BMC methods最新文献

{"title":"A dual-fluorescence assay for gene delivery vehicle screening in macrophages with an inflammation-inducible reporter construct.","authors":"Allie Ivy, Shelby N Bess, Shilpi Agrawal, Varun Kochar, Abbey L Stokes, Timothy J Muldoon, Christopher E Nelson","doi":"10.1186/s44330-025-00030-x","DOIUrl":"https://doi.org/10.1186/s44330-025-00030-x","url":null,"abstract":"<p><strong>Background: </strong>Macrophages are a promising target for therapeutics in various applications such as regenerative medicine and immunotherapy for cancer. Due to their plastic nature, macrophages can switch from a non-activated state to activated with the smallest environmental change. For macrophages to be effective in their respective applications, screening for phenotypic changes is necessary to elucidate the cell response to different delivery vehicles, vaccines, small molecules, and other stimuli.</p><p><strong>Methods: </strong>We created a sensitive and dynamic high-throughput screening method for macrophages based on the activation of NF-κB. For this reporter, we placed an mRFP1 fluorescence gene under the control of an inflammatory promoter, which recruits NF-κB response elements to promote expression during the inflammatory response in macrophages. We characterized the inflammatory reporter based on key markers of an inflammatory response in macrophages including TNF-α cytokine release and immunostaining for inflammatory and non-inflammatory cell surface markers. We compared gene delivery and inflammation of several clinically relevant viral vehicles and commercially available non-viral vehicles. Statistical analysis between groups was performed with a one-way ANOVA with post-hoc Tukey's test.</p><p><strong>Results: </strong>The reporter macrophages demonstrated a dynamic range after LPS stimulation with an EC50 of 0.61 ng/mL that was highly predictive of TNF-α release. Flow cytometry revealed heterogeneity between groups but confirmed population level shifts in pro-inflammatory markers. Finally, we demonstrated utility of the reporter by showing divergent effects with various leading gene delivery vehicles.</p><p><strong>Discussion: </strong>This screening technique developed here provides a dynamic, high-throughput screening technique for determining inflammatory response by mouse macrophages to specific stimuli. The method presented here provides insight into the inflammatory response in mouse macrophages to different viral and non-viral gene delivery methods and provides a tool for high-throughput screening of novel vehicles.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-025-00030-x.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"2 1","pages":"8"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12062070/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144056919","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":"scAI-SNP: a method for inferring ancestry from single-cell data.","authors":"Sung Chul Hong, Francesc Muyas, Isidro Cortés-Ciriano, Sahand Hormoz","doi":"10.1186/s44330-025-00029-4","DOIUrl":"10.1186/s44330-025-00029-4","url":null,"abstract":"<p><strong>Background: </strong>Collaborative efforts, such as the Human Cell Atlas, are rapidly accumulating large amounts of single-cell data. To ensure that single-cell atlases are representative of human genetic diversity, we need to determine the ancestry of the donors from whom single-cell data are generated. Self-reporting of race and ethnicity, although important, can be biased and is not always available for the datasets already collected.</p><p><strong>Methods: </strong>Here, we introduce scAI-SNP, a tool to infer ancestry directly from single-cell genomics data. To train scAI-SNP, we identified 4.5 million ancestry-informative single-nucleotide polymorphisms (SNPs) in the 1000 Genomes Project dataset across 3201 individuals from 26 population groups. For a query single-cell dataset, scAI-SNP uses these ancestry-informative SNPs to compute the contribution of each of the 26 population groups to the ancestry of the donor from whom the cells were obtained.</p><p><strong>Results: </strong>Using diverse single-cell datasets with matched whole-genome sequencing data, we show that scAI-SNP is robust to the sparsity of single-cell data, can accurately and consistently infer ancestry from samples derived from diverse types of tissues and cancer cells, and can be applied to different modalities of single-cell profiling assays, such as single-cell RNA-seq and single-cell ATAC-seq.</p><p><strong>Discussion: </strong>Finally, we argue that ensuring that single-cell atlases represent diverse ancestry, ideally alongside race and ethnicity, is ultimately important for improved and equitable health outcomes by accounting for human diversity.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-025-00029-4.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"2 1","pages":"10"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12089154/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144121847","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":"iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation.","authors":"Sarah Lecinski, Jamieson A L Howard, Chris MacDonald, Mark C Leake","doi":"10.1186/s44330-025-00023-w","DOIUrl":"10.1186/s44330-025-00023-w","url":null,"abstract":"<p><strong>Background: </strong>Cells employ myriad regulatory mechanisms to maintain protein homeostasis, termed proteostasis, to ensure correct cellular function. Dysregulation of proteostasis, which is often induced by physiological stress and ageing, often results in protein aggregation in cells. These aggregated structures can perturb normal physiological function, compromising cell integrity and viability, a prime example being early onset of several neurodegenerative diseases. Understanding aggregate dynamics <i>in vivo</i> is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are not fully understood.</p><p><strong>Methods: </strong>Here, we report an improved methodology for production of fluorescent aggregates in model budding yeast which can be detected, tracked and quantified using fluorescence microscopy in live cells. This new openly-available technology, iPAR (inducible Protein Aggregation Reporter), involves monomeric fluorescent protein reporters fused to a ∆ssCPY* aggregation biomarker, with expression controlled under the copper-regulated <i>CUP1</i> promoter.</p><p><strong>Results: </strong>Monomeric tags overcome challenges associated with non-physiological reporter aggregation, whilst <i>CUP1</i> provides more precise control of protein production. We show that iPAR and the associated bioimaging methodology enables quantitative study of cytoplasmic aggregate kinetics and inheritance features <i>in vivo</i>. We demonstrate that iPAR can be used with traditional epifluorescence and confocal microscopy as well as single-molecule precise Slimfield millisecond microscopy. Our results indicate that cytoplasmic aggregates are mobile and contain a broad range of number of iPAR molecules, from tens to several hundred per aggregate, whose mean value increases with extracellular hyperosmotic stress.</p><p><strong>Discussion: </strong>Time lapse imaging shows that although larger iPAR aggregates associate with nuclear and vacuolar compartments, we show directly, for the first time, that these proteotoxic accumulations are not inherited by daughter cells, unlike nuclei and vacuoles. If suitably adapted, iPAR offers new potential for studying diseases relating to protein oligomerization processes in other model cellular systems.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-025-00023-w.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"2 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11958454/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143775259","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":"Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods","authors":"S. Robinson, Mark Samuels, William Jones, Nicolas A. Stewart, Murat Eravci, N. Mazarakis, D.C. Gilbert, Giles Critchley, G. Giamas","doi":"10.1186/s44330-024-00007-2","DOIUrl":"https://doi.org/10.1186/s44330-024-00007-2","url":null,"abstract":"","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141714041","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mini-MEndR: a miniaturized 96-well predictive assay to evaluate muscle stem cell-mediated repair.","authors":"Nitya Gulati, Sadegh Davoudi, Bin Xu, Saifedine T Rjaibi, Erik Jacques, Justin Pham, Amir Fard, Alison P McGuigan, Penney M Gilbert","doi":"10.1186/s44330-024-00005-4","DOIUrl":"10.1186/s44330-024-00005-4","url":null,"abstract":"<p><strong>Background: </strong>Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair.</p><p><strong>Methods: </strong>The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7⁺ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair \"in a dish\". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines.</p><p><strong>Results: </strong>The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7⁺ cell expansion) to a known modulator of MuSC mediated repair (p38/β MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu.</p><p><strong>Discussion: </strong>The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-024-00005-4.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"1 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11173370/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141319492","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}