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

Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels,but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected,leading to liver-selective pharmacology,when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality,as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441,which reduces glucose levels and improves lipid profiles in an animal model of diabetes. View Publication

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study,we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single,living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique,it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research. View Publication

T cell homeostasis is crucial for maintaining an efficient and balanced T cell immunity. The interaction between TCR and self peptide (sp) MHC ligands is known to be the key driving force in this process,and it is believed to be functionally and mechanistically different from that initiated by the antigenic TCR stimulation. Yet,very little is known about the downstream signaling events triggered by this TCR-spMHC interaction and how they differ from those triggered by antigenic TCR stimulation. In this study,we show that T cell conditional ablation of MEKK3,a Ser/Thr kinase in the MAPK cascade,causes a significant reduction in peripheral T cell numbers in the conditional knockout mice,but does not perturb thymic T cell development and maturation. Using an adoptive mixed transfer method,we show that MEKK3-deficient T cells are severely impaired in lymphopenia-induced cell proliferation and survival. Interestingly,the Ag-induced T cell proliferation proceeds normally in the absence of MEKK3. Finally,we found that the activity of ERK1/2,but not p38 MAPK,was attenuated during the lymphopenia-driven response in MEKK3-deficient T cells. Together,these data suggest that MEKK3 may play a crucial selective role for spMHC-mediated T cell homeostasis. View Publication

BACKGROUND: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study,we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. RESULTS: Colony formation and sulforhodamine B (IC50 textless 1 nM) assays,and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature,termed rapamycin metagene index (RMI),of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset,RMI did not correlate with tumor size or lymph node status. High (textgreater75th percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis,RMI (P = 0.029),tumor size (P = 0.015) and lymph node status (P = 0.001) were prognostic. In van 't Veer study,RMI was not associated with the time to develop distant metastasis (P = 0.41). In the Wang dataset,RMI predicted time to disease relapse (P = 0.009). CONCLUSION: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. View Publication

In this study,we have identified a novel member of the AMPK family,namely Sucrose non-fermenting related kinase (Snrk),that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart,and brain,and in cell types such as endothelial cells,smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts,and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts,and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally,adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis,and shows an autonomous role for SNRK during mammalian development. View Publication

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening,biomarker discovery,and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here,we describe the (1) retroviral reprogramming strategies in the generation of human iPSC,(2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality,and (3) protocol of cardiac differentiation by modulation of Wnt signaling and $\$-catenin pathway. View Publication

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However,further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-$\gamma$. In addition,we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-$\beta$ receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells,LTBR induced profound transcriptional and epigenomic remodelling,leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-$\kappa$B pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and ?? T cells,highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes. View Publication

The spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture,cell–cell interactions,and spatial expression patterns,they are limited to the multicellular scale. We present Bento,a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains,annotating localization patterns,and quantifying gene–gene colocalization. We demonstrate MERFISH,seqFISH +,Molecular Cartography,and Xenium datasets. Bento is part of the open-source Scverse ecosystem,enabling integration with other single-cell analysis tools.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03217-7. View Publication

Erythropoiesis,the essential process of hematopoietic stem cell development into erythrocytes,is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver,neonatal spleen,and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen,short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype,demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation,including hemoglobinization,cell condensation,and enucleation,and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells. View Publication