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Influenza infection predisposes individuals to secondary pneumonia caused by a range of pathogens, including both bacterial and fungal organisms. Neutrophils are critical effector cells during infection. In this study, we analyzed the transcriptional pathways of lung neutrophils isolated from mouse models of influenza-associated pulmonary aspergillosis (IAPA) and post-influenza methicillin-resistant Staphylococcus aureus (MRSA) pneumonia to examine the immunopathological mechanisms underlying post-influenza super-infection. Pathways associated with neutrophil chemotaxis and degranulation were inhibited in IAPA compared to singular A. fumigatus infection and pathways associated with neutrophil recruitment and phagocytosis were inhibited in IAPA compared to singular influenza infection. Pathways associated with neutrophil recruitment and degranulation were inhibited in post-influenza MRSA pneumonia compared to singular MRSA infection and pathways associated with cytokine signaling were inhibited in post-influenza MRSA pneumonia compared to singular influenza infection. When the 2 types of super-infection were directly compared, pathways related to cytokine induction and neutrophil function were inhibited in IAPA neutrophils compared to post-influenza MRSA pneumonia. These data demonstrate that influenza causes neutrophil dysfunction, predisposing to secondary fungal and bacterial infections.

Gastrointestinal (GI) dysfunction, characterized by severe diarrhea and dehydration, is a central contributor to morbidity and mortality in filovirus disease in patients, yet the role of the epithelium in this clinical outcome remains poorly defined. Here, we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses, including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover, infection impaired adenylate cyclase signaling and CFTR-mediated ion transport, providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract, causing intense diarrhea and life-threatening dehydration. Yet, how these viruses affect the gut remains poorly understood. In this study, we used human mini-guts—small, three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues, disrupt key barrier structures, and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks.

Retinal neurogenesis is mediated by the coordinated activities of a complex gene regulatory network (GRN) of transcription factors (TFs) in multipotent retinal progenitor cells (RPCs). How this GRN mechanistically guides neural competence remains poorly understood. In this study, we present integrated transcriptional, genetic and genomic analyses to uncover the regulatory mechanisms of SOX2, a key factor in establishing neural identity in RPCs. We show that SOX2 is preferentially enriched in the RPC-specific enhancer landscape associated with essential regulators of retinogenesis. Disruption of SOX2 expression impairs retinogenesis, marked by a selective loss of enhancer activity near genes essential for RPC proliferation and lineage specification. We identified the RPC transcription factor VSX2 as a binding partner for SOX2 and, together, SOX2 and VSX2 co-target a core, retina-specific chromatin repertoire characterized by enhanced TF binding and robust chromatin accessibility. This cooperative binding establishes a shared SOX2-VSX2 transcriptional code that promotes the expression of crucial regulators of neurogenesis while repressing the acquisition of alternative lineage cell fate. Our data illuminate fundamental biological insights on how transcription factors act in concert to drive chromatin-based genetic programs underlying retinal neural identity.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population, causing infectious mononucleosis, susceptibility to autoimmune diseases and multiple malignancies of epithelial or B cell-origin. EBV infects epithelial cells and B cells through interaction between viral glycoproteins and different host receptors, but it has remained unknown whether a common receptor mediates infection of its two major host cell targets. Here, we establish R9AP as a crucial EBV receptor for entry into epithelial and B cells. R9AP silencing or knockout, R9AP-derived peptide and R9AP monoclonal antibody each significantly inhibit, whereas R9AP overexpression promotes, EBV uptake into both cell types. R9AP binds directly to the EBV glycoprotein gH/gL complex to initiate gH/gL-gB-mediated membrane fusion. Notably, the interaction of R9AP with gH/gL is inhibited by the highly competitive gH/gL-neutralizing antibody AMMO1, which blocks EBV epithelial and B cell entry. Moreover, R9AP mediates viral and cellular membrane fusion in cooperation with EBV gp42-human leukocyte antigen class II or gH/gL-EPHA2 complexes in B cells or epithelial cells, respectively. We propose R9AP as the crucial common receptor of B cells and epithelial cells and a potential prophylactic and vaccine target for EBV.

Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens, including parasites. These ETs function to trap, immobilize, and eventually kill pathogens. In line with this, monocytes and macrophages have been shown to release ETs, known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii, this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods: Primary human and bovine monocytes, monocytic THP-1 cells, and THP-1 cell-derived macrophages (M0-, M1-, and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), immunofluorescencemicroscopy, and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally, the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6, and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion: Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore, the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs, although this was a rare event. In primary human monocytes, an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS), primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells, THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS, all THP-1 cell-derived macrophages showed an enhanced release of IL-1β, and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent, MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites.

Pancreatic β cells derived from human induced pluripotent stem cells (hiPSCs) represent a promising therapeutic avenue in regenerative medicine for diabetes treatment. However, current differentiation protocols lack the specificity and efficiency required to reliably produce fully functional β cells, limiting their clinical applicability. Epigenetic barriers, such as histone modifications, may hinder proper differentiation and the acquisition of essential maturation markers in these cells. Methods: hiPSCs were cultured under feeder-free conditions and subjected to lentiviral transduction with shRNA constructs to silence KDM4A. Differentiation into pancreatic β-like cells was performed using stepwise protocols, with or without doxycycline supplementation, to evaluate the effect of KDM4A suppression. Gene expression was quantified by RT-qPCR, protein expression was assessed by western blotting and immunofluorescence, and functional insulin release was determined by glucose-stimulated insulin secretion (GSIS) assays. Statistical analysis was conducted using unpaired two-tailed Student’s t-tests, with significance set at p < 0.05. Results: A reduction in pancreatic development proteins was observed in the different differentiation states evaluated, after blocking KDM4A expression. Knockdown of KDM4A significantly reduced the expression of pancreatic β-cell genes, such as PDX1, Nkx6.1, and Ins, by 50% compared to WT iPSCs differentiated under the same conditions. Similarly, glucose-stimulated insulin secretion was reduced by approximately 80% in KDM4A-deficient β-like cells. Conclusions: These results emphasize the critical role of histone demethylation in hiPSC differentiation toward β cells. Our findings identify KDM4A as a key epigenetic regulator, suggesting that its modulation could enhance the generation of functional β cells for regenerative medicine in diabetes.

The utility of measuring real-time cellular bioenergetics of peripheral blood mononuclear cells (PBMCs) as biomarkers in disease monitoring, such as the bioenergetic health index, is of emerging interest. However, various experimental factors can impact the accuracy and reproducibility of these measurements. Methods: : PBMC bioenergetics were probed in real-time using extracellular flux analysis to identify optimal seeding density and injection protocol. Using a modified protocol, we assessed the extent to which blood processing time and isolation method (SepMate™ vs. EasySep™ Direct) influence PBMC bioenergetics under basal and stimulated conditions. Advanced metabolic control analysis including mitochondrial and glycolytic ATP supply flux, respiratory control ratio, bioenergetic health index, and mitochondrial toxicity index were used to identify and quantify PBMC bioenergetics. Results: Measures of metabolic profiling such as mitochondrial respiration, glycolytic activity, ATP supply flux, and respiratory control ratio were significantly diminished in PBMCs due to blood processing delay (48–72 hours) and were influenced by isolation method. Extended blood processing time significantly lowered T cell activation capacity in PBMCs, evidenced by decreased responses of mitochondrial and glycolytic ATP supply to CD3/CD28 activation. Discussion/Conclusion: This study demonstrates that key experimental variables including blood processing time and isolation method critically affect the reliability and biological relevance of PBMC metabolic assessments, highlighting the importance of protocol standardisation for accurate bioenergetic biomarker measurements.

Embryonic stem cell (ESC)-derived cardiomyocytes are a key resource for studying cardiac development and advancing regenerative therapies. Beclin1 (Becn1), a core regulator of autophagy and cardiac morphogenesis, has an undefined role in cardiomyocyte lineage specification. This study aims to investigate the regulatory function of Becn1 during cardiac differentiation from both mouse and human ESCs. Methods: Mouse and human ESCs were differentiated into cardiomyocytes through established embryoid body (EB) formation or monolayer differentiation protocols. Stable Becn1 knockdown was achieved using short hairpin RNA (shRNA). Cardiomyocyte differentiation efficiency was evaluated by flow cytometry, immunocytochemistry, and contraction assays. Differentiation cardiomyocyte function was evaluated by sarcomere arrangement, calcium transients, and microelectrode array (MEA). Transcriptomic profiling was conducted by bulk RNA sequencing, and pathway dynamics were analyzed using qPCR and western blotting. Results: Becn1 expression declined over the course of differentiation. Knockdown of Becn1 significantly enhanced cardiomyocyte yield and promoted earlier onset of contractile activity, accompanied by increased expression of cardiac-specific markers. Mechanistically, Becn1 deficiency elicited a biphasic Wnt signaling response, characterized by early activation during mesodermal induction followed by suppression at later stages of differentiation. This shift was accompanied by sustained BMP pathway activation. Notably, Becn1-deficient ESCs underwent efficient cardiac differentiation in the absence of exogenous VEGF or FGF, with BMP signaling compensating for their omission. These findings were recapitulated in human ESCs, where BECN1 knockdown supported Wnt modulators-independent cardiomyocyte differentiation through coordinated modulation of Wnt and BMP pathways. Conclusions: Becn1 serves as a negative regulator of cardiac lineage commitment by orchestrating stage-specific Wnt/BMP signaling dynamics. Silencing Becn1 enhances cardiomyocyte differentiation and enables growth factor-independent lineage progression. These findings offer a novel approach to improve the efficiency and scalability of stem cell-derived cardiomyocyte production for regenerative applications.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma, immunosuppressive tumor microenvironment (TME), and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence, infiltration, and function in PDAC. Methods: We established a direct reprogramming strategy to generate cytotoxic NK cells (1 F-NKs) by targeting BCL11B, a transcription factor essential for T cell lineage commitment, using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1 F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition. Results: BCL11B depletion enabled the generation of CD56brightCD16bright 1 F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into BCL11B locus generated MSLN-1 F-NKs with stable antigen specific activity. A genome-wide screen identified PKMYT1 as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1, sensitizing PDACs to 1 F-NK cytotoxicity. In PDAC xenograft models, 1 F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably, this triple combination elicited a synergistic antitumor effect, outperforming each monotherapy or dual combination. Conclusions: This study presents a synergistic immunotherapy platform that integrates NK reprogramming, CAR engineering, and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors.

Cancer stem cells were prominent responsible for cancer initiation, metastasis, and invasion as well as therapeutic resistance in colorectal cancer (CRC). The extracellular axon guidance factor netrin-1 has been found to be overexpressed in several malignant cancers such as glioma, lung cancers, and colorectal cancer. However, the role of netrin-1 on cancer stemness in CRC remains unveiled. Our study revealed high expression of netrin-1 in colorectal cancer tissues and its ability to promote cancer stemness by interacting with receptors UNC5B and neogenin on murine colorectal cancer cell. Mechanistically, the netrin-1-UNC5B/neogenin axis activates the downstream NF-κB and ERK1/2 signaling pathways, reinforcing the stemness properties of tumor cells, and further exacerbating tumor progression. Clinically, netrin-1 expression associated with poor survival and high CD133 expression in patients with CRC. Taken together, these results suggest that netrin-1 blockade could be a compelling therapeutic strategy to improve the poor outcomes and trigger cancer stemness inhibition in CRC treatment.

Chemotherapy-induced peripheral neuropathy (CIPN) affects up to two-thirds of cancer patients undergoing cytotoxic chemotherapy. Here, we used human iPSC-derived sensory neurons (iPSC-DSN) to model CIPN in vitro. Administration of various chemotherapeutic agents (i.e., paclitaxel, vincristine, bortezomib and cisplatin) at clinically applicable concentrations resulted in reduced cell viability, axonal degeneration, electrophysiological dysfunction and increased levels of phosphorylated c-Jun in iPSC-DSN. Transcriptomic analyses revealed that the upregulation of c-Jun strongly correlated with the expression of genes of neuronal injury, apoptosis and inflammatory signatures. To test whether c-Jun plays a central role in the development of CIPN, we applied the small molecule inhibitor of the Jun N-terminal kinase, SP600125, to iPSC-DSN treated with neurotoxic chemotherapy. c-Jun inhibition prevented chemotherapy-induced neurotoxicity by preserving cell viability, axonal integrity and electrophysiological function of iPSC-DSN. These findings identify c-Jun as a key mediator of CIPN pathophysiology across multiple drug types and present preclinical evidence that c-Jun inhibition is an attractive therapeutic target to prevent CIPN.

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.