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Burkholderia pseudomallei, the cause of melioidosis, forms biofilms that facilitate survival, alter antimicrobial susceptibility and promote disease recurrence. Neutrophils contribute to bacterial eradication through phagocytosis, degranulation and neutrophil extracellular traps (NETs). However, NETs are demonstrably insufficient to eradicate B. pseudomallei. This study has revealed the ability of NET fragments containing DNA to elevate B. pseudomallei biofilm formation, as evidenced by crystal-violet staining and confocal microscopy. Further investigation demonstrated that 15 mM N-acetylcysteine (NAC), efficiently suppressed NETs stimulated by B. pseudomallei and effectively prevented B. pseudomallei from forming NET-associated biofilm in the presence of polymorphonuclear leukocytes. Remarkably, we demonstrated that NAC has antibacterial properties against five clinical B. pseudomallei isolates through kinetic growth monitoring for 24 h. Interestingly, 15 mM NAC inhibits NET production and improves neutrophil-mediated killing through phagocytosis and degranulation, considerably diminishing survival of B. pseudomallei. Our findings suggest that NAC, a multifaceted therapeutic agent, holds significant potential as an adjunctive treatment against B. pseudomallei infection. NAC not only inhibits NETs but also enhances neutrophil functionality and exhibits remarkable antibacterial activity against B. pseudomallei. These properties may contribute to more effective eradication of B. pseudomallei by reducing biofilm formation associated with NETs and improving overall neutrophil-mediated immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13506-1.

AbstractPancreatic ductal adenocarcinoma (PDAC), one of the most aggressive forms of pancreatic cancer, is associated with poor survival outcomes and currently ranks as the third leading cause of cancer-related death in the United States. Despite its clinical significance, the mechanisms of PDAC development and progression remain, in part, poorly understood. In this study, we provide evidence of a novel role of sorting nexin 10 (SNX10), a member of the sorting nexin family, in the regulation of KRAS-induced pancreatic carcinogenesis. We demonstrate that SNX10 is downregulated in PDAC, especially in advanced cases. Furthermore, mutational analysis revealed SNX10 genetic alterations in PDAC cases. Functional studies demonstrated that SNX10 overexpression in human PDAC cells inhibited cell proliferation and colony formation. Moreover, SNX10 overexpression induced G1-phase cell-cycle arrest and decreased KRAS signaling activity. Using a novel Snx10 knockout mouse crossed with a Kras-driven PDAC model, we observed reduced survival, increased tumor cell proliferation, enhanced aggression, and heightened inflammation. Collectively, these findings highlight SNX10 as a tumor suppressor candidate in PDAC and underscore its promise as a foundation for new therapeutic approaches.Significance:SNX10 plays a crucial role in reducing pancreatic tumorigenesis. This discovery offers valuable insights into PDAC’s biology and the development of new effective treatments.

Porcine epidemic diarrhoea virus (PEDV) is the most widespread porcine coronavirus worldwide, causing high mortality and a high incidence rate among piglets. The molecular mechanisms by which PEDV regulates epithelial cell function and differentiation, as well as its disruption of the intestinal mucosal barrier, are not yet fully understood. This study reveals that PEDV infection reduces the number of goblet cells and impairs the intestinal barrier integrity in newborn piglets. Regarding the pathways involved in the differentiation of intestinal stem cells (ISCs), PEDV infection concurrently activates the Notch and MAPK pathways while suppressing the Wnt/β-catenin pathway in the intestines of piglets. Furthermore, in vitro experiments using intestinal monolayer organoid models showed that PEDV infection hinders goblet cell differentiation by activating the Notch signalling pathway. Additionally, the PEDV-encoded ORF3 protein, which is crucial for activating the Notch pathway, inhibits goblet cell differentiation in PEDV-infected intestinal monolayer organoids. This study offers new insights into the mechanisms underlying intestinal mucosal barrier dysfunction induced by PEDV infection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13567-025-01599-5.

Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34+ hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients. Gene editing of hematopoietic stem and progenitor cells offers promise as a curative treatment for chronic granulomatous disease (CGD). Here, the authors develop a D10A Cas9n based gene editing strategy to treat CGD with no detectable off-target activity or chromosomal translocations.

Umbilical cord blood (UCB) units are an alternative source of human hematopoietic stem cells (HSCs) for allogeneic stem cell transplants. A large quantity of HSCs is needed but the low number of accessible cells from UCB has been a significant limitation. Improving the ex vivo growth of HSCs while preserving their functioning is required. Here, we report that andrographolide (AP) enhanced the expansion of human UCB-derived HSCs (HSPCs) and pro-moted primitive HSCs (CD34+CD38−CD90+). AP also improved HSC functionality, evidenced by increased growth of colony-forming units and multilineage differentiation. AP upregulated genes involved in the Wnt/β-catenin and Notch signaling pathways. AP also modulated signaling pathways involved in HSC self-renewal, proliferation, survival, and differentiation, demonstrated by Nanostring analysis. The results of this study suggest that andrographolide enhances ex vivo UCB-HSC expansion while maintaining functionality and has potential for treatment of hematological diseases.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15647-9.

Increased micro- and nanoplastic (MNP) pollution poses significant health risks, yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood. Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods, enabling multi-parameter single cell analysis. We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an ‘apical-out’ topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake. Using a phasor analysis approach, we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy. The resulting ‘fluorescence lifetime barcoding’ enabled distinguishing of different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total cell energy budget and epithelial inflammation. We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems. Fluorescence Lifetime Imaging Microscopy (FLIM) combined with pristine model NIR MNPs visualizes the internalization and biological impact of the nanoplastics in live small intestinal organoids

Despite robust antibody responses, immunity induced by acellular pertussis vaccine (DTaP) wanes over time and risk of pertussis seems to be lower in children who receive whole-cell vaccine (DTP) as their first dose. To interrogate the early immunologic response to pertussis vaccine, we enrolled 56 healthy infants who received either DTP or DTaP at 2-, 4-, 6-, and 18-months of age. RNA-sequencing and ribosome profiling of PBMC were performed prior to vaccination (Day 1) and on either Day 2 or Day 8. Pathway enrichment analysis on Days 2 and 8 showed enrichment of TLR-signaling and FcϒR-mediated phagocytosis among DTP recipients. DTP also led to increases in IRAK-4 and IL-1ß. After booster vaccination, a higher frequency of PT-specific B-cells was observed in DTP- vs. DTaP recipients. These data provide insights into the early immunologic responses to pertussis vaccine and may guide next-generation pertussis vaccine development.

Pleural mesothelioma (PM) is an aggressive, asbestos-linked cancer with limited treatment options and a poor prognosis. Lactate dehydrogenase B (LDHB) converts lactate to pyruvate, and its silencing reduces mitochondrial metabolism, particularly nucleotide synthesis. However, whether and a role of LDHB in PM is unclear. This study aimed to investigate the effects of silencing LDHB in PM cells and their response to chemotherapy. LDHB was silenced using siRNA transfection and inducible shRNA constructs. Proliferation, colony formation, and cell viability were assessed, while DNA damage was analyzed through ɣH2AX levels. Compared to normal mesothelial cells, LDHB was highly expressed in PM cell lines. LDHB inhibition significantly reduced proliferation, cell viability, and colony formation, indicating its crucial role in PM cells. Additionally, LDHB silencing significantly increased nuclear DNA damage accumulation as indicated by elevated ɣH2AX levels, which was reversed by nucleotide supplementation. In vivo, LDHB inhibition reduced tumor growth and enhanced cisplatin’s therapeutic efficacy. LDHB silencing increased ɣH2AX levels, which were further elevated with cisplatin treatment. Our results highlight LDHB as a novel therapeutic target in PM, where its inhibition induces DNA damage and improves the efficacy of cisplatin therapy.

Given the local and systemic adverse reactions associated with whole-cell pertussis vaccines combined with diphtheria and tetanus toxoids (DTP), acellular pertussis vaccines combined with the same toxoids (DTaP) were developed in the 1990s. In comparison to DTP, DTaP vaccines demonstrated reduced reactogenicity and equivalent or improved immunogenicity and efficacy. However, there has been a resurgence of pertussis disease, particularly in DTaP-vaccinated children, suggesting that immunity wanes more quickly with DTaP vaccination. To elucidate the differences in immune responses to DTP and DTaP vaccines, we employed a systems biology-based strategy to compare global changes in gene expression following primary vaccination with either DTP or DTaP. We used RNA-Seq and ribosome profiling (RP) to identify transcriptional and translational signatures, respectively, in peripheral blood mononuclear cells (PBMCs) collected from 50 infant recipients of DTP or DTaP at two time-points (baseline (pre-vaccination at Day 1) and either Day 2 or 8 post-vaccination). We also used standard serologic methods to assess immunogenicity, and correlated these results with transcriptional and translational signatures. Here, we provide a detailed description of the rationale, experimental design, methodology, and enrollment procedures used. Given the technical complexity of our approach, our objective is to fill knowledge gaps, describe key quality metrics, and support future publications. In brief, we recovered 4-12 million PBMCs (average 8.9 million) with 99% viability per 2.5 mL blood sample, enabling excellent nucleic acid recovery yields for the preparation of high-quality sequencing libraries. In turn, these generated RNA-Seq and RP datasets with sufficient genome coverage breadth and depth to enable differential gene expression analyses, demonstrating the validity of this approach to study pertussis vaccine immunology specifically, and its utility to characterize mechanisms of the human immune response to vaccination generally.

The telomerase enzyme is essential for telomere maintenance. Pathogenic variants in telomere-associated genes have been associated with critical telomere shortening, resulting in telomere biology disorders (TBD) such as bone marrow failure, idiopathic pulmonary fibrosis, and dyskeratosis congenita. The TBDs are clinically heterogeneous and families with TBD often experience an earlier onset and increased symptom severity for each generation. Consensus guidelines have identified certain genetic variants as pathogenic or likely pathogenic, but many are classified as variants of uncertain significance (VUS) in the absence of additional supporting evidence. The pathogenicity of a VUS in genes encoding the telomerase complex could be evaluated by in vitro telomerase activity (TA) measurement. We have developed a functional TA assay in patient-derived T-cells based on the Telomeric Repeat Amplification Protocol (TRAP) combined with qPCR. TA was significantly lower in six TBD patients with a TERT or TERC variant compared to controls (0.11 versus 0.54, p < 0.001). Four patients had a TA of more than three standard deviations below the mean of controls, strongly supporting pathogenicity of the variants. In summary, functional analysis of TA in patient-derived cells could support pathogenic evaluation in clinical diagnostics and reduce the number of reported VUS for TBD patients.

Although lithium (Li) is a widely used treatment for bipolar disorder, its exact mechanisms of action remain elusive. Research has shown that the two stable Li isotopes, which differ in their mass and nuclear spin, can induce distinct effects in both in vivo and in vitro studies. Since sodium (Na+) channels are the primary pathway for Li+ entry into cells, we examined how Li+ affects the current of Na+ channels using whole-cell patch-clamp techniques on SH-SY5Y neuroblastoma cells and human iPSC-derived cortical neurons. Our findings indicate that mammalian Na+ channels in both neuronal models studied here display no selectivity between Na+ and Li+, unlike previously reported bacterial Na+ channels. We observed differences between the two neuronal models in three measured parameters (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{V}_{\text{h}\text{a}\text{l}\text{f}},\:{G}_{\text{m}\text{a}\text{x}},\:z$$\end{document}). We saw no statistically significant differences between any ions in SH-SY5Y cells, but small differences in the half-maximum activation potential (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{V}_{\text{h}\text{a}\text{l}\text{f}}$$\end{document}) between Na+ and 6Li+ and between 7Li+ and 6Li+ were found in iPSC-derived cortical neurons. Although Na+ channels are widely expressed and important in neuronal function, the very small differences observed in this work suggest that Li+ regulation through Na+ channels is likely not the primary mechanism underlying Li+ isotope differentiation.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12893-9.

BackgroundExtracellular Vesicles (EVs) derived from mesenchymal stem cells (MSCs) have gained recognition as promising therapeutic and drug delivery agents in regenerative medicine. However, their clinical application is limited by donor variability, low scalability, and inconsistent therapeutic quality. To overcome these challenges, a robust and standardized production platform is urgently needed.MethodsWe developed a scalable biomanufacturing strategy by generating and expanding MSCs from extended pluripotent stem cells (EPSC) using a suspension bioreactor culture system. A fixed-bed bioreactor was integrated for automated, continuous expansion of iMSCs and downstream EV harvesting. EVs were isolated through a streamlined protocol and characterized for size, morphology, surface markers, and bioactivity. Therapeutic efficacy was assessed in a bleomycin-induced pulmonary fibrosis mouse model.ResultsiMSC-derived EVs (iMSC-EVs) exhibited comparable characteristics to primary MSC-EVs, including a size distribution of 70–80 nm, cup-shaped morphology, and expression of canonical EV markers (CD63, CD81, TSG101). iMSCs were expanded for up to 20 days in 3D culture, yielding > 5 × 10⁸ cells per batch using a suspension bioreactor culture system and producing ~ 1.2 × 10¹³ EV particles/day in a fixed-bed bioreactor. In vivo, iMSC-EVs significantly reduced Ashcroft fibrosis scores and bronchoalveolar lavage fluid protein levels in bleomycin-injured lungs, with therapeutic efficacy comparable to primary MSC-EVs.ConclusionsThis study establishes a scalable and standardized platform for producing high-quality iMSC-EVs using bioreactor-based systems. Our approach addresses key limitations in traditional EV production and sets the stage for AI-integrated, fully automated, GMP-compliant manufacturing of therapeutic EVs suitable for clinical translation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04507-y.