Reporter.","authors":"Katie G Vineall, Danielle L Schmitt","doi":"10.21769/BioProtoc.5746","DOIUrl":"10.21769/BioProtoc.5746","url":null,"abstract":"<p><p>Phosphatase and tensin homolog-induced kinase 1 (PINK1) is a serine/threonine kinase that plays a key role in mitophagy initiation. Loss-of-function autosomal recessive mutations in PINK1 cause early onset Parkinson's disease (EOPD). Current approaches for studying PINK1 function depend on bulk techniques that can only provide snapshots of activity and could miss the dynamics and cell-to-cell heterogeneity of PINK1 activity or provide an indirect readout of PINK1 activity. Here, we present a protocol using our newly developed phase separation-based PINK1 biosensor (PINK1-SPARK) to observe real-time activity of endogenous PINK1 in single cells. Following transfection of live cells with PINK1-SPARK, cells are treated with mitochondrial depolarizing agents and visualized using widefield or confocal fluorescence microscopy, either following the same cells over time for time-lapse imaging of PINK1 activity or end-point measurements. Thus, PINK1-SPARK is a new tool that enables the measurement of PINK1 activity in single live cells, allowing for further elucidation of the role of PINK1 in mitophagy and cell function. Key features • Detailed protocol for use of PINK1-SPARK, a new PINK1 biosensor introduced in Vineall et al. [1]. • PINK1-SPARK, based on phase separation, has a high signal-to-noise, enabling robust detection of PINK1 activity in multiple cell types under multiple activating conditions. • Enables measurement of real-time endogenous PINK1 activation at the single-cell level.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5746"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403116/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611143","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 Vivo Light-Sheet Imaging of Senescence Reporter Activity in a Transparent Killifish.","authors":"Birgit Perner, Christoph Englert","doi":"10.21769/BioProtoc.5762","DOIUrl":"10.21769/BioProtoc.5762","url":null,"abstract":"<p><p>Aging is associated with progressive accumulation of senescent cells, which contribute to tissue dysfunction and organismal decline. Conventional approaches for assessing cellular senescence, such as histological or immunofluorescence analyses of fixed tissue sections and flow cytometry, require tissue collection, thereby precluding longitudinal in vivo studies. To enable the analysis of cellular senescence in a living vertebrate model, we have previously generated a <i>cdkn1a (p21</i>)-driven GFP reporter line that was established in the transparent <i>klara</i> background of <i>Nothobranchius furzeri</i>. Here, we describe a protocol for in vivo light-sheet microscopy of the reporter activity as readout for senescence-associated cell cycle arrest with single-cell resolution. The procedure involves anesthesia and mounting of fish for stable positioning within the imaging chamber, with particular attention to animal welfare considerations. It further includes the acquisition of three-dimensional image stacks and subsequent image processing. The workflow allows monitoring of GFP-positive cells in intact living killifish at different developmental stages. Although imaging depth remains limited despite organismal transparency, this method provides high-resolution volumetric imaging with minimal phototoxicity and enables analysis of senescence dynamics in a short-lived vertebrate model. It is currently performed as a terminal procedure under approved ethical regulations, but longitudinal imaging would also be possible with additional ethical authorization. Key features • This protocol allows intravital imaging of fluorescent reporter activity in intact, anesthetized small teleost fish using light-sheet microscopy. • Two complementary mounting strategies enable stable imaging across different developmental stages. • A customized mounting support is rapidly prepared using UV-curable adhesive that fits directly into the standard Lightsheet Z.1 sample holder (Zeiss). • Monitoring of vital parameters enables continuous assessment of the animal's physiological state during imaging and early detection of stress or compromised well-being.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5762"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611151","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":"Iodine Staining of Glycogen Storage in <i>Caenorhabditis elegans</i>.","authors":"Hiba Daghar, Éric Samarut, Alex J Parker","doi":"10.21769/BioProtoc.5745","DOIUrl":"10.21769/BioProtoc.5745","url":null,"abstract":"<p><p>Glycogen is a highly conserved macromolecule across species, and its visualization provides critical insights into both physiological processes and disease states. Existing approaches for glycogen imaging in <i>Caenorhabditis elegans</i> rely primarily on traditional microscopy slides, which introduce variability in image acquisition and downstream data analysis, limit throughput, and require substantial hands-on time and technical expertise. Here, we present a standardized, cost-effective, and high-throughput imaging method that enables efficient visualization and quantification of glycogen in <i>C. elegans</i>. Our approach utilizes a custom-designed three-dimensional pad containing two to four chambers, allowing control and experimental samples to be processed simultaneously under identical conditions. Worms are exposed to iodine crystals, ensuring uniform staining while minimizing reagent use and handling variability. Imaging is performed using a simple binocular microscope, and analysis is conducted in Fiji, making the workflow accessible to laboratories with minimal specialized equipment or training. This method also reduces technical variability, shortens turnaround time, and requires only basic reagents and expertise, making it well-suited for both research and teaching laboratories. Importantly, the platform is readily adaptable to other nematode species and scalable for large-scale genetic or pharmacological screening applications. Together, this workflow minimizes technical variability and provides a robust platform for comparative glycogen analysis in <i>C. elegans</i>. Key features • Standardized glycogen staining method allows less data variability. • Provides a quantitative tool to measure glycogen buildup in <i>C. elegans</i>. • Requires a customized pad for imaging. • Macro is available for batch processing.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5745"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403113/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611128","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 of Mononucleated and Binucleated Hepatocytes by Flow Cytometry.","authors":"Yusuke V Watanabe, Masaki Nishikawa, Yasuyuki Sakai, Takeshi Katsuda","doi":"10.21769/BioProtoc.5760","DOIUrl":"10.21769/BioProtoc.5760","url":null,"abstract":"<p><p>Polyploid hepatocytes are one of the unique features of the liver. Some polyploid hepatocytes have chromosomes in a single nucleus (e.g., 1x4n, 1x8n), while others separate their chromosomes into two nuclei (e.g., 2x2n, 2x4n). In ploidy research, hepatocytes are typically sorted according to their cellular ploidy, revealing their contribution to tumorigenesis and cellular senescence. However, the conventional sorting method fails to distinguish 1x4n from 2x2n, or 1x8n from 2x4n cells, leaving it unclear whether hepatocytes with the same cellular ploidy but different nuclear configurations are identical or phenotypically different. Here, we describe a detailed protocol for fractionating mononucleated and binucleated hepatocytes. First, we present the method for isolating primary mouse hepatocytes and staining them with the DNA dye Hoechst 33342. Flow cytometry is then used to detect fluorescence differences between mononucleated and binucleated hepatocytes. This protocol enables the discrimination of hepatocyte subpopulations with identical cellular ploidy, providing a useful tool to investigate the functional heterogeneity of polyploid hepatocytes. Key features • This protocol summarizes a method for isolating primary mouse hepatocytes. • This protocol enables us to isolate mononucleated and binucleated subpopulations from mixed primary hepatocytes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5760"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611123","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":"Ex Vivo Assessment of Extracellular Acidification Rate in Murine Intestinal Tissue.","authors":"Alexander F Lesser, Mitchell L Drumm","doi":"10.21769/BioProtoc.5749","DOIUrl":"10.21769/BioProtoc.5749","url":null,"abstract":"<p><p>Seahorse metabolic assays are now widely utilized across numerous fields for performing functional assessments of glycolysis and mitochondrial function in adherent or suspension cell culture samples. Seahorse assays measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as a means of assessing glycolysis and mitochondrial function, respectively. Currently, the vast majority of Seahorse metabolic assays are performed using in vitro samples due to the current established standardized method. However, a uniform approach to assess real-time functional measurements of glycolysis and mitochondrial function in ex vivo tissue samples remains elusive. In particular, this protocol was designed to assess glycolysis in ex vivo murine intestinal samples through ECAR measurements using the Agilent Seahorse XFe24 platform with corresponding Islet Capture microplates and screens. This protocol was developed to provide functional measurements of glycolytic metabolism in murine intestinal tissue samples. This protocol details a method to assess glycolysis in tissue samples and represents the next stage of ex vivo metabolic methods to complement existing standardized in vitro approaches. While this protocol was developed to assess ECAR in ex vivo murine intestinal samples, the same approach can be applied to assessing mitochondrial respiration through measurements of OCR in other tissue types. Overall, this protocol expands the purview of Seahorse metabolic assays through the inclusion of tissue samples and provides the framework to interrogate organ-level metabolism in the context of systemic nutrient metabolism and physiology. Key features • Protocol for applying Seahorse metabolic assays to tissue samples. • Specifically designed to assess glycolysis through extracellular acidification rate (ECAR) in ex vivo murine intestinal samples. • Provides functional metabolism data to complement gene and protein expression data. • Protocol can be adapted to measure mitochondrial function through assessments of oxygen consumption rate (OCR) in other tissue types.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5749"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611182","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}