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

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

Oxidative stress serves as an important regulator of both apoptosis and metabolic reprogramming in tumor cells. Chaetocin,a histone methyltransferase inhibitor,is known to induce ROS generation. As elevating basal ROS level sensitizes glioma cells to apoptosis,the ability of Chaetocin in regulating apoptotic and metabolic adaptive responses in glioma was investigated. Chaetocin induced glioma cell apoptosis in a ROS-dependent manner. Increased intracellular ROS induced (i) Yes-associated protein 1 (YAP1) expression independent of the canonical Hippo pathway as well as (ii) ATM and JNK activation. Increased interaction of YAP1 with p73 and p300 induced apoptosis in an ATM-dependent manner. Chaetocin induced JNK modulated several metabolic parameters like glucose uptake,lactate production,ATP generation,and activity of glycolytic enzymes hexokinase and pyruvate kinase. However,JNK had no effect on ATM or YAP1 expression. Coherent with the in vitro findings,Chaetocin reduced tumor burden in heterotypic xenograft glioma mouse model. Chaetocin-treated tumors exhibited heightened ROS,pATM,YAP1 and pJNK levels. Our study highlights the coordinated control of glioma cell proliferation and metabolism by ROS through (i) ATM-YAP1-driven apoptotic pathway and (ii) JNK-regulated metabolic adaptation. The elucidation of these newfound connections and the roles played by ROS to simultaneously shift metabolic program and induce apoptosis could provide insights toward the development of new anti-glioma strategies. View Publication

Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs,we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post-transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR-203 has been extensively studied in human tumors,but has not been characterized in hESCs. We show that miR-203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Tendon-bone junctions (TBJs) are frequently injured,especially in athletic settings. Healing of TBJ injuries is slow and is often repaired with scar tissue formation that compromises normal function. This study explored the feasibility of using kartogenin (KGN),a biocompound,to enhance the healing of injured TBJs. We first determined the effects of KGN on the proliferation and chondrogenic differentiation of rabbit bone marrow stromal cells (BMSCs) and patellar tendon stem/progenitor cells (PTSCs) in vitro. KGN enhanced cell proliferation in both cell types in a concentration-dependent manner and induced chondrogenic differentiation of stem cells,as demonstrated by high expression levels of chondrogenic markers aggrecan,collagen II and Sox-9. Besides,KGN induced the formation of cartilage-like tissues in cell cultures,as observed through the staining of abundant proteoglycans,collagen II and osteocalcin. When injected into intact rat patellar tendons in vivo,KGN induced cartilage-like tissue formation in the injected area. Similarly,when KGN was injected into experimentally injured rat Achilles TBJs,wound healing in the TBJs was enhanced,as evidenced by the formation of extensive cartilage-like tissues. These results suggest that KGN may be used as an effective cell-free clinical therapy to enhance the healing of injured TBJs. View Publication

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level,it results in drastic chromatin changes to erase the existing somatic memory" and to establish the pluripotent state. Accordingly View Publication

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation,caloric restriction,high-intensity exercise,or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however,the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB,but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate,suppresses activation of the NLRP3 inflammasome in response to urate crystals,ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family,CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11,inflammasome activation. Mechanistically,BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK),reactive oxygen species (ROS),autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle,and independently of uncoupling protein-2 (UCP2),sirtuin-2 (SIRT2),the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo,BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome,familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome. View Publication

The role of intermediate methylation states in DNA is unclear. Here,to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity,we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers,exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation,are predominantly allele-independent and are conserved across individuals and between mouse and human,suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons,highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. View Publication

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably,several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP,but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site,revealing a mechanism of filovirus inhibition. View Publication

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here,we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions,resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions,which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. View Publication

Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation,CSR requires activation-induced cytidine deaminase (AID),whose expression is restricted to B cells,as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice,we identified a B cell-intrinsic role for Rab7,a small GTPase involved in intracellular membrane functions,in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription,as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed,in vivo and in vitro,normal proliferation and survival,normal Blimp-1 expression and plasma cell differentiation,as well as intact activation of the noncanonical NF-κB,p38 kinase,and ERK1/2 kinase pathways. They,however,were defective in AID expression and CSR in vivo and in vitro,as induced by CD40 engagement or dual TLR1/2-,TLR4-,TLR7-,or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells,CSR was rescued by enforced AID expression. These findings,together with our demonstration that Rab7-mediated canonical NF-κB activation,as critical to AID induction,outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR,likely by promoting assembly of signaling complexes along intracellular membranes. View Publication

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo,yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5,an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore,Ncx1 heartbeat mutants,as well as static cultures of AGM,exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures,transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. View Publication

Tools for rapid and efficient transgenesis in safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE View Publication