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Venoms comprise highly sophisticated bioactive molecules modulating ion channels,receptors,coagulation factors,and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research,we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis,pore formation,and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements,we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic,ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms,we found that the pore-forming latrotoxin ?-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes,while the lytic bee peptide melittin is not selective. Furthermore,evaluation of snake venoms showed that Elapid species induced rapid membrane lysis,while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits,aligning with clinical observations at venom level,beyond discriminating among ion pore-forming,membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic,cardiotoxic and neurotoxic effects,and assist in identifying venom components that hold the potential to benefit humankind. Graphical abstractImage 1 Highlights•Optimization of bioassays to study venoms strengthens the discovery of novel drugs and envenomation treatments•We developed a high-content bioassay measuring DNA and [Ca2+]i that investigates multiple mechanisms in venom biology•This bioassay monitored membrane integrity,ion channels and ion pore formation to unravel venom's mechanism of action•We found the latrotoxin ?-Lt1a strikingly prefers neuron-like cells while the ?-helical melittin is non-selective•Evaluation of Elapid and Viper snake venoms demonstrates that this bioassay predicts the phylogeny and clinical findings View Publication

The emergence of the primitive streak,representing an organizing center for gastrulation,marks the mesendodermal lineage specification from epiblast,in which the epiblast cells undergo highly organized collective behaviors to form mesendodermal cells properly. Cell death is observed at the peri-gastrulation stage,especially in the primitive streak region. However,the dynamic and regulatory mechanism of cell death in the primitive streak formation is unclear. Here,we observed that a quick inhibition of the fast elevated cell death is coinciding with an accumulation of ?-catenin during the early stage of primitive streak induction from human embryonic stem cells (hESCs). Deficiency of ?-catenin in hESCs does not affect their self-renewal but cause robust cell death after primitive streak induction,while neuroectodermal differentiation remains unchanged. Overexpression of full-length ?-catenin in ?-catenin-deficient hESCs restores the cell death restriction during induction of primitive streak. Mechanistically,the ?-catenin-restricted cell death during primitive streak is transcription-independent. The accumulated ?-catenin traps casein kinase-1 in ?-catenin destruction complex following WNT activation via its ARM repeat domain,resulting in the inhibition of mTORC1 by stabilizing DEPTOR,subsequently attenuates mitochondrial translocation of p53 and enhances mitophagy to promote cell survival. Consistently,mTORC1 inhibition by rapamycin or RAD001 attenuates the cell death in ?-catenin-deficient cells during induction of primitive streak. In addition,only the ?-catenin retains activations of cell death restriction and transcriptional activity can promote hESCs to successfully differentiate into primitive streak and cardiomyocytes,suggesting that ?-catenin-restricted cell death safeguards the fate transition during the primitive streak induction via offering a crucial window for the accumulation of ?-catenin to induce lineage-specific genes. These findings provide new insights into the function and mechanisms by which ?-catenin coordinates the cell death and early lineage commitment. View Publication

BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass,irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications,neurodegeneration,and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity,homeostasis,and repair. Here,we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome,their modulation,and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ? 5 Gy) were treated with iMSC CM,endothelial cell viability,adherence,spheroid compactness,and proangiogenic sprout formation were significantly ameliorated,and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently,iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore,iMSC CM suppressed IR-induced NF?B activation,TNF-? release,and ROS production in THP1 cells. Additionally,iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3,iMSCs,or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2,IL6,IL8/CXCL8,ANG (Angiogenin),GRO?/CXCL1,and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors,the PI3K/AKT/mTOR modulator PRAS40 and ?-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism,translation,mitochondrial respiration,DNA damage repair,and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03847-5. View Publication

BackgroundGraft-versus-host disease (GVHD) and relapse are major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Metabolites play crucial roles in immune regulation,but their causal relationships with GVHD and relapse remain unclear.MethodsWe utilized genetic variants from genome-wide association studies (GWAS) of 309 known metabolites as instrumental variables to evaluate their causal effects on acute GVHD (aGVHD),gut GVHD,chronic GVHD (cGVHD),and relapse in different populations. Multiple causal inference methods,heterogeneity assessments,and pleiotropy tests were conducted to ensure result robustness. Multivariable MR analysis was performed to adjust for potential confounders,and validation MR analysis further confirmed key findings. Mediation MR analysis was employed to explore indirect causal pathways.ResultsAfter correction for multiple testing,we identified elevated pyridoxate and proline levels as protective factors against grade 3–4 aGVHD (aGVHD3) and relapse,respectively. Conversely,glycochenodeoxycholate increased the risk of aGVHD3,whereas 1-stearoylglycerophosphoethanolamine had a protective effect. The robustness and stability of these findings were confirmed by multiple causal inference approaches,heterogeneity,and horizontal pleiotropy analyses. Multivariable MR analysis further excluded potential confounding pleiotropic effects. Validation MR analyses supported the causal roles of pyridoxate and 1-stearoylglycerophosphoethanolamine,while mediation MR revealed that pyridoxate influences GVHD directly and indirectly via CD39 + Tregs. Pathway analyses highlighted critical biochemical alterations,including disruptions in bile acid metabolism and the regulatory roles of vitamin B6 derivatives. Finally,clinical metabolic analyses,including direct fecal metabolite measurements,confirmed the protective role of pyridoxate against aGVHD.ConclusionsOur findings provide novel insights into the metabolic mechanisms underlying GVHD and relapse after allo-HSCT. Identified metabolites,particularly pyridoxate,may serve as potential therapeutic targets for GVHD prevention and management.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. Key points1. Strong evidence has found the protective effect of pyridoxate against aGVHD and has been validated in patients.2. Mediated MR analysis suggests that pyridoxate may reduce aGVHD risk by increasing CD39+ Tregs level.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12916-025-04026-w. View Publication

The development of hematopoietic cell lineages is a highly complex process governed by a delicate interplay of various transcription factors. The expression of these factors is influenced,in part,by epigenetic signatures that define each stage of cell differentiation. In particular,the formation of B lymphocytes depends on the sequential silencing of stemness genes and the balanced expression of interdependent transcription factors,along with DNA rearrangement. We have investigated the impact of Dido3 deficiency,a protein involved in chromatin status readout,on B cell differentiation within the hematopoietic compartment of mice. Our findings revealed significant impairments in the successive stages of B cell development. The absence of Dido3 resulted in remarkable alterations in the expression of essential transcription factors and differentiation markers,which are crucial for orchestrating the differentiation process. Additionally,the somatic recombination process,responsible for generation of antigen receptor diversity,was also adversely affected. These observations highlight the vital role of epigenetic regulation,particularly the involvement of Dido3,in ensuring proper B cell differentiation. This study reveals new mechanisms underlying disruptive alterations,deepening our understanding of hematopoiesis and may potentially lead to insights that aid in the development of therapeutic interventions for disorders involving aberrant B cell development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-025-01394-x. View Publication

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD),characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here,we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes,as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils,which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT,which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation,focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus,LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls,likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation,which could potentially be exploited therapeutically to reduce excessive inflammation in CGD. View Publication

Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here,we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells,and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells,trans-epithelial electrical resistance (TEER) ({\textgreater}400 Ohms.cm2),and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents ({\textgreater}3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time,this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore,this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF,ASL,ion channel function,presence of differentiated cells,TEER) when developing in vitro lung models. View Publication

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that,in human,the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution,we observe a continuous but polarised organisation of the 49f+ compartment,where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics,whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state,distinct from lymphoid-primed multipotent progenitors,representing the earliest entry point into lymphoid commitment. View Publication

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However,the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4,Sox2,c-Myc,and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog,TDGF,and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction,small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology,gene expression,long-term self-renewal ability,and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation. ?? 2008 Elsevier B.V. All rights reserved. View Publication

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis,a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD),an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally,we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels,with a concomitant increase of the RIP1 protein. Together,these two related proteins form the necrosome,the complex responsible for induction of necroptotic cell death. Interestingly,we found that caspase-8 mRNA,a known negative regulator of necroptosis,was transiently decreased following OGD. Importantly,we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia,specifically in the CA1 area of the hippocampus. Moreover,we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together,our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury. View Publication

BACKGROUND Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity,despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive,only cataloging heterogeneity at one point in time,and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity,and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS We introduce the programmable Polaris microfluidic lab-on-chip for single-cell sequencing,which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1,such as an altered oxidative stress response,have a large paracrine signaling component. Furthermore,we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation,both associated with the oxidative stress response and altered proteostasis. Interestingly,SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'),with no known co-regulation. CONCLUSION As single-cell methods continue to mature,so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture,live-cell imaging,and single-cell sequencing,we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages,for example,react to inflammation and form treatment resistant HIV reservoirs. View Publication