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AGGF1 is an angiogenic factor with G-Patch and FHA domains 1 described by our group. Gain-of-function mutations in AGGF1 cause Klippel-Trenaunay syndrome, whereas somatic loss-of-function mutations cause cancer. Paraspeckles are small membraneless subnuclear structures with a diameter of 0.5-1 μm, and composed of lncRNA NEAT1 as the scaffold and three core RNA-binding proteins NONO, PSPC1, and PSF. Here, we show that AGGF1 is a key regulatory and structural component of paraspeckles that induces paraspeckle formation, forms an outside rim of paraspeckles, wraps around the NONO/PSF/PSPC1/NEAT1 core, and regulates the size and number of paraspeckles. AGGF1-paraspeckles are larger (1 μm) than conventional paraspeckles. RNA-FISH in combination with immunostaining shows that AGGF1, NONO, and NEAT1_2 co-localize in 20.58% of NEAT1_2-positive paraspeckles. Mechanistically, AGGF1 interacts with NONO, PSF, and HNRNPK, and upregulates NEAT1_2, a longer, 23 kb NEAT1 transcript with a key role in regulation of paraspeckle size and number. RNA-immunoprecipitation shows that AGGF1 interacts with NEAT1, which may be another possible mechanism underlying the formation of AGGF1-paraspeckles. NEAT1_2 knockdown reduces the number and size of AGGF1-paraspeckles. Functionally, AGGF1 regulates alternative RNA splicing as it decreases the exon skipping/inclusion ratio in a CD44 model. AGGF1 is also localized in some nuclear foci without NEAT1 or NONO, suggesting that AGGF1 is an important liquid-liquid phase separation (LLPS) driver for other types of AGGF1-positive nuclear condensates (referred to as AGGF1-bodies). Our results identify a special type of AGGF1-coated paraspeckles and provide important insights into the formation, structure, and function of paraspeckles.

Background Non‐small cell lung cancer (NSCLC) is one of the most malignant cancers worldwide and its pathogenesis is not completely clear. In this study, we explored the functions and mechanisms of exosomes transferring miR‐3180‐3p in NSCLC progression. Methods The expression levels of miR‐3180‐3p in NSCLC tissues and paracarcinoma tissues was obtained from the GEO database (GEO: GSE53882). Exosomes derived from A549 cells were identified. Proliferation, migration and invasion were measured after treatment with exosomal miR‐3180‐3p or transfection using miR‐3180‐3p mimics. The relationship between miR‐3180‐3p and forkhead box P4 (FOXP4) was predicted using a bioinformatic tool and measured using a dual‐luciferase reporter gene assay and western blotting. Finally, a mouse xenograft model of NSCLC cells was established to verify the function of exosomal miR‐3180‐3p in vivo. Results We found that miR‐3180‐3p decreased in both NSCLC cell lines and patient tissues. Overexpression of miR‐3180‐3p or treatment with exosomal miR‐3180‐3p significantly suppressed cell proliferation and metastasis in NSCLC cell lines. Subsequently, we found miR‐3180‐3p downregulated FOXP4 protein expression levels. Furthermore, the volumes and weights of nude mouse tumors expressing exosomal miR‐3180‐3p were significantly reduced. Conclusions Exosomal miR‐3180‐3p suppresses NSCLC progression by downregulating FOXP4 expression. Key points Significant findings of the study We found that exosomal miR‐3180‐3p suppressed NSCLC progression and also identified a miR‐3180‐3p target gene. These findings provide a foundation to determine innovative therapeutic strategies. What this study adds This study contributes to research investigating exosomal containing miRNAs., We found that exosomal miR‐3180‐3p suppressed NSCLC progression and also identified a miR‐3180‐3p target gene.

Atrial fibrillation (AF) affects >30 million individuals worldwide. However, no genetic mutation from human patients with AF has been linked to inflammation. Here, we show that AF-associated human variant p.Ile138Thr in natriuretic peptide A (NPPA) encoding the atrial natriuretic peptide (ANP) causes inflammation, fibroblast activation, atrial fibrosis, and AF in knock-in (KI) rats. Variant p.Ile138Thr inhibits the interaction between ANP and its receptor natriuretic peptide receptor A and reduces intracellular cGMP levels. RNA sequencing and follow-up analyses showed that mutant ANP (mANP) activates multiple innate immunity pathways, including TNF-α, NF-κB, and IL-1β signaling. mANP induces differentiation of cardiac fibroblasts (CFs) to myofibroblasts and promotes CF proliferation and fibrosis. These results suggest that NPPA variant p.Ile138Thr causes AF by activating TNF-α, NF-κB, and IL-1β signaling, inflammation, and fibrosis. Multiple computational programs suggest that p.Ile138Thr is damaging or deleterious. Based on the 2015 American College of Medical Genetics and Genomics Standards and Guidelines, p.Ile138Thr can be classified as a likely pathogenic variant. Variant p.Ile138Thr was found only in Asian people in the Genome Aggregation Database and Exome Aggregation Consortium database at an averaged frequency of 0.026%. An estimated 1.15 million Asian people carry the variant and might be at risk of AF. The KI rats may provide an inflammation-based, genetic animal model for AF valuable for testing anti-inflammation or other therapies for AF.-Cheng, C., Liu, H., Tan, C., Tong, D., Zhao, Y., Liu, X., Si, W., Wang, L., Liang, L., Li, J., Wang, C., Chen, Q., Du, Y., Wang, Q. K., Ren, X. Mutation in NPPA causes atrial fibrillation by activating inflammation and cardiac fibrosis in a knock-in rat model.

Vascular hyperpermeability caused by distorted endothelial cell-cell junctions is associated with the no-reflow phenomenon after opening of the occluded vessels in patients with coronary artery disease (CAD), the leading cause of death worldwide. Coronary no-reflow is observed in ∼30% of CAD patients after percutaneous coronary stenting and is associated with a worse prognosis at follow-up and a higher incidence of death. However, limited tools are available to control vascular hyperpermeability and no-reflow. Losartan, an angiotensin II (Ang II) receptor blocker acting on the Ang II type-1 receptor (AT1R) subtype, is a prescription drug for treating hypertension. Here we show that in a murine model of ischemia and reperfusion (I/R), losartan blocked vascular hyperpermeability and decreased infarct size, hemorrhages, edema, and inflammation. Mechanistically, losartan-mediated inhibition of vascular hyperpermeability is mediated by the inhibition of phosphorylation of Src and vascular endothelial cadherin (VE-cadherin), which increases VEGF receptor 2 (VEGFR2)-Src-VE-cadherin complex formation, resulting in increased cell surface VE-cadherin and inhibition of vascular hyperpermeability. On the other hand, hypoxia and reoxygenation increased the phosphorylation levels of Src and VE-cadherin and reduced the formation of the VEGFR2-Src-VE-cadherin complex, which led to reduced cell surface VE-cadherin and increased vascular hyperpermeability; all were inhibited by losartan. These data suggest that losartan may be used for blocking vascular hyperpermeability associated with I/R.-Li, Y., Yao, Y., Li, J., Chen, Q., Zhang, L., Wang, Q. K. Losartan protects against myocardial ischemia and reperfusion injury via vascular integrity preservation.

Atrial fibrillation (AF) affects 33.5 million individuals worldwide. It accounts for 15% of strokes and increases risk of heart failure and sudden death. The voltage-gated cardiac sodium channel complex is responsible for the generation and conduction of the cardiac action potential, and composed of the main pore-forming α-subunit Na(v)1.5 (encoded by the SCN5A gene) and one or more auxiliary β-subunits, including Na(v)β1 to Na(v)β4 encoded by SCN1B to SCN4B, respectively. We and others identified loss-of-function mutations in SCN1B and SCN2B and dominant-negative mutations in SCN3B in patients with AF. Three missense variants in SCN4B were identified in sporadic AF patients and small nuclear families; however, the association between SCN4B variants and AF remains to be further defined. In this study, we performed mutational analysis in SCN4B using a panel of 477 AF patients, and identified one nonsynonymous genomic variant p.Gly8Ser in four patients. To assess the association between the p.Gly8Ser variant and AF, we carried out case-control association studies with two independent populations (944 AF patients vs. 9,81 non-AF controls in the first discovery population and 732 cases and 1,291 controls in the second replication population). Significant association was identified in the two independent populations and in the combined population (p = 4.16 × 10(−4), odds ratio [OR] = 3.14) between p.Gly8Ser and common AF as well as lone AF (p = 0.018, OR = 2.85). These data suggest that rare variant p.Gly8Ser of SCN4B confers a significant risk of AF, and SCN4B is a candidate susceptibility gene for AF.

N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.

Aims MOG1 is a small protein that can bind to small GTPase RAN and regulate transport of RNA and proteins between the cytoplasm and nucleus. However, the in vivo physiological role of mog1 in the heart needs to be fully defined. Methods Mog1 knockout zebrafish was generated by TALEN. Echocardiography, histological analysis, and electrocardiograms were used to examine cardiac structure and function. RNA sequencing and real-time RT-PCR were used to elucidate the molecular mechanism and to analyse the gene expression. Isoproterenol was used to induce cardiac hypertrophy. Whole-mount in situ hybridization was used to observe cardiac morphogenesis. Results Mog1 knockout zebrafish developed cardiac hypertrophy and heart failure (enlarged pericardium, increased nppa and nppb expression and ventricular wall thickness, and reduced ejection fraction), which was aggravated by isoproterenol. RNAseq and KEGG pathway analyses revealed the effect of mog1 knockout on the pathways of cardiac hypertrophy, dilatation and contraction. Mechanistic studies revealed that mog1 knockout decreased expression of tbx5, which reduced expression of cryab and hspb2, resulting in cardiac hypertrophy and heart failure. Overexpression of cryab, hspb2 and tbx5 rescued the cardiac oedema phenotype of mog1 KO zebrafish. Telemetry electrocardiogram monitoring showed QRS and QTc prolongation and a reduced heart rate in mog1 knockout zebrafish, which was associated with reduced scn1b expression. Moreover, mog1 knockout resulted in abnormal cardiac looping during embryogenesis because of the reduced expression of nkx2.5, gata4 and hand2. Conclusion Our data identified an important molecular determinant for cardiac hypertrophy and heart failure, and rhythm maintenance of the heart.

The phlda3 gene encodes a small, 127-amino acid protein with only a PH domain, and is involved in tumor suppression, proliferation of islet β-cells, insulin secretion, glucose tolerance, and liver injury. However, the role of phlda3 in vascular development is unknown. Here, we show that phlda3 overexpression decreases the expression levels of hemangioblast markers scl, fli1, and etsrp and intersegmental vessel (ISV) markers flk1 and cdh5, and disrupts ISV development in tg(flk1:GFP) and tg(fli1:GFP) zebrafish. Moreover, phlda3 overexpression inhibits the activation of protein kinase B (AKT) in zebrafish embryos, and the developmental defects of ISVs by phlda3 overexpression were reversed by the expression of a constitutively active form of AKT. These data suggest that phlda3 is a negative regulator of hemangioblast specification and ISV development via AKT signaling.

A genomic variant in the human ADTRP [androgen-dependent tissue factor (TF) pathway inhibitor (TFPI) regulating protein] gene increases the risk of coronary artery disease, the leading cause of death worldwide. TFPI is the TF pathway inhibitor that is involved in coagulation. Here, we report that adtrp and tfpi form a regulatory axis that specifies primitive myelopoiesis and definitive hematopoiesis, but not primitive erythropoiesis or vasculogenesis. In zebrafish, there are 2 paralogues for adtrp (i.e., adtrp1 and adtrp2). Knockdown of adtrp1 expression inhibits the specification of hemangioblasts, as shown by decreased expression of the hemangioblast markers, etsrp, fli1a, and scl; blocks primitive hematopoiesis, as shown by decreased expression of pu.1, mpo, and l-plastin; and disrupts the specification of hematopoietic stem cells (definitive hematopoiesis), as shown by decreased expression of runx1 and c-myb. However, adtrp1 knockdown does not affect erythropoiesis during primitive hematopoiesis (no effect on gata1 or h-bae1) or vasculogenesis (no effect on kdrl, ephb2a, notch3, dab2, or flt4). Knockdown of adtrp2 expression does not have apparent effects on all markers tested. Knockdown of adtrp1 reduced the expression of tfpi, and hematopoietic defects in adtrp1 morphants were rescued by tfpi overexpression. These data suggest that the regulation of tfpi expression is one potential mechanism by which adtrp1 regulates primitive myelopoiesis and definitive hematopoiesis.—Wang, L., Wang, X., Wang, L., Yousaf, M., Li, J., Zuo, M., Yang, Z., Gou, D., Bao, B., Li, L., Xiang, N., Jia, H., Xu, C., Chen, Q., Wang, Q. K. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.

We report on the remarkable potential of highly epitaxial and pure (001)-oriented CeO2 thin films grown on conducting Nb-doped SrTiO3 (NSTO) substrates by laser molecular beam epitaxy for nonvolatile memory application. Resistive switching (RS) devices with the structure of Au/epi-CeO2/NSTO exhibit reversible and steady bipolar RS behaviour with large high/low resistance ratio and a narrow dispersion of the resistance values. Detailed analysis of the conduction mechanisms reveals that the trapping/detrapping processes and oxygen vacancies migration play important roles in the switching behaviour. In the light of XPS measurement results, the CeO2/NSTO interface with oxygen vacancies or defects is responsible for the RS effect. Furthermore, a model is proposed to explain this resistance switching behaviour. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Restless legs syndrome (RLS, MIM 102300) is a common neurological disorder characterized by an urge to move the limbs and is usually accompanied by uncomfortable sensations that worsen with rest, improve with movement, and are worse in the evening or night.1 It can impair sleep and adversely affects health and quality of life. Epidemiological studies of RLS revealed a prevalence rate of 5 to 12% in Caucasian populations, which is much higher than the rate in Asian and African populations (0.1–0.6%), suggesting a possible role for racial or ethnic factors in the occurrence of RLS.2 Furthermore, RLS is as much as twice more common in females than in males.3 RLS is associated with a positive family history in 45 to 65% of patients, and twin studies showed a high concordant rate in monozygotic twins.4 Our familial aggregation analysis showed a correlation rate of 0.17 between first degree relatives and heritability of 0.60.5 All these results suggest that genetic factors play an important role in the pathogenesis of RLS. Genome-wide linkage studies have identified six genetic loci for RLS, including RLS1 on chromosome 12q12–q21,6 RLS2 on 14q13–21,7 RLS3 on 9p24-p22,5 RLS4 on 2q33,8 RLS5 on 20p13, and RLS6 on 16p12.1.9,10 Till date, no disease-causing mutations from these loci have been reported. A recent genome-wide association study (GWAS) in a Caucasian population showed that two single nucleotide polymorphisms (SNPs), including rs1975197 and rs4626664 located within introns of the PTPRD gene, were significantly associated with RLS.11 The PTPRD gene is located within the RLS3 locus reported by our group.5 In this study, we took advantage of the 15 large families used for identifying the RLS3 locus to independently assess whether SNPs in PTPRD (ENST00000381196) are associated with RLS using a family-based sibling transmission disequilibrium test (Sib-TDT). We also evaluated the association using a population- based association study in a cohort of 189 sporadic patients and 560 controls. To the best of knowledge, this is the first study to replicate the association between variants in PTPRD gene and RLS. Furthermore, we scanned the sequences of all coding exons and boundaries between exons and introns of PTPRD gene in the 15 probands from the 15 different families to identify potential disease-causing mutations.

We report the identification of a novel 12 bp deletion of the pre-mRNA splicing gene PRPF31 in a large Chinese family with autosomal dominant retinitis pigmentosa (adRP). This mutation results in the deletion of four amino acids (DeltaH(111)K(112)F(113)I(114)) including H(111), an amino acid residue that is highly conserved throughout evolution. The 12 bp deletion co-segregates with the disease phenotype in 19 RP patients in the family, but is not present in unaffected relatives or 100 normal individuals. Our data indicate that the novel 12 bp deletion in PRPF31 causes retinitis pigementosa in this Chinese adRP family. In contrast to the incomplete penetrance observed in most adRP families linked to chromosome band 19q13.4 (RP11), the 12 bp PRPF31 deletion identified in this study appears to show high penetrance. These data expand the spectrum of PRPF31 mutations causing adRP, and confirm the role of PRPF31 in the pathogenesis of RP.

Obsessive-compulsive disorder (OCD) is a chronic psychiatric disease characterized by recurrent obsessions, compulsions, or both. The prevalence rate of OCD is 2.1% in the general population. Here we report cytogenetic analysis of 26 patients affected with OCD. In one male patient (OCD-K33), we identified a fragile X chromosome by cytogenetic analysis with 21% of cells demonstrating a fragile site at Xq27-q28. Polymerase chain reaction (PCR) and Southern blot analysis demonstrated that the molecular basis of the OCD-K33 fragile X chromosome was expansion of the CCG repeat at FRAXE. The number of the expanded repeats was estimated to be more than 300 copies, qualifying it as a full FRAXE mutation. Further analysis of the family members of OCD-K33 revealed another member with a full FRAXE mutation (630-1,200 copies of the CCG repeat), who had the clinical phenotype of speech impairment, and two other members with normal phenotypes and no FRAXE expansion. The two FRAXE expansions lead to complete methylation at the CCG repeat. The co-segregation of the full FRAXE mutation with apparent neurologic disorders in the same family provides further support to the notion that FRAXE is a genetic neurologic condition. Our findings expand the spectrum of clinical phenotypes associated with FRAXE mutations.

Heart disease (cardiovascular disease) is the leading cause of death in developed countries. Genetic factors play an important role in the pathogenesis of heart disease. The revolutionary advances in molecular genetics have provided new insights into the responsible genes and pathogenic mechanisms of heart disease, including congenital heart defects, arrhythmias, cardiomyopathies, heart failure, and coronary artery disease. Keywords: cardiac genetics; heart structure and function; heart development; congenital heart defects; cardiac arrhythmias