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

Organoids are self-assembled 3D cellular structures that resemble organs structurally and functionally,providing in vitro platforms for molecular and therapeutic studies. Generation of organoids from human cells often requires long and costly procedures with arguably low efficiency. Prediction and selection of cellular aggregates that result in healthy and functional organoids can be achieved by using artificial intelligence-based tools. Transforming images of 3D cellular constructs into digitally processable data sets for training deep learning models requires labeling of morphological boundaries,which often is performed manually. Here,we report an application named OrganoLabeler,which can create large image-based data sets in a consistent,reliable,fast,and user-friendly manner. OrganoLabeler can create segmented versions of images with combinations of contrast adjusting,K-means clustering,CLAHE,binary,and Otsu thresholding methods. We created embryoid body and brain organoid data sets,of which segmented images were manually created by human researchers and compared with OrganoLabeler. Validation is performed by training U-Net models,which are deep learning models specialized in image segmentation. U-Net models,which are trained with images segmented by OrganoLabeler,achieved similar or better segmentation accuracies than the ones trained with manually labeled reference images. OrganoLabeler can replace manual labeling,providing faster and more accurate results for organoid research free of charge. View Publication

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

INTRODUCTION: Alzheimer's disease (AD) pathology is complex and involves mitochondrial dysfunction. There are emerging therapies targeting mitochondrial function in clinical trials for AD. This highlights the need for biomarkers that measure mitochondrial function. METHODS: We determined the utility of a novel blood‐based mitochondrial biomarker,the mitochondrial functional index (MFI),in the context of AD in a pilot study.RESULTS: In vitro and in vivo models of AD had a reduced MFI. MFI was lower in human AD subjects and APOE ????4 carriers. Receiver operating characteristic analysis showed MFI had a higher area under the curve than other plasma biomarkers. The MFI biomarker correlated with the Mini‐Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR) scale. DISCUSSION: This study highlights the potential utility of MFI as a functional blood‐based mitochondrial biomarker to interrogate energy metabolism. Ongoing studies are examining the relationship of MFI with brain energy metabolism outcomes. Highlights: The MFI biomarker is reduced in cell and animal models of AD. The MFI biomarker is reduced in human AD subjects and APOE ε4 carriers. The MFI biomarker can discriminate between subjects with normal cognition and AD with better performance than other plasma biomarkers. The MFI biomarker correlates with cognitive scores. View Publication

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However,the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes,particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here,we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations,we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems,which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity,we use an arrangement relying on an FEP film,Immersol W,water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution,supports large tile-scan acquisitions,and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost,plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes. View Publication

Baculovirus,an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems,is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering,we developed SHOT 2.0,an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56,with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow,enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform,we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types,including iPSCs and liver cancer cells,with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox,providing a flexible platform for genome editing and future gene delivery. 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