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

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML),we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues,and developed an algorithm to identify potential targets expressed in leukemia stem cells,but not in normal CD34+CD38- hematopoietic cells,T cells,or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19,we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML. View Publication

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication

INTRODUCTION The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED Key talks delivered at the 'CiPA on my mind' session,held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS),are summarized. Issues and potential solutions relating to crucial constituents [e.g.,biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes),study platforms,drug solutions,and data analysis] of CiPA core assays are critically examined. DISCUSSION In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy,systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories,it can be implemented as qualified regulatory procedure. View Publication

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia,which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC),a critical regulator of transcriptional elongation,suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions,as part of SEC and the polymerase-associated factor complex (PAFc),are associated with the BET family of acetyl-lysine recognizing,chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia,via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family,GSK1210151A (I-BET151),has profound efficacy against human and murine MLL-fusion leukaemic cell lines,through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is,at least in part,due to the inhibition of transcription at key genes (BCL2,C-MYC and CDK6) through the displacement of BRD3/4,PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value,providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally,the efficacy of I-BET151 against human leukaemia stem cells is demonstrated,providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

MicroRNAs (miRNAs/miRs) are small,endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling,we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs,120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses,we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet,miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion,we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation,miR-139 expression is dispensable for influenza-specific CTL responses. View Publication

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior,drug response,and cell–matrix interactions. Here,we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates,facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation,GelMA preparation,bioprinting,and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research,including studies of metastasis,drug resistance,cancer cell lipid metabolism,and TME-associated hypoxia. View Publication

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization,including disrupted nucleocytoplasmic transport (NCT); however,the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons,which is sufficient to cause NCT deficits,nuclear pore abnormalities,and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further,GDE2 deficits are evident in human neural cell models of ALS,which display erroneous Wnt activation that,when inhibited,increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease. Synopsis Nuclear exclusion of TDP-43 is observed in various pathologies,but the physiological mechanisms that ensure its nuclear localization are not well-known. This work shows that inhibition of persistent Wnt activation in neurons by GDE2 prevents TDP-43 nuclear exclusion. GDE2 inhibits canonical Wnt signaling in adult postmitotic neurons.Sustained activation of canonical Wnt signaling in neurons disrupts the nuclear pore complex,impairs nucleocytoplasmic transport,and results in TDP-43 nuclear exclusion.iPS neurons from patients with C9orf72 ALS show decreased GDE2 expression and increased activation of canonical Wnt signaling.Inhibition of Wnt activation mitigates TDP-43 dysfunction in C9orf72 iPS neurons. GDE2 maintains TDP-43 nuclear localization by inhibiting Wnt activation in neurons. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

Eosinophils,a type of granulocyte derived from myeloid precursors in the bone marrow,are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation,tissue homeostasis,and host defense,while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions,their specific expression and functions within eosinophils have not been thoroughly investigated,largely due to the reliance on tissue homogenates. In an effort to address this gap,we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs,ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology,we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly,ITGB2-AS1 deficiency led to impaired eosinophil differentiation,as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins,including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally,ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions,with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function,positioning it as a novel regulator in eosinophil biology. View Publication

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii,a parasite that naturally travels from the human gut to the central nervous system (CNS),can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems,the rhoptries and dense granules,to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells,brain organoids and in vivo,and probe protein activity using imaging,pull-down assays,scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept,we demonstrate GRA16-mediated brain delivery of the MeCP2 protein,a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system,we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology,Biologics,Synthetic biology View Publication

Metastasis is one of the leading causes of cancer-related death worldwide. Fascin,a protein that bundles actin filaments to produce protrusions in cancer cells,plays a significant role in the enhancement of cell migration. This protein has been shown that the overexpression of this protein is related to the appearance of different types of cancer,such as colorectal cancer. In this study,we conducted in silico screening of the Enamine library,a compound library with a broad chemical space. Using a ligand-based virtual screening approach based on the pharmacophore model of G2,we identified the predicted inhibitors. First,these compounds were validated by physicochemical analysis. Differential scanning calorimetry (DSF) was used to study the binding between the predicted compounds and fascin protein,followed by an F-actin bundling assay to determine which compounds inhibited the bundling function of fascin. Z1362873773,which exhibited binding to fascin and inhibited F-actin bundling,was further tested in cell cultures to assess its effects on cancer cell viability and migration as well as in organoid models to evaluate potential cytotoxicity. Finally,we established a protocol that can be applied to discover anti-fascin agents from diverse compound libraries. A new molecule has been identified with considerable fascin inhibitory and migration-arresting capacity,which may lead to the development of new therapies to treat cancer. The online version contains supplementary material available at 10.1038/s41598-025-96457-x. Subject terms: Biochemistry,Biophysics,Cancer,Drug discovery,Molecular biology,Virtual drug screening View Publication