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

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic $\$-cells. In other systems,Musashi genes regulate cell fate via Notch signaling,which we recently showed regulates $\$-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms,as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of $\$-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1,while it decreased Ins1 and Ins2 expression,revealing a molecular link between ER stress and $\$-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1,stimulate proliferation,inhibit apoptosis and reduce insulin expression,whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together,these results demonstrate overlapping,but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and $\$-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory $\$-cell proliferation and the loss of $\$-cell gene expression seen in specific phases of the progression to type 2 diabetes. View Publication

Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin,a potent antihyperglycaemic agent now recommended as the first-line oral therapy for T2D (Type 2 diabetes). The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production,mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. In addition,the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase),a cellular metabolic sensor,providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. The demonstration that respiratory chain complex I,but not AMPK,is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. Beyond its effect on glucose metabolism,metformin has been reported to restore ovarian function in PCOS (polycystic ovary syndrome),reduce fatty liver,and to lower microvascular and macrovascular complications associated with T2D. Its use has also recently been suggested as an adjuvant treatment for cancer or gestational diabetes and for the prevention in pre-diabetic populations. These emerging new therapeutic areas for metformin will be reviewed together with recent findings from pharmacogenetic studies linking genetic variations to drug response,a promising new step towards personalized medicine in the treatment of T2D. View Publication

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent,stem cell-derived cardiomyocytes,including contraction force,rate,duration,and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy,and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive,inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents,or as a platform for studying cardiomyocyte biology. View Publication

Large-scale cancer genomics projects are profiling hundreds of tumors at multiple molecular layers,including copy number,mRNA and miRNA expression,but the mechanistic relationships between these layers are often excluded from computational models. We developed a supervised learning framework for integrating molecular profiles with regulatory sequence information to reveal regulatory programs in cancer,including miRNA-mediated regulation. We applied our approach to 320 glioblastoma profiles and identified key miRNAs and transcription factors as common or subtype-specific drivers of expression changes. We confirmed that predicted gene expression signatures for proneural subtype regulators were consistent with in vivo expression changes in a PDGF-driven mouse model. We tested two predicted proneural drivers,miR-124 and miR-132,both underexpressed in proneural tumors,by overexpression in neurospheres and observed a partial reversal of corresponding tumor expression changes. Computationally dissecting the role of miRNAs in cancer may ultimately lead to small RNA therapeutics tailored to subtype or individual. View Publication

BACKGROUND Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor,FV,must be proteolytically activated by α-thrombin to FVa to function in prothrombinase,the platelet molecule is released from α-granules in a partially activated state,obviating the need for proteolytic activation. OBJECTIVES The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes,plasma-derived FV is proteolytically processed to form the platelet-derived pool. METHODS & RESULTS Subsequent to FV endocytosis,a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain,and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va. CONCLUSIONS Taken together,these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool. View Publication

Metformin is the first-line drug treatment for type 2 diabetes. Globally,over 100 million patients are prescribed this drug annually. Metformin was discovered before the era of target-based drug discovery and its molecular mechanism of action remains an area of vigorous diabetes research. An improvement in our understanding of metformin's molecular targets is likely to enable target-based identification of second-generation drugs with similar properties,a development that has been impossible up to now. The notion that 5' AMP-activated protein kinase (AMPK) mediates the anti-hyperglycaemic action of metformin has recently been challenged by genetic loss-of-function studies,thrusting the AMPK-independent effects of the drug into the spotlight for the first time in more than a decade. Key AMPK-independent effects of the drug include the mitochondrial actions that have been known for many years and which are still thought to be the primary site of action of metformin. Coupled with recent evidence of AMPK-independent effects on the counter-regulatory hormone glucagon,new paradigms of AMPK-independent drug action are beginning to take shape. In this review we summarise the recent research developments on the molecular action of metformin. View Publication

The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors,yet excessive immune reactivity is prevented under homeostasis. The intestinal microbiome can influence host susceptibility to extra-intestinal autoimmune disorders. Here we report that polysaccharide A (PSA),a symbiosis factor for the human intestinal commensal Bacteroides fragilis,protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE),an animal model for multiple sclerosis,through Toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39(+) CD4 T-cell subset by PSA. Ablation of CD39 signalling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further,CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3(+) CD4 Tregs. Importantly,CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination. View Publication

Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However,as recently reported by us and another group,tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules,we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report,tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin,we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin,we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore,tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast,other microtubule-targeting drugs,such as vincristine,paclitaxel,and colchicine,could not suppress the growth of cells overexpressing ABC transporters. Moreover,the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors. View Publication

There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover,proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells,acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein,we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4,and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog,but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion,our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however,efficient colony formation requires proteasome activity. Therefore,discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. View Publication

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins,the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins,and as extension of previous work (Palomo et al.,2014),a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. View Publication

Summary Rett syndrome (RTT) is caused by mutations of MECP2,a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show,using an isogenic human embryonic stem cell model of RTT,that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells. View Publication

Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1),a key component of the stem cell niche,regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate and whether these actions are related to its lineage skewing effects are poorly understood. Here we demonstrate that,although DL1 activates signal transducer and activator of transcription 3 signaling similarly to the gp130-activating cytokine interleukin-6 (IL-6),it has opposite effects on myeloid cell production. Mechanistically,these different outcomes are attributable to a DL1-mediated reduction in membrane (m)-bound IL-6 receptor (R) expression,converting progenitor cells from being directly IL-6 responsive to requiring both IL-6 and soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL-6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action in which DL1 changes cytokine receptor distributions on hematopoietic cells,altering feedback networks and their impact on stem cell fate. View Publication