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BACKGROUND: RNA sequencing (RNA-seq) is widely used for gene expression profiling and quantification. Quantitative RNA sequencing usually requires cell counting and spike-in, which is not always applicable to many samples. Here, we present a novel quantitative RNA sequencing method independent of spike-ins or cell counting, named siqRNA-seq, which can be used to quantitatively profile gene expression by utilizing gDNA as an internal control. Single-stranded library preparation used in siqRNA-seq profiles gDNA and cDNA with equal efficiency. RESULTS: To quantify mRNA expression levels, siqRNA-seq constructs libraries for total nucleic acid to establish a model for expression quantification. Compared to Relative Quantification RNA-seq, siqRNA-seq is technically reliable and reproducible for expression profiling but also can sequence reads from gDNA which can be used as an internal reference for accurate expression quantification. Applying siqRNA-seq to investigate the effects of actinomycin D on gene expression in HEK293T cells, we show the advantages of siqRNA-seq in accurately identifying differentially expressed genes between samples with distinct global mRNA levels. Furthermore, we analyzed factors influencing the downward trend of gene expression regulated by ActD using siqRNA-seq and found that mRNA with m6A modification exhibited a faster decay rate compared to mRNA without m6A modification. Additionally, applying this technique to the quantitative analysis of seven tumor cell lines revealed a high degree of diversity in total mRNA expression among tumor cell lines. CONCLUSIONS: Collectively, siqRNA-seq is a spike-in independent quantitative RNA sequencing method, which creatively uses gDNA as an internal reference to absolutely quantify gene expression. We consider that siqRNA-seq provides a convenient and versatile method to quantitatively profile the mRNA landscape in various samples.
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ARN Mensajero , Análisis de Secuencia de ARN , Humanos , ARN Mensajero/genética , Células HEK293 , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.
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Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.
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Picea , Reguladores del Crecimiento de las Plantas , Reguladores del Crecimiento de las Plantas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Picea/genética , Sacarosa/farmacología , Sacarosa/metabolismo , Proliferación Celular , Ácido 2,4-Diclorofenoxiacético/farmacología , Semillas , Técnicas de Embriogénesis Somática de Plantas/métodosRESUMEN
Programmed DNA double-strand break (DSB) formation is a crucial step in meiotic recombination, yet techniques for high-efficiency and precise mapping of the 3' ends of DSBs are still in their infancy. Here, we report a novel technique, named DNA End tailing and sequencing (DEtail-seq), which can directly and ultra-efficiently characterize the 3' ends of meiotic DSBs with near single-nucleotide resolution in a variety of species, including yeast, mouse, and human. We find that the 3' ends of meiotic DSBs are stable without significant resection in budding yeast. Meiotic DSBs are strongly enriched in de novo H3K4me3 peaks in the mouse genome at leptotene stage. We also profile meiotic DSBs in human and find DSB hotspots are enriched near the common fragile sites during human meiosis, especially at CCCTC-binding factor (CTCF)-associated enhancers. Therefore, DEtail-seq provides a powerful method to detect DSB ends in various species, and our results provide new insights into the distribution and regulation of meiotic DSB hotspots.
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Roturas del ADN de Doble Cadena , Proteínas de Saccharomyces cerevisiae , Ratones , Humanos , Animales , Saccharomyces cerevisiae/genética , Recombinación Homóloga , Proteínas de Saccharomyces cerevisiae/genética , Meiosis/genéticaRESUMEN
This study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of [Formula: see text] 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which [Formula: see text] 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.
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Picea/embriología , Semillas/crecimiento & desarrollo , Germinación , Picea/fisiologíaRESUMEN
BACKGROUND: Human pluripotent stem cell-derived limbal stem cells (hPSC-derived LSCs) provide a promising cell source for corneal transplants and ocular surface reconstruction. Although recent efforts in the identification of LSC markers have increased our understanding of the biology of LSCs, much more remains to be characterized in the developmental origin, cell fate determination, and identity of human LSCs. The lack of knowledge hindered the establishment of efficient differentiation protocols for generating hPSC-derived LSCs and held back their clinical application. RESULTS: Here, we performed a time-course single-cell RNA-seq to investigate transcriptional heterogeneity and expression changes of LSCs derived from human embryonic stem cells (hESCs). Based on current protocol, expression heterogeneity of reported LSC markers were identified in subpopulations of differentiated cells. EMT has been shown to occur during differentiation process, which could possibly result in generation of untargeted cells. Pseudotime trajectory analysis revealed transcriptional changes and signatures of commitment of hESCs-derived LSCs and their progeny-the transit amplifying cells. CONCLUSION: Single-cell RNA-seq revealed time-course expression changes and significant transcriptional heterogeneity during hESC-derived LSC differentiation in vitro. Our results demonstrated candidate developmental trajectory and several new candidate markers for LSCs, which could facilitate elucidating the identity and developmental origin of human LSCs in vivo.
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Aim: To identify differential mRNA and ncRNA expression profiles and competing endogenous RNA-associated regulatory networks during the progression of atherosclerosis (AS). Materials & methods: We systematically analyzed whole-transcriptome sequencing of samples from different stages of AS to evaluate their long noncoding RNA (lncRNA), circular RNA (circRNA), miRNA and mRNA profiles. Results: We constructed three AS-related competing endogenous RNA regulatory networks of differentially expressed circRNAs, lncRNAs, miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the circRNAs in the network were enriched in lipid metabolic processes and participated in the PPAR signaling pathway. Furthermore, lncRNAs were related to receptor activity, myofibrils and cardiovascular system development. Conclusion: The current findings further clarified the regulatory mechanisms at different stages of AS and may provide new ideas and targets for AS.
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Aterosclerosis/genética , Redes Reguladoras de Genes/genética , MicroARNs/genética , ARN Circular/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Transcriptoma/genética , Animales , Aterosclerosis/patología , Biología Computacional , Ontología de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. METHODS: We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. RESULTS: MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. CONCLUSION: This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.
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Células Madre Mesenquimatosas , Diferenciación Celular , Células Cultivadas , Humanos , Inmunomodulación , Transducción de SeñalRESUMEN
BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are multipotent cells with a promising application potential in regenerative medicine and immunomodulation. However, MSCs cultured in vitro exhibit functional heterogeneity. The underlying molecular mechanisms that define MSC heterogeneity remain unclear. METHODS: We investigated the gene expression profile via single-cell RNA sequencing (scRNA-seq) of human primary Wharton's jelly-derived MSCs (WJMSCs) cultured in vitro from three donors. We also isolated CD142+ and CD142- WJMSCs based on scRNA-seq data and compared their proliferation capacity and "wound healing" potential in vitro. Meanwhile, we analyzed publicly available adipose-derived MSC (ADMSCs) scRNA-seq data and performed transcriptome comparison between WJMSCs and ADMSCs at the single-cell level. RESULTS: GO enrichment analysis of highly variable genes (HVGs) obtained from WJMSCs revealed that these genes are significantly enriched in extracellular region with binding function, involved in developmental process, signal transduction, cell proliferation, etc. Pathway analysis showed that these HVGs are associated with functional characteristics of classic MSCs, such as inflammation mediated by chemokine and cytokine signaling, integrin signaling, and angiogenesis. After regressing out the batch and cell cycle effects, these HVGs were used for dimension reduction and clustering analysis to identify candidate subpopulations. Differentially expressed gene analysis revealed the existence of several distinct subpopulations of MSCs that exhibit diverse functional characteristics related to proliferation, development, and inflammation response. In line with our data, sorted CD142+ and CD142- WJMSCs showed distinct proliferation capacity as well as "wound healing" potential. Although WJMSCs and ADMSCs were derived from different tissues and were displaying different differentiation potencies, their HVGs were largely overlapped and had similar functional enrichment. CONCLUSION: HVGs identified in MSCs are associated with classic MSC function. Regarding therapeutic potential, these genes are associated with functional characteristics, on which the MSC clinical application were theoretically based, such as development and inflammation response. Altogether, these HVGs hold the potential to be used as candidate markers for further potency association studies.
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Células Madre Mesenquimatosas , Gelatina de Wharton , Diferenciación Celular , Proliferación Celular/genética , Células Cultivadas , Humanos , RNA-Seq , Cordón UmbilicalRESUMEN
OBJECTIVE: Despite the current antiatherosclerotic and antithrombotic therapies, the incidence of advanced atherosclerosis-associated clinical events remains high. Whether long noncoding RNAs (lncRNAs) affect the progression of atherosclerosis and whether they are potential targets for the treatment of advanced atherosclerosis are poorly understood. Approach and Results: The progression of atherosclerotic lesions was accompanied by dynamic alterations in lncRNA expression, as revealed by RNA sequencing and quantitative polymerase chain reaction. Among the dynamically changing lncRNAs, we identified a novel lncRNA, lncRNA Associated with the Progression and Intervention of Atherosclerosis (RAPIA), that was highly expressed in advanced atherosclerotic lesions and in macrophages. Inhibition of RAPIA in vivo not only repressed the progression of atherosclerosis but also exerted atheroprotective effects similar to those of atorvastatin on advanced atherosclerotic plaques that had already formed. In vitro assays demonstrated that RAPIA promoted proliferation and reduced apoptosis of macrophages. A molecular sponge interaction between RAPIA and microRNA-183-5p was demonstrated by dual-luciferase reporter and RNA immunoprecipitation assays. Rescue assays indicated that RAPIA functioned at least in part by targeting the microRNA-183-5p/ITGB1 (integrin ß1) pathway in macrophages. In addition, the transcription factor FoxO1 (forkhead box O1) could bind to the RAPIA promoter region and facilitate the expression of RAPIA. CONCLUSIONS: The progression of atherosclerotic lesions was accompanied by dynamic changes in the expression of lncRNAs. Inhibition of the pivotal lncRNA RAPIA may be a novel preventive and therapeutic strategy for advanced atherosclerosis, especially in patients resistant or intolerant to statins.
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Aterosclerosis/terapia , Expresión Génica , Macrófagos/metabolismo , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Animales , Apoptosis/efectos de los fármacos , Aterosclerosis/genética , Aterosclerosis/prevención & control , Atorvastatina/farmacología , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Proteína Forkhead Box O1/metabolismo , Humanos , Integrina beta1/metabolismo , Macrófagos/química , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , MicroARNs/metabolismo , MicroARNs/farmacología , Regiones Promotoras Genéticas/fisiología , Células RAW 264.7 , ARN Largo no Codificante/fisiologíaRESUMEN
Cardiac fibrosis is a common pathological condition that contributes to the progression of many cardiac diseases. Circular RNAs (circRNAs) are emerging as new regulators of cardiac fibrosis. However, the expression and function of circRNAs in cardiac fibrosis remain largely unknown. The present study aims to investigate the circRNA expression profile and identify the roles of circRNAs in cardiac fibrosis. Transforming growth factor-ß1 (TGF-ß1) was used to establish an in vitro model of cardiac fibrosis in cardiac fibroblasts. CircRNA sequencing revealed that a total of 283 circRNAs were aberrantly expressed in fibrotic cardiac fibroblasts, with 79 upregulated and 204 downregulated. The expression changes of randomly selected circRNAs were validated by real-time PCR. A circRNA-based competing endogenous RNA network 1755 nodes and 30394 edges was established, and module analysis was conducted using the plug-in MCODE. KEGG pathway enrichment analysis was performed for mRNAs involved in the top three enriched modules. The results showed that these mRNAs were enriched in cardiac fibrosis-related signalling pathways, including the 'TGF-beta signaling pathway', 'MAPK signaling pathway', 'AMPK signaling pathway', and 'PI3K-Akt signaling pathway'. The predicted ceRNAs and bioinformatics analysis revealed the potential role of circRNAs in cardiac fibrosis, which would provide useful information for understanding the mechanism and finding effective prevention and treatment targets for cardiac fibrosis.
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Fibroblastos/patología , ARN Circular/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Biología Computacional , Regulación hacia Abajo , Fibrosis/patología , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Regulación hacia ArribaRESUMEN
The transformation of biomass using steam gasification is a chemical route to facilitate changes in organic or residue supported carbonaceous substances addicted to carbon mono-oxide, hydrogen including carbon-di-oxide, etc. However, to commercialize the method of steam gasification, the hurdles persist during the gasification as well as downstream processing. This article delivers a summary of the different approaches that are described in the previous studies to achieve H2 refinement and adaptation within the gasifier system. These include advanced aspects in the research and development of biomass gasification (alike advancements under the gasification operation). The upshot of diverse operating conditions like steam flow rate, operating temperature, moisture content, gasifier agents, residence time, biomass to air, steam to biomass, equivalence ratio, etc. towards the execution of biomass gasifier. This review accomplishes that the interdependence of several issues must be considered in point to optimise the producer gas.
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Hidrógeno , Vapor , Biomasa , Carbono , TemperaturaRESUMEN
Here, we report the synthesis of copper-manganese alloy (CuMnO2) using graphitic carbon nitride (gCN) as a novel support material. The successful formation of CuMnO2-gCN was confirmed through spectroscopic, optical, and other characterization techniques. We have applied this catalyst as the energy storage material in the alkaline media and it has shown good catalytic behavior in supercapacitor applications. The CuMnO2-gCN demonstrates outstanding electrocapacitive performance, having high capacitance (817.85 A·g-1) and well-cycling stability (1000 cycles) when used as a working electrode material for supercapacitor applications. For comparison, we have also used the gCN and Cu2O-gCN for supercapacitor applications. This study proposes a simple path for the extensive construction of self-attaining double metal alloy with control size and uniformity in high-performance energy-storing materials.
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BACKGROUND AND AIMS: Plaque progression increases the risk of a cardiovascular event. This study aims to determine whether intraplaque neovascularization (NV) associates with a greater risk of plaque progression. METHODS: Baseline and 12-month follow-up IVUS was used in combination with baseline OCT to assess 164 non-culprit plaques in 118 CAD patients. A generalized estimating equation approach with exchangeable correlation structure was used to correct for the dependency of repeated measurements. RESULTS: Patients were divided into two groups according to NV (52 patients with 62 NV plaques, 66 patients with 102 non-NV plaques). Non-culprit plaques in the NV group exhibited a more frequent occurrence of TCFA (pâ¯=â¯0.004), macrophage (pâ¯=â¯0.005), cholesterol crystal (pâ¯=â¯0.012), calcification (pâ¯=â¯0.030), thinner fibrous cap thickness (FCT) [(86.8⯱â¯55.1) vs. (127.4⯱â¯70.1) µm, pâ¯=â¯0.015], larger lipid arc [(219.5⯱â¯66.9) vs. (179.8⯱â¯61.4), pâ¯=â¯0.002] compared to the non-NV group. A large change in percent atheroma volume (PAV), plaque plus media cross-sectional area (P&M CSA), plaque volume, and plaque burden was observed from baseline to follow-up in the NV group. Changes in P&M CSA, plaque volume, and plaque burden showed significant differences in fibroatheroma with NV. Intraplaque NV could predict a high risk of plaque progression despite statin therapy [OR 6.521 (95% CI 2.457-17.308), pâ¯<â¯0.001]. CONCLUSIONS: NV might attenuate the benefits of statin therapy in plaque progression. This study may provide a new basis for anti-angiogenic strategies to prevent atherosclerotic plaque progression.
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Angiografía Coronaria/métodos , Enfermedad de la Arteria Coronaria/diagnóstico , Vasos Coronarios/diagnóstico por imagen , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Placa Aterosclerótica/diagnóstico , Tomografía de Coherencia Óptica/métodos , Ultrasonografía Intervencional/métodos , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Vasos Coronarios/efectos de los fármacos , Progresión de la Enfermedad , Estudios de Seguimiento , Humanos , Neovascularización Patológica/diagnóstico , Placa Aterosclerótica/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
AIMS: This study aimed to investigate the progression and vascular shrinkage of vulnerable plaque lesions with a plaque burden at least 70% among patients with coronary artery disease by optical coherence tomography (OCT) and intravascular ultrasound (IVUS). METHODS: Fifty-six OCT-identified vulnerable plaques from 47 patients were included among coronary angiography-identified nonculprit/nontarget lesions. Serial IVUS images were used to assess plaque progression and vascular shrinkage. RESULTS: Thirty-five small vulnerable plaques (plaque burden <70%, group A) and 21 large vulnerable plaques (plaque burden ≥70%, group B) were identified. The IVUS results at baseline show that mean plaque areas (Pâ<â0.001) and the percentage atheroma volume (PAV) (Pâ<â0.0001) were greater and the minimal lumen area (Pâ<â0.0001) was smaller in group B. The absolute and relative changes in the PAV and mean plaque area from baseline to follow-up were not significantly different. However, the lesions exhibited vessel shrinkage [the mean external elastic membrane (EEM) area (Pâ=â0.02) and mean lumen area (Pâ=â0.03) were significantly smaller in group B] from baseline to follow-up. Patients in group B also exhibited clinical events (recurrent angina symptoms) during the follow-up period. Positive correlations were found between changes in the mean plaque area and the mean EEM area in large vulnerable plaques (râ=â0.61, Pâ<â0.0001) and between changes in the mean EEM area and the mean lumen area in large vulnerable plaques (râ=â0.61, Pâ<â0.0001). CONCLUSION: Vulnerable plaque progression was not different between small and large vulnerable plaques. However, large vulnerable plaque lesions tended to exhibit vascular shrinkage, which is possible a cause of coronary artery lumen loss in patients with large vulnerable plaques.
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Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Placa Aterosclerótica , Tomografía de Coherencia Óptica , Ultrasonografía Intervencional , Angiografía Coronaria , Progresión de la Enfermedad , Femenino , Fibrosis , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Recurrencia , Rotura Espontánea , Factores de TiempoRESUMEN
Herein, well-defined Pd nanoparticles (NPs) developed on Ni substrate (Pd NPs/Ni) are synthesized via a facile galvanic replacement reaction (GRR) route performed in ethaline-based deep eutectic solvent (DES). For comparison, a Pd NPs/Ni composite is also prepared by the GRR method conducted in an aqueous solution. The Pd NPs/Ni obtained from the ethaline-DES is catalytically more active and durable for the methanol electro-oxidation reaction (MOR) than those of the counterpart derived from conventional aqueous solution and commercial Pd/C under alkaline media. Detailed kinetic analysis indicates that the unique solvent environment offered by ethaline plays vital roles in adjusting the reactivity of the active species and their mass transport properties to control over the genesis of the Pd NPs/Ni nanocomposite. The resulting Pd NPs/Ni catalyst possesses a homogeneous dispersion of Pd NPs with a strong Pd (metal)-Ni (support) interaction. This structure enhances the charge transfer between the support and the active phases, and optimizes the adsorption energy of OH- and CO on the surface, leading to superior electrocatalytic performance. This work provides a novel GRR strategy performed in ethaline-DES to the rational design and construction of advanced metal/support catalysts with strong interaction for improving the activity and durability for MOR.
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BACKGROUND: Obesity is causally associated with atherosclerosis, and adipose tissue (AT)-derived exosomes may be implicated in the metabolic complications of obesity. However, the precise role of AT-exosomes in atherogenesis remains unclear. We herein aimed to assess the effect of AT-exosomes on macrophage foam cell formation and polarization and subsequent atherosclerosis development. METHODS AND RESULTS: Four types of exosomes isolated from the supernatants of ex vivo subcutaneous AT and visceral AT (VAT) explants that were derived from wild-type mice and high-fat diet (HFD)-induced obese mice were effectively taken up by RAW264.7 macrophages. Both treatment with wild-type VAT exosomes and HFD-VAT exosomes, but not subcutaneous AT exosomes, markedly facilitated macrophage foam cell generation through the downregulation of ATP-binding cassette transporter (ABCA1 and ABCG1)-mediated cholesterol efflux. Decreased expression of liver X receptor-α was also observed. Among the 4 types of exosomes, only HFD-VAT exosomes significantly induced M1 phenotype transition and proinflammatory cytokine (tumor necrosis factor α and interleukin 6) secretion in RAW264.7 macrophages, which was accompanied by increased phosphorylation of NF-κB-p65 but not the cellular expression of NF-κB-p65 or IκB-α. Furthermore, systematic intravenous injection of HFD-VAT exosomes profoundly exacerbated atherosclerosis in hyperlipidemic apolipoprotein E-deficient mice, as indicated by the M1 marker (CD16/32 and inducible nitric oxide synthase)-positive areas and the Oil Red O/Sudan IV-stained area, without affecting the plasma lipid profile and body weight. CONCLUSIONS: This study demonstrated a proatherosclerotic role for HFD-VAT exosomes, which is exerted by regulating macrophage foam cell formation and polarization, indicating a novel link between AT and atherosclerosis in the context of obesity.
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Enfermedades de la Aorta/patología , Aterosclerosis/patología , Exosomas/trasplante , Células Espumosas/patología , Grasa Intraabdominal/trasplante , Obesidad/patología , Placa Aterosclerótica , Grasa Subcutánea/trasplante , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Colesterol/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exosomas/metabolismo , Exosomas/patología , Células Espumosas/metabolismo , Grasa Intraabdominal/metabolismo , Grasa Intraabdominal/patología , Receptores X del Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Obesidad/metabolismo , Fenotipo , Fosforilación , Células RAW 264.7 , Grasa Subcutánea/metabolismo , Grasa Subcutánea/patología , Factores de Tiempo , Factor de Transcripción ReIA/metabolismoRESUMEN
Human embryonic stem cells (hESCs) have the potential to form any cell type in the body, making them attractive cell sources in drug screening, regenerative medicine, disease and developmental processes modeling. However, not all hESC lines have the equal potency to generate desired cell types in vitro. Significant variations have been observed for the differentiation efficiency of various human ESC lines. The precise underpinning molecular mechanisms are still unclear. In this work, we compared transcriptome variations of four hESC lines H7, HUES1, HUES8 and HUES9. We found that hESC lines have different gene expression profiles, and these differentially expressed genes (DEGs) are significantly enriched in developmental processes, such as ectodermal, mesodermal and endodermal development. The enrichment difference between hESC lines was consistent with its lineage bias. Among these DEGs, some pluripotency factors and genes involved in signaling transduction showed great variations as well. The pleiotropic functions of these genes in controlling hESC identity and early lineage specification, implicated that different hESC lines may utilize distinct balance mechanisms to maintain pluripotent state. When the balance is broken in a certain environment, gene expression variation between them could impact on their different lineage specification behavior.
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Diferenciación Celular , Células Madre Embrionarias/metabolismo , Transcriptoma , Células Cultivadas , Células Madre Embrionarias/citología , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismoRESUMEN
Super-enhancers (SEs) are large clusters of transcriptional enhancers that drive expression of genes controlling cell identity. They play important roles in development, disease initiation and progression such as tumorigenesis. Compared to typical enhancers (TEs), most key oncogenes in tumor cells are driven by SEs, and common diseases such as Alzheimer's disease related variations are enriched in SEs. SEs hold great promising in key oncogenes identification and diseases-associated variants discovery. In this review, we first summarize how to identify enhancers on a genome-wide scale, and then introduce the concept of SEs and the methodology for SEs identification. Lastly, we describe SEs' main structural and functional properties and discuss its application in the future research.