Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

Influenza Virus Down-Modulates G6PD Expression and Activity to Induce Oxidative Stress and Promote Its Replication

none10no: Influenza virus infection induces oxidative stress in host cells by decreasing the intracellular content of glutathione (GSH) and increasing reactive oxygen species (ROS) level. Glucose-6-phosphate dehydrogenase (G6PD) is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH) that is used to regenerate the reduced form of GSH, thus restoring redox homeostasis. Cells deficient in G6PD display elevated levels of ROS and an increased susceptibility to viral infection, although the consequences of G6PD modulation during viral infection remain to be elucidated. In this study, we demonstrated that influenza virus infection decreases G6PD expression and activity, resulting in an increase in oxidative stress and virus replication. Moreover, the down regulation of G6PD correlated with a decrease in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor that regulates the expression of the antioxidant response gene network. Also down-regulated in influenza virus infected cells was sirtuin 2 (SIRT2), a NADPH-dependent deacetylase involved in the regulation of G6PD activity. Acetylation of G6PD increased during influenza virus infection in a manner that was strictly dependent on SIRT2 expression. Furthermore, the use of a pharmacological activator of SIRT2 rescued GSH production and NRF2 expression, leading to decreased influenza virus replication. Overall, these data identify a novel strategy used by influenza virus to induce oxidative stress and to favor its replication in host cells. These observations furthermore suggest that manipulation of metabolic and oxidative stress pathways could define new therapeutic strategies to interfere with influenza virus infection.openDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, LuciaDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, Luci

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment

Posphodiesterase 5 regulates the beating rate in murine neonatal cardiomyocytes

Heart rate is fi nely regulated by the sympathetic nervous system through beta adrenoreceptors (βARs) signaling. β-ARs stimulation induces cAMP/cGMP synthesis whereas phosphodiesterases (PDE) catalyze the hydrolysis of the cyclic nucleotides, however their precise interaction is not well defi ned (1, 2). The experimental model used to investigate the role of PDE5 inhibition on the heart function are the spontaneously beating cardiac myocytes from neonatal mice. Preliminary experiments were performed to determine whether neonatal cardiomyocyte cultures show the same PDEs expression pattern of adult mice hearts. We analyzed through RT-PCR and WB experiments the mRNA and protein levels of PDE1C, PDE2, PDE3A, PDE4 and PDE5 and we observed that are expressed both in hearts and in cultured neonatal cardiomyocytes. These data suggest that the cardiomyocyte is a suitable model to investigate the PDEs role in cardiac function. Experiments performed to evaluate the contraction rate stimulated by β-AR signaling activation show: that PDE5 is a positive modulator through hydrolysis of cGMP, and the inhibition of PDE5 causes a positive chronotropic eff ects reduction by the PDE2 activation through the increase of cGMP level. Notably, the use of PDE2 knockout mice reverts the negative chronotropic eff ects obtained by PDE5 inhibition. Finally, we observed that PDE5 selectively impacts heart rate interfering with β2AR signaling in neonatal cardiac myocytes, with little or no eff ect on β1AR signaling. These data show a novel role of PDE5 on the sympathetic regulation of cardiac beating and highlight the mechanicistic pathways of the long term eff ect of PDE5 inhibition in cardiac hypertrophy

PDE5 inhibition counteracts β- adrenergic induction of cardiac hypertrophy

The b-adrenoreceptors play important roles in cardiovascular function regulation mediated by the sympathetic nervous system. It is known that sustained b-adrenergic stimulations promotes cardiac hypertrophy (Oleg et al., 2007). Recently an antihypertrophic role of sildenafil, that acts as a phosphodiesterase 5 (PDE5) inhibitor, has been demonstrated in mice where hypertrophy was mechanically induced (Takimoto et al., 2005). We report the results obtained on a cellular system of cardiac hypertrophy in vitro. By using three-dimensional cultures of mouse ventricular cardiomyocytes (Xiang et al., 2005) and isolated cardiomyocytes we show that: 1) these cells express levels of PDE5 comparable with the ones in normal heart, 2) treatment of the cultures with the b-adrenoreceptors agonist isoproterenol induces cell hypertrophy accompanied by an increment of the level of PDE5 expression and 3) sildenafil prevents the development of such hypertrophy through specific b-adrenoreceptors and signaling pathways 4) the inhibition of other members of PDE family might contribute to the prevention of hypertrophy following b-adrenergic stimulation. In summary, we present a test system that may contribute to clarify intracellular signaling pathways leading to cardiac hypertrophy and to identify molecular targets, like the ones involved in PDE5 activity, on which to steer the development of new drugs and to design new clinical therapies

Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I–Mediated Antiviral Responses in Primary Human Dendritic Cells

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5′ triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)–stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5

Analyzing Election Sentiments in Tweets with Gated Recurrent Units (GRU)

Sentiment analysis, a key task in natural language processing, is important for detecting the emotional tone portrayed in text. In this study, we focus on implementing a Gated Recurrent   Unit (GRU) model to analyze attitudes within the 2020 Donald Trump Election tweets dataset. By setting the GRU model with carefully selected parameters, the aim of the study is to unveil the inherent sentiment patterns in the dataset. To develop the sentiment analysis model, the study devised a three phase methodology which that include data preprocessing, feature selection using correlation matrix, and lastly the implementation of GRU. Futhermore, we provided the outcomes of our experiment, evaluating the model's performance through important measures such as accuracy, precision, and recall. Notably, our data exhibit an exceptional accuracy rate of 93%, verifying the model's power to appropriately categorize attitudes. Additionally, both recall and precision receive outstanding ratings of 94% and 96%, indicating the model's skill in distinguishing both positive and negative attitudes. This inquiry emphases the effective usage of the GRU model in sentiment analysis, shedding light on the emotional nuances within the 2020 Donald Trump dataset and enriching our understanding of sentiments during the election period

Evaluating Soft Keyboard Layouts by Using Paper Mockups

Two experiments to compare text-entry were carried out using Soft Keyboard Layouts. Paper mockups were used and we carried out manual timing in a relaxed situation, even though within a classroom environment. Volunteer students were paired and made to work in such groups, serving as participants and also helping out in the timing process. One set of students served as participants at first and the other timed them. They then switched roles halfway and exchanged the tasks of participation and timing. Participants tapped the phrase “the quick brown fox jumps over the lazy dog” on each keyboard layouts five times using stylus. The time for word entry was measured and used to determine the speeds; which were found to be 24.8 to 34.3 for QWERTY Keyboard Layout and 11.9 to 14.8 for Opti Layout. We discussed the advantages and shortfalls of the methods of evaluation

The global impact of the coronavirus pandemic

The coronavirus pandemic has engulfed the nations of the world for the first five months of 2020 and altered the pace, fabric and nature of our lives. In this overview accompanying the Special Issue of Cytokine & Growth Factor Reviews, we examine some of the many social and scientific issues impacted by SARS-CoV2 - personal lives, economy, scientific communication, the environment. International members of Istituto Pasteur in Rome and INITIATE, the Marie Curie Training Network reflect on the lasting global impact of the coronavirus pandemic.Medical Microbiolog