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

IntroductionCurrent antiretroviral therapy (ART) for HIV infection reduces plasma viral loads to undetectable levels and has increased the life expectancy of people with HIV (PWH). However,this increased lifespan is accompanied by signs of accelerated aging and a higher prevalence of age-related comorbidities. Tat (Trans-Activator of Transcription) is a key protein for viral replication and pathogenesis. Tat is encoded by 2 exons,with the full-length Tat ranging from 86 to 101 aa (Tat101). Introducing a stop codon in position 73 generates a 1 exon,synthetic 72aa Tat (Tat72). Intracellular,full-length Tat activates the NF-κB pro-inflammatory pathway and increases antiapoptotic signals and ROS generation. These effects may initiate a cellular senescence program,characterized by cell cycle arrest,altered cell metabolism,and increased senescence-associated secretory phenotype (SASP) mediator release However,the precise role of HIV-Tat in inducing a cellular senescence program in CD4+ T-cells is currently unknown.MethodsJurkat Tetoff cell lines stably transfected with Tat72,Tat101,or an empty vector were used. Flow cytometry and RT-qPCR were used to address senescence biomarkers,and 105 mediators were assessed in cell supernatants with an antibody-based membrane array. Key results obtained in Jurkat-Tat cells were addressed in primary,resting CD4+ T-cells by transient electroporation of HIV-Tat-FLAG plasmid DNA.ResultsIn the Jurkat cell model,expression of Tat101 increased the levels of the senescence biomarkers BCL-2,CD87,and p21,and increased the release of sCD30,PDGF-AA,and sCD31,among other factors. Tat101 upregulated CD30 and CD31 co-expression in the Jurkat cell surface,distinguishing these cells from Tat72 and Tetoff Jurkats. The percentage of p21+,p16+,and γ-H2AX+ cells were higher in Tat-expressing CD4+ T-cells,detected as a FLAG+ population compared to their FLAG- (Tat negative) counterparts. Increased levels of sCD31 and sCD26 were also detected in electroporated CD4+ T-cell supernatants.DiscussionIntracellular,full-length HIV-Tat expression increases several senescence biomarkers in Jurkat and CD4+ T-cells,and SASP/Aging mediators in cell supernatants. Intracellular HIV-Tat may initiate a cellular senescence program,contributing to the premature aging phenotype observed in PWH. Graphical Abstract View Publication

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However,its impact on the immune landscape of the tumor microenvironment (TME) remains unexplored. Here,we investigated the role of CD24a,the murine CD24 gene,in tumor progression and TME immune dynamics in a murine triple-negative breast cancer (TNBC) model.MethodsClustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 knockout technology was employed to generate CD24a knockout in the murine TNBC cell line 4T1. Flow cytometry was utilized to analyze the immune cell populations,including myeloid-derived suppressor cells (MDSCs),natural killer cells,T cells,and macrophages,within tumors,spleens,and bone marrow in the orthotopic mouse 4T1 breast cancer model. Immunofluorescence (IF) staining was used to detect the immune cells in tumor sections. High-speed confocal was used to perform three-dimensional (3D) mapping of immune cells in the 4T1 orthotopic tumors.ResultsKnocking out CD24a significantly reduced tumor growth kinetics and prolonged mouse survival in vivo. Flow cytometry and IF analysis of tumor samples revealed that CD24a loss significantly promoted the infiltration of M1 macrophages and cytotoxic CD8+ T cells into the TME while reducing the recruitment and expansion of granulocytic MDSCs (gMDSCs). In vitro coculture experiments showed that CD24a deficiency significantly enhanced macrophage‐mediated phagocytosis and CD8⁺ T cell-mediated cytotoxicity,effects that were partially reversed by re‐expression of CD24a. Moreover,in vivo depletion of macrophages and CD8+ T cells reverted the delayed tumor growth caused by CD24a knockout,underscoring their critical role in tumor growth suppression associated with CD24a knockout. 3D mapping of immune cells in the TME confirmed the anti-tumor immune landscape in the CD24a knockout 4T1 tumors. Furthermore,in vitro analysis showed that CD24a loss upregulated macrophage colony-stimulating factor expression while suppressed levels of CXCL1,CXCL5,and CXCL10,chemokines known to recruit gMDSCs,further providing a molecular basis for enhanced macrophage recruitment and diminished gMDSC accumulation.ConclusionsOur findings suggest that CD24a may regulate immune suppression within the TNBC TME. Targeting CD24a enhances macrophage- and CD8⁺ T cell-mediated anti-tumor immune responses and is associated with a shift in the TME toward a more immunogenic state,thereby suppressing tumor growth. These results may support CD24 as a promising immunotherapeutic target for TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-025-01165-3. View Publication

Understanding diseases as the result of continuous transitions from a healthy system is more realistic than understanding them as discrete states. Here,we designed the spectrum formation approach (SFA),a machine learning-based method that extracts key features contributing to disease state continuity. We applied the SFA to transcriptomic data from patients with progressive liver disease and neurodegenerative movement disorders to examine its effectiveness in identifying biologically relevant gene sets. The SFA identified transcription factors that potentially regulate liver inflammation and voluntary movement. In neurodegenerative disorders,the SFA also identified genes regulated by ETS-1,with unclear effects on movement. In functional assessment using human iPSC-derived neurons,ETS-1 overexpression disrupted dopamine receptor balance,reduced GABA-producing enzyme levels,and promoted cell death. These findings suggest that the SFA enables the discovery of regulatory factors capable of modifying disease states and provides a framework for the continuity-based interpretation of biological systems. Subject terms: Diseases,Pathogenesis,Signs and symptoms View Publication

Alpha-synuclein (aSyn) post-translational modifications (PTM),especially phosphorylation at serine 129 and C-terminal truncations,are highly enriched in Lewy bodies (LB),Lewy neurites,and other pathological aggregates in Parkinson’s disease and synucleinopathies. However,the precise role of these PTM in pathology formation,neurodegeneration,and pathology spreading remains unclear. Here,we systematically investigated the role of post-fibrillization C-terminal aSyn truncations in regulating uptake,processing,seeding,and LB-like inclusion formation using a neuronal seeding model that recapitulates LB formation and neurodegeneration. We show that C-terminal cleavage of aSyn fibrils occurs rapidly post exogenous fibril internalization and during intracellular LB-like inclusion formation. Blocking cleavage of internalized fibrils does not affect seeding,but inhibiting enzymes such as calpains 1 and 2 alters LB-like inclusion formation. We show that C-terminal truncations,along with other PTMs,regulate fibril interactome remodeling,shortening,lateral association,and packing. These findings reveal distinct roles of C-terminal truncations at different aggregation stages on the pathway to LB formation,highlighting the need for consideration of stage‑specific strategies to target aSyn proteolytic cleavages. View Publication

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a complex and multifactorial etiology,making it challenging to treat. While recent advances in immunomodulatory biologics,such as antitumor necrosis factor-α (TNF-α) antibodies,have shown moderate success,systemic administration of antibody therapeutics may lead to several adverse effects,including the risk of autoimmune disorders due to systemic cytokine depletion. Transient RNA interference using exogenous short interfering RNA (siRNA) to regulate target gene expression at the transcript level offers an alternative to systemic immunomodulation. However,siRNAs are susceptible to premature degradation and have poor cellular uptake. Graphene oxide (GO) nanoparticles have been shown to be effective nanocarriers for biologics due to their reduced cytotoxicity and enhanced bioavailability. In this study,we evaluate the therapeutic efficacy of GO mediated TNF-α_siRNA using in vitro models of chronic inflammation generated by treating murine small intestines (enteroids) and large intestines (colonoids) with inflammatory agents IL-1β,TNF-α,and LPS. The organotypic mouse enteroids and colonoids developed an inflammatory phenotype similar to that of IBD,characterized by impaired epithelial homeostasis and an increased production of inflammatory cytokines such as TNF-α,IL-1β,and IL-6. We assessed siRNA delivery to these inflamed organoids using three different GO formulations. Out of the three,small-sized GO with polymer and dendrimer modifications (smGO) demonstrated the highest transfection efficiency,which led to the downregulation of inflammatory cytokines,indicating an attenuation of the inflammatory phenotype. Moreover,the transfection efficiency and inflammation-ameliorating effects could be further enhanced by increasing the TNF-α_siRNA/smGO ratio from 1:1 to 3:1. Overall,the results of this study demonstrate that ex vivo organoids with disease-specific phenotypes are invaluable models for assessing the therapeutic potential of nanocarrier-mediated drug and biologic delivery systems. View Publication

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications,but has long-term cytotoxic effects. Trehalose,a natural disaccharide,has been proposed as a non-toxic cryoprotectant. However,the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs,which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally,the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery,offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods. Subject terms: Membrane biophysics,Biomedical engineering View Publication

Acute myeloid leukemia (AML) is a deadly hematopoietic malignancy. Although many patients achieve complete remission with standard induction therapy,a combination of cytarabine and anthracycline,~40% of patients have induction failure. These refractory patients pose a treatment challenge,as they do not respond to salvage therapy or allogeneic stem cell transplant. Herein,we show that AML patients who experience induction failure have elevated expression of the NF-κB target gene tumor necrosis factor alpha-induced protein-3 (TNFAIP3/A20) and impaired necroptotic cell death. A20 High AML are resistant to anthracyclines,while A20 Low AML are sensitive. Loss of A20 in AML restores sensitivity to anthracycline treatment by inducing necroptosis. Moreover,A20 prevents necroptosis in AML by targeting the necroptosis effector RIPK1,and anthracycline-induced necroptosis is abrogated in A20 High AML. These findings suggest that NF-κB-driven A20 overexpression plays a role in failed chemotherapy induction and highlights the potential of targeting an alternative cell death pathway in AML. Subject terms: Acute myeloid leukaemia,Cancer therapeutic resistance View Publication

Brain organoids have been proposed as suitable human brain model candidates for a variety of applications. However,the lack of appropriate maturation limits the transferability of such functional tools. Here,we present a method to facilitate neuronal maturation by integrating astrocyte-secreted factors into hPSC-derived 2D and 3D neural culture systems. We demonstrate that protein- and nutrient-enriched astrocyte-conditioned medium (ACM) accelerates neuronal differentiation with enlarged neuronal layer and the overproduction of deep-layer cortical neurons. We captured the elevated changes in the functional activity of neuronal networks within ACM-treated organoids using comprehensive electrophysiological recordings. Furthermore,astrocyte-secreted cues can induce lipid droplet accumulation in neural cultures,offering protective effects in neural differentiation to withstand cellular stress. Together,these data indicate the potential of astrocyte secretions to promote neural maturation. Subject terms: Neurological models,Neuronal development View Publication

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1,CCM2,or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years,it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1,CCM2,or CCM3 knockout,single-cell RNA sequencing,and high-content imaging,we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions,inactivation of CCM1,CCM2,or CCM3 also led to specific gene expression patterns in neuronal,mesenchymal,and endothelial cell populations,respectively. Taking advantage of the different fluorescent labels of the hiPSCs,we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast,CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally,the excellent scalability of blood vessel organoid differentiation in a 96-well format further supports their use in high-throughput drug discovery and other biomedical research studies. The online version contains supplementary material available at 10.1007/s10456-025-09985-5. View Publication

Cytolysin A (ClyA) is a pore‐forming protein from a strongly silenced gene in non‐pathogenic Escherichia coli,including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction,ClyA‐expressing bacteria display a cytolytic phenotype. However,it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli ‐host interactions in vivo. Here,we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA + E. coli were internalised into cultured colon cancer cells. The OMV‐associated ClyA caused reduced levels of cancer‐activating proteins such as H3K27me3,CXCR4,STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non‐pathogenic E. coli can influence the stability of the EZH2 protein,reducing its activity in epigenetic regulation,causing elevated level of the tumour suppressor protein p53. View Publication

Monoclonal antibody-based immune checkpoint inhibitors,which have brought breakthrough effects in cancer treatments,are expected to assist in the treatment of viral diseases. However,antibody therapies may cause immune-related side effects,such as inflammation and pneumonia,due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group),namely compound 2 (molecular weight: 456.6),from our library of sulfur-containing protein–protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1,with the dissociation rate constant (K D ) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably,oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33,H1N1 subtype) caused a significant increase in the neutralizing antibody titers,as well as recovery from influenza-induced pneumonia. Overall,2 provides insight for the development of therapeutic drugs against early viral infections,with both virus titer-reducing and antibody titer-boosting effects. Moreover,2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development,Pharmacology,Screening,Structure-based drug design View Publication

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis,binding and uptake of exosomes are well characterized4,5,delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology,we show that these two lipid receptors,which interact in the late endosome9,are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway,which Ebola virus exploits to infect cells9,mediates HAV infection by exo-HAV,which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen,but not the endosomal uncoating of HAV particles contained in the exosomes,is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV,infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses,and call for a reassessment of the role of envelope glycoproteins in infection. View Publication