搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition associated with the inflammatory activation of alveolar macrophages. Here,we examined the role of circVAPA in regulating inflammasome activation and macrophage inflammatory polarization in an ARDS model.MethodscircVAPA expression levels were analyzed in macrophages isolated from healthy controls and patients with ARDS. In vitro cell models of mouse alveolar macrophages and an in vivo mouse ARDS model were established through Lipopolysaccharide (LPS) stimulation. The effects of circVAPA knockdown on macrophage inflammatory polarization,inflammasome activation,and pulmonary tissue damage were investigated in both cell and animal models. The interaction between circVAPA and downstream factors was verified through a luciferase reporter assay and by silencing circVAPA.ResultscircVAPA upregulation in alveolar macrophages was associated with the inflammation in ARDS patients. circVAPA was also upregulated in LPS-stimulated mouse alveolar macrophages (MH-S cells). Additionally,circVAPA knockdown attenuated the inflammatory activation of MH-S cells and reduced the expression of pyroptosis-related proteins. circVAPA silencing also mitigated the inflammatory effects of LPS-stimulated MH-S cells on lung epithelial cells (MLE-12),and alleviated the inflammatory damage in the pulmonary tissue of ARDS mouse model. We further showed that miR-212-3p/Sirt1 axis mediated the functional role of circVAPA in the inflammatory polarization of MH-S cells.ConclusionOur data suggest that circVAPA promotes inflammasome activity and macrophage inflammation by modulating miR-212-3p/Sirt1 axis in ARDS. Targeting circVAPA may be employed to suppress the inflammatory activation of alveolar macrophages in ARDS.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10735-024-10312-3. View Publication

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However,it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here,Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells,type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium. View Publication

Donor-specific antibodies (DSAs) targeting mismatched human leukocyte antigen (HLA) molecules are one of the principal threats to long-term graft survival in solid organ transplantation. However,many patients with long-term circulating DSAs do not manifest rejection responses,suggesting a degree of heterogeneity in their pathogenicity and related functional activity. Immunologic risk stratification of transplant recipients is complicated by challenges intrinsic to defining alloantibody responses that are potentially pathogenic versus those that are not. Thus,a comprehensive understanding of how human alloantibodies target and interact with donor HLA molecules is vital for the development and evaluation of new strategies aimed at reducing antibody-mediated rejection responses. In this study,we employ hydrogen–deuterium exchange–mass spectrometry (HDX–MS),molecular dynamics (MD) simulations,and advanced biochemical and biophysical methodologies to thoroughly characterize a panel of human monoclonal alloantibodies and define the influence of Fc-region biology,antibody binding kinetics,target antigen density,and structural characteristics on their ability to potentiate the forms of immune effector mechanisms that are strongly implicated in transplant rejection. Our findings have significant implications for our understanding of the key biological determinants that underlie the pathogenicity or lack thereof of human alloantibodies. View Publication

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids,organoids,tumoroids,or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies,vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints,we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids,as well as blood vessel organoids generated from pluripotent stem cells,cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids,as they successfully provide intravascular perfusion to these structures. We find that organoid growth,maturation,and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics. Subject terms: Stem-cell biotechnology,Tissue engineering,Biomedical engineering,Induced pluripotent stem cells,Microfluidics View Publication

Current anticancer therapies cannot eliminate all cancer cells,which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N -methyltransferase 9 (PRMT9),whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML),eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response,suppressing cell survival. Notably,PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase,which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall,we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy. Subject terms: Cancer,Tumour immunology View Publication

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level,ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However,whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here,we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules,neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43 Tg -ALS mice,ATXN2-Q33 causes reduced motor function,NMJ alterations,neuron degeneration and altered in vitro stress granule dynamics. Furthermore,gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together,these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy. Subject terms: Amyotrophic lateral sclerosis,Molecular neuroscience,Cellular neuroscience View Publication

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform,called UniMat,which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered,permeable membrane-based platform. Using UniMat,we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably,kidney organoids within UniMat show improved maturation,showing increased expression of nephron transcripts,more in vivo-like cell-type balance,enhanced vascularization,and better long-term stability. Moreover,UniMat’s design offers a more standardized organoid model for disease modeling and drug testing,as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence,UniMat presents a valuable platform for organoid technology,with potential applications in organ development,disease modeling,and drug screening. Subject terms: Nanofabrication and nanopatterning,Biomaterials,Stem-cell biotechnology View Publication

Pancreatic cancer is one of the most malignant cancers,and limited therapeutic options are available. The induction of ferroptosis is considered to be a novel,promising strategy that has potential in cancer treatment,and ferroptosis inducers may be new options for eradicating malignant cancers that are resistant to traditional drugs. The exact mechanism underlying the function of sorcin in the initiation and progression of pancreatic cancer remains unclear. The expression of sorcin in cancer tissues was assessed by analyzing TCGA,GEO and immunohistochemical staining data,and the function of sorcin in the induction of ferroptosis in pancreatic cancer cells was investigated. The mechanism underlying the function of sorcin was revealed through proteomics,co-IP,Ch-IP,and luciferase assays. Natural product screening was subsequently performed to screen for products that interact with sorcin to identify new ferroptosis inducers. We first showed that sorcin expression was positively correlated with the survival and tumor stages of patients with pancreatic cancer,and we revealed that sorcin inhibited ferroptosis through its noncalcium binding function. Furthermore,we discovered that sorcin interacted with PAX5 in the cytoplasm and inhibited PAX5 nuclear translocation,which in turn decreased FBXL12 protein expression and then reduced ALDH1A1 ubiquitination,thus inhibiting ferroptosis. Moreover,an in-house natural product screen revealed that celastrol inhibited the interaction of sorcin and PAX5 by directly binding to the Cys194 residue of the sorcin protein; disruption of the sorcin-PAX5 interaction promoted the nuclear translocation of PAX5,induced the expression of FBXL12,increased the ubiquitylation of ALDH1A1,and eventually induced ferroptosis in pancreatic cancer cells. In this study,we revealed the mechanism of action of sorcin,which is a druggable target for inducing ferroptosis,we identified celastrol as a novel agent that induces ferroptosis,and we showed that disrupting the sorcin-PAX5 interaction is a promising therapeutic strategy for treating pancreatic cancer. The online version contains supplementary material available at 10.1186/s13045-025-01680-8. View Publication

The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST,its efficiency remains low. Here,we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST -positive hPSCs from ~ 5 to ~ 43.7%,providing a robust method for stabilizing XCI in female hPSCs for diverse applications. The online version contains supplementary material available at 10.1186/s13287-025-04501-4. View Publication

During viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi's sarcoma-associated herpesvirus interacts and inhibits the central cell death effector,Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins,like vFlip,on liver physiology in vivo,mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level,hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably,embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice,culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis. View Publication

Cerebrospinal fluid (CSF) is a vital liquid,providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP),a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here,we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally,the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes. View Publication

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT),a key enzyme in NAD+ salvage,and loss of NAMPT activity alters their inflammatory potential. However,the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase,which led to consumption of NAD+. In this setting,increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway. View Publication