Improvement of Cardiac Function in Mouse Myocardial Infarction after Transplantation of Epigenetically-Modified Bone Marrow Progenitor Cells

OBJECTIVE: To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice. METHODS AND RESULTS: We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells. CONCLUSION: Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells

Mitochondrial polymorphisms in rat genetic models of hypertension

Hypertension is a complex trait that has been studied extensively for genetic contributions of the nuclear genome. We examined mitochondrial genomes of the hypertensive strains: the Dahl Salt-Sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR), and the Albino Surgery (AS) rat, and the relatively normotensive strains: the Dahl Salt-Resistant (R) rat, the Milan Normotensive Strain (MNS), and the Lewis rat (LEW). These strains were used previously for linkage analysis for blood pressure (BP) in our laboratory. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S and R rats. However, variants were detected among the mitochondrial genomes of the various hypertensive strains, S, SHR, and AS, and also among the normotensive strains R, MNS, and LEW. A total of 115, 114, 106, 106, and 16 variations in mtDNA were observed between the comparisons S versus LEW, S versus MNS, S versus SHR, S versus AS, and SHR versus AS, respectively. Among the 13 genes coding for proteins of the electron transport chain, 8 genes had nonsynonymous variations between S, LEW, MNS, SHR, and AS. The lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes. The variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes

Digital Signature : Comparative study of its usage in developed and developing countries

The online trading is growing widely day by day, which makes safety the biggest concern while carrying out trading by electronic means. As many other operations can be done with digital environment and internet, operation that provides identity validation should also be added to the digital environment. When data are transferred, the user should make sure that there are no changes in the original data while transferring them from sender to receiver. And it has also become necessary to authenticate the users often to ensure security and to avoid fraud. There are lot of different ways of online identification, in which digital signature is considered to be one of the powerful way of authentication. So, the online user use digital signature to authenticate the sender and to maintain the integrity of the document sent. In this paper, a study is carried out to identify the usage of digital signature and the perspective of people towards it in developed and developing countries and a survey is taken to support the theory

Digital Signature : Comparative study of its usage in developed and developing countries

The online trading is growing widely day by day, which makes safety the biggest concern while carrying out trading by electronic means. As many other operations can be done with digital environment and internet, operation that provides identity validation should also be added to the digital environment. When data are transferred, the user should make sure that there are no changes in the original data while transferring them from sender to receiver. And it has also become necessary to authenticate the users often to ensure security and to avoid fraud. There are lot of different ways of online identification, in which digital signature is considered to be one of the powerful way of authentication. So, the online user use digital signature to authenticate the sender and to maintain the integrity of the document sent. In this paper, a study is carried out to identify the usage of digital signature and the perspective of people towards it in developed and developing countries and a survey is taken to support the theory

Vibration Analysis of Industrial Drive for Broken Bearing Detection Using Probabilistic Wavelet Neural Network

A reliable monitoring of industrial drives plays a vital role to prevent from the performance degradation of machinery. Today’s fault detection system mechanism uses wavelet transform for proper detection of faults, however it required more attention on detecting higher fault rates with lower execution time. Existence of faults on industrial drives leads to higher current flow rate and the broken bearing detected system determined the number of unhealthy bearings but need to develop a faster system with constant frequency domain. Vibration data acquisition was used in our proposed work to detect broken bearing faults in induction machine. To generate an effective fault detection of industrial drives, Biorthogonal Posterior Vibration Signal-Data Probabilistic Wavelet Neural Network (BPPVS-WNN) system was proposed in this paper. This system was focused to reducing the current flow and to identify faults with lesser execution time with harmonic values obtained through fifth derivative. Initially, the construction of Biorthogonal vibration signal-data based wavelet transform in BPPVS-WNN system localizes the time and frequency domain. The Biorthogonal wavelet approximates the broken bearing using double scaling and factor, identifies the transient disturbance due to fault on induction motor through approximate coefficients and detailed coefficient. Posterior Probabilistic Neural Network detects the final level of faults using the detailed coefficient till fifth derivative and the results obtained through it at a faster rate at constant frequency signal on the industrial drive. Experiment through the Simulink tool detects the healthy and unhealthy motor on measuring parametric factors such as fault detection rate based on time, current flow rate and execution time

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility

<div><p>Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both <i>in vitro</i> and <i>in vivo</i> into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.</p></div

Differentiation of hf-iPSCs and he-iPSCs into neuronal cells.

<p>(A, B) hf-iPSCs and he-iPSCs were cultured under neuronal differentiation culture conditions for 21 days show significantly higher expression neuronal mRNA transcripts of Olig2 and MAP2 than the control cells. Fold expression was calculated as the ratio of he-iPSCs expression-to-parent control cells expression. Each bar represents the mean ± SEM of three replicated experiments. *p<0.05, **p<0.01. (C, D) The immunofluorescence staining of hf-iPSC and he-iPSC-derived neuronal cultures expressed neuron specific marker PGP9.5 (red) and astrocytes specific marker GFAP (green). (E<b>)</b> The qRT-PCR data show that the hf-iPSC-derived endothelial cells express the mRNA transcripts of CD31 and VE-Cadherin. (F1<b>)</b> The VE-Cadherin mRNA expression was further supported by the immunofluorescence analysis of VE-Cadherin protein expression. (F2<b>)</b> The tube formation assay showed that hf-iPSCs were capable of differentiating into endothelial cells under specific culture conditions. (G<b>)</b> The qRT-PCR data show that the endothelial cells derived from he-iPSCs express gene transcripts of CD31 and VE-Cadherin. Data are expressed as mean ± SEM, n = 3, *p<0.05. (H1<b>)</b> The VE-Cadherin mRNA expression was further supported by the immunofluorescence analysis of VE-Cadherin protein expression. (H2<b>)</b> The he-iPSCs were cultured under endothelial differentiation medium forms tubes and capillaries.</p

Characterization of pluripotency in hf-iPSCs and he-iPSCs.

<p>Quantitative real-time PCR array-based expression pattern of 86 pluripotent genes. (A, B) Among the 86 genes, 44 genes in hf-iPSCs and 49 genes in he-iPSCs were significantly up regulated in he-iPSCs at passage 3 (P3), which are represented in red color. (C, D) The selected up regulated pluripotent genes from qRT-PCR array that showed more than 100 fold mRNA expression in hf-iPSCs and he-iPSCs. Each bar represents the mean ± SEM of three replicated experiments. Fold expression was calculated as the ratio of hf-iPSCs expression-to-parent control cells expression. The hf-iPSCs and he-iPSCs at P3 cells were further analyzed by immunofluorescence staining. (E) The immunofluorescence microscopic image shows the hf-iPSCs were stained positive for the Oct4 and Nanog protein expression. (F) Similarly, the he-iPSCs were also positive for Oct4 and Nanog protein expression at P3. (G) The Western analysis showed that the endogenous Oct4, Sox2 and Nanog genes are getting activated and expressing higher levels of proteins when compared to control parent cells. Histone 3 serves as a protein loading control. Representative images are from three repeated experiments.</p