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

T cell dysfunction is a pivotal driving factor in autoimmune diseases,yet its underlying regulatory mechanisms remain incompletely understood. The role of long non-coding RNAs (lncRNAs) in immune regulation has gradually been recognized,although their functional mechanisms in T cells remain elusive. This study focuses on lncBADR (LncRNA Branched-chain Amino acids Degradation Regulator),elucidating its mechanism by which it regulates branched-chain amino acids (BCAAs) metabolism to influence T cell effector functions. Mice with specific knockout of lncBADR (T celllncBADR−/−) exhibited markedly ameliorated experimental autoimmune encephalomyelitis (EAE) symptoms. Mechanistic investigations revealed that lncBADR inhibits BCAAs degradation by binding to the enzymes Mccc1 and Pcca,leading to the accumulation of BCAAs within T-cells. This,in turn,activates the mTOR-Stat1 signaling pathway,promoting IFN-γ secretion and exacerbating EAE pathology. In contrast,knockout of lncBADR restored BCAAs degradation,significantly reducing IFN-γ secretion in T cells and suppressing their pathogenic functions. Further studies demonstrated that high-BCAAs feeding partially reversed the protective effects of lncBADR knockout,indicating that lncBADR plays a crucial role in autoimmune inflammation by regulating BCAAs metabolism. This study offers new insights into targeting lncBADR or modulating BCAAs metabolism as potential therapeutic strategies for autoimmune diseases. View Publication

Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells,especially relapsed and refractory (R/R)-AML,present high GPX4 levels and enzyme activities,pharmacological inhibition of GPX4 alone has limited application in AML. Thus,whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. Lipid reactive oxygen species (ROS),malondialdehyde (MDA),and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC),ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3),or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally,we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells,especially those with MLL rearrangements,than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects,low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis,and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically,DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK,suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition,DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally,DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients,especially for R/R-AML. The online version contains supplementary material available at 10.1186/s40164-024-00489-4. View Publication

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

Epithelial-to-mesenchymal transition (EMT),cancer stem cells (CSCs),and colorectal cancer (CRC) therapy resistance are closely associated. Prior reports have demonstrated that sphingosine-1-phosphate (S1P) supports stem cells and maintains the CSC phenotype. We hypothesized that the EMT inducer SNAI1 drives S1P signaling to amplify CSC self-renewal capacity and chemoresistance. CRC cell lines with or without ectopic expression of SNAI1 were used to study the role of S1P signaling as mediators of cancer stemness and 5-fluorouracil (5FU) chemoresistance. The therapeutic ability of sphingosine kinase 2 (SPHK2) was assessed using siRNA and ABC294640,a SPHK2 inhibitor. CSCs were isolated from patient-derived xenografts (PDXs) and assessed for SPHK2 and SNAI1 expression. Ectopic SNAI1 expressing cell lines demonstrated elevated SPHK2 expression and increased SPHK2 promoter activity. SPHK2 inhibition with siRNA or ABC294640 ablated in vitro self-renewal and sensitized cells to 5FU. CSCs isolated from CRC PDXs express increased SPHK2 relative to the non-CSC population. Combination ABC294640/5FU therapy significantly inhibited tumor growth in mice and enhanced 5FU response in therapy-resistant CRC patient-derived tumor organoids (PDTOs). SNAI1/SPHK2 signaling mediates cancer stemness and 5FU resistance,implicating S1P as a therapeutic target for CRC. The S1P inhibitor ABC294640 holds potential as a therapeutic agent to target CSCs in therapy refractory CRC. View Publication

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However,the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells,and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover,MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC. View Publication

Functional cellular heterogeneity in tumours often underlies incomplete response to therapy and relapse. Previously,we demonstrated that the growth of the paediatric brain malignancy,sonic hedgehog subgroup medulloblastoma,is rooted in a dysregulated developmental hierarchy,the apex of which is defined by characteristically quiescent SOX2 + stem-like cells. Integrating gene expression and chromatin accessibility patterns in distinct cellular compartments,we identify the transcription factor Olig2 as regulating the stem cell fate transition from quiescence to activation,driving the generation of downstream neoplastic progenitors. Inactivation of Olig2 blocks stem cell activation and tumour output. Targeting this rare OLIG2-driven proliferative programme with a small molecule inhibitor,CT-179,dramatically attenuates early tumour formation and tumour regrowth post-therapy,and significantly increases median survival in vivo. We demonstrate that targeting transition from quiescence to proliferation at the level of the tumorigenic cell could be a pivotal medulloblastoma treatment strategy. Subject terms: Cancer stem cells,Mechanisms of disease,Cancer therapy View Publication

Cerebral organoids (COs) are valuable tools for studying the intricate interplay between glial cells and neurons in brain development and disease,including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and inducing pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly with the neuronal progenitors. Compared with conventional COs,CO-iMs were more efficient at generating CD45 + /CD11b + /Iba-1 + microglia and presented a physiologically relevant proportion of microglia (~ 7%). CO-iMs presented substantially increased expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs are susceptible to HIV infection,resulting in a significant increase in several pro-inflammatory cytokines/chemokines,which are abrogated by the addition of antiretrovirals. Thus,CO-iM is a robust model for deciphering neuropathogenesis,neuroinflammation,and viral infections of brain cells in a 3D culture system. The online version contains supplementary material available at 10.1186/s12974-025-03353-2. View Publication

Biological mechanisms are inherently dynamic,requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales,making them unsuitable for studying dynamic processes or characterizing essential genes,where chronic depletion can cause cell death. We compare five inducible protein degradation systems—dTAG,HaloPROTAC,IKZF3,and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency,basal degradation,target recovery after ligand washout,and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However,AID 2.0’s higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations,we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants,including S210A,that significantly enhance the overall degron efficiency. The resulting degron system,named AID 2.1,maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout,enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts. Subject terms: Genetic engineering,CRISPR-Cas9 genome editing,Peptides View Publication

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation,allowing symbiotic co-existence with the intestinal microbiota. However,upon acute events such as epithelial damage and concomitant influx of microbes,these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here,we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly,these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition,Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together,our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling. View Publication

The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However,the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here,we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological,immunological,and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions,however,viral replication and the clinical course of infection are attenuated. Thus,Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion. View Publication

Our understanding of the signalling pathways regulating early human development is limited,despite their fundamental biological importance. Here,we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors,along with IGF1 ligand. Consequently,we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions,we derive several pluripotent stem cell lines that express pluripotency-associated genes,retain high viability and a normal karyotype,and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos,and in both primed and na{\{i}}ve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche." View Publication

Immunotherapy is innovating clinical cancer management. Nevertheless,only a small fraction of patient's benefit from current immunotherapies. To improve clinical management of cancer immunotherapy,it is critical to develop strategies for response monitoring and prediction. In this study,we describe inducible T-cell costimulator (ICOS) as a conserved mediator of immune response across multiple therapy strategies. ICOS expression was evaluated by flow cytometry,89Zr-DFO-ICOS mAb PET/CT imaging was performed on Lewis lung cancer models treated with different immunotherapy strategies,and the change in tumor volume was used as a read-out for therapeutic response. ImmunoPET imaging of ICOS enabled sensitive and specific detection of activated T cells and early benchmarking of immune response. A STING (stimulator of interferon genes) agonist was identified as a promising therapeutic approach in this manner. The STING agonist generated significantly stronger immune responses as measured by ICOS ImmunoPET and delayed tumor growth compared with programmed death-1 checkpoint blockade. More importantly,ICOS ImmunoPET enabled early and robust prediction of therapeutic response across multiple treatment regimens. These data show that ICOS is an indicator of T-cell-mediated immune response and suggests ICOS ImmunoPET as a promising strategy for monitoring,comparing,and predicting immunotherapy success in cancer. SIGNIFICANCE: ICOS ImmunoPET is a promising strategy to noninvasively predict and monitor immunotherapy response.See related commentary by Choyke,p. 2975. View Publication