Epicardial CCM2 Promotes Cardiac Development and Repair Via its Regulation on Cytoskeletal Reorganization.

The epicardium provides epicardial-derived cells and molecular signals to support cardiac development and regeneration. Zebrafish and mouse studies have shown that ccm2, a cerebral cavernous malformation disease gene, is essential for cardiac development. Endocardial cell-specific deletion of Ccm2 in mice has previously established that Ccm2 is essential for maintenance of the cardiac jelly for cardiac development during early gestation. The current study aimed to explore the function of Ccm2 in epicardial cells for heart development and regeneration. Through genetic deletion of Ccm2 in epicardial cells, our in vivo and ex vivo experiments revealed that Ccm2 is required by epicardial cells to support heart development. Ccm2 regulates epicardial cell adhesion, cell polarity, cell spreading, and migration. Importantly, the loss of Ccm2 in epicardial cells delays cardiac function recovery and aggravates cardiac fibrosis following myocardial infarction. Molecularly, Ccm2 targets the production of cytoskeletal and matrix proteins to maintain epicardial cell function and behaviors. Epicardial Ccm2 plays a critical role in heart development and regeneration via its regulation of cytoskeleton reorganization.

Genome-wide nonessential gene identification of Autographa californica multiple nucleopolyhedrovirus.

The Baculovirus Expression Vector System (BEVS) is an insect cell-based heterologous protein expression system that possesses powerful potential in the development of protein drugs and vaccines. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely-used vector in BEVS with 151 open reading frames (ORFs) containing essential and nonessential genes. Deletion of nonessential genes has many advantages including increased foreign gene insertion. In this study, the λ red recombination system was used to knock out genes in a modified AcMNPV that carried an enhanced yellow fluorescent protein (eYFP) at the Ac126-Ac127 locus. Eighty genes were almost completely deleted respectively and 69 gene knockout AcMNPVs (KOVs) were obtained to evaluate their infection efficiency. After infecting Spodoptera frugiperda 9 (Sf9) cells, 51 KOVs including 62 genes showed similar infectivity as wide type (WT) and hence were defined as nonessential genes. However, 18 KOVs produced fewer infectious virions, indicating that these genes were influential in the production of progeny viruses. Combining our research with previous studies, a desired minimal AcMNPV genome containing 86 ORFs and all of the homologous regions (hrs) was brought up, facilitating genetic modification of baculovirus vectors and improvement of recombinant protein expression in the future.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRß. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells.

CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FK506-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.

LRP6 downregulation promotes cardiomyocyte proliferation and heart regeneration.

The adult mammalian heart is thought to be a terminally differentiated organ given the postmitotic nature of cardiomyocytes. Consequently, the potential for cardiac repair through cardiomyocyte proliferation is extremely limited. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor that is required for embryonic heart development. In this study we investigated the role of LRP6 in heart repair through regulation of cardiomyocyte proliferation. Lrp6 deficiency increased cardiomyocyte cell cycle activity in neonatal, juvenile and adult mice. Cardiomyocyte-specific deletion of Lrp6 in the mouse heart induced a robust regenerative response after myocardial infarction (MI), led to reduced MI area and improvement in left ventricular systolic function. In vivo genetic lineage tracing revealed that the newly formed cardiomyocytes in Lrp6-deficient mouse hearts after MI were mainly derived from resident cardiomyocytes. Furthermore, we found that the pro-proliferative effect of Lrp6 deficiency was mediated by the ING5/P21 signaling pathway. Gene therapy using the adeno-associated virus (AAV)9 miRNAi-Lrp6 construct promoted the repair of heart injury in mice. Lrp6 deficiency also induced the proliferation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our study identifies LRP6 as a critical regulator of cardiomyocyte proliferation, which may lead to the development of a novel molecular strategy to promote myocardial regeneration and repair.

Epigenetic Control of a Local Chromatin Landscape.

Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. Decades of research have associated this process of chromatin regulation and gene expression with human diseased states. With the goal of understanding how chromatin dysregulation contributes to disease, as well as preventing or reversing this type of dysregulation, a multidisciplinary effort has been launched to control the epigenome. Chemicals that alter the epigenome have been used in labs and in clinics since the 1970s, but more recently there has been a shift in this effort towards manipulating the chromatin landscape in a locus-specific manner. This review will provide an overview of chromatin biology to set the stage for the type of control being discussed, evaluate the recent technological advances made in controlling specific regions of chromatin, and consider the translational applications of these works.

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.

Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.

Synthesis, anti-cancer evaluation of benzenesulfonamide derivatives as potent tubulin-targeting agents.

A series of benzenesulfonamide derivatives were synthesized and evaluated for their anti-proliferative activity and interaction with tubulin. These new derivatives showed significant activities against cellular proliferative and tubulin polymerization. Compound BA-3b proved to be the most potent compound with IC50 value ranging from 0.007 to 0.036 µM against seven cancer cell lines, and three drug-resistant cancer cell lines, which indicated a promising anti-cancer agent. The target tubulin was also verified by dynamic tubulin polymerization assay and tubulin intensity assay.

Superposition of an incoherent magnetic field inhibited EGF receptor clustering and phosphorylation induced by a 1.8 GHz pulse-modulated radiofrequency radiation.

PURPOSE: The present study was conducted to investigate the effect of a temporally incoherent ('noise') magnetic field (MF) on radiofrequency radiation (RFR)-induced epidermal growth factor (EGF) receptor clustering and phosporylation in cultured cells. MATERIALS AND METHODS: Human amniotic epithelial (FL) cells were exposed for 15 min to either a 1.8 GHz RFR (modulated at 217 Hz), a 2 µT incoherent MF, or concurrently to the RFR and incoherent MF. Epidermal growth factor treatment severed as the positive control. Epidermal growth factor receptor clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining, and phosphorylation of EGF receptors was measured by western blot technology. RESULTS: Exposure of FL cells to the 1.8 GHz RFR at SAR (specific absorption rate) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min induced EGF receptor clustering and enhanced phosphorylation on tyrosine-1173 residue, whereas exposure to RFR at SAR of 0.1 W/kg for 15 min did not significantly cause these effects. Exposure to a 2 µT incoherent MF for 15 min did not significantly affect clustering and phosphorylation of EGF receptor in FL cells. When superimposed, the incoherent MF completely inhibited EGF receptor clustering and phosphorylation induced by RFR at SAR of 0.5, 1.0, and 2.0 W/kg, but did not inhibit the effects induced at SAR of 4.0 W/kg. CONCLUSION: Based on the data of the experiment, it is suggested that membrane receptors could be one of the main targets by which RFR interacts with cells. An incoherent MF could block the interaction to a certain extent.

A 1.8-GHz radiofrequency radiation induces EGF receptor clustering and phosphorylation in cultured human amniotic (FL) cells.

PURPOSE: Many studies have shown that exposure to radiofrequency radiation (RFR) could activate cellular signal transduction pathways. In the present research, we investigated the effects of exposure to a 1.8-GHz RFR at different intensities on epidermal growth factor (EGF) receptor clustering and phosphorylation in human amniotic (FL) cells. MATERIALS AND METHODS: Receptor clustering on cellular membrane surface was analyzed using immunofluorescence assessed by confocal microscopy, and phosphorylation of EGF receptors was measured by western blot technology. EGF treatment served as a positive control. RESULTS: The results showed that, compared with sham exposure, exposure to RFR at specific absorption rate (SAR) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min significantly induced EGF receptor clustering and enhanced phosphorylation on the tyrosine-1173 residue in FL cells, whereas exposure to a SAR 0.1 W/kg radiation for 15 min did not cause a significant effect. CONCLUSION: Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets that RFR interacts with cells, and the dose-rate threshold, in the case of EGF receptors, is between SAR of 0.1 and 0.5 W/kg. The results indicate a sigmoid dependence of RFR effects on intensity.