Bio-protocol最新文献

{"title":"Evaluating <i>Arabidopsis</i> Primary Root Growth in Response to Osmotic Stress Using an In Vitro Osmotic Gradient Experimental System.","authors":"Selene Píriz-Pezzutto, Mauro Martínez-Moré, Maria Martha Sainz, Omar Borsani, Mariana Sotelo-Silveira","doi":"10.21769/BioProtoc.5397","DOIUrl":"10.21769/BioProtoc.5397","url":null,"abstract":"<p><p>The root meristem navigates the highly variable soil environment where water availability limits water absorption, slowing or halting growth. Traditional studies use uniform high osmotic potentials, poorly representing natural conditions where roots gradually encounter increasing osmotic potentials. Uniform high osmotic potentials reduce root growth by inhibiting cell division and shortening mature cell length. This protocol describes a simple and effective in vitro system using a gradient mixer that generates a vertical gradient in an agar gel based on the principle of communicating vessels, exploiting gravity to generate a continuous mannitol concentration gradient (from 0 to 400 mM mannitol) reaching osmotic potentials of -1,2 MPa. It enables long-term <i>Arabidopsis</i> root growth analysis under progressive water deficit, improving phenotyping and molecular studies in soil-like conditions. Key features • Novel approach: Unique method to evaluate primary root growth in <i>Arabidopsis</i> under increasing osmotic potentials. • Osmotic gradient system: Simulating a gradual osmotic gradient in the root growth zone while maintaining aerial tissues under control conditions. • Sustained growth: <i>Arabidopsis</i> Col-0 and <i>ttl1</i> mutant seedlings maintain proper root growth for 25 days, even at osmotic potentials as low as -1.2 MPa. • Enhanced growth rates: Roots grown in the osmotic gradient exhibit higher growth rates than those in homogeneous high osmotic potential conditions. • Phenotypic observation: <i>ttl1</i> seedlings grown in the osmotic gradient do not show the typical swelling phenotype observed at extreme osmotic potentials (-1.2 MPa).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5397"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304458/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755393","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 Beads as a Focus Fiduciary to Aid Software-Based Autofocus Accuracy in Microscopy.","authors":"Isabel Gibson, Elizabeth Julie Osterlund, Ray Truant","doi":"10.21769/BioProtoc.5388","DOIUrl":"10.21769/BioProtoc.5388","url":null,"abstract":"<p><p>Brightfield microscopy is an ideal application for studying live cell systems in a minimally invasive manner. This is advantageous in long-term experiments to study dynamic cellular processes such as stress response. Depending on the sample type and preparation, the inherent qualities of brightfield microscopy, being very low contrast, can contribute to technical issues such as focal drift, sequencing lags, and complete failure of software autofocus systems. Here, we describe the use of microbeads as a focus aid for long-term live cell imaging to address these autofocus issues. This protocol is inexpensive to implement, without extensive additional sample preparation, and can be used to capture focused images of transparent cells in a label-free manner. To validate this protocol, a widefield inverted microscope was used with software-based autofocus to image overnight in time-lapse format, demonstrating the use of the beads to prevent focal drift in long-term experiments. This improves autofocus accuracy on relatively inexpensive microscopes without using hardware-based focus aids. To validate this protocol, the KNIME logistics software was used to train a random forest model to perform binary image classification. Key features • Label-free live cell imaging in time-lapse format. • Troubleshooting software autofocus for brightfield mode.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5388"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304461/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755412","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":"Virus Isolation and Rice Protoplast Infection.","authors":"Yu Huang, Zhirui Yang, Yi Li","doi":"10.21769/BioProtoc.5383","DOIUrl":"10.21769/BioProtoc.5383","url":null,"abstract":"<p><p>Rice (<i>Oryza sativa</i>), a staple crop sustaining half of humanity's caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV-host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening. Key features • The protocol purifies virus particles of RSV with strong infection capacity, which can directly infect rice protoplasts when co-transfected with exogenous plasmids for functional studies. • This transgene-independent approach accelerates antiviral determinant profiling from several months to three days.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5383"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755413","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 Novel Composite Method of Post-stroke Epilepsy Induction.","authors":"Yiting Guo, Raymond Tak Fai Cheung","doi":"10.21769/BioProtoc.5387","DOIUrl":"10.21769/BioProtoc.5387","url":null,"abstract":"<p><p>The global burden of stroke has increased in the past several decades, and post-stroke epilepsy (PSE) is a common complication. Contrasted with the advancement in knowledge of stroke pathophysiology, the exact pathogenesis of PSE is unclear. Various animal stroke models have been utilized to investigate the underlying mechanisms of PSE, but the success rate of PSE induction is low. To address this limitation, a novel PSE model was established in the rat by inducing status epilepticus using lithium-pilocarpine one week after photothrombotic stroke. Successful indication of status epilepticus and mortality rate at three days after status epilepticus were the main measurements. Potential usefulness of this model was also illustrated by preliminary results on locomotor activity, exploratory behavior, and anxiety level evaluated using the open-field test, as well as mossy fiber sprouting (MFS) in the hippocampal dentate granule cells using Zinc transporter 3 immunofluorescence staining at 8 weeks after PSE induction. This novel composite method of PSE induction may facilitate future studies on the pathogenesis and treatment of PSE. Key features • We developed a new PSE model in the rat by combining the photothrombotic model of stroke and lithium-pilocarpine (LIP) model of temporal lobe epilepsy (TLE). • In our novel rat PSE model, 94% of rats achieved status epilepticus, and mortality rate at 3 days was 25%. • The PSE rats appeared to have a decreased anxiety level on the open-field test and MFS in the hippocampal dentate granule cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5387"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304455/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755391","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":"Fast TV-PRO-seq: Accelerated and Streamlined Protocol for Timing RNA Polymerase Pausing.","authors":"Jie Zhang, Zhixian Liang, Mingxin Sun, Daniel Hebenstreit, Shaohui Zhang","doi":"10.21769/BioProtoc.5395","DOIUrl":"10.21769/BioProtoc.5395","url":null,"abstract":"<p><p>Transcriptional pausing dynamically regulates spatiotemporal gene expression during cellular differentiation, development, and environmental adaptation. Precise measurement of pausing duration, a critical parameter in transcriptional control, has been challenging due to limitations in resolution and confounding factors. We introduce Fast TV-PRO-seq, an optimized protocol built on time-variant precision run-on sequencing (TV-PRO-seq), which enables genome-wide, single-base resolution mapping of RNA polymerase II pausing times. Unlike standard PRO-seq, Fast TV-PRO-seq employs sarkosyl-free biotin-NTP run-on with time gradients and integrates on-bead enzymatic reactions to streamline workflows. Key improvements include (1) reducing experimental time from 4 to 2 days, (2) reducing cell input requirements, and (3) improved process efficiency and simplified command-line operations through the use of bash scripts. Key features • Reduces experimental duration from 4 to 2 days via on-bead enzymatic reactions and streamlined workflows. • Enables single-nucleotide resolution pausing time mapping using time-variant biotin-NTP run-on with saturation kinetics. • Compatible with reduced cell input (10⁶-10⁸ cells) and sarkosyl-free conditions for improved experimental feasibility. • Integrates bash scripts and simplified commands for enhanced reproducibility and reduced computational complexity.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5395"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12305362/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755395","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":"Western Blotting and Immunoprecipitation of Native Human PIEZO1 Channels.","authors":"Jinyuan Vero Li, Zijing Zhou, Charles D Cox","doi":"10.21769/BioProtoc.5385","DOIUrl":"10.21769/BioProtoc.5385","url":null,"abstract":"<p><p>PIEZO1 is a mechanically activated ion channel essential for mechanotransduction and downstream signaling in almost all organ systems. Western blotting is commonly used to study the expression, stability, and post-translational modifications of proteins. However, as a large transmembrane protein, PIEZO1 contains extensive hydrophobic regions and undergoes post-translational modifications that increase its propensity for nonspecific protein-protein interactions. As a result, conventional sample preparation methods seem unsuitable for PIEZO1. For example, heating and sonicating transmembrane proteins exposes hydrophobic regions, leading to aggregation, improper detergent interactions, and loss of solubility, ultimately compromising their detection in western blots. To address these challenges, we developed a western blot protocol optimized for human PIEZO1 by preparing lysates consistently at lower temperatures and incorporating strong reducing and alkylation reagents into the western blot lysis buffer to ensure proper protein solubilization and minimal cross-linking. Using the same antibody, we also developed an immunoprecipitation protocol with optimized detergents to maintain the solubilization of native human PIEZO1, enabling the discovery of a new family of auxiliary subunits. Key features • Simple modifications to the standard RIPA buffer prevent protein aggregates of large transmembrane proteins. • Minimal protein degradation and cross-linking by modifying cell lysis conditions and protein extraction process. • Clear separation of glycosylated and non-glycosylated PIEZO1 by SDS-PAGE.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5385"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304459/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755417","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":"ZnCl₂ Precipitation-Assisted Sample Preparation for Proteomic Analysis.","authors":"Qiqing He, Qingjing Chen, Dongxue Wang, Fuchu He","doi":"10.21769/BioProtoc.5398","DOIUrl":"10.21769/BioProtoc.5398","url":null,"abstract":"<p><p>This manuscript details protocols for the ZnCl₂ precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl₂ at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, such as sodium dodecyl sulfate (SDS), Triton X-100, and urea, at high concentrations in solution from protein solutions prior to trypsin digestion. It is a practical, robust, and cost-effective approach for proteomic sample preparation. It has been observed that 90.2% of the proteins can be recovered from lysates by incubating with an equal volume of ZPB at room temperature for 10 min. In 1 h of data-dependent acquisition (DDA) analysis on an Exploris 480, 4,037 proteins and 25,626 peptides were quantified from 1 μg of mouse small intestine proteins, reaching a peak of 4,500 proteins and up to 30,000 peptides with 5 μg of input. Additionally, ZASP outperformed other common sample preparation methods such as sodium deoxycholate (SDC)-based in-solution digestion, acetone precipitation, filter-aided sample preparation (FASP), and single-pot, solid-phase-enhanced sample preparation (SP3). It demonstrated superior performance in protein (4,456 proteins) and peptide identification (29,871 peptides), lower missing cleavage rates (16.3%), and high reproducibility (Pearson correlation coefficients of 0.96 between replicates) with similar protein distributions and cellular localization patterns. Significantly, the cost of ZASP per sample with 100 μg of protein as input is lower than 30 RMB, including the expense of trypsin. Key features • ZASP is a user-friendly method that enables efficient, sensitive, and cost-effective proteomic analysis in a common biochemistry lab through simple incubation and centrifugation. • ZASP enables 90% protein recovery from lysates with various trypsin and LC-MS-incompatible reagents by incubating at room temperature for 10 min. • ZASP enables unbiased proteomic analysis of diverse biological samples, including cells, optimal cutting temperature (OCT)-embedded tissues, and formalin-fixed paraffin-embedded (FFPE) tissues.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5398"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304474/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755419","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 Fluorescence-Based Flippase Assay to Monitor Lipid Transport by Drs2-Cdc50.","authors":"Inja M Van Der Linden, Sara Abad Herrera, Cédric Montigny, Guillaume Lenoir, Thomas Günther Pomorski, Huriye D Uzun","doi":"10.21769/BioProtoc.5393","DOIUrl":"10.21769/BioProtoc.5393","url":null,"abstract":"<p><p>Flippases, a functionally distinct group of transmembrane proteins that flip lipids from the extracellular or luminal side to the cytosolic side of biological membranes, are key players in many important physiological processes, such as membrane trafficking and cellular signaling. To study the function of these membrane proteins under chemically defined conditions, reconstituting them into artificial vesicles is a crucial and effective approach. There are various methods for protein reconstitution involving different detergents and detergent removal techniques to integrate membrane proteins into artificial vesicles. In this protocol, we describe the reconstitution of the yeast flippase complex Drs2-Cdc50, which translocates phosphatidylserine across membranes of the trans-Golgi network at the expense of ATP hydrolysis. The flippase complex is incorporated into liposomes using a zwitterionic detergent, followed by detergent removal via dialysis-a gentle and effective strategy that helps preserve protein function. To evaluate the activity of the reconstituted flippase complex, two complementary assays are employed: (1) a fluorescence-based quenching assay to measure lipid transport; and (2) an ATPase assay using an ATP-regenerating system to measure ATP hydrolysis. Together, these methods provide a robust platform for analyzing the functional reconstitution of Drs2-Cdc50 in a defined membrane environment. Key features • Provides a gentle reconstitution approach via detergent removal by dialysis. • Measures ATPase activity using an NADH-coupled assay with an ATP-regenerating system. • Assesses lipid flippase activity with a sodium dithionite-based assay with fluorescent lipid derivatives. • Provides a well-defined experimental setup for direct characterization of lipid flippases.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5393"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755390","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":"Fixation and Expansion Microscopy of <i>Xenopus</i> Egg Extract Spindles.","authors":"Gabriel Guilloux, Maiko Kitaoka, Karel Mocaer, Claire Heichette, Laurence Duchesne, Rebecca Heald, Thierry Pecot, Romain Gibeaux","doi":"10.21769/BioProtoc.5396","DOIUrl":"10.21769/BioProtoc.5396","url":null,"abstract":"<p><p>In vitro systems based on <i>Xenopus</i> egg extracts have elucidated many aspects of spindle assembly. Still, numerous unknowns remain, particularly concerning the variation in spindle morphologies. The <i>X. laevis</i> and <i>X. tropicalis</i> egg extract systems, which recapitulate diverse spindle sizes and architectures, serve as ideal tools to investigate the regulation of spindle morphometrics. However, fully understanding spindle architectural differences is hindered by the spindle's size and high microtubule density. Indeed, classical fluorescence microscopy lacks the resolution to detail the organization of spindle microtubules, and although electron tomography can distinguish individual microtubules, segmenting thousands of microtubules and tracking them across dozens of sections remains an unachieved challenge. Therefore, we set out to apply expansion microscopy to the study of <i>Xenopus</i> egg extract spindles. During this process, we realized that optimizing spindle fixation as well was crucial to preserve microtubule integrity. Here, we present an optimized fixation and expansion microscopy protocol that enables the study of spindle architecture in egg extracts of both <i>X. laevis</i> and <i>X. tropicalis</i>. Our method retains the fluorescence of rhodamine tubulins added to the extracts and allows for both pre- and post-expansion immunofluorescence analysis. Key features • Expansion of optimally fixed spindle assembled from egg extracts of <i>X. leavis, X. tropicalis</i>, and possibly others. • Retains the fluorescence of the rhodamine-tubulin that copolymerizes with endogenous <i>Xenopus</i> tubulin within spindle microtubules, allowing their imaging without immunolabeling. • Compatible with both pre- and post-expansion immunolabeling to increase labeling possibilities. • Optimized spindle fixation that best preserves microtubule integrity for expansion and can also be used without expansion for regular immunofluorescence experiments.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5396"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304471/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755396","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":"Metabolite Production and Extraction of Indole Compound From the Tomato Endophyte <i>Streptomyces</i> sp. VITGV100.","authors":"Veilumuthu Pattapulavar, Sathiyabama Ramanujam, Sanjivkumar Muthusamy, Shweta Panchal, John Godwin Christopher","doi":"10.21769/BioProtoc.5386","DOIUrl":"10.21769/BioProtoc.5386","url":null,"abstract":"<p><p>Endophytic actinomycetes, particularly <i>Streptomyces</i> species, have gained significant attention due to their potential to produce novel bioactive compounds. In this study, we isolated and characterized an endophytic <i>Streptomyces</i> sp. VITGV100 from the tomato plant (<i>Lycopersicon esculentum</i>), employing the direct streak method and whole-genome sequencing. A genome analysis was done to uncover its biosynthetic potential and identify indole-type compounds. The strain's secondary metabolite production was evaluated through GC-MS analysis, and its antimicrobial activity was tested against selected human pathogenic bacteria. Our protocol outlines a comprehensive approach, describing the isolation and extraction of metabolites and genome mining for indole-type compounds. This isolate has potential pharmaceutical applications, accelerating the discovery of novel indole-type bioactive compounds. Key features • A systematic approach for isolating <i>Streptomyces</i> sp. VITGV100 from <i>Lycopersicon esculentum</i>, including surface sterilization and selective culturing on ISP2 medium. • Whole-genome sequencing and annotation: High-throughput Illumina sequencing followed by genome assembly, annotation, and functional characterization to explore biosynthetic potential and metabolic pathways. • Secondary metabolite profiling and bioactivity screening: GC-MS-based identification of bioactive metabolites, followed by antibacterial activity assessment against human pathogens using well-diffusion assays. • Identification of biosynthetic gene clusters (BGCs) using antiSMASH 6.0.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5386"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755408","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}