BMC methods最新文献

{"title":"BLASE: bulk linkage analysis for single cell experiments - teasing out the secrets of bulk transcriptomics with trajectory analysis.","authors":"Andrew McCluskey, Toby Kettlewell, Adrian M Smith, Rhiannon Kundu, David A Gunn, Thomas D Otto","doi":"10.1186/s44330-026-00082-7","DOIUrl":"10.1186/s44330-026-00082-7","url":null,"abstract":"<p><strong>Background: </strong>Transcriptomics has profoundly improved our knowledge of cells. The advent of single-cell transcriptomics has enabled researchers to investigate the changes which individual cells undergo, such as cell type differentiation. Bulk RNA-seq is more practical in both cost and ease, but cannot elucidate cell type specific trajectories. Deconvolution methods can estimate cell types in RNA-seq data, but there is a need for methods characterising their position on a trajectory.</p><p><strong>Methods: </strong>We present a new method called BLASE, which discretises the pseudotime of a scRNA-seq trajectory, and then uses Spearman correlation to infer the closest matching period of pseudotime to a bulk RNA-seq sample. Bootstrapping provides confidence intervals around the correlation, and subsequently informs \"strong\" calls. BLASE can discretise pseudotime using several different methods, provides heuristics for hyperparameter selection, and enables the visualisation of results.</p><p><strong>Results: </strong>BLASE performs correctly and outperforms other tools. In simulated scRNA-seq data it accurately identified the correct pseudotime bin for 10 pseudobulked pseudotime bins, whereas other tools we tested ranged in accuracy from around 40-90%. In experimental data, BLASE correctly mapped all pseudobulked pseudotime bins, compared to a range of around 20-75%. We tested BLASE on use cases with published single-cell and bulk transcriptomics. On 10x Visium spatial data, BLASE could resolve the spatio-temporal process of keratinocyte differentiation. When mapping a time-course of 48 hour microarray data to the <i>Plasmodium falciparum</i> lifecycle, BLASE correctly identified the predominant cell type of synchronised cells in 48/48 samples (30 strong). Finally, BLASE identified a developmental rate difference in <i>P. falciparum</i> grown with or without heat shock conditions. Of 345 genes differentially expressed, 142 were attributed to developmental rate differences. The remaining 203 should represent the true signal better, and revealed new gene ontology terms.</p><p><strong>Discussion: </strong>BLASE outperforms existing tools in simulated and experimental data. It can be used to a) annotate scRNA-seq data from existing RNA-seq, b) identify progress of RNA-seq data through a process captured in scRNA-seq, and c) be used to correct developmental differences in differential expression analysis. BLASE is released as an open-source R package under the GPL3 license, and is available on Bioconductor.</p><p><strong>Clinical trial: </strong>Not applicable.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7619496/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149043362","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":"Automated workflow for genotyping individual transposon library variants.","authors":"Lorea Alejaldre, Ana-Mariya Anhel, Lewis Grozinger, Ángel Goñi-Moreno","doi":"10.1186/s44330-025-00054-3","DOIUrl":"10.1186/s44330-025-00054-3","url":null,"abstract":"<p><strong>Background: </strong>The fields of genome engineering and synthetic biology rely on high-throughput screening to accelerate research and increase reproducibility. This often requires the use of costly equipment such as flow cytometers, specialized microfluidic devices or liquid handlers. Despite the availability of population-level screening methods, genotyping individual clones in transposon libraries remains a necessary but frequently labor-intensive task well suited for liquid-handling platforms. Although open-source automation is democratizing access to liquid handlers, it requires a minimum level of programming expertise or the availability of validated protocols to implement experimental workflows. To address these challenges, we present a step-by-step automated workflow designed for individual variant genotyping of transposon insertion libraries using an open-source OT-2 liquid handler and a custom Python package to annotate sequencing results.</p><p><strong>Methods: </strong>The automated protocol comprises six modular steps that can be used separately or integrated into different workflows: 1) colony picking into selective media, 2) counter-selection using the backbone and integrated antibiotic resistances and preparation of glycerol stocks pre-cultures, 3) colony counter-selection and 4) control PCR assays to filter spurious integration events, 5) arbitrary PCR assays and 6) sequencing and annotation using a custom Python package. This protocol is demonstrated using standardized vectors adhering to the Standard European Vector Architecture (SEVA) guidelines and standardized Lab Automation Protocol (LAP) scripts to ensure reproducibility and portability.</p><p><strong>Results: </strong>The automated workflow was validated using a transposon insertion library in Pseudomonas putida KT2440, enabling reliable genotyping of individual variants within 4-5 days. No spurious integrations were detected, and genomic insertion sites were successfully identified for 40 out of 46 variants, demonstrating robustness, accessibility, and scalability of the open-source workflow.</p><p><strong>Discussion: </strong>This automated workflow enables reliable genotyping while reducing hands-on time and minimizing human error. Its modular, standardized design and validated protocols promote protocol reuse and sharing while broadening access to open-source laboratory automation. While the throughput of the workflow is limited by the deck capacity of the liquid handler, it can be scaled by subsequent runs or parallel processing on multiple liquid handlers. In addition, the workflow can be further streamlined through integration of an OT-2 adapted colony picker. Overall, this open-source workflow provides a robust foundation for scalable and reproducible genotyping of individual variants in genome engineering and synthetic biology applications.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7618916/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147501133","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":"Optimizing post-transplantation detection of subcutaneously transplanted islets using dithizone staining.","authors":"James Lu, Matthew Ishahak, Marlie M Maestas, Jeffrey R Millman","doi":"10.1186/s44330-026-00075-6","DOIUrl":"10.1186/s44330-026-00075-6","url":null,"abstract":"<p><strong>Background: </strong>Pancreatic islet transplantation is a promising therapeutic strategy to restore insulin independence in patients with type 1 diabetes mellitus. In addition to primary human islets, human pluripotent stem cell-derived islets have shown clinical promise. However, optimizing transplantation sites and improving methods for standardized islet dosing and post-transplant graft localization are key limitations. The subcutaneous space offers an alternative site for transplantation due to its accessibility and minimally invasive nature; nevertheless, poor vascularization and difficulty locating engrafted islets limit its experimental utility. The use of dithizone, a zinc-chelating dye that selectively binds the high intracellular zinc concentrations of insulin-producing β-cells, may enable rapid graft localization through selective staining. Here, we investigate whether dithizone staining can reliably localize transplanted islet grafts within the subcutaneous space of murine models.</p><p><strong>Methods: </strong>SC-islets were generated from HUES8 stem cells and functionally validated using glucose-stimulated insulin secretion. Islet volume was standardized prior to transplantation using automated islet equivalent quantification with the BioRep Islet Cell Counter. Approximately 4,000 islet equivalents of either SC-islets or primary human islets were transplanted into the subcutaneous space of immunodeficient mice. Graft function was assessed longitudinally via human C-peptide measurements. Four weeks post-transplantation, dithizone staining was applied to harvested skin tissue to localize islet grafts, and immunohistochemical validation was performed.</p><p><strong>Results: </strong>Automated quantification of islet equivalents enabled consistent dosing across transplantation groups. Transplanted islets maintained function in vivo, as demonstrated by detectable human C-peptide levels. Dithizone staining produced clear and selective red labeling of insulin-producing β-cell grafts within the subcutaneous tissue, enabling reliable localization and recovery of engrafted islets. Immunohistochemical analyses confirmed the presence of insulin-expressing cells at isolated islet grafts.</p><p><strong>Discussion: </strong>Dithizone staining is a rapid, cost-effective approach to identify subcutaneous islet grafts and enables downstream analyses, addressing a significant limitation in islet transplantation research. When combined with standardized pre-transplant islet quantification, this approach provides a cohesive and reproducible framework for evaluating subcutaneous islet transplantation. Future studies could complement these results by assessing the potential effects of dithizone exposure and exploring its applicability across other transplantation sites and imaging modalities.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-026-00075-6.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"23"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13180754/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147978445","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":"Integrated protocol for concurrent generation of microglial and neuronal cells in human cerebral organoids.","authors":"Yukyeong Lee, Joshua L Roffman, Rakesh Karmacharya","doi":"10.1186/s44330-026-00062-x","DOIUrl":"10.1186/s44330-026-00062-x","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Microglia, the resident immune cells in the brain, play essential roles in synaptic pruning, neurodevelopment, and the pathogenesis of neuropsychiatric disorders. Since neuronal cells and microglia arise from different embryonic lineages, brain organoids differentiated from human induced pluripotent stem cells (iPSCs) do not typically contain microglial cells, limiting their utility in interrogating neuroimmune interactions in neurodevelopmental and in disease. Reliable methods to generate brain organoids with integrated microglial cells can model important in vivo cellular interactions in an in vitro system.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;We developed a method to generate microglia-containing cerebral organoids (MG-COs) from human iPSCs using concurrent induction of neuronal and microglial lineages by leveraging the respective patterning molecules for those two lineages during the early stages of embryoid body formation. The resulting MG-COs were characterized with systematic morphological and transcriptomic analyses at specific stages of the differentiation process to confirm lineage-specific differentiation. The transcriptome profiles of MG-COs were compared to cerebral organoids (COs) differentiated without microglial cells to delineate gene expression differences that arise in the setting of concurrent microglia differentiation during development and growth of the organoids. Neuronal network activity in the MG-COs and COs was assessed using microelectrode array (MEA) experiments.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;MG-COs generated with the protocol resulted in organoids that had a robust population of stable microglial cells integrated in the organoids, as evidenced by the presence of canonical microglial markers alongside neuronal and glial markers in the MG-COs. Transcriptomic profiling of MG-COs identified the presence of immune- and synapse-related genes, including those involved in microglial activation, synaptic plasticity, and neurotransmission in the MG-COs, underscoring the potential of MG-COs as a platform for modeling neuroimmune during neurodevelopment. MG-COs showed a robust response to the inflammatory cytokine IL-17a, which is known to activate microglia. MG-COs demonstrated significantly enhanced neuronal activity earlier in development, when compared to COs, with increased spontaneous firing activity and synchronized bursting in the MG-COs.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Discussion: &lt;/strong&gt;We developed a robust protocol for generating MG-COs from human iPSCs through concurrent induction of microglial and neuronal cells during embryoid body formation, enabling the growth of cerebral organoids with integrated microglial cells. MG-COs generated using the protocol give rise to three-dimensional structures comprising neuronal, glial, and microglial cells, as evidenced by immunocytochemistry and gene expression. MG-COs exhibited similar composition of neuronal and glial cells, when compared to COs, except for the ab","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13267931/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148267756","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":"metaIVP: an integrative metavirome focused metagenomic processing pipeline.","authors":"Kalyan Sahu, Qiuming Yao","doi":"10.1186/s44330-026-00090-7","DOIUrl":"https://doi.org/10.1186/s44330-026-00090-7","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Metagenomic studies increasingly rely on complex, multi-tool pipelines to recover and characterize viral and non-viral genomes from mixed microbial communities. While these pipelines enable high-resolution genome recovery, limited functionality in downstream post-processing workflows and insufficient logging structures often hinder reproducibility, error tracing, and selective re-analysis. These challenges are particularly critical in metaviral analyses, where viral and non-viral genomes must be processed using distinct methodologies. To address these limitations, we introduce metaIVP, a modular, integrative, and flexible framework designed to systematically manage genome content purification, re-binning, quality assessment, and downstream analyses of viral and non-viral metagenomic contexts.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;The metaIVP framework is organized into hierarchical modules, each governed by dedicated log files that explicitly control execution state and re-runnability. Contig-level and bin-level analytical and purification steps are implemented as essential modules to isolate genome contents, followed by separate viral and non-viral post-processing workflows. Viral workflows incorporate contamination detection, genome quality evaluation, host prediction, and virus-specific binning. Non-viral analyses include genome binning, alignment and mapping statistics, genome quality assessment, and replication rate estimation. Checkpoints are explicitly defined such that deletion of selected module- or sub-module-level logs enables targeted re-execution of specific analytical steps without rerunning the full pipeline. All analyses are integrated to depict a comprehensive system in the metagenomic samples, with focus on the metaviromic information.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;The usage of metaIVP was demonstrated using both a well-controlled human gut virome dataset and a geographically structured environmental metavirome dataset, showing its broad applicability across host-associated and environmental systems. The pipeline effectively separates viral and non-viral genomic content, improves viral bin purity, and preserves sample-specific functional, taxonomic, and host-association features after virome enrichment. Compared with recent state-of-the-art approaches, metaIVP achieves comparable performance, particularly when optional re-binning with vRhyme is applied, while maintaining a higher fraction of high-confidence viral bins.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Discussion: &lt;/strong&gt;The metaIVP addresses a key gap in metavirome analysis by jointly characterizing viral and non-viral genomic components and supporting integrative downstream analyses within a single framework. Its user-friendly, modular, and controllable design allows flexible execution and provides a foundation for incorporating additional downstream analytical tools as metavirome methodologies continue to evolve.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Supplementary information: &lt;/strong&gt;The","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"37"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13473319/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148763751","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 bone and cancer cells co-culture methodology to model breast cancer metastasis to bone ex vivo.","authors":"Ricardo J Romero-Moreno, Courtney L Flatt, Madeline P Sheeley, Xiaotong Alex Lin, Glen L Niebur, Laurie E Littlepage","doi":"10.1186/s44330-026-00085-4","DOIUrl":"10.1186/s44330-026-00085-4","url":null,"abstract":"<p><strong>Background: </strong>One of the major challenges in late-stage cancer research is studying the direct interactions between cancer cells and the metastatic tissue. Metastasis is an inefficient multistep process in which only a small subset of cancer cells successfully colonizes the new microenvironment. Bone is the most common tissue for breast and prostate cancer metastasis and the third most common site across all cancer types. We present an ex vivo co-culture protocol for modeling bone metastasis that enables direct investigation of cancer cell interactions with the native bone and marrow microenvironment.</p><p><strong>Methods: </strong>The procedure involves the extraction and preparation of mouse long bones (femurs and tibiae), injection of cancer cells into the bone marrow cavity, maintenance of the co-culture, and sample processing for downstream analysis. The protocol supports functional readouts, including bioluminescence imaging of tumor cell viability, conditioned media sampling, and downstream tissue analysis, such as immunohistochemistry or micro-computed tomography (µCT). The culture setup procedure can be completed in a single day, and daily media maintenance thereafter requires minimal time. Final sample processing times vary, depending on the analysis completed.</p><p><strong>Discussion: </strong>The system maintains viable and metabolically active bone, marrow, and cancer cells for up to four weeks and preserves the heterogeneity of the bone microenvironment. It is highly adaptable for various cancer types and bone genotypes and enables high-throughput functional interrogation of cancer colonization in a complex microenvironment. The protocol requires basic expertise in mammalian cell culture and mouse dissection and is suitable for researchers studying metastasis, tumor-stroma interactions, or preclinical therapeutic screening in the bone microenvironment.</p><p><strong>Clinical trial number: </strong>Not applicable.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"36"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461694/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726727","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 methodological framework for constructing opioid agonist therapy episodes in administrative health data.","authors":"Kiana Yazdani, Cassidy Tam, Christopher C Fisher, Scott D Emerson, Jason Trigg, Katherine W Kooij, Mary A De Vera, Fidel Vila-Rodriguez, Robert S Hogg, Julio S G Montaner, Viviane Dias Lima","doi":"10.1186/s44330-026-00064-9","DOIUrl":"10.1186/s44330-026-00064-9","url":null,"abstract":"<p><strong>Background: </strong>Opioid Agonist Therapy (OAT) is the most effective intervention to reduce overdose risk, and administrative health data are now increasingly used to study OAT outcomes. However, current methods for constructing OAT episodes rely heavily on fixed permissible gaps and often overlook switches between medications, concurrent therapies, and the temporal complexity of dispensing patterns. We aimed to develop a robust episode-construction framework that more precisely reflects real-world OAT use within administrative health data.</p><p><strong>Methods: </strong>We analyzed OAT dispensations from people living with HIV in British Columbia (2010-2020). Episodes were constructed using three components: Allen's interval algebra, temporal margins ([Formula: see text]), and drug-specific permissible gaps. Allen's algebra characterized the temporal relation between two dispensations as <i>meets</i> (one starts when another ends), <i>before</i> (a positive gap), <i>overlaps</i> (partial overlap), <i>starts</i> (same start, earlier end), <i>finishes</i> (later start, same end), <i>contains</i> (one fully encloses another), or <i>equals</i> (identical start and end). Temporal margins distinguished short from long overlaps within the same OAT medication and differentiated switches from concurrent therapy when different OATs overlapped. Permissible gaps defined the maximum interruption allowed before episode discontinuation and were mapped onto Allen's <i>before</i> relation. Using this combined framework, we identified monotherapy (single-OAT use), transition (medication switches), multitherapy (concurrent OAT use), and transition-multitherapy episodes.</p><p><strong>Results: </strong>The <i>before</i> relation predominated across all OATs, particularly for methadone (99.43%). The <i>equals</i> relation was notably prevalent for buprenorphine (21.46%), slow-release oral morphine (20.87%), and injectable OAT (1.91%). To build monotherapy episodes, we applied [Formula: see text]=7 days, while [Formula: see text]=14 days differentiated transition from multitherapy episodes. We identified 10,386 episodes: 93.53% monotherapy, 3.72% transition, 2.27% multitherapy, and < 1% transition-multitherapy.</p><p><strong>Conclusion: </strong>We propose an episode-building framework based on Allen's relations, temporal margins, and permissible gaps that fine-tune OAT classification in administrative health data. The method is transferable to other settings and populations with suitable parameter adjustments.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-026-00064-9.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"11"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12979340/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147470682","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 simple and versatile fluorescence-based method to enhance prime editing in human pluripotent stem cells.","authors":"Carlye Frisch, Salma Leyasi, William W Kostes, Stefan J Tekel, Kylie Standage-Beier, Gayathri Srinivasan, Xiao Wang, David A Brafman","doi":"10.1186/s44330-026-00077-4","DOIUrl":"10.1186/s44330-026-00077-4","url":null,"abstract":"<p><strong>Background: </strong>Prime editing is a versatile and precise genome-editing technique that enables targeted modifications with unprecedented accuracy. Unlike other CRISPR-based methods, prime editing does not induce double-stranded DNA breaks, thereby minimizing genomic instability and off-target effects. This technology allows for precise insertions, deletions, and substitutions, surpassing the limitations of conventional genome-editing approaches. However, a major challenge remains-low editing efficiency in human pluripotent stem cells (hPSCs), which restricts its broader application in stem cell research and regenerative medicine.</p><p><strong>Methods: </strong>This protocol describes Prime-Induced Nucleotide Engineering using a Transient Reporter for Editing Enrichment (PINE-TREE), a fluorescence-based system designed to enhance prime editing efficiency in hPSCs. The step-by-step methodology details plasmid construction, transfection, and clonal isolation strategies, facilitating real-time enrichment of prime-edited cells. Additionally, the protocol includes comprehensive methods for evaluating off-target effects and confirming the maintenance of pluripotency, ensuring precision and reliability in edited cell lines.</p><p><strong>Discussion: </strong>PINE-TREE significantly improves the efficiency of prime editing in hPSCs, addressing key limitations associated with low editing rates. By enabling real-time fluorescence-based enrichment, this system enhances the isolation of successfully edited cells, reducing the need for labor-intensive selection processes. Moreover, its adaptability across multiple cell types and compatibility with various prime editing strategies make it a valuable tool for researchers in genome engineering, disease modeling, and regenerative medicine.</p><p><strong>Clinical trial number: </strong>Not applicable.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13367918/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148451259","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":"FedDA-TSformer: Federated Domain Adaptation with vision TimeSformer for left ventricle segmentation on gated myocardial perfusion SPECT image.","authors":"Yehong Huang, Chen Zhao, Rochak Dhakal, Min Zhao, Guang-Uei Hung, Zhixin Jiang, Weihua Zhou","doi":"10.1186/s44330-026-00057-8","DOIUrl":"10.1186/s44330-026-00057-8","url":null,"abstract":"<p><strong>Background: </strong>Accurate assessment of left ventricular function is essential for diagnosing and managing cardiovascular disease. Gated myocardial perfusion SPECT (MPS) enables simultaneous evaluation of perfusion and function, but reliable contour extraction is challenged by image noise, resolution limits, and anatomical variability. Multi-center validation is further restricted by data privacy concerns, underscoring the need for robust and privacy-preserving contouring methods.</p><p><strong>Methods: </strong>In this study, we propose a novel approach, FedDA-TSformer, which integrates Federated Domain Adaptation with the TimeSformer model for the task of left ventricle segmentation using MPS images. The proposed model captures spatial and temporal features through a Divide-Space-Time-Attention mechanism, which ensures spatial-temporal consistency in predictions across multi-center datasets. To facilitate domain adaptation, we employ a local maximum mean discrepancy (LMMD) loss to align model outputs across data from three different institutions. This strategy effectively combines federated learning and domain adaptation to enhance model generalization while ensuring data security.</p><p><strong>Results: </strong>We evaluated FedDA-TSformer on a dataset comprising 150 subjects collected from three hospitals, with each cardiac cycle divided into eight gates. The model achieved Dice Similarity Coefficients (DSC) of 0.842 and 0.907 for left ventricular endocardium and epicardium segmentation, respectively.</p><p><strong>Discussion: </strong>FedDA-TSformer provides a robust, privacy-preserving solution for multi-center left ventricular segmentation, outperforming traditional FedAvg in handling domain shifts. By leveraging the TimeSformer architecture and domain adaptation mechanisms, the framework ensures spatial-temporal consistency and data security across heterogeneous clinical sites. Despite current limitations regarding communication overhead and its focus on a small SPECT-only dataset, this study establishes a scalable foundation for collaborative cardiac diagnosis. Future work will prioritize model compression, asynchronous updates, and cross-modality generalization to CT and MRI to enhance its practicality in resource-constrained environments.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12862020/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146115397","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":"Road map for primary hepatocyte qualification in human liver organ models.","authors":"Mahboubeh Varmazyad, Dillon C Gavlock, Michael W Castiglione, Richard DeBiasio, Gregory LaRocca, Celeste Reese, Lawrence A Vernetti, Mark Schurdak, Andrew M Stern, D Lansing Taylor, Mark T Miedel, Jacquelyn A Brown","doi":"10.1186/s44330-026-00058-7","DOIUrl":"10.1186/s44330-026-00058-7","url":null,"abstract":"<p><strong>Background: </strong>Microphysiological systems (MPS) are powerful tools for modeling human organ function and evaluating therapeutic interventions. The performance and translational relevance of MPS are highly dependent on the quality and suitability of the cells used. Given the expanding array of primary and stem cell-derived sources, there is a critical need for systematic frameworks to guide cell selection, particularly for disease-specific applications such as Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD).</p><p><strong>Methods: </strong>We developed a phased, stepwise methodology to evaluate and select primary human hepatocytes for use in a liver MPS model of MASLD. The protocol incorporates assessments of cell source quality, plating efficiency, viability, baseline liver function, and responsiveness to disease-inducing conditions. Quantitative metrics and pass/fail criteria were applied at each stage to ensure consistent and reproducible evaluation across cell lots.</p><p><strong>Results: </strong>Application of the protocol enabled effective triaging of hepatocyte sources, distinguishing cell lots with superior plating performance and functional profiles. Selected hepatocytes exhibited robust expression of liver-specific markers, maintained metabolic activity, and demonstrated disease-relevant phenotypes under MASLD-inducing conditions.</p><p><strong>Discussion: </strong>This structured evaluation framework facilitates the identification of high-quality hepatocytes for MPS liver models, improving reproducibility and disease modeling accuracy. While this protocol was tailored for MASLD, the approach is adaptable to other liver diseases or applications. Limitations include potential variability in donor tissue availability and the need for standardization across laboratories.</p><p><strong>Clinical trial number: </strong>Not applicable.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1186/s44330-026-00058-7.</p>","PeriodicalId":519945,"journal":{"name":"BMC methods","volume":"3 1","pages":"6"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12883510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146159733","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}