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1.
Cell Rep ; 27(13): 3780-3789.e4, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242412

RESUMO

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.

2.
Cancer Res ; 79(11): 3001-3006, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31015229

RESUMO

Increasing evidence has suggested a role for adenosine-to-inosine RNA editing in carcinogenesis. However, the clinical utility of RNA editing remains limited because functions of the vast majority of editing events remain largely unexplored. To help the cancer research community investigate functional consequences of individual editing events, we have developed a user-friendly bioinformatic resource, The Cancer Editome Atlas (TCEA; http://tcea.tmu.edu.tw). TCEA characterizes >192 million editing events at >4.6 million editing sites from approximately 11,000 samples across 33 cancer types in The Cancer Genome Atlas. Clinical information, miRNA expression, and alteration in miRNA targeting modulated through RNA editing are also integrated into TCEA. TCEA supports several modules to search, analyze, and visualize the cancer editome, providing a solid basis for investigating the oncogenic mechanisms of RNA editing and expediting the identification of therapeutic targets in cancer. SIGNIFICANCE: This user-friendly bioinformatic resource reduces the barrier to analyzing the huge and complex cancer RNA editome that cancer researchers face and facilitates the identification of novel therapeutic targets in cancer.

3.
J Am Coll Cardiol ; 72(16): 1923-1925, 2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-30309469
4.
Artigo em Inglês | MEDLINE | ID: mdl-30219887

RESUMO

Aims: The management of anticoagulation therapy in patients with atrial fibrillation (AF) and cancer is challenging due to increased thrombotic and bleeding risks. We sought to determine the safety and efficacy of rivaroxaban in patients with AF and a history of cancer. Methods and Results: ROCKET AF randomized 14,264 patients with AF to rivaroxaban or warfarin with a median follow-up of 1.9 years. Cox regression models were used to assess the association between cancer history and clinical outcomes, and the relative treatment effect of rivaroxaban versus warfarin in these patients. A total of 640 patients enrolled in ROCKET AF had a history of cancer, with the most common types being prostate (28.6%), colorectal (16.1%), and breast (14.7%) cancer. Patients with a history of cancer were older, more frequently male, more likely to have prior VKA use, and had higher rates of overall bleeding (HR 1.30 95% CI 1.16-1.47; p < 0.0001) and non-cardiovascular death (HR 1.47 95% CI 1.04-2.07; p = 0.031) compared with those with no cancer history. There were no significant associations between cancer history and stroke, venous thromboembolism, or myocardial infarction. The relative efficacy of rivaroxaban versus warfarin for prevention of stroke/systemic embolism was similar in those with and without a history of cancer (interaction p-value=0.21). Conclusion: In ROCKET AF, a history of cancer was associated with a higher risk of bleeding and non-cardiovascular death, but not ischemic events. The relative efficacy and safety of rivaroxaban compared with warfarin were not significantly different in patients with and without a history of cancer. The results of this study are exploratory and should be taken in context of the study population, which may not be generalizable to those with advanced malignancy. Further investigation is needed to understand optimal anticoagulation strategies in patients with AF and cancer.

5.
Am J Hematol ; 93(12): 1451-1460, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30144150

RESUMO

Sickle cell disease (SCD) nephropathy and lower estimated glomerular filtration rate (eGFR) are risk factors for early mortality. Furthermore, rate of eGFR decline predicts progression to end-stage renal disease in many clinical settings. However, factors predicting renal function decline in SCD are poorly documented. Using clinical, laboratory, genetic, and metabolomic data, we evaluated predictors of renal function decline in a longitudinal cohort of 288 adults (mean age 33.0 years). In 193 subjects with 5-year follow-up data, mean rate of eGFR decline was 2.35 mL/min/1.73 m2 /year, nearly twice that of African American adults overall. Hyperfiltration was prevalent at baseline (61.1%), and 36.8% of subjects experienced rapid eGFR decline (≥3 mL/min/1.73 m2 /year). Severe Hb genotype; proteinuria; higher platelet and reticulocyte counts, and systolic BP; and lower Hb level and BMI were associated with rapid decline. A risk scoring system was created using these 7 variables and was highly predictive of rapid eGFR decline, with odds of rapid decline increasing 1.635-fold for every point increment (P < 0.0001). Rapid eGFR decline was also associated with higher organ system severity score and peak creatinine. Additionally, two metabolites (asymmetric dimethylarginine and quinolinic acid) were associated with rapid decline. Further investigation into longitudinal SCD nephropathy (SCDN) trajectory, early markers of SCDN, and tools for risk stratification should inform interventional studies targeted to slowing GFR decline and improving SCD outcomes.

6.
Mol Cell ; 71(6): 956-972.e9, 2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-30146317

RESUMO

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

7.
Am Heart J ; 200: 102-109, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29898836

RESUMO

BACKGROUND: We investigated the impact of polyvascular disease in patients enrolled in ROCKET AF. METHODS: Cox regression models were used to assess clinical outcomes and treatment effects of rivaroxaban compared with warfarin in patients with atrial fibrillation and coronary, peripheral, or carotid artery disease, or any combination of the 3. RESULTS: A total of 655 (4.6%) patients had polyvascular disease (≥2 disease locations), and 3,391 (23.8%) had single-arterial bed disease. Patients with polyvascular disease had similar rates of stroke/systemic embolism but higher rates of cardiovascular and bleeding events when compared with those without vascular disease. Use of rivaroxaban compared with warfarin was associated with higher rates of stroke in patients with polyvascular disease (hazard ratio [HR] 2.41, 95% CI 1.05-5.54); however, this was not seen in patients with single-bed (HR 0.90, 95% CI 0.64-1.28) or no vascular disease (HR 0.85, 95% CI 0.69-1.04; interaction P = .058). There was a significant interaction for major or nonmajor clinically relevant bleeding in patients with polyvascular (HR 1.23, 95% CI 0.91-1.65) and single-bed vascular disease (HR 1.30, 95% CI 1.13-1.49) treated with rivaroxaban compared with warfarin when compared with those without vascular disease (HR 0.95, 95% CI 0.87-1.04; interaction P = .0006). Additional antiplatelet therapy in this population did not improve stroke or cardiovascular outcomes. CONCLUSION: The use of rivaroxaban compared with warfarin was associated with a higher risk of stroke and bleeding in patients with polyvascular disease enrolled in ROCKET AF. Further studies are needed to understand the optimal management of this high-risk population.

8.
Nat Protoc ; 13(6): 1253-1274, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29748649

RESUMO

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

9.
Cancer Lett ; 420: 156-167, 2018 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-29410067

RESUMO

Paclitaxel is widely used in the combination chemotherapy for many cancers including esophageal squamous cell carcinoma (ESCC). However, the paclitaxel resistance occurs frequently in treating ESCC and the mechanism is not fully understood yet. The heterogeneity of gene expression within the drug-resistant cancer cells may be one of the major factors contributing to its resistance. In the present study, we successfully induced paclitaxel resistance in ESCC cell line KYSE-30 through low dose and long-term treatment of paclitaxel. Gene expression profiles were measured utilizing population RNA-seq and single-cell RNA-seq (scRNA-seq). 37 single cells from KYSE-30 cells and 73 single cells from paclitaxel resistant KYSE-30 cells (Taxol-R) were subjected to scRNA-seq. Weighted gene co-expression network analysis (WGCNA) of scRNA-seq data revealed two major subpopulations in both KYSE-30 and Taxol-R cancer cells. Two subpopulations based on the KRT19 expression levels in KYSE-30 cells exhibited different paclitaxel sensitivity, suggesting the existence of an intrinsic paclitaxel resistance in KYSE-30 cells. In addition, the Taxol-R cells that acquired the resistance to paclitaxel through induction were characterized with higher expressions of proteasomes but a lower expression of HIF-1 signaling genes. Furthermore, we showed that carfilzomib (CFZ), a proteasome inhibitor, could attenuate the paclitaxel resistance in Taxol-R cancer cells through activating the HIF-1 signaling. Our new finding may pave a way leading to an improvement in the treatment on cancers including ESCC by combining CFZ with paclitaxel as a novel approach for cancer therapy.

10.
Prog Cardiovasc Dis ; 60(4-5): 514-523, 2018 Jan - Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29339167

RESUMO

In patients with non-valvular atrial fibrillation (NVAF), oral anticoagulation is important for prevention of stroke and systemic embolism (SE). While Vitamin K antagonists (VKAs) have historically been the standard of care, these medications are limited by numerous food and drug interactions with onerous requirements for frequent monitoring and dose adjustments. Over the past decade, several novel oral anticoagulants (NOACs) have been developed to directly inhibit factor IIa/thrombin (dabigatran) or activated factor X (apixaban, rivaroxaban, edoxaban). These medications have been shown to be at least as effective as warfarin for stroke prevention in NVAF with more favorable safety profiles. However, their advantages are underscored by a lack of specific antidotes and assays quantifying their anticoagulant effects. This paper addresses the use of NOACs compared to VKAs in patients with NVAF, with a special focus on high-risk populations, including the elderly, those with renal disease, diabetes mellitus, coronary artery disease, and previous stroke. The current literature surrounding special clinical scenarios including the treatment of bleeding, perioperative management, and the use of NOACs in cardioversion and catheter ablation will be also discussed.


Assuntos
Anticoagulantes , Fibrilação Atrial/tratamento farmacológico , Acidente Vascular Cerebral/prevenção & controle , Administração Oral , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/classificação , Fibrilação Atrial/complicações , Humanos , Conduta do Tratamento Medicamentoso , Risco Ajustado/métodos , Acidente Vascular Cerebral/etiologia , Resultado do Tratamento
12.
J Mol Cell Cardiol ; 112: 104-113, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28935506

RESUMO

Endothelial glycolysis plays a critical role in the regulation of angiogenesis. We investigated the role of Sirtuin 3 (SIRT3) on endothelial cell (EC) glycolytic metabolism, angiogenesis, and diastolic function. Our aim was to test the hypothesis that loss of SIRT3 in ECs impairs endothelial glycolytic metabolism and angiogenesis and contributes to myocardial capillary rarefaction and the development of diastolic dysfunction. Using SIRT3 deficient ECs, SIRT3 was found to regulate a metabolic switch between mitochondrial respiration and glycolysis. SIRT3 knockout (KO)-ECs exhibited higher mitochondrial respiration and reactive oxygen species (ROS) formation. SIRT3 knockout (KO)-ECs exhibited a reduction in the expression of glycolytic enzyme, PFKFB3, and a fall in glycolysis and angiogenesis. Blockade of PFKFB3 reduced glycolysis and downregulated expression of VEGF and Angiopoietin-1 (Ang-1) in ECs. Deletion of SIRT3 in ECs also impaired hypoxia-induced expression of HIF-2α, VEGF, and Ang-1, as well as reduced angiogenesis. In vivo, endothelial-specific SIRT3 KO (ECKO) mice exhibited a myocardial capillary rarefaction together with a reduced coronary flow reserve (CFR) and diastolic dysfunction. Histologic study further demonstrated that knockout of SIRT3 in ECs significantly increased perivascular fibrosis in the coronary artery. These results implicate a role of SIRT3 in modulating endothelial function and cardiac function. Ablation of SIRT3 leads to impairment of EC glycolytic metabolism and angiogenic signaling, which may contribute to coronary microvascular rarefaction and diastolic dysfunction in SIRT3 ECKO mice.


Assuntos
Diástole , Deleção de Genes , Glicólise , Coração/fisiopatologia , Neovascularização Fisiológica , Sirtuína 3/metabolismo , Animais , Capilares/metabolismo , Hipóxia Celular , Circulação Coronária , Células Endoteliais/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos , Consumo de Oxigênio , Fenótipo , Fosfofrutoquinase-2/metabolismo , Transdução de Sinais , Sirtuína 3/deficiência , Volume Sistólico
13.
Am J Cardiol ; 120(10): 1837-1840, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28886856

RESUMO

The safety of intravenous thrombolysis in patients taking rivaroxaban has not been well established. We retrospectively analyzed the outcomes of all patients who received thrombolytic therapy in the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). A review of medical and adverse event records for patients receiving thrombolytic therapy while enrolled in ROCKET AF was performed to determine their baseline characteristics, indications for thrombolysis, and type of agent used. Safety end points were 30-day post-thrombolytic rates of stroke, bleeding, and mortality. A total of 28 patients in ROCKET AF received thrombolytic therapy, with 19 patients on rivaroxaban and 9 patients on warfarin. Ischemic stroke was the most common indication for thrombolysis (n = 10), and alteplase was the most commonly used fibrinolytic agent (n = 14). Of the 19 patients in the rivaroxaban group, there were 2 nonfatal bleeding events and 2 deaths, mostly occurring when thrombolytic therapy was administered within 48 hours of the last rivaroxaban dose. Of the 9 patients in the warfarin group, there was 1 nonfatal bleeding event and 3 deaths, most occurring when thrombolytic therapy was administered outside of 48 hours from the last warfarin dose. In conclusion, these observations suggest that careful assessment of the time since the last dose may be of clinical significance in patients on novel oral anticoagulants who require emergent thrombolysis.


Assuntos
Fibrilação Atrial/tratamento farmacológico , Embolia/prevenção & controle , Rivaroxabana/administração & dosagem , Acidente Vascular Cerebral/prevenção & controle , Terapia Trombolítica/métodos , Vitamina K/antagonistas & inibidores , Varfarina/administração & dosagem , Administração Oral , Idoso , Fibrilação Atrial/complicações , Fibrilação Atrial/mortalidade , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Embolia/epidemiologia , Embolia/etiologia , Inibidores do Fator Xa/administração & dosagem , Feminino , Seguimentos , Humanos , Incidência , Masculino , Estudos Retrospectivos , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologia , Taxa de Sobrevida/tendências , Fatores de Tempo , Resultado do Tratamento , Estados Unidos/epidemiologia
14.
Sci Rep ; 7(1): 4649, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28680048

RESUMO

Previous studies have reported inconsistent results of the associations of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) with incident type 2 diabetes (diabetes hereafter). We aimed to resolve the controversy by taking nonalcoholic fatty liver disease (NAFLD) into account. The study population comprised 132,377 non-diabetic individuals (64,875 men and 67,502 women) aged 35-79 who had two or more health examinations during 1996-2014. A total of 6,555 incident diabetes (3,734 men and 2,821 women) were identified, on average, over 5.8 years of follow-up. Cox regression was used to calculate the hazard ratio (HR) for incident diabetes, adjusting for classical confounders. The risk of incident diabetes was significantly associated with NAFLD [HR = 2.08 (men) and 2.65 (women)]. Elevated ALT, AST, GGT and ALP were also significantly associated with the increased risk of diabetes, with HRs of 1.27, 1.23, 1.58 and 1.37, respectively, in men, and 1.56, 1.18, 1.48 and 1.44, respectively in women. Our results suggest that NAFLD, ALT, AST, GGT and ALP are independent predictors for incident diabetes in both men and women.

15.
Tumour Biol ; 39(6): 1010428317705573, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28651487

RESUMO

Surgery is the most effective treatment for breast cancer patients. However, some patients developed recurrence and distant metastasis after surgery. Adjuvant therapy is considered for high-risk patients depending on several prognostic markers, and lymphovascular invasion has become one of such prognostic markers that help physicians to identify the risk for distant metastasis and recurrence. However, the mechanism of lymphovascular invasion in breast cancer remains unknown. This study aims to unveil the genes and pathways that may involve in lymphovascular invasion in breast cancer. In total, 108 breast cancer samples were collected during surgery and microarray analysis was performed. Significance analysis of the microarrays and limma package for R were used to examine differentially expressed genes between lymphovascular invasion-positive and lymphovascular invasion-negative cases. Network and pathway analyses were mapped using the Ingenuity Pathway Analysis and the Database for Annotation, Visualization and Integrated Discovery. In total, 86 differentially expressed genes, including 37 downregulated genes and 49 upregulated genes were identified in lymphovascular invasion-positive patients. Among these genes, TNFSF11, IL6ST, and EPAS1 play important roles in cytokine-receptor interaction, which is the most enriched pathway related to lymphovascular invasion. Moreover, the results also suggested that an imbalance between extracellular matrix components and tumor micro-environment could induce lymphovascular invasion. Our study evaluated the underlying mechanisms of lymphovascular invasion, which may further help to assess the risk of breast cancer progression and identify potential targets of adjuvant treatment.


Assuntos
Neoplasias da Mama/genética , Metástase Linfática/genética , Proteínas de Neoplasias/biossíntese , Recidiva Local de Neoplasia/genética , Transcriptoma/genética , Adulto , Idoso , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Biologia Computacional , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Resultado do Tratamento
17.
J Cell Mol Med ; 21(9): 1967-1978, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28266128

RESUMO

Accumulating evidence demonstrates that hypoxia-inducible factor (HIF-α) hydroxylase system has a critical role in vascular remodelling. Using an endothelial-specific prolyl hydroxylase domain protein-2 (PHD2) knockout (PHD2EC KO) mouse model, this study investigates the regulatory role of endothelial HIF-α hydroxylase system in the development of renal fibrosis. Knockout of PHD2 in EC up-regulated the expression of HIF-1α and HIF-2α, resulting in a significant decline of renal function as evidenced by elevated levels of serum creatinine. Deletion of PHD2 increased the expression of Notch3 and transforming growth factor (TGF-ß1) in EC, thus further causing glomerular arteriolar remodelling with an increased pericyte and pericyte coverage. This was accompanied by a significant elevation of renal resistive index (RI). Moreover, knockout of PHD2 in EC up-regulated the expression of fibroblast-specific protein-1 (FSP-1) and increased interstitial fibrosis in the kidney. These alterations were strongly associated with up-regulation of Notch3 and TGF-ß1. We concluded that the expression of PHD2 in endothelial cells plays a critical role in renal fibrosis and vascular remodelling in adult mice. Furthermore, these changes were strongly associated with up-regulation of Notch3/TGF-ß1 signalling and excessive pericyte coverage.


Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Rim/irrigação sanguínea , Rim/patologia , Deleção de Sequência , Remodelação Vascular , Animais , Artérias/patologia , Arteríolas/patologia , Pressão Sanguínea , Fibrose , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Camundongos Knockout , Pericitos/metabolismo , Pericitos/patologia , Fenótipo
18.
Artigo em Inglês | MEDLINE | ID: mdl-28149327

RESUMO

BACKGROUND: Intrauterine exposure to maternal smoking is linked to impaired executive function and behavioral problems in the offspring. Maternal smoking is associated with reduced fetal brain growth and smaller volume of cortical gray matter in childhood, indicating that prenatal exposure to tobacco may impact cortical development and manifest as behavioral problems. Cellular development is mediated by changes in epigenetic modifications such as DNA methylation, which can be affected by exposure to tobacco. RESULTS: In this study, we sought to ascertain how maternal smoking during pregnancy affects global DNA methylation profiles of the developing dorsolateral prefrontal cortex (DLPFC) during the second trimester of gestation. When DLPFC methylation profiles (assayed via Illumina, HM450) of smoking-exposed and unexposed fetuses were compared, no differentially methylated regions (DMRs) passed the false discovery correction (FDR ≤ 0.05). However, the most significant DMRs were hypomethylated CpG Islands within the promoter regions of GNA15 and SDHAP3 of smoking-exposed fetuses. Interestingly, the developmental up-regulation of SDHAP3 mRNA was delayed in smoking-exposed fetuses. Interaction analysis between gestational age and smoking exposure identified significant DMRs annotated to SYCE3, C21orf56/LSS, SPAG1 and RNU12/POLDIP3 that passed FDR. Furthermore, utilizing established methods to estimate cell proportions by DNA methylation, we found that exposed DLPFC samples contained a lower proportion of neurons in samples from fetuses exposed to maternal smoking. We also show through in vitro experiments that nicotine impedes the differentiation of neurons independent of cell death. CONCLUSIONS: We found evidence that intrauterine smoking exposure alters the developmental patterning of DNA methylation and gene expression and is associated with reduced mature neuronal content, effects that are likely driven by nicotine.


Assuntos
Encéfalo/metabolismo , Metilação de DNA , Exposição Materna , Fumar , Encéfalo/patologia , Feminino , Desenvolvimento Fetal/genética , Feto/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Idade Gestacional , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Gravidez , Segundo Trimestre da Gravidez , Regiões Promotoras Genéticas , Succinato Desidrogenase/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
19.
Int J Cardiol ; 236: 413-422, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28131704

RESUMO

RATIONALE: Vascular maturation plays an important role in wound repair post-myocardial infarction (MI). The Notch3 is critical for pericyte recruitment and vascular maturation during embryonic development. OBJECTIVE: This study is to test whether Notch3 deficiency impairs vascular maturation and blunts cardiac functional recovery post-MI. APPROACH AND RESULTS: Wild type (WT) and Notch3 knockout (Notch3KO) mice were subjected to MI by the ligation of left anterior descending coronary artery (LAD). Cardiac function and coronary blood flow reserve (CFR) were measured by echocardiography. The expression of angiogenic growth factor, pericyte/capillary coverage and arteriolar formation were analyzed. Loss of Notch3 in mice resulted in a significant reduction of pericytes and small arterioles. Notch3 KO mice had impaired pericyte/capillary coverage and CFR compared to WT mice. Notch3 KO mice were more prone to ischemic injury with larger infarcted size and higher rates of mortality. The expression of CXCR-4 and VEGF/Ang-1 was significantly decreased in Notch3 KO mice. Notch3 KO mice also had few NG2+/Sca1+ and NG2+/c-kit+ progenitor cells in the ischemic area and exhibited worse cardiac function recovery at 2weeks after MI. These were accompanied by a significant reduction of pericyte/capillary coverage and arteriolar maturation. Furthermore, Notch3 KO mice subjected to MI had increased intracellular adhesion molecule-2 (ICAM-2) expression and CD11b+ macrophage infiltration into ischemic areas compared to that of WT mice. CONCLUSION: Notch3 mutation impairs recovery of cardiac function post-MI by the mechanisms involving the pre-existing coronary microvascular dysfunction conditions, and impairment of pericyte/progenitor cell recruitment and microvascular maturation.


Assuntos
Circulação Coronária/fisiologia , Vasos Coronários/metabolismo , Microvasos/metabolismo , Isquemia Miocárdica/metabolismo , Receptor Notch3/deficiência , Recuperação de Função Fisiológica/fisiologia , Animais , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Microvasos/fisiopatologia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia
20.
Science ; 355(6325)2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28082412

RESUMO

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/fisiologia , Animais , Linhagem da Célula/genética , Células Cultivadas , Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Feminino , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Transcrição Genética , Ativação Viral/genética
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