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

SUMMARY Increasing evidence suggests that the mechanics of chromatin and nucleoplasm regulate gene transcription and nuclear function. However,how the chromatin and nucleoplasm sense and respond to forces remains elusive. Here,we employed a strategy of applying forces directly to the chromatin of a cell via a microinjected 200-nm anti-H2B-antibody-coated ferromagnetic nanoparticle (FMNP) and an anti-immunoglobulin G (IgG)-antibody-coated or an uncoated FMNP. The chromatin behaved as a viscoelastic gel-like structure and the nucleoplasm was a softer viscoelastic structure at loading frequencies of 0.1–5 Hz. Protein diffusivity of the chromatin,nucleoplasm,and RNA polymerase II (RNA Pol II) and RNA Pol II activity were upregulated in a chromatin-stretching-dependent manner and stayed upregulated for tens of minutes after force cessation. Chromatin stiffness increased,but the mechanomemory duration of chromatin diffusivity decreased,with substrate stiffness. These findings may provide a mechanomemory mechanism of transcription upregulation and have implications on cell and nuclear functions. Graphical abstract In brief Rashid et al. show that chromatin and nucleoplasm in cells behave as viscoelastic materials. Chromatin stretching mediates the mechanomemory of chromatin and nucleoplasm diffusivity as well as of RNA polymerase II activity. The mechanomemory of RNA polymerase II activity provides a mechanism for sustained transcription upregulation tens of minutes after force cessation. View Publication

Clinical studies have shown that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is associated with aggressive periodontitis and can potentially trigger or exacerbate rheumatoid arthritis (RA). However,the mechanism is poorly understood. Here,we show that systemic infection with A. actinomycetemcomitans triggers the progression of arthritis in mice anti-collagen antibody-induced arthritis (CAIA) model following IL-1β secretion and cell infiltration in paws in a manner that is dependent on caspase-11-mediated inflammasome activation in macrophages. The administration of polymyxin B (PMB),chloroquine,and anti-CD11b antibody suppressed inflammasome activation in macrophages and arthritis in mice,suggesting that the recognition of lipopolysaccharide (LPS) in the cytosol after bacterial degradation by lysosomes and invasion via CD11b are needed to trigger arthritis following inflammasome activation in macrophages. These data reveal that the inhibition of caspase-11-mediated inflammasome activation potentiates aggravation of RA induced by infection with A. actinomycetemcomitans. This work highlights how RA can be progressed by inflammasome activation as a result of periodontitis-associated bacterial infection and discusses the mechanism of inflammasome activation in response to infection with A. actinomycetemcomitans. View Publication

The prognosis for adult B-cell acute lymphoblastic leukemia remains unfavorable,especially in the context of relapsed and refractory disease. Exploring the molecular mechanisms underlying disease progression holds significant promise for improving clinical outcomes. In this investigation,utilizing single-cell transcriptome sequencing technology,we discerned a correlation between Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) and the progression of B-cell acute lymphoblastic leukemia. Our findings reveal a significant upregulation of UHRF1 in cases of relapsed and refractory B-cell acute lymphoblastic leukemia,thereby serving as a prognostic indicator for poor outcomes. Both deletion of UHRF1 or overexpression of its downstream target secreted frizzled-related protein 5 (SFRP5) resulted in the inhibition of leukemia cell proliferation,promoting cellular apoptosis and induction of cell cycle arrest. Our results showed that UHRF1 employs methylation modifications to repress the expression of SFRP5,consequently inducing the WNT5A-P38 MAPK-HK2 signaling axis,resulting in the augmentation of lactate,the critical metabolic product of aerobic glycolysis. Furthermore,we identified UM164 as a targeted inhibitor of UHRF1 that substantially inhibits P38 protein phosphorylation,downregulates HK2 expression,and reduces lactate production. UM164 also demonstrated antileukemic activity both in vitro and in vivo. In summary,our investigation revealed the molecular mechanisms of epigenetic and metabolic reprogramming in relapsed and refractory B-cell acute lymphoblastic leukemia and provides potential targeted therapeutic strategies to improve its inadequate prognosis. View Publication

ObjectiveB lymphocytes play a crucial role in immunity but also contribute to the pathogenesis of various diseases. Cannabis plants produce numerous biologically active compounds,including cannabinoids. The two most studied phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids exert diverse and potent biological effects primarily through the endocannabinoid system (ECS),which also plays a key role in mature B-cell function. Both the immune system and the ECS undergo age-related changes that lead to a clinically significant decline in function.MethodsThis study compares the effects of THC and CBD on B-cell activity in young and aged mice. Murine B lymphocytes were activated using lipopolysaccharide (LPS) and interleukin-4 (IL-4),and the impact of cannabinoid treatments was assessed in terms of cell phenotype,proliferation,antibody secretion,tumor necrosis factor-alpha (TNFα) secretion,extracellular signal-regulated kinase (ERK) phosphorylation,and the cellular metabolome.ResultsBoth THC and CBD exhibited dose-dependent inhibitory effects on B-cell activation in young and aged mice. However,we show here,for the first time,that the treatments induce distinct metabolic profiles. Although some metabolites,such as glucose-6-phosphate,pentose phosphate pathway (PPP) and nucleotide metabolites,were reduced by both cannabinoids,THC selectively reduced the levels of a distinct set of amino acids,while only CBD increased the levels of Citrulline and Allantoin. Additionally,the effects of THC and CBD differed between young and aged B cells,suggesting that age-related changes in the ECS may influence cannabinoid sensitivity.ConclusionsThese findings provide insights into the distinct mechanisms by which THC and CBD regulate immune activation and may open the door for investigating the mechanisms behind cannabinoids effects on the immune system. They also highlight the need for further research into phytocannabinoid-based therapies,particularly in age-specific contexts. Given the immunoregulatory properties of cannabinoids,especially CBD,tailored therapeutic strategies may enhance their clinical applications View Publication

Interleukin-6 (IL-6) is a multifunctional cytokine that plays important roles in inflammation. Several studies have shown that IL-6 regulates various aspects of T cell function,including the differentiation of CD4+ T cells into the pro-inflammatory Th17 subset. Given the tight link between T cell metabolism and function,and the role of IL-6 in regulating cellular metabolism across tissues,we investigated the role of IL-6 signaling in Th17 cell metabolism. Using T cell specific IL-6 receptor (IL-6R) conditional knockout mice and littermate controls,we found that IL-6R signaling regulates the proportions of CD4+ and CD8+ T cells and drives CD4+ T cell differentiation into Th17 cells. We also found that IL-6R signaling is required for Th17 cell glycolytic metabolism. In T cell-specific IL-6R knockout mice,Th17 cells had reduced glucose uptake and glycolysis,as well as decreased expression of key glycolytic enzymes,while showing increased basal oxygen consumption. However,we also found that IL-6R signaling enhanced oxidative capacity and mitochondrial coupling efficiency in Th17 T cells. Importantly,inhibition of lactate dehydrogenase using FX11 selectively impaired Th17 cell differentiation with minimal effects on Treg cells. These findings suggest that targeting metabolic pathways regulated by IL-6R signaling can selectively inhibit inflammatory Th17 responses,offering a potential strategy for controlling IL-6 mediated inflammation. View Publication

Cannabinoid receptor 1 (CB1) has been implicated in multiple inflammatory diseases by regulating pro-inflammatory mediators or altering immune cell polarization. However,the expression and direct functional role of CB1 in T cells remain largely unexplored. Here,we demonstrate that primary murine T cells express CB1 and that its novel agonist,BI-5756,directly increases the frequencies of regulatory T cells (Tregs) in primary murine pan T cells after activation. In addition,BI-5756 exhibits an in vivo protective effect against graft-versus-host disease (GvHD),an allogeneic T cell-mediated inflammatory complication after allogeneic hematopoietic cell transplantation (allo-HCT),resulting in an improved overall survival with enhanced platelet recovery and reconstitution of bone marrow-derived B and T cells. BI-5756 also directly suppresses tumor cell growth and upregulates MHC I,MHC II,and CD80 on tumor cells,which may subsequently enhance T cell-mediated anti-tumor responses in mixed lymphocyte reaction with A20 cells. The ability of BI-5756 to increase Tregs was significantly abrogated by rimonabant,a potent and selective CB1 antagonist,suggesting that the immunomodulatory effect of BI-5756 is mediated via CB1. In summary,BI-5756,a potent CB1 agonist,increases Tregs while preserving anti-tumor responses in vitro and effectively reduces GvHD in vivo. View Publication

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However,the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package,factR2,which offers a comprehensive suite of AS-NMD analysis functions. Using this tool,we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably,this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. The online version contains supplementary material available at 10.1186/s13059-024-03305-8. View Publication

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy,contingent upon the species is crucial to circumvent transgene silencing,necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly,transgene silencing occurred while using the CAG promoter,contrary to conventional understanding,whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations,confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally,GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats,thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2,mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations,with implications for future research in gene-engineered rat models. The online version contains supplementary material available at 10.1186/s12917-024-04123-7. View Publication

Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal expansion of myeloid progenitor cells. Despite recent advancements in the treatment of AML,relapse still remains a significant challenge,necessitating the development of innovative therapies to eliminate minimal residual disease. One promising approach to address these unmet clinical needs is natural killer (NK) cell immunotherapy. To implement such treatments effectively,it is vital to comprehend how AML cells escape the NK-cell surveillance. Signal transducer and activator of transcription 3 (STAT3),a component of the Janus kinase (JAK)-STAT signaling pathway,is well-known for its role in driving immune evasion in various cancer types. Nevertheless,the specific function of STAT3 in AML cell escape from NK cells has not been deeply investigated. In this study,we unravel a novel role of STAT3 in sensitizing AML cells to NK-cell surveillance. We demonstrate that STAT3-deficient AML cell lines are inefficiently eliminated by NK cells. Mechanistically,AML cells lacking STAT3 fail to form an immune synapse as efficiently as their wild-type counterparts due to significantly reduced surface expression of intercellular adhesion molecule 1 (ICAM-1). The impaired killing of STAT3-deficient cells can be rescued by ICAM-1 overexpression proving its central role in the observed phenotype. Importantly,analysis of our AML patient cohort revealed a positive correlation between ICAM1 and STAT3 expression suggesting a predominant role of STAT3 in ICAM-1 regulation in this disease. In line,high ICAM1 expression correlates with better survival of AML patients underscoring the translational relevance of our findings. Taken together,our data unveil a novel role of STAT3 in preventing AML cells from escaping NK-cell surveillance and highlight the STAT3/ICAM-1 axis as a potential biomarker for NK-cell therapies in AML. View Publication

Besides neutralizing antibodies,which are considered an important measure for vaccine immunogenicity,Fc-mediated antibody functions can contribute to antibody-mediated protection. They are strongly influenced by structural antibody properties such as subclass and Fc glycan composition. We here applied a systems serology approach to dissect humoral immune responses induced by MVA-MERS-S,an MVA-vectored vaccine against the Middle East respiratory syndrome coronavirus (MERS-CoV). Building on preceding studies reporting the safety and immunogenicity of MVA-MERS-S,our study highlights the potential of a late boost,administered one year after prime,to enhance both neutralizing and Fc-mediated antibody functionality compared to the primary vaccination series. Distinct characteristics were observed for antibodies specific to the MERS-CoV spike protein S1 and S2 subunits,regarding subclass and glycan compositions as well as Fc functionality. These findings highlight the benefit of a late homologous booster vaccination with MVA-MERS-S and may be of interest for the design of future coronavirus vaccines. Subject areas: Cell biology,Immune response,Immunology,Virology View Publication

Streptococcus pneumoniae is a global priority respiratory pathogen that kills over a million people annually. The pore-forming cytotoxin,pneumolysin (PLY) is a major virulence factor. Here,we found that recombinant PLY as well as wild-type pneumococcal strains,but not the isogenic PLY mutant,upregulated the shedding of extracellular vesicles (EVs) harboring membrane-bound toxin from human THP-1 monocytes. PLY-EVs induced cytotoxicity and hemolysis dose-dependently upon internalization by recipient monocyte-derived dendritic cells. Proteomics analysis revealed that PLY-EVs are selectively enriched in key inflammatory host proteins such as IFI16,NLRC4,PTX3,and MMP9. EVs shed from PLY-challenged or infected cells induced dendritic cell maturation and primed them to infection. In vivo,zebrafish administered with PLY-EVs showed pericardial edema and mortality. Adoptive transfer of bronchoalveolar-lavage-derived EVs from infected mice to healthy recipients induced lung damage and inflammation in a PLY-dependent manner. Our findings identify that host EVs released during infection mediate pneumococcal pathogenesis. Subject areas: Microbiology,Bacteriology,Cell biology View Publication

Isobaric labeling-based mass spectrometry (ILMS) has been widely used to quantify,on a proteome-wide scale,the relative protein abundance in different biological conditions. However,large-scale ILMS data sets typically involve multiple runs of mass spectrometry,bringing great computational difficulty to the integration of ILMS samples. We present zMAP,a toolset that makes ILMS intensities comparable across mass spectrometry runs by modeling the associated mean-variance dependence and accordingly applying a variance stabilizing z-transformation. The practical utility of zMAP is demonstrated in several case studies involving the dynamics of cell differentiation and the heterogeneity across cancer patients. The online version contains supplementary material available at 10.1186/s13059-024-03382-9. View Publication