Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

Segmental aneuploidies with 1 Mb resolution in human preimplantation blastocysts.

PURPOSE: This study aimed to investigate the spectrum and characteristics of segmental aneuploidies (SAs) of <10 megabase (Mb) length in human preimplantation blastocysts. METHODS: Preimplantation genetic testing for aneuploidy was performed in 15,411 blastocysts from 5171 patients using a validated 1 Mb resolution platform. The characteristics and spectrum of SAs, including the incidence, sizes, type, inheritance pattern, clinical significance, and embryo distribution, were studied. RESULTS: In total, 6.4% of the 15,411 blastocysts carried SAs of >10 Mb, 4.9% of embryos had SAs ranging between 1 to 10 Mb, and 84.3% of 1 to 10 Mb SAs were <5 Mb in size. Inheritance pattern analysis indicated that approximately 63.8% of 1 to 10 Mb SAs were inherited and were predominantly 1 to 3 Mb in size. Furthermore, 18.4% of inherited SAs and 51.9% de novo 1 to 10 Mb SAs were pathogenic or likely pathogenic (P/LP). Different from whole-chromosome aneuploidies, reanalysis indicated that 50% of the de novo 1 to 10 Mb SAs and 70% of the >10 Mb SAs arose from mitotic errors. CONCLUSION: Based on the established platform, 1 to 10 Mb SAs are common in blastocysts and include a subset of P/LP SAs. Inheritance pattern analysis and clinical interpretation based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines contributed to determine the P/LP SAs.

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

The 90-day prognostic value of copeptin in acute intracerebral hemorrhage.

The aim of the study was to assess the 90-day prognostic value of copeptin in a group of Chinese patients with acute intracerebral hemorrhage (ICH). In this study, all consecutive patients with first-ever ICH from 2010 to 2012 were recruited to participate in the study. On admission, plasma copeptin levels were measured by enzyme-linked immunosorbent assay. The Glasgow Coma Scale (GCS) and Hemphill ICH scores were assessed on admission blinded to plasma copeptin levels. For the assessment of functional outcome at 90 days, Modified Rankin Scale was used. During the study period, 271 patients were diagnosed as ICH and were included in the analysis. The median GCS score on admission was 11 points. Patients with an unfavorable outcomes and non-survivors had significantly increased plasma copeptin levels on admission (P < 0.001 for both). Copeptin was an independent prognostic marker of functional outcome and death [odds ratio 3.45 (95 % confidence intervals: 1.85-6.99) and 3.66 (2.42-8.28), respectively, P < 0.001 for both, adjusted for age, the hematoma volume and other predictors] in patients with ICH. In receiver operating characteristic curve analysis, copeptin could improve the Hemphill score in predicting 90-day functional outcome [area under the curve (AUC) of the combined model, 0.83; 95 % CI 0.74-0.90; P < 0.001] and mortality (AUC of the combined model, 0.88; 95 % CI 0.82-0.93; P < 0.001). In conclusion, our study suggests that copeptin levels are a useful tool to predict unfavorable functional outcome and mortality 90 days after ICH and have a potential to assist clinicians.

C/EBP-α ameliorates CCl(4)-induced liver fibrosis in mice through promoting apoptosis of hepatic stellate cells with little apoptotic effect on hepatocytes in vitro and in vivo.

CCAAT enhancer binding protein-α (C/EBP-α) is a transcript factor that regulates adipocyte differentiation and induces apoptosis in hepatic stellate cells (HSCs) in vivo and in vitro. However, the effect of C/EBP-α on hepatocytes in vivo remains unknown. This study investigated whether C/EBP-α exerts different apoptotic effects on hepatocytes and HSCs in vitro and in vivo. An adenovirus vector-expressing C/EBP-α gene was constructed, and a rat hepatic stellate cell lines (HSC-T6) and hepatocytes were transfected. A CCl(4)-induced liver fibrosis model in mice was also utilized. C/EBP-α induced apoptosis in hepatocytes and HSCs, but a significant difference between these cell types was observed in vitro. The mitochondrial pathway was involved in the apoptotic process and was predominant in HSC-T6 apoptosis. In the CCl(4)-induced mice liver fibrosis model, the administration of Ad-C/EBP-α decreased extracellular matrix deposition, including collagen and hydroxyproline content, and γ-GT levels, a marker of liver damage, were reduced significantly. Immunohistochemistry and TUNEL assay results showed an increase of apoptosis in HSCs, but hepatocytes were less affected. C/EBP-α induced differential apoptotic effects in hepatocytes and HSCs in vitro and in vivo. This differential effect could be a potential target for the treatment of hepatic fibrosis with little hepatic toxicity.

Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells.

Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.