Transforming growth factor-β1 (TGF-β1) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β1 may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β1-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β1 to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β1 promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β1 in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β1-induced autophagy is required for the fibrogenic response in hATMyofbs.
Eukaryotic chromosomes are folded into hierarchical domains, forming functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks contributing to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for the identification of nucleolar associated domains (NADs). Here we have established DamID- and HiC-based methodologies to generate accurate genome-wide maps of NADs in embryonic stem cells (ESCs) and neural progenitor cells (NPCs), revealing layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs show higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation into NPCs, chromosomes around the nucleolus acquire a more compact, rigid architecture with neural genes moving away from nucleoli and becoming unlocked for later activation. Further, histone modifications and the interaction strength within A and B compartments of NADs and LADs in ESCs set the choice to associate with NL or nucleoli upon dissociation from their respective compartments during differentiation. The methodologies here developed will make possible to include the nucleolar contribution in nuclear space and genome function in diverse biological systems.
The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.
The antimicrobial effects of garlic, ginger, carrot and turmeric pastes against Escherichia coli O157:H7 in laboratory buffer and model food system were investigated. Turmeric paste, fresh carrot, ginger and garlic pastes from roots, and commercial ginger and garlic paste were heated alone or with buffered peptone water (BPW) or ground beef at 70 degrees C for 7 min. All samples were inoculated with a three strain cocktail of overnight cultures of E. coli O157: H7 and stored at 4 degrees C and 8 degrees C for 2 weeks. Each paste exhibited different antimicrobial effects alone and in ground beef or BPW at 4 degrees C and 8 degrees C for 2 weeks. Commercial ginger paste and fresh garlic paste showed the strongest antimicrobial activity with complete inactivation of E. coli O157:H7 in the paste at 3 days at 4 degrees C and 8 degrees C. Carrot and turmeric pastes did not show any antimicrobial activity both at 4 degrees C and 8 degrees C. Commercial garlic showed antimicrobial activity at both 4 degrees C and 8 degrees C (about 1 log CFU/g reduction) in the paste. However, fresh ginger paste showed antimicrobial activity only at 8 degrees C. Only commercial ginger paste had antimicrobial activity in BPW at 4 degrees C for 2 weeks. However, commercial ginger paste showed antimicrobial activity in ground beef at 3 days and after (about 1-2 log CFU/g) compared to control samples at 8 degrees C for 2 weeks. Fresh garlic paste showed antimicrobial activity only in BPW (1.3 log CFU/g) at 8 degrees C. These results indicate that the antimicrobial activity of these pastes is decreased in ground beef and laboratory buffer.
The incidence of heart failure with concomitant cardiac fibrosis is very high in developed countries. Fibroblast activation in heart is causal to cardiac fibrosis as they convert to hypersynthetic cardiac myofibroblasts. There is no known treatment for cardiac fibrosis. Myofibroblasts contribute to the inappropriate remodeling of the myocardial interstitium, which leads to reduced cardiac function and ultimately heart failure. Elevated levels of autophagy have been linked to stress-induced ventricular remodeling and other cardiac diseases. Previously, we had shown that TGF-β1 treatment of human atrial fibroblasts both induced autophagy and enhanced the fibrogenic response supporting a linkage between the myofibroblast phenotype and autophagy. We now demonstrate that with in vitro culture of primary rat cardiac fibroblasts, inhibition of autophagy represses fibroblast to myofibroblast phenoconversion. Culturing unpassaged cardiac fibroblasts for 72 hours on plastic tissue culture plates is associated with elevated α-smooth muscle actin (α-SMA) expression. This activation parallels increased microtubule-associated protein 1A/1B-light chain 3 (LC-3β II) protein expression. Inhibition of autophagy with bafilomycin-A1 (Baf-A1) and chloroquine (CQ) in cardiac fibroblasts significantly reduces α-SMA and extracellular domain A fibronectin (ED-A FN) protein vs untreated controls. Myofibroblast cell migration and contractility were significantly reduced following inhibition of autophagy. These data support the possibility of a causal link between cardiac fibroblast-to-myofibroblast phenoconversion and autophagy.
Nucleosome positioning maps of several organisms have shown that Transcription Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly positioned nucleosomes. Using genome-wide nucleosome maps and histone variant occupancy in the mouse liver, we show that the majority of genes were associated with a single prominent H2A.Z containing nucleosome in their promoter region. We classified genes into clusters depending on the proximity of H2A.Z to the TSS. The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z was positioned closer to the TSS of genes with higher expression levels. We confirmed this relation between the proximity of H2A.Z and expression level in the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over seven consecutive nucleosomes, was correlated with expression. Further, a nucleosome was positioned over the TSS of silenced genes while it was displaced to expose the TSS in highly expressed genes. Our results suggest that gene expression levels in vivo are determined by accessibility of the TSS and proximity of H2A.Z.
MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3′ Untranslated Regions (3′ UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.
Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.
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