Actin mRNA Research Articles

RNA interference is induced in the glassy winged sharpshooter Homalodisca vitripennis by actin dsRNA

The glassy winged sharpshooter, Homalodisca vitripennis, is an unusually robust and efficient leafhopper vector of Xylella fastidiosa. X. fastidiosa is the causative agent of Pierce's disease, almond scorch, citrus variegated chlorosis and other serious plant diseases. The present study was conducted to establish whether RNA interference (RNAi) was induced in nymphal H. vitripennis that were injected with actin dsRNAs and other dsRNAs. A dramatic reduction in target H. vitripennis actin mRNAs and the formation of small interfering RNAs (siRNAs), hallmarks of RNAi, were found following the injection of actin dsRNAs. Quantitative reverse transcription PCR indicated an 80% reduction in actin mRNA levels by 5 days post-injection. Western blot analysis showed a dramatic drop in actin protein levels by 3 days post-injection. Biological effects such as incomplete nymphal-adult ecdysis and > 95% mortality were also found following the injection of fifth-instar nymphs with actin dsRNA. Dramatic reductions in target mRNA levels were also found following the injection of other dsRNAs into fifth-instar H. vitripennis. The findings indicate that RNAi is induced in post-embryonic leafhoppers by dsRNA. The present system can be used to screen potential gene-silencing targets that can be used for reducing the vector competence of H. vitripennis and other leafhoppers.

The Response of Vocal Fold Fibroblasts and Mesenchymal Stromal Cells to Vibration

Illumination of cellular changes caused by mechanical forces present within the laryngeal microenvironment may well guide strategies for tissue engineering the vocal fold lamina propria. The purpose of this study was to compare the response of human vocal fold fibroblasts (hVFF) and bone marrow mesenchymal stem cells (BM-MSC) to vibratory stimulus. In order to study these effects, a bioreactor capable of vibrating two cell seeded substrates was developed. The cell seeded substrates contact each other as a result of the sinusoidal frequency, producing a motion similar to the movement of true vocal folds. Utilizing this bioreactor, hVFF and BM-MSC were subjected to 200 Hz vibration and 20% strain for 8 hours. Immunohistochemistry (Ki-67 and TUNEL) was performed to examine cell proliferation and apoptosis respectively, while semi-quantitative RT-PCR was used to assess extracellular matrix related gene expression. HVFF significantly proliferated (p = 0.011) when subjected to 200 Hz vibration and 20% strain, while BM-MSC did not (p = 1.0). A statistically significant increase in apoptosis of BM-MSC (p = 0.0402) was observed under the experimental conditions; however high cell viability (96%) was maintained. HVFF did not have significantly altered apoptosis (p = 0.7849) when subjected to vibration and strain. Semi-quantitative RT-PCR results show no significant differences in expression levels of collagen I (BM-MSC p = 0.1951, hVFF p = v0.3629), fibronectin (BM-MSC p = 0.1951, hVFF p = 0.2513), and TGF-β1 (BM-MSC p = 0.2534, hVFF p = 0.6029) between vibratory and static conditions in either cell type. Finally, smooth muscle actin mRNA was not present in either vibrated or static samples, indicating that no myofibroblast differentiation occurred for either cell type. Together, these results demonstrate that BM-MSC may be a suitable alternative to hVFF for vocal fold tissue engineering. Further investigation into a larger number of gene markers, protein levels, increased number of donors and vibratory conditions are warranted.

Fractal Dimension as a Measure of Altered Actin Cytoskeleton in MC3T3-E1 Cells Under Simulated Microgravity Using 3-D/2-D Clinostats

Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.

Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure

The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Osteopontin regulates α‐smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.

MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis

BackgroundMicroRNAs (miRNAs) are important in hepatic pathophysiology and the development of liver cancer.ObjectiveTo explore miRNAs that are regulated with the progression of liver fibrosis caused by chronic liver disease.DesignThe regulated...

Quantifying Actin mRNA Expression with Super Resolution Fluorescence Microscopy

The copy number of a specific mRNA and its subcellular localization in cells is a mode by which cells can regulate protein synthesis. Beta-actin mRNA is transported to filopodia upon serum stimulation, regions of cell growth and movement where actin polymerization is essential (1). The copy number of mRNA molecules directly translates to the amount of actin monomers that are synthesized. Quantifying the actin mRNA copy numbers provides a measure of how polymerization of actin is regulated.To this extent, we apply stochastic super-resolution microscopy with photoswitchable fluorescent probes combined with single-molecule localization (2,3) for quantitative studies of mRNA concentration and localization in eukaryotic cells. For this purpose, we apply various labelling approaches for mRNA, including well-established methods such as fluorescence in-situ hybridization (FISH) (4). Focussing on actin mRNA, we derive quantitative information on copy numbers, and we apply coordinate-based algorithms to analyze our single-molecule super-resolution data.References(1) Ben-Ari et al. (2010) J Cell Sci, 123, 1761-1774.(2) Betzig et al. (2006) Science, 313, 1642-1645.(3) Heilemann et al. (2008) Angew. Chemie, 47, 6172-6176.(4) Levskey, J. M.; Singer, R. (2003) Journal of Cell Science, 116, 2833-2838.

Hepatoprotective Effects of Phloridzin on Hepatic Fibrosis Induced by Carbon Tetrachloride against Oxidative Stress-Triggered Damage and Fibrosis in Rats

The present study was to study the hepatoprotective effects of phloridzin (PHL) on hepatic fibrosis induced by carbon tetrachloride (CCl₄) in rats, on the basis of this investigation, the possible mechanism of PHL was elucidated. Male Sprague Dawley (SD) rats were randomly divided into six groups: control, model, PHL-L, PHL-M, PHL-H and colchine. All rats except control group were intraperitoneally injected with CCl₄, and control rats were injected with olive oil, twice a week for eight weeks. At the same time, the rats were orally given homologue drugs once a day, respectively. Hepatoprotective effects of PHL were evaluated by liver weight indexes, biochemical values, total antioxidant capacity and total-superoxide dismutase, histopathological observations, hepatic fibrosis, and the hepatic fibrosis relative gene and protein expressions. PHL significantly improved hepatic function; remarkably decreased serum hyaluronic acid (HA), transforming growth factor-β1 (TGF-β1), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver tissues hydroxyproline, malondialdehyde (MDA) levels, increased glutathione peroxidase (GSH-Px), total-antioxygen capacity (T-AOC) and total-superoxide dismutase (T-SOD) contents of liver tissues; Real-time polymerase chain reaction (PCR) and immunohisto-chemical results showed PHL might markedly reverse the up-regulated mRNA and protein expressions of the α-smooth muscle actin (SMA), TGF-β1 and tissue inhibitor of metalloproteinase-1 (TIMP1), up-regulate the matrix metalloproteinase-1 (MMP1) mRNA and protein expressions. Histopathological observations provided supportive evidence for biochemical analyses and the hepatic fibrosis relative gene and protein expressions, and with the dose of PHL increasing, the aforesaid improvement became more and more strong. The studies demonstrated that PHL exerted beneficially hepatoprotective effects on hepatic fibrosis induced by CCl₄, mainly enhancing antioxidant capacity of liver organizations, reduce the level of lipid peroxidation induced by CCl₄, and protect hepatocyte membranes from damage, and alleviate hepatic fibrosis.

Magnesium chenoursodeoxycholic acid ameliorates carbon tetrachloride-induced liver fibrosis in rats

This study was designed to investigate the hepatoprotective effects of magnesium chenoursodeoxycholic acid (Mg-CUD), a magnesium trihydrate salt of ursodeoxycholic acid and chenodeoxycholic acid, in carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Rats were treated with CCl(4) dissolved in olive oil (0.5 mL/kg, twice a week) intraperitoneally for eight weeks. Mg-CUD was administered orally at 15.625, 31.25, 62.5 and 125 mg/kg once a day. Chronic CCl(4) administration induced increases in serum transforming growth factor-β1, hepatic hydroxyproline content and serum alanine aminotransferase activity. Mg-CUD attenuated these increases. The levels of α-smooth muscle actin protein and mRNA expression were increased by chronic CCl(4) exposure and Mg-CUD attenuated these increases. Mg-CUD suppressed increases in matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 mRNA expression and elevation of oxidative stresses by attenuating lipid peroxidation and enhancing reduced glutathione/oxidized glutathione ratio. The overexpression of toll-like receptor 4 and increased nuclear translocation of nuclear factor-κB and phosphorylated c-Jun, a component of activator protein 1, were suppressed by Mg-CUD. Furthermore, CCl(4) increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and cyclooxygenase (COX)-2. Mg-CUD attenuated the levels of TNF-α, IL-6 and COX-2, while it augmented the level of IL-10. Our results suggest that Mg-CUD may prevent liver fibrosis by modulating collagen accumulation and inflammatory signaling pathways.

Hepcidin knockout mice as a model of iron-overload associated liver fibrosis development

Background and aims: Hepcidin is the central regulator of iron homeostasis. Disrupted hepcidin signalling results in hereditary hemochromatosis and iron overload in chronic liver disorders. While the association between iron overload and development of end-stage liver disease is well established, the understanding of the underlying pathomechanisms is hampered by the lack of a suitable animal model. To circumvent this problem, we studied hepcidin-knockout (KO) mice as a model of iron-overload associated liver disease. Methods: 6 and 12 month-old hepcidin-KO and -wild type (WT) mice fed 3% iron carbonyl-containing diet (Fe-diet) since four weeks of age were compared to age-matched WT and KO animals kept on standard diet. The liver phenotype was quantified serologically as well as morphometrically based on hematoxylin & eosin, Prussian blue and Sirius red stainings. Liver iron content was determined by atomic mass absorption. Liver fibrosis development was determined by collagen RT-PCR and hydroxyproline assay. Results: 6 month Fe-fed hepcidin KO mice (compared to WTs) exhibited (i) increased iron liver contents (2543±114 vs. 1493±136 p<0,005); (ii) elevated AST and serum iron levels (AST: KO 261±15, WT 142±34 p<0,05; serum iron: KO 95±3, WT 46±5, p<0,0005); (iii) increased liver inflammation and hepatocellular apoptosis; and (iv) elevated markers of hepatic stellate cell activation (collagen and α-smooth muscle actin mRNA), but no detectable liver fibrosis. 12 month Fe-fed hepcidin KO mice (compared to WTs) also showed increased liver injury markers as well as marked iron accumulation. In addition, they developed significantly stronger liver fibrosis than their wildtype counterparts as suggested by collagen RT-PCR, hydroxyproline assay and morphometrical evaluation. Conclusions: Iron-fed hepcidin KOs develop not only a mild chronic liver injury, but also liver fibrosis and therefore represent unique tool to study the mechanism of iron overload-related liver diseases

ZBP1 and Ribosome Association with β Actin mRNA Measured by Fluorescence Fluctuation Spectroscopy

Intracellular mRNA localization plays an important role in post transcriptional gene regulation by targeting protein synthesis to a specific cellular compartment. mRNA localization has been extensively investigated with fluorescence in situ hybridization and live cell imaging in various model organisms. However, quantitative measurement of the translation of localizing mRNA with high spatiotemporal precision remains challenging. β-actin mRNA has been shown to localize to the leading edge of primary fibroblasts and provides an excellent model system to address this problem. The localization involves a cis-acting zipcode sequence and a trans-acting zipcode binding protein (ZBP1). Here we employ fluorescence fluctuation spectroscopy (FFS) to investigate the localization of β-actin mRNA and its association with ribosome subunits in live cell. Two technical advances make this experiment possible. First, we have developed a mouse model in which the β-actin gene has 24XMS2 sequences inserted. Furthermore, this mouse has been crossed with a ZBP1 knock out mouse. We generate primary mouse embryonic fibroblasts from these mice. By expressing GFP-MS2 capsid protein in the cell, all endogenous β-actin mRNA are fluorescently labeled. In addition, mCherry labeled ZBP1 or ribosome subunit are introduced into the cells to visualize their interaction with the β-actin mRNA by FFS. Second, we have developed a dual-color brightness analysis tool, specifically hetero-species partition analysis, to measure the interaction between the GFP-MS2 labeled mRNA and another mCherry labeled factor. We systematically measured the interaction between the β-actin mRNA and the ZBP1/ribosome in the perinuclear region and leading edge. Our data shows that β-actin mRNA binds more ZBP1 but less ribosome in the perinuclear region than in the leading edge, which supports the model that ZBP1 represses β-actin mRNA association with ribosome in the nuclear periphery until it reaches the leading edge.

Noninvasive detection of neural progenitor cells in living brains by MRI

The presence of pericytes in brain regions undergoing repair is evident of the recruitment of bone marrow-derived multipotent regenerative cells to the neurovascular unit during angiogenesis. At present, post mortem sampling is the only way to identify them. Therefore, such cell typing is inadequate for preserving neural progenitor cells for any meaningful stem cell therapy. We aimed to target cerebral pericytes in vivo using dual gene transcript-targeted MRI (GT-tMRI) in male C57black6 mice after a 60-min bilateral carotid artery occlusion (BCAO). We attached superparamagnetic iron oxide nanoparticles (SPIONs) to phosphorothioate-modified micro-DNA that targets actin or nestin mRNA. Because BCAO compromises the blood-brain barrier (BBB) and induces expression of α-smooth muscle (αSM)-actin and nestin antigens by pericytes in new vessels, we delivered pericyte-specific magnetic resonance contrast agents (SPION-actin or SPION-nestin at 4 mg Fe/kg) by i.p. injection to C57black6 mice that had experienced BCAO. We demonstrated that the surge in cerebral iron content by inductively coupled plasma-mass spectrometry matched the increase in the frequency of relaxivity. We also found that SPION-nestin was colocalized in αSM- actin- and nestin-expressing pericytes in BCAO-treated C57black6 or transgenic mice [B6.Cg-Tg(CAG-mRFP1) 1F1Hadj/J, expressing red fluorescent protein by actin promoter]. We identified pericytes in the repair patch in living brains after BCAO with a voxel size of 0.03 mm(3). The presence of electron-dense nanoparticles in vascular pericytes in the region of BBB injury led us to draw the conclusion that GT-tMRI can noninvasively reveal neural progenitor cells during vascularization.

Effect of Udenafil on Portal Venous Pressure and Hepatic Fibrosis in Rats

The purpose of this study was to investigate the therapeutic efficacy of udenafil (CAS 268203-93-6), a phosphodiesterase type 5 (PDE5) inhibitor, on bile duct ligation (BDL)-induced portal hypertension. Udenafil was given orally to rats at dose levels of 1, 5 or 25 mg/kg/day for 3 weeks in order to examine the chronic effect on portal venous pressure (PVP). Udenafil was also given orally to investigate the sequential change of PVP in BDL animals. The effect of udenafil on hepatic stellate cell activation and fibrotic change-related protein mRNA expression were examined. In a pharmacokinetic study, the pharmacokinetic parameters in sham-operated rats and BDL rats were compared. Three-week udenafil treatment decreased PVP by approximately 30% compared to the vehicle group. In a single oral administration study, the PVP of the udenafil treated group was lower than that of the control group throughout the experimental period. Compared to control, udenafil suppressed the expression of procollagen type I and alpha-smooth muscle actin mRNA. In the pharmacokinetic study, the AUC of udenafil in BDL rats was approximately 5 times higher than that in sham-operated rats. The results of this study suggest that udenafil has beneficial effects on portal hypertension and the effect may well be attributed to its anti-fibrogenic activity.

Abstract P023: HDAC Inhibition Promotes Cardiogenesis and the Survival of Embryonic Stem Cells Through Proteasome-Dependent Pathway

Objectives: Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Methods and Results: Using the well-established mouse CGR8 mouse ESC culture, we evaluated the impact of HDAC inhibition and proteasome pathway on the cell death, viability and apoptosis in ESCs in response to oxidant stress (100μ mol/L hydrogen peroxides). We demonstrated that HDAC inhibitors, both TSA (50 nmol/L) and sodium butyrate (200 μ mol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs. HDAC inhibition reduced the cleaved caspase 3, 6, 9, PARP and TUNE-positive ESCs, which were abrogated with MG132 (0.5 μ mol/L), a specific proteasome inhibitor. Furthermore, we employed in vitro “hanging drop” methods to carry out two weeks of embryoid bodies (EB) culture to assess the effect of HDAC inhibition and proteasome pathway on cardiogenesis. HDAC inhibition stimulates the growth of EB, which is associated with faster spontaneous rhythmic contraction. HDAC inhibition increased the up-regulation of GATA4, MEF2, NKX2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. Immunostaining analysis demonstrates that trichostatin A and sodium butyrate resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows the activation of MEF2. Conclusions: Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicit the survival pathway, which is dependent on specific HDAC4 degradation and subsequent MEF2 activation of ESCs.

Abstract P194: CCAAT/Enhancer-Binding Protein Beta Negatively Regulates Hemodynamic Stress-Induced Cardiac Hypertrophy

The CCAAT/enhancer-binding proteins (C/EBP) beta is a member of family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Previous reports showed that the protein and mRNA levels of C/EBP beta were increased in rat ischemia-reperfused or human failing hearts. Recent study indicates an important role of C/EBP beta in physiological cardiac hypertrophic responses. In the present study, we generated cardiac-specific C/EBP beta-deficient mice (CKO) to elucidate its in vivo function in pathological cardiac hypertrophy. We crossed floxed C/EBP beta mice with mice expressing Cre recombinase in cardiac-specific manner. Echocardiographic and physiological analyses revealed that CKO showed no cardiac phenotypes under basal conditions at 10 weeks old, indicating that the C/EBP beta is not essential for mouse heart development. Then, we subjected CKO and control mice (CTL) to pressure overload by means of transverse aortic constriction (TAC). In wild-type mouse hearts, the expression level of C/EBP beta was increased after TAC. One week after TAC, CKO showed left ventricle (LV) hypertrophy (LV/body weight, CKO 4.64 ± 0.08 mg/g versus CTL 4.14 ± 0.06 mg/g, p&lt;0.01, LV mass index, CKO 106.1 ± 1.7 versus CTL 98.5 ± 1.8, p&lt;0.05), without showing contractile dysfunction. Cross-sectional area of cardiomyocytes was increased in CKO after TAC (CKO 373.6 ± 2.1 μm 2 versus CTL 300.5 ± 3.6 μm 2 , p&lt;0.01). The increase in the atrial natriuretic factor (ANF) or alpha skeletal actin mRNA expression, molecular markers for cardiac remodeling, was observed in CKO hearts. Furthermore, overexpression of C/EBP beta in isolated neonatal rat cardiomyocytes inhibited an increase in 3 H-leucine uptake induced by phenylephrine stimulation. C/EBP beta is expressed as three distinct protein isoforms, namely FL, LAP, and LIP, which are encoded by a single gene, but transcribed from different initiation sites. Luciferase reporter gene assay showed that overexpression of LAP isoform attenuated the transcriptional activity of ANF induced by phenylephrine, but not LIP. Thus, we conclude that C/EBP beta attenuates the pathological cardiac hypertrophy induced by hemodynamic stresses.

Abstract 16720: Induction of Notch Activity Promotes a Cardiac Differentiated Phenotype in Adult Cardiac Progenitor Cells

Introduction: Notch is a critical mediator of heart development, stem cell self-renewal and specification; appropriate timing and levels of Notch signaling are essential for proper cell fate determination. However, constitutive activation of Notch does not recapitulate the physiological signal driving cardiac differentiation. Hypothesis: Controlled pulses of Notch activation enhance cardiac differentiation in CPCs. Methods: Ligand-regulated or tetracycline inducible versions of intracellular Notch designed to control timing and activation level were engineered into cultured adult cardiac progenitor cells (CPCs). Stable CPC lines were created carrying constructs for GFP (CPCe), ligand-regulated intracellular Notch (CPCeK), or tetracycline inducible intracellular Notch (CPCtetKmER). Induction was performed using 1 µM tamoxifen and/or 1 µM doxycycline with proliferation measured over a period of 6 days. Markers of Notch signaling and cardiac differentiation were assessed by quantitative PCR, immunoblotting, or immunostaining up to 6 days after induction. Results: Increased levels of hey1, jagged1, cardiac troponin T (cTnT) and smooth muscle actin (SMA) transcripts were observed following one day of Notch induction in CPCeK and CPCtetKmER. Changes indicative of differentiation were present in CPCeKs and CPCtetKmERs following one week after Notch induction including flattened and enlarged morphology, elevated levels of smooth muscle actin (SMA) and jagged1 mRNA, and expression of SMA protein. Proliferation decreased after three days of sustained Notch signaling. Conclusions: Notch signaling promotes CPCs to acquire phenotypic characteristics of smooth muscle or myocyte differentiation. Engineering CPCs allows for appropriate “physiologic” temporal control to regulate the potent cardiogenic activity of Notch signaling. Regulation of Notch activity in CPCs utilizing temporal and reversible expression will enhance commitment and differentiation when utilized in conjunction with cell based therapy for heart disease, thereby enhancing myocardial regeneration and repair.

HDAC inhibition promotes cardiogenesis and the survival of embryonic stem cells through proteasome‐dependent pathway

Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Using the well-established mouse ESC culture, we demonstrated that HDAC inhibitors, both trichostatin A (TSA,50 nmol/L) and sodium butyrate (NaB, 200 µmol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs in response to oxidant stress. HDAC inhibition reduced the cleaved caspases 3, 6, 9, PARP, and TUNEL positive ESCs, which were abrogated with MG132 (0.5 µmol/L), a specific proteasome inhibitor. Furthermore, HDAC inhibition stimulates the growth of embryoid bodies (EB), which are associated with a faster spontaneous rhythmic contraction. HDAC inhibition increases the up-regulation of GATA4, MEF2C, Nkx2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. TSA and NaB resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows for the activation of MEF2. Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicits the survival pathway.

Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each targeted viroid were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, GYSVd-1, AGVd, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.

Effects of cardiotrophin-1 on differentiation and maturation of rat bone marrow mesenchymal stem cells induced with 5-azacytidine in vitro

BackgroundCardiotrophin-1 (CT-1) is a cytokine involved in the growth and survival of cardiac cells that stimulates cardiomyogenesis in pluripotent murine embryonic stem (ES) cells. But it is not known whether...

Aclidinium inhibits human lung fibroblast to myofibroblast transition

BackgroundFibroblast to myofibroblast transition is believed to contribute to airway remodelling in lung diseases such as asthma and chronic obstructive pulmonary disease. This study examines the role of aclidinium, a...