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Staphylococcus aureus is a food-borne pathogen that quickly forms biofilm on meat contact surfaces and thus poses a serious threat to the safety of the meat industry. This study evaluated the attachment, survival, and growth of S. aureus biofilm with exposure to environmental factors in the meat industry by simulated ready-to-eat (RTE) cooked beef product contamination scenarios. The results indicated that the meat-borne S. aureus biofilm formation dynamic could be divided into four different phases: initial adhesion (4-12 h), exponential (12-24 h), slow growth (1-3 days), and stationary (3-7 days). Meat-borne S. aureus has strong adhesion and biofilm formation ability, and its biofilm exhibits persistence, high-intensity metabolic activity, aerotaxis, and strain heterogeneity. This study has also demonstrated that in the long-term existence of meat-borne S. aureus biofilm on stainless steel and plexiglass surfaces (>7 days, 7.2-8.8 log CFU/cm2 ), expose to RTE cooked beef products, may cause it to become high-risk contaminated food. Meat-borne S. aureus that forms a dense and rough concave-convex in the shape of biofilm architecture was observed by scanning electron microscopy, consisting of complex components and adhesion of living and dead cells. This was further confirmed by the meat-borne S. aureus biofilm on the stainless steel surface by attenuated total reflectance Fourier transformed infrared spectroscopy, and the dominant peaks in biofilm spectra were mainly associated with proteins, polysaccharides, amino acid residues, and phospholipids (>50%). These findings may help in the identification of the main sources of contamination within the meat industry and the subsequent establishment of strategies for biofilm prevention and removal. PRACTICAL APPLICATION: This study revealed the meat-borne S. aureus biofilm formation mechanism and found that it exhibited strong colonization and biofilm-forming ability, which can persist on the contact surfaces of ready-to-eat beef products. These initial findings could provide information on the behavior of meat-borne S. aureus biofilm attached to meat contact surfaces under conditions commonly encountered in meat environments, which help to support the determination of the main sources of contamination within the meat industry and the subsequent establishment of strategies for biofilm prevention and removal. It was also helpful in controlling biofilm contamination and improving meat safety to minimize it.
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Manipulación de Alimentos , Staphylococcus aureus , Animales , Bovinos , Manipulación de Alimentos/métodos , Cinética , Acero Inoxidable , Biopelículas , Microbiología de AlimentosRESUMEN
The additive engineering strategy promotes the efficiency of solution-processed perovskite solar cells (PSCs) over 25%. However, compositional heterogeneity and structural disorders occur in perovskite films with the addition of specific additives, making it imperative to understand the detrimental impact of additives on film quality and device performance. In this work, the double-edged sword effects of the methylammonium chloride (MACl) additive on the properties of methylammonium lead mixed-halide perovskite (MAPbI3-x Clx ) films and PSCs are demonstrated. MAPbI3-x Clx films suffer from undesirable morphology transition during annealing, and its impacts on the film quality including morphology, optical properties, structure, and defect evolution are systematically investigated, as well as the power conversion efficiency (PCE) evolution for related PSCs. The FAX (FA = formamidinium, X = I, Br, and Ac) post-treatment strategy is developed to inhibit the morphology transition and suppress defects by compensating for the loss of the organic components, a champion PCE of 21.49% with an impressive open-circuit voltage of 1.17 V is obtained, and remains over 95% of the initial efficiency after storing over 1200 hours. This study elucidates that understanding the additive-induced detrimental effects in halide perovskites is critical to achieve the efficient and stable PSCs.
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Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50-150 µm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.
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Células Madre Pluripotentes Inducidas , Femenino , Humanos , Metafase , Oocitos , Folículo Ovárico , Oogénesis/genéticaRESUMEN
The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.
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Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células Madre Pluripotentes Inducidas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Metilación de ADN , Epigénesis Genética , Células Madre Pluripotentes Inducidas/citología , Histona Demetilasas con Dominio de Jumonji/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción SOXB1/genética , PorcinosRESUMEN
Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2+) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo.
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Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Células Madre Embrionarias de Ratones/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Animales , Benzamidas/farmacología , Sistemas CRISPR-Cas , Células Cultivadas , Medios de Cultivo , Proteínas de Unión al ADN/genética , Dimetindeno/farmacología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Desarrollo Embrionario/genética , Técnicas de Inactivación de Genes , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Minociclina/farmacología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Factores de Transcripción/genética , TransfecciónRESUMEN
Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos, although the molecular mechanism underlying such an expanded 2CLC potency remains elusive. We examine global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We find that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with activation of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by DUX and post-transcriptionally represses ZMYM2 and its partner LSD1, and ZMYM2 recruits LSD1/HDAC corepressor complex to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the previously unappreciated DUX-miR-344-Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.
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Retrovirus Endógenos , MicroARNs , Animales , Retrovirus Endógenos/genética , Ratones , MicroARNs/genética , Células Madre Embrionarias de Ratones , CigotoRESUMEN
Objective: To present a patient with anti-CV2 autoimmune encephalitis admitted for Parkinson-like symptoms and bilateral leukoencephalopathy. Case report: The patient was admitted for Parkinson-like symptoms combined with loss of taste. Serum anti-CV2 antibody was positive. Cranial magnetic resonance imaging revealed bilateral leukoencephalopathy. Breast cancer was detected by positron emission tomography (PET) and ultrasound. Immunotherapy was not performed. Modified radical mastectomy revealed a pT1cN0M0 breast cancer, positive for estrogen and progesterone receptors, and HER2 negative. The resting tremors disappeared by 1 week after surgery. The modified Rankin score (mRS) was four before surgery, and decreased to one at 9 months after surgery. Conclusion: Anti-CV2 autoimmune encephalitis can present as Parkinsonism with bilateral leukoencephalopathy on MRI. PET scanning can be useful to reveal an occult cancer. Treatment of the cancer may improve the paraneoplastic neurological syndrome without the need of immunosuppressive therapy.
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OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.
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Endodermo/citología , Endodermo/embriología , Porcinos/embriología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Técnicas de Cocultivo , Perros , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Expresión Génica , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Porcinos/genética , Porcinos/metabolismo , Quimera por TrasplanteRESUMEN
Coastal sea turtle stranding and bycatch are common phenomena worldwide and have received more attention in recent years. They are caused by both natural and anthropogenic factors. One thousand and seventy-two turtles were reported to be victims of these phenomena from March 1997 to November 2019 in Taiwan. Number of stranding/bycatch were variable and infrequent for the first 14 years, but increased each year after 2012 and peaked in 2019 with 217 cases. Most turtles were juveniles to subadults. All five of Taiwan's species were reported in stranding and bycatch records, and the green turtle was reported the most common. The main reported seasons lasted from winter to spring, when the weather changes dramatically. The sex ratio (female: male) ranged from 7 in the hawksbill turtle to 0.7 in the olive ridley, with an average of 2.4 for all species. Green turtles were the dominant stranded species, and more loggerhead turtles were by-caught. The hotspots were the towns of Dougou and Tochen in Yilan County, and Gongliao District in New Taipei City, located in NE coast of Taiwan respectively. Stranding was the more common of the two phenomena reported, and 80% of all stranded turtles were subadult green turtles. Eighty percent of all stranded/bycaught turtles were dead. Pond-nets were the fishing gear that accounted for the most bycatch, and captured mainly living young and subadult green turtles as well as subadult loggerhead turtles. The hotspots for bycatch were the towns of Dongou and Tochen in Yilan County. The Coast Guard and concerned citizen were the main sources of reports. This is the first study to analyze the long-term stranding/bycatch of sea turtles in Taiwan.
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Porcine OTX2 was found to be highly activated in porcine iPS cells (piPSCs) that were reported by different laboratories worldwide. To reveal the regulatory function of OTX2 in porcine reprogrammed cells, we screened porcine miRNA-seq databases and found two miRNAs, miR-1343 and miR-545, that could specifically bind to 3'UTR of OTX2 and suppress endogenous OTX2 expression in piPSCs. Knockdown of OTX2 by miR-1343 and miR-545 could significantly increase the expression of SOX2 and ESRRB, but did not alter the expressions of OCT4 and KLF4, and improve the pluripotency of piPSCs. The promoter-based assays showed that OTX2 potentially bound to the promoter region of SOX2 and ESRRB and suppressed their expression. On the other hand, SOX2 could interact with OTX2 promoter. Ectopic expression of SOX2 could significantly decrease OTX2 promoter activity, showing that there is a negative feedback loop between SOX2 and OTX2. Additionally, SOX2 and ESRRB significantly stimulated miR-1343 expression in piPSCs, but OTX2 down regulated the expression of miR-1343 in either direct or indirect manners. In summary, this study demonstrates that there is a regulatory network mediated by miR-1343, in which downregulation of OTX2 by miR-1343 can elevate the expression of pluripotent genes that were then sustain the pluripotency of piPSCs.
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Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Factores de Transcripción Otx/genética , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , Regiones no Traducidas 3' , Animales , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Regiones Promotoras Genéticas , Porcinos , TranscriptomaRESUMEN
At present, the experimental technique to produce human red blood cells in vitro is complicated, and in order to optimize the induction steps, human pluripotent stem cells were differentiated into red blood cells through two induction steps. First, human pluripotent stem cells (including Rh negative A type umbilical cord mesenchymal stem cells (hUCMSCRh-A) and human iPS cells (hiPS)) were differentiated into CD31+ and CD34+ cells in BVF medium. PCR and flow cytometry were used to exam the expression of CD31 and CD34. We found that hUCMSCRh-A derived CD31+ and CD34+ cells were 5.3% and 22.7%, respectively; hiPS derived CD31+ and CD34+ cells were 31.2% and 8.2%, respectively. For the second induction step, the obtained CD31+ and CD34+ cells were differentiated into mature erythrocytes for 36 days under the addition of various growth factors. Through Giemsa staining, we found that the obtained mature erythrocytes were similar in morphology and size to normal human erythrocytes, and some obtained erythrocytes were enucleated. Globin expression was detected by real time RT-PCR, and the expression of ß-globin was more than 20%. The obtained erythrocytes are collected into the centrifuge tube, and then erythrocytes were naturally settled and showed the red color. Our findings provide a novel and effective method for the quantity generation of human red blood cells in vitro.
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Diferenciación Celular , Eritrocitos/citología , Células Madre Pluripotentes Inducidas/citología , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
Preparation of mouse embryonic fibroblast (MEF) feeder cells to maintain pluripotent stem cells (PSCs) is time consuming and involved in animal issues. Here, we demonstrated a novel method to prepare feeder cells with high efficiency, timesaving, and low costs. MEFs in 3 × 104 cell/cm2 were fixed by methanol for 5 min and air drying for 5 min. Thereafter, the methanol fixed MEF cells (MT-MEF) were able to be used directly to culture PSCs or stored at room temperature for the future usage. PSCs cultured on MT-MEF could be continuously expanded for over 40 passages with the naïve pluripotency. MT-MEFs could also be used to maintain human and pig iPSCs. Moreover, methanol fixed MEFs' culture dish was able to be reused for at least 4 times, and to be applied for antibiotic resistant screening assay to establishing stable transfected PSC lines. Alternatively, the immortalized cell lines, for instance NIH3T3 cells, could also be fixed by methanol and used as feeder cells to maintain PSCs. Thus, this novel means of methanol fixed feeder cells can completely replace the mitomycin C and gamma radiation treated MEF feeder cells, and be used to maintain PSCs derived from mouse as well as other animal species.
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Técnicas de Cultivo de Célula , Células Nutrientes , Animales , Fibroblastos , Metanol , Ratones , Células 3T3 NIHRESUMEN
Derivation of bona fide porcine pluripotent stem cells is still a critical issue because porcine embryonic stem cells (ESCs) are not available yet, and most of the culture conditions to maintain porcine induced pluripotent stem cells (piPSCs) are based on conditions for mouse and human iPS cells. In this study, we generated a doxycycline-inducible porcine iPS cell line (DOX-iPSCs) and used it to screen the optimal culture condition to sustain the self-renewal of piPSCs. We found that LIF and b-FGF were required for porcine cell reprogramming, but were not essential cytokines for maintaining the self-renewal and pluripotency of piPSCs. A serum-free 3i medium, which includes three inhibitors CHIR99021, SB431542, and PD0325901, three cytokines BMP4, SCF, and IL-6, and human platelet lysates (PL), was made through serious selections. In 3i condition, the doxycycline-inducible iPSCs could be passaged for a long term without the addition of doxycycline, and the flattened morphology of intermediate state piPSCs could convert to the naïve-like morphology with the increase in endogenous pluripotent gene expressions. Additionally, pPSC cell line isolated from 5.5 days blastocysts could be sustained in 3i medium and the expression of endogenous pluripotent genes OCT4, ESRRB, and STELLA was significantly increased. Our finding directed a new reprogramming strategy by using 3i condition to maintain and convert primed piPSCs into naïve-like pluripotent state. A combination of traditional LIF/b-FGF conditions and 3i condition may help us to find out an appropriate reprogramming approach to generate the naïve state of porcine iPSCs.
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In post-transcriptional mRNA modification, m6A has been observed in a wide range of eukaryotes. METTL3, as a component of methyltransferase complex for m6A modification, regulates mouse naïve pluripotency and influences mRNA stability, especially affecting the expression level of the key pluripotent transcription factors. To reveal the expression pattern of the porcine METTL3 gene, we analyzed METTL3 expression level in different porcine tissues, somatic cells, and induced pluripotent stem cells (piPSCs) by RT-PCR. To identify the function of METTL3 for regulation of the expression of porcine pluripotent genes, we cloned a 1 859-bp coding sequence of METTL3 and synthesized a shRNA against METTL3. When knocking down METTL3 expression in piPSCs, the cell type of piPSCs became naïve-like morphology, alkaline phosphatase activity was increased, and expression level of pluripotent genes NANOG, OCT4 and LIN28A was significantly elevated. In addition, piPSCs cultured in medium containing 10 mmol/L cycloleucine for 48 h exhibited the similar result as that knocked down METTL3. These findings set the stage for optimization of piPS culture condition and further study on the roles of m6A in piPSCs.
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Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Metiltransferasas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Metiltransferasas/genética , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas de Unión al ARN/genética , PorcinosRESUMEN
Unipotent spermatogonial stem cells (SSCs) can be efficiently reprogrammed into pluripotent stem cells only by manipulating the culture condition, without introducing exogenous reprogramming factors. This phenotype raises the hypothesis that the endogenous transcription factors (TFs) in SSCs may facilitate reprogramming to acquire pluripotency. In this study, we screened a pool of SSCs TFs (Bcl6b, Lhx1, Foxo1, Plzf, Id4, Taf4b, and Etv5), and found that oncogene Etv5 could dramatically increase the efficiency of induced pluripotent stem cells (iPSCs) generation when combined with Yamanaka factors. We also demonstrated that Etv5 could promote mesenchymal-epithelial transition (MET) at the early stage of reprogramming by regulating Tet2-miR200s-Zeb1 axis. In addition, Etv5 knockdown in mouse embryonic stem cells (mESCs) could decrease the genomic 5hmC level by downregulating Tet2. Furthermore, the embryoid body assay revealed that Etv5 could positively regulate primitive endoderm specification through regulating Gata6 and negatively regulate epiblast specification by inhibiting Fgf5 expression. In summary, our findings provide insights into understanding the regulation mechanisms of Etv5 under the context of somatic reprogramming, mESCs maintenance, and differentiation.
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Reprogramación Celular , Proteínas de Unión al ADN/genética , Endodermo/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/genética , Animales , Diferenciación Celular , Proliferación Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Embrión de Mamíferos , Endodermo/citología , Endodermo/crecimiento & desarrollo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Embrionarias de Ratones/citología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.
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Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos/fisiología , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/fisiología , Animales , Células Cultivadas , Cromatina/genética , Metilación de ADN , Dioxigenasas , Epigénesis Genética/fisiología , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Unión ProteicaRESUMEN
Oct4 is an important transcription factor for maintaining self-renewal and pluripotency of pluripotent stem cells (PSCs). Human OCT4 can be alternatively spliced and generate OCT4a, OCT4b, and OCT4b1. In this study, we discovered the novel Oct4 variants of Oct4b' and Oct4b1-3 in mouse PSCs for the first time. The expression of Oct4b variants, especially for Oct4b', was down regulated along with the downregulation of Oct4a when stem cells were differentiated. We also found four Oct4 translational products that were differentially expressed in mouse PSCs under the different culture conditions. The constructs of Oct4b2 and Oct4b3 could be alternatively spliced into Oct4b and Oct4b' when constructs were transiently transfected in NIH3T3 cells. Oct4b' encoded a 189 aa protein, and Oct4b could generate three distinct proteins including Oct4b-246aa, Oct4b-221aa, and Oct4b-189aa. The Oct4b variants could be alternatively translated in different type cells under the control of internal ribosome entry site (IRES) element that is within 5' upstream sequence of Oct4b. These findings provide new insights into reconsidering Oct4 variants expression and its additional role in maintaining the pluripotency of stem cells.
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Empalme Alternativo/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Isoformas de Proteínas/genética , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Ratones , Células 3T3 NIH , Especificidad de Órganos/genéticaRESUMEN
The estrogen-related receptor b (ESRRB) is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency. In mouse ES cells, overexpression of ESRRB can maintain LIF-independent self-renewal in the absence of Nanog. However, the fundamental features of porcine ESRRB remain elusive. In this study, we revealed the expression profiles of ESRRB in both porcine pluripotent stem cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor that enhanced porcine pluripotent gene activation. Addition of ESRRB into the cocktail of core pluripotent factors Oct4, Sox2, Klf4, and c-Myc (OSKM + E) could significantly enhance the reprograming efficiency and the formation of alkaline phosphatase positive colonies. Conversely, knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes, minimized the alkaline phosphatase activity, and initiated the porcine induced pluripotent stem cell differentiation. Therefore, porcine ESRRB is a crucial transcription factor to improve the self-renewal of piPSCs.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Receptores de Estrógenos/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Autorrenovación de las Células , Células Cultivadas , Técnicas de Reprogramación Celular , Regulación del Desarrollo de la Expresión Génica , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Receptores de Estrógenos/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Sus scrofa , TransfecciónRESUMEN
Previous evidences have proved that porcine-induced pluripotent stem cells (piPSCs) could be induced to distinctive metastable pluripotent states. This raises the issue of whether there is a common transcriptomic profile existing among the piPSC lines at distinctive state. In this study, we performed conjoint analysis of small RNA-seq and mRNA-seq for three piPSC lines which represent LIF dependence, FGF2 dependence and LFB2i dependence, respectively. Interestingly, we found there are 16 common microRNAs which potentially target 13 common mRNAs among the three piPSC lines. Dual-luciferase reporter assay validated that miR-370, one of the 16 common microRNAs, could directly target the 3'UTR of LIN28A. When the differentiation occurred, miR-370 could be activated in piPSCs and switched off the expression of LIN28A. Ectopic expression of miR-370 in piPSCs could reduce LIN28A expression, decrease the alkaline phosphatase activity, slow down the proliferation, and further cause the downregulation of downstream pluripotent genes (OCT4, SOX2, NANOG, SALL4 and ESRRB) and upregulation of differentiation relevant genes (SOX9, JARID2 and JMJD4). Moreover, these phenotypes caused by miR-370 could be rescued by overexpressing LIN28A. Collectively, our findings suggest that a set of common miRNA-mRNA interactions exist among the distinct piPSC lines, which orchestrate the self-renewal and differentiation of piPSCs independent of their metastable pluripotent states.
Asunto(s)
Reprogramación Celular/genética , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/genética , Factores de Transcripción/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/metabolismo , Anotación de Secuencia Molecular , Transducción de Señal , Porcinos , Factores de Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
Learning from a few examples and generalizing to markedly different situations are capabilities of human visual intelligence that are yet to be matched by leading machine learning models. By drawing inspiration from systems neuroscience, we introduce a probabilistic generative model for vision in which message-passing-based inference handles recognition, segmentation, and reasoning in a unified way. The model demonstrates excellent generalization and occlusion-reasoning capabilities and outperforms deep neural networks on a challenging scene text recognition benchmark while being 300-fold more data efficient. In addition, the model fundamentally breaks the defense of modern text-based CAPTCHAs (Completely Automated Public Turing test to tell Computers and Humans Apart) by generatively segmenting characters without CAPTCHA-specific heuristics. Our model emphasizes aspects such as data efficiency and compositionality that may be important in the path toward general artificial intelligence.