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

Autism Spectrum Disorder (ASD) is a developmental disorder characterized by impaired social communication and repetitive behaviors. In recent years,a pharmacological mouse model of ASD involving maternal administration of valproic acid (VPA) has become widely used. Newborn pups in this model show an abnormal balance between excitatory and inhibitory (E/I) signaling in neurons and exhibit ASD-like behavior. However,the molecular basis of this model and its implications for the pathogenesis of ASD in humans remain unknown. Using quantitative secretome analysis,we found that the level of leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was upregulated in the conditioned medium of VPA model neurons. This upregulation was associated with excitatory synaptic organizer activity. The secreted form of the extracellular domain of Lingo2 (sLingo2) is produced by the transmembrane metalloprotease ADAM10 through proteolytic processing. sLingo2 was found to induce the formation of excitatory synapses in both mouse and human neurons,and treatment with sLingo2 resulted in an increased frequency of miniature excitatory postsynaptic currents in human neurons. These findings suggest that sLingo2 is an excitatory synapse organizer involved in ASD,and further understanding of the mechanisms by which sLingo2 induces excitatory synaptogenesis is expected to advance our understanding of the pathogenesis of ASD. Subject terms: Autism spectrum disorders,Neuroscience View Publication

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death worldwide. Although immune checkpoint inhibitors (ICIs) have shown remarkable clinical efficacy,they can also induce a paradoxical cancer acceleration,known as hyperprogressive disease (HPD),whose causative mechanisms are still unclear. This study investigated the mechanisms of ICI resistance in an HPD-NSCLC model. Two primary cell cultures were established from samples of a NSCLC patient,before ICI initiation (“baseline”,NSCLC-B) and during HPD (“hyperprogression”,NSCLC-H). The cell lines were phenotypically and molecularly characterized through immunofluorescence,Western Blotting and RNA-Seq analysis. To assess cell plasticity and aggressiveness,cellular growth patterns were evaluated both in vitro and in vivo through 2D and 3D cell growth assays and patient-derived xenografts establishment. In vitro investigations,including the evaluation of cell sensitivity to interferon-gamma (IFN-γ) and cell response to PD-L1 modulation,were conducted to explore the influence of these factors on cell plasticity regulation. NSCLC-H exhibited increased expression of specific CD44 isoforms and a more aggressive phenotype,including organoid formation ability,compared to NSCLC-B. Plastic changes in NSCLC-H were well described by a deep transcriptome shift,that also affected IFN-γ–related genes,including PD-L1. IFN-γ–mediated cell growth inhibition was compromised in both 2D-cultured NSCLC-B and NSCLC-H cells. Further,the cytokine induced a partial activation of both type I and type II IFN-pathway mediators,together with a striking increase in NSCLC-B growth in 3D cell culture systems. Finally,low IFN-γ doses and PD-L1 modulation both promoted plastic changes in NSCLC-B,increasing CD44 expression and its ability to produce spheres. Our findings identified plasticity as a relevant hallmark of ICI-mediated HPD by demonstrating that ICIs can modulate the IFN-γ and PD-L1 pathways,driving tumor cell plasticity and fueling HPD development. The online version contains supplementary material available at 10.1186/s12967-024-06023-8. View Publication

Chromothripsis,the chaotic shattering and repair of chromosomes,is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN),we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%,driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A,a serine threonine kinase,is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo,and represents a druggable axis. Collectively,these findings define chr. 21amp as a prognostic biomarker in BP-MPN,and link chromothripsis to a therapeutic target. Subject terms: Leukaemia,DNA sequencing View Publication

JAK (Janus Kinase) inhibitors,such as ruxolitinib,were introduced a decade ago for treatment of myeloproliferative neoplasms (MPN). To evaluate ruxolitinib’s impact on MPN clonal evolution,we interrogate a myelofibrosis patient cohort with longitudinal molecular evaluation and discover that ruxolitinib is associated with clonal outgrowth of RAS pathway mutations. Single-cell DNA sequencing combined with ex vivo treatment of RAS mutated CD34 + primary patient cells,demonstrates that ruxolitinib induces RAS clonal selection both in a JAK/STAT wild-type and hyper-activated context. RAS mutations are associated with decreased transformation-free and overall survival only in patients treated with ruxolitinib. In vitro and in vivo competition assays demonstrate increased cellular fitness of RAS- mutated cells under ruxolitinib or JAK2 knock-down,consistent with an on-target effect. MAPK pathway activation is associated with JAK2 downregulation resulting in enhanced oncogenic potential of RAS mutations. Our results prompt screening for pre-existing RAS mutations in JAK inhibitor treated patients with MPN. Subject terms: Myeloproliferative disease,Cancer therapeutic resistance,Tumour heterogeneity,Cancer genetics View Publication

Metformin rejuvenates adult rat oligodendrocyte progenitor cells (OPCs) allowing more efficient differentiation into oligodendrocytes and improved remyelination,and therefore is of interest as a therapeutic in demyelinating diseases such as multiple sclerosis (MS). Here,we test whether metformin has a similar effect in human stem cell derived-OPCs. We assess how well human monoculture,organoid and chimera model culture systems simulate in vivo adult human oligodendrocytes,finding most close resemblance in the chimera model. Metformin increases myelin proteins and/or sheaths in all models even when human cells remain fetal-like. In the chimera model,metformin leads to increased mitochondrial area both in the human transplanted cells and in the mouse axons with associated increase of mitochondrial function/metabolism transcripts. Human oligodendrocytes from MS brain donors treated pre-mortem with metformin also express similar transcripts. Metformin’s brain effect is thus not cell-specific,alters metabolism in part through mitochondrial changes and leads to more myelin production. This bodes well for clinical trials testing metformin for neuroprotection. Subject terms: Oligodendrocyte,Multiple sclerosis,Multiple sclerosis,Regeneration and repair in the nervous system View Publication

Antibiotic resistance is a threat to human health,yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies,we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN . Unexpectedly,efflux pump mutations increased P. aeruginosa virulence during infection; however,neither the prevalence of mexEFoprN inactivating mutations in real human infections,nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for virulence enhancing mutations. Using genome sequencing of clinical isolates,we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq,metabolomics,genetic approaches,and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity. Subject terms: Antimicrobial resistance,Pathogens,Bacteriology,Molecular evolution View Publication

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here,we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP,termed the wing region. One mAb targeting the GP2 wing,MR228,showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb,MR235,to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs,resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection. View Publication

Type III interferon-lambda (IFN-$\lambda$) plays a critical role against infection,particularly in mucosal infection in the respiratory and gastrointestinal tract. Our study and other previous studies have shown that porcine IFN-$\lambda$ more efficiently curtails the infection of porcine epidemic diarrhea virus (PEDV) in the intestine epithelia than type I IFN,whereas IFN-$\lambda$3 exerts a more potent effect than IFN-$\lambda$1. However,the underlying mechanism remains elusive,and in particular,the transcriptional profile induced by IFN-$\lambda$3 has not been reported. Here,to resolve the mechanism responsible for the disparity between IFN-$\lambda$3 and type I IFN in anti-mucosal virus infection,we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-$\lambda$3 resulted in the differential expression of 983 genes. In contrast,IFN-$\alpha$ only modified the expression of 134 genes,and 110 of these genes were also observed in the response to IFN-$\lambda$3. A transcriptional enrichment analysis indicated that IFN-$\lambda$3 or IFN-$\alpha$ regulates multiple cellular processes and that IFN-$\lambda$3 activates more robust signaling pathways,particularly the antiviral Jak-STAT signaling pathway,than IFN-$\alpha$. Furthermore,we verified the RNA-Seq results through an RT-qPCR analysis of IPEC-J2 cells and porcine enteroids. Moreover,transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by IFN-$\lambda$3 significantly inhibited PEDV infection. Collectively,the data showed that IFN-$\lambda$3 induces a unique transcriptional profile that does not completely overlap with that induced by IFN-$\alpha$ and strongly elicits a set of genes responsible for the antiviral activity of IFN-$\lambda$3. These findings provide important knowledge regarding the elicited ISGs of type I and III IFNs in restricting porcine intestinal viral infection. View Publication

The mechanism of Fas antigen-mediated apoptosis is at present unclear. We show here that the 100,000 x g supernatant from cell lysates prepared from anti-Fas-stimulated JUR-KAT T cells,induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors,TPCK and DCI,and by the interleukin 1-beta-converting enzyme inhibitor,VAD-FMK. In addition,chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK,but not by DCI or VAD-FMK. These results suggest that Fas-mediated apoptosis in T cells involves the activation of a cascade of proteases. View Publication

Although arsenic trioxide (As(2)O(3)) is an effective therapy in acute promyelocytic leukemia (APL),its use in other malignancies is limited by the toxicity of concentrations required to induce apoptosis in non-APL tumor cells. We looked for agents that would synergize with As(2)O(3) to induce apoptosis in malignant cells,but not in normal cells. We found that trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),a widely known antioxidant,enhances As(2)O(3)-mediated apoptosis in APL,myeloma,and breast cancer cells. Treatment with As(2)O(3) and trolox increased intracellular oxidative stress,as evidenced by heme oxygenase-1 (HO-1) protein levels,c-Jun terminal kinase (JNK) activation,and protein and lipid oxidation. The synergistic effects of trolox may be specific to As(2)O(3),as trolox does not add to toxicity induced by other chemotherapeutic drugs. We explored the mechanism of this synergy using electron paramagnetic resonance and observed the formation of trolox radicals when trolox was combined with As(2)O(3),but not with doxorubicin. Importantly,trolox protected nonmalignant cells from As(2)O(3)-mediated cytotoxicity. Our data provide the first evidence that trolox may extend the therapeutic spectrum of As(2)O(3). Furthermore,the combination of As(2)O(3) and trolox shows potential specificity for tumor cells,suggesting it may not increase the toxicity associated with As(2)O(3) monotherapy in vivo. View Publication

OBJECTIVES An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE),potentially contributing to autoantigen externalisation,type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However,another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model,MRL/lpr. Here,we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. METHODS NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors,Cl-amidine and the newly described BB-Cl-amidine. NET formation,endothelial function,interferon signature,nephritis and skin disease were examined in treated mice. RESULTS Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function,while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys,while protecting against skin disease. CONCLUSIONS PAD inhibition reduces NET formation,while protecting against lupus-related damage to the vasculature,kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken. View Publication

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However,efficient stem cell to beta cell differentiation has proven difficult,possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals,we isolated pancreatic and gastrointestinal stromal cells,and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas,whereas stroma-specific activation of signaling via loss of Hedgehog regulators,Sufu and Spop,impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly,inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation,altogether revealing the requirement for organ-specific regulation of stromal niche signals. View Publication