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A novel series of 3,4-dihydroxychalcones was synthesized to evaluate their effects against 5-lipoxygenase and cyclooxygenase. Almost all compounds exhibited potent inhibitory effects on 5-lipoxygenase with antioxidative effects,and some also inhibited cyclooxygenase. The 2',5'-disubstituted 3,4-dihydroxychalcones with hydroxy or alkoxy groups exhibited optimal inhibition of cyclooxygenase. We found that 2',5'-dimethoxy-3,4-dihydroxychalcone (37; HX-0836) inhibited cyclooxygenase to the same degree as flufenamic acid and 5-lipoxygenase,more than quercetin. Finally,these active inhibitors of 5-lipoxygenase inhibited arachidonic acid-induced mouse ear edema more than phenidone. View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

This paper presents a 360°,size-adjustable microelectrode array (MEA) system for the long-term electrophysiological monitoring of cerebral organoids derived from human pluripotent stem cells. The system consists of eight independently positionable multielectrode probes,each carrying eight electrodes arranged vertically. This configuration resulted in 64 recording channels surrounding the organoid. The multielectrode probes were mounted on custom-designed miniature manipulators with three degrees of freedom. This setup enabled positioning and contact with organoids of varying sizes (approximately 1–3.7 mm in diameter). The design allowed circumferential access and facilitated standard incubator-based cultivation without disrupting the recording setup. Fabricated using flexible printed circuit technology,this MEA system offers relatively low production costs. It is also amenable to widespread implementation in laboratory settings. Experimental results demonstrated the successful recording of neuronal activity,including spike detection and signal stability,over 2 weeks of continuous organoid culture. These results suggests that the three-dimensional system provides broad spatial coverage and supports long-term monitoring for basic biomedical research. It also holds potential for future applications such as biohybrid computing. View Publication

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine,including individualized,patient-specific stem cell-based treatments. There are,however,few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues,ideally comprising direct-write printing of cells for encapsulation,proliferation,and differentiation. Here,such a method,employing a clinically amenable polysaccharide-based bioink,is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically,There are extrusion printed the bioink including iPSCs,alginate (Al; 5% weight/volume [w/v]),carboxymethyl-chitosan (5% w/v),and agarose (Ag; 1.5% w/v),crosslinked the bioink in calcium chloride for a stable and porous construct,proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm,ectoderm,and mesoderm,or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined,scalable,and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. View Publication

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS),but GWAS do not reveal causal variants,effector genes,or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C,Hi-C,ATAC-seq,and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features,and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific,but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types,suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However,trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this,we pharmacologically validated squalene synthase,a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE,as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms,and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression,function,and pathology across multiple,distinct tissues and cell types. View Publication

We have established an in vitro 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular,we reported that the mature model,entirely constituted of primary cells of human origin,develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable Haemophilus influenzae results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary,we propose our in vitro model as a reliable and potentially customizable system to study mid/long term host-pathogen processes. View Publication

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR),a compound that activates 5'-AMP-activated protein kinase (AMPK),has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study,we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes,in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently,closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation,assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle,in which AMPK stimulation promotes glucose transport to provide ATP as a fuel,AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes,inhibiting triacylglycerol synthesis,to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may,therefore,give insight into the mechanism of insulin action. View Publication

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways,the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK,i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose,heat shock),AICAR does not perturb the cellular contents of ATP,ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation,causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase),and almost total cessation of two of the known target pathways,i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase,previously demonstrated in cell-free assays,also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade. View Publication

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK),a key regulator for controlling energy metabolism,is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest,without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers,Nanog and stage-specific embryonic antigen-1,in the presence of leukemia inhibitory factor,without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently,an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation,an effect inhibited by MG132,a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs,it increased expression levels of erythroid cell lineage markers (Ter119,GATA1,Klf1,Hbb-b,and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR,endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs. View Publication

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However,because of 5-azacytidine's general toxicity,other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that,when present in DNA,it inhibited DNA methylation,led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here,the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed. View Publication