An induced pluripotent stem cell line carrying a silencing-resistant calcium reporter allele.

Calcium indicators are sensitive tools to image neural activity. However, their use in human induced pluripotent stem cells (iPSC)-derived neurons is limited by silencing of the transgene. We generated the iPSC line MSE2336A carrying heterozygous insertion in the safe-harbor locus AAVS1 of the ultrasensitive protein calcium sensor (GCaMP6) under the control of CAG promoter and UCOE to maintain robust transgene expression in differentiated cells.

CRISPRpas: programmable regulation of alternative polyadenylation by dCas9.

Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance.

Proteomic analysis of mitochondrial biogenesis in cardiomyocytes differentiated from human induced pluripotent stem cells.

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts.

Widespread transcript shortening through alternative polyadenylation in secretory cell differentiation.

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy.

An alternative mitophagy pathway mediated by Rab9 protects the heart against ischemia.

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3.

Interferon regulatory factor 5 (IRF5) suppresses hepatitis C virus (HCV) replication and HCV-associated hepatocellular carcinoma.

Hepatitis C virus (HCV) infection is a major risk factor for the development of chronic liver disease. The disease typically progresses from chronic HCV to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and death. Chronic inflammation associated with HCV infection is implicated in cirrhosis and HCC, but the molecular players and signaling pathways contributing to these processes remain largely unknown.

Effect of densely ionizing radiation on cardiomyocyte differentiation from human-induced pluripotent stem cells.

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.

Transient receptor potential cation 3 channel regulates melanoma proliferation and migration.

Melanoma has an extremely poor prognosis due to its rapidly progressive and highly metastatic nature. Several therapeutic drugs have recently become available, but are effective only against melanoma with specific BRAF gene mutation. Thus, there is a need to identify other target molecules.

Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling.

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma.

Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration.

Store-operated Ca(2+) entry (SOCE) is a major mechanism of Ca(2) (+) import from extracellular to intracellular space, involving detection of Ca(2+) store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca(2+) channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor.

'Reduced malignancy as a mechanism for longevity in mice with adenylyl cyclase type 5 disruption'.

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside.

Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase.

Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-β-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF).

Gβγ subunits inhibit Epac-induced melanoma cell migration.

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied.

Caloric restriction reduces growth of mammary tumors and metastases.

We investigated the effects of caloric restriction (CR) on growth of tumors and metastases in the 4T1 mammary tumor model and found that CR, compared with normal diet, reduced the growth of mammary tumors and metastases and the total number of metastases that originated both spontaneously from the primary tumor and also experimentally from i.v. injection of the tumor cells.

Epac1 promotes melanoma metastasis via modification of heparan sulfate.

Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma.

Exchange protein directly activated by cyclic AMP increases melanoma cell migration by a Ca2+-dependent mechanism.

Melanoma has a poor prognosis due to its strong metastatic ability. Although Ca(2+) plays a major role in cell migration, little is known about the role of Ca(2+) in melanoma cell migration. We recently found that the exchange protein directly activated by cyclic AMP (Epac) increases melanoma cell migration via a heparan sulfate-related mechanism.

Epac increases melanoma cell migration by a heparan sulfate-related mechanism.

Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma.

Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy.

Although it has been shown that Epac1 mRNA is expressed ubiquitously and Epac2 mRNA predominantly in the brain and endocrine tissues, developmental and pathophysiological changes of these molecules have not been characterized. Developmental changes were analyzed in murine heart, brain, kidneys, and lungs by RT-PCR analysis, which revealed more drastic developmental changes of Epac2 mRNA than Epac1. Only the Epac2 mRNA in kidney showed a transient expression pattern with dramatic decline into adulthood.