Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
Stem Cell Res ; 56: 102524, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34481189

RESUMO

Alpha-smooth muscle actin (α-SMA) is encoded by ACTA2 and is a key protein in the cellular contractile system of various mesodermal cell types, including hepatic stellate cells (HSCs), smooth muscle cells, and cardiomyocytes. α-SMA, which is a key protein in the development of hepatic fibrosis, is widely used as a reliable marker of HSC activation. Here, we generated an ACTA2-EGFP reporter human induced pluripotent stem cell line, KITi001-C-41, using a CRISPR/Cas9-based knock-in system. These reporter hiPSC lines can be used for lineage tracing of mesodermal cells and for screening of HSC activation factors.

2.
Biomedicines ; 9(7)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34356912

RESUMO

Encoded hydrogel microparticles mounting DNA probes are powerful tools for high-performance microRNA (miRNA) detection in terms of sensitivity, specificity, and multiplex detection capability. However, several particle rinsing steps in the assay procedure present challenges for rapid and efficient detection. To overcome this limitation, we encapsulated dense magnetic nanoparticles to reduce the rinsing steps and duration via magnetic separation. A large number of magnetic nanoparticles were encapsulated into hydrogel microparticles based on a discontinuous dewetting technique combined with degassed micromolding lithography. In addition, we attached DNA probes targeting three types of miRNAs related to preeclampsia to magnetically encoded hydrogel microparticles by post-synthesis conjugation and achieved sensitivity comparable to that of conventional nonmagnetic encoded hydrogel microparticles. To demonstrate the multiplex capability of magnetically encoded hydrogel microparticles while maintaining the advantages of the simplified rinsing process when addressing multiple samples, we conducted a triplex detection of preeclampsia-related miRNAs. In conclusion, the introduction of magnetically encoded hydrogel microparticles not only allowed efficient miRNA detection but also provided comparable sensitivity and multiplexed detectability to conventional nonmagnetic encoded hydrogel microparticles.

3.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
4.
EMBO Rep ; 22(5): e51656, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33913240

RESUMO

Liquid-liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus-responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.


Assuntos
Proteínas Intrinsicamente Desordenadas , Plantas/genética
5.
JAMA ; 324(16): 1640-1650, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33107944

RESUMO

Importance: Current guidelines recommend ticagrelor as the preferred P2Y12 platelet inhibitor for patients with acute coronary syndrome (ACS), primarily based on a single large randomized clinical trial. The benefits and risks associated with ticagrelor vs clopidogrel in routine practice merits attention. Objective: To determine the association of ticagrelor vs clopidogrel with ischemic and hemorrhagic events in patients undergoing percutaneous coronary intervention (PCI) for ACS in clinical practice. Design, Setting, and Participants: A retrospective cohort study of patients with ACS who underwent PCI and received ticagrelor or clopidogrel was conducted using 2 United States electronic health record-based databases and 1 nationwide South Korean database from November 2011 to March 2019. Patients were matched using a large-scale propensity score algorithm, and the date of final follow-up was March 2019. Exposures: Ticagrelor vs clopidogrel. Main Outcomes and Measures: The primary end point was net adverse clinical events (NACE) at 12 months, composed of ischemic events (recurrent myocardial infarction, revascularization, or ischemic stroke) and hemorrhagic events (hemorrhagic stroke or gastrointestinal bleeding). Secondary outcomes included NACE or mortality, all-cause mortality, ischemic events, hemorrhagic events, individual components of the primary outcome, and dyspnea at 12 months. The database-level hazard ratios (HRs) were pooled to calculate summary HRs by random-effects meta-analysis. Results: After propensity score matching among 31 290 propensity-matched pairs (median age group, 60-64 years; 29.3% women), 95.5% of patients took aspirin together with ticagrelor or clopidogrel. The 1-year risk of NACE was not significantly different between ticagrelor and clopidogrel (15.1% [3484/23 116 person-years] vs 14.6% [3290/22 587 person-years]; summary HR, 1.05 [95% CI, 1.00-1.10]; P = .06). There was also no significant difference in the risk of all-cause mortality (2.0% for ticagrelor vs 2.1% for clopidogrel; summary HR, 0.97 [95% CI, 0.81-1.16]; P = .74) or ischemic events (13.5% for ticagrelor vs 13.4% for clopidogrel; summary HR, 1.03 [95% CI, 0.98-1.08]; P = .32). The risks of hemorrhagic events (2.1% for ticagrelor vs 1.6% for clopidogrel; summary HR, 1.35 [95% CI, 1.13-1.61]; P = .001) and dyspnea (27.3% for ticagrelor vs 22.6% for clopidogrel; summary HR, 1.21 [95% CI, 1.17-1.26]; P < .001) were significantly higher in the ticagrelor group. Conclusions and Relevance: Among patients with ACS who underwent PCI in routine clinical practice, ticagrelor, compared with clopidogrel, was not associated with significant difference in the risk of NACE at 12 months. Because the possibility of unmeasured confounders cannot be excluded, further research is needed to determine whether ticagrelor is more effective than clopidogrel in this setting.


Assuntos
Síndrome Coronariana Aguda/cirurgia , Clopidogrel/efeitos adversos , Intervenção Coronária Percutânea , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Ticagrelor/efeitos adversos , Síndrome Coronariana Aguda/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Aspirina/administração & dosagem , Estudos de Casos e Controles , Causas de Morte , Clopidogrel/administração & dosagem , Bases de Dados Factuais/estatística & dados numéricos , Dispneia/induzido quimicamente , Feminino , Hemorragia/induzido quimicamente , Humanos , Isquemia/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Metanálise em Rede , Pontuação de Propensão , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Recidiva , República da Coreia , Estudos Retrospectivos , Acidente Vascular Cerebral/epidemiologia , Ticagrelor/administração & dosagem , Estados Unidos
6.
J Bacteriol ; 202(17)2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32571966

RESUMO

Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentus IMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen. Chemotaxis of S. meliloti cells toward alfalfa roots mediates this symbiosis. The present study establishes programmed proteolysis as a factor in the maintenance of the S. meliloti chemotaxis system. Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S. meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response. While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S. meliloti In addition, our study should alert about the cautionary use of epitope tags for protein quantification.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/química , Ciclo Celular/fisiologia , Deleção de Genes , Movimento , Processamento de Proteína Pós-Traducional , Proteólise
7.
Nat Neurosci ; 23(7): 854-868, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424286

RESUMO

General anesthesia (GA) can produce analgesia (loss of pain) independent of inducing loss of consciousness, but the underlying mechanisms remain unclear. We hypothesized that GA suppresses pain in part by activating supraspinal analgesic circuits. We discovered a distinct population of GABAergic neurons activated by GA in the mouse central amygdala (CeAGA neurons). In vivo calcium imaging revealed that different GA drugs activate a shared ensemble of CeAGA neurons. CeAGA neurons also possess basal activity that mostly reflects animals' internal state rather than external stimuli. Optogenetic activation of CeAGA potently suppressed both pain-elicited reflexive and self-recuperating behaviors across sensory modalities and abolished neuropathic pain-induced mechanical (hyper-)sensitivity. Conversely, inhibition of CeAGA activity exacerbated pain, produced strong aversion and canceled the analgesic effect of low-dose ketamine. CeAGA neurons have widespread inhibitory projections to many affective pain-processing centers. Our study points to CeAGA as a potential powerful therapeutic target for alleviating chronic pain.


Assuntos
Anestésicos Gerais/farmacologia , Núcleo Central da Amígdala/efeitos dos fármacos , Neurônios GABAérgicos/efeitos dos fármacos , Dor/fisiopatologia , Animais , Feminino , Masculino , Camundongos , Vias Neurais/efeitos dos fármacos , Percepção da Dor/efeitos dos fármacos , Percepção da Dor/fisiologia
8.
FASEB J ; 34(7): 9141-9155, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32421247

RESUMO

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.


Assuntos
Citocromo P-450 CYP1A1/química , Fluorescência , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Preparações Farmacêuticas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Diferenciação Celular , Citocromo P-450 CYP1A1/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Transdução de Sinais
9.
Chem Commun (Camb) ; 56(36): 4950-4953, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32239050

RESUMO

We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Complexos Multienzimáticos/genética , Proteína 9 Associada à CRISPR/metabolismo , Complexos Multienzimáticos/metabolismo , Streptococcus pyogenes/enzimologia
10.
Transl Stroke Res ; 11(6): 1296-1305, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32306239

RESUMO

Prediction of outcome after stroke may help clinicians provide effective management and plan long-term care. We aimed to develop and validate a score for predicting good functional outcome available for hospitals after ischemic stroke using linked data. A total of 22,005 patients with acute ischemic stroke from the Clinical Research Center for Stroke Registry between July 2007 and December 2014 were included in the derivation group. We assessed functional outcomes using a modified Rankin scale (mRS) score at 3 months after ischemic stroke. We identified predictors related to good 3-month outcome (mRS score ≤ 2) and developed a score. External validations (geographic and temporal validations) of the developed model were performed. The prediction model performance was assessed using the area under the receiver operating characteristic curve (AUC) and the calibration test. Stroke severity, sex, stroke mechanism, age, pre-stroke mRS, and thrombolysis/thrombectomy treatment were identified as predictors for 3-month good functional outcomes in the S-SMART score (total 34 points). Patients with higher S-SMART scores had an increased likelihood of a good outcome. The AUC of the prediction score was 0.805 (0.798-0.811) in the derivation group and 0.812 (0.795-0.830) in the geographic validation group for good functional outcome. The AUC of the model was 0.812 (0.771-0.854) for the temporal validation group. Moreover, they had good calibration. The S-SMART score is a valid and useful tool to predict good functional outcome following ischemic stroke. This prediction model may assist in the estimation of outcomes to determine care plans after stroke.

11.
Biochem Biophys Res Commun ; 521(4): 874-879, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31708105

RESUMO

The laminar structure, a unique feature of the mammalian cerebrum, is formed by a number of genes in a highly complex process. The pyramidal neurons that make up each layer of the cerebrum are functionally characterized by specific gene expressions. In particular, Cux1 and Cux2, which are specifically expressed in layer II-IV neurons, are known to regulate dendritic branching, spine morphology, and synapse formation. However, it is still unknown how their expression is regulated transcriptionally. Here we constructed Cux2-mCherry transgenic mice that reproduce the cortical layer II-IV-specific expression of Cux2, a member of the Cut/Cux/CDP family, using BAC transgenesis and a variety of coordinated cortical layer markers that are known to date. Our immunohistochemistry analysis shows that mCherry was expressed in cortical layer II-IV and the corpus callosum in the same way as endogenous Cux2 without ectopic expression. We also identified a region of 220 bp that is highly conserved in mammals and controls specific cerebral expression of Cux2, using comparative genome analysis and in vivo reporter assays. Furthermore, we confirm that Lhx2, whose expression in cortical layer II-IV is similar to that of the Cux2 enhancer, can act as a transcriptional activator. These results suggest that cortical layer II-IV expression of Cux2 can be regulated by the interaction of Cux2-E1 and Lhx2, and that their failure to co-regulate is associated with neurodevelopmental disorders such as autism and schizophrenia.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Córtex Cerebral/fisiologia , Cromossomos Artificiais Bacterianos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Luminescentes/genética , Camundongos Transgênicos , Células Piramidais/metabolismo , Fatores de Transcrição/metabolismo
12.
J Clin Lab Anal ; 33(2): e22685, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30298531

RESUMO

BACKGROUND: Currently, three commercial in vitro diagnostic matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are widely used in clinical laboratories. The ASTA MicroIDSys system (ASTA Inc, South Korea) is a newly developed MALDI-TOF MS system used for the identification of pathogenic microorganisms. In the present study, we assessed the performance of the ASTA MALDI-TOF MS system for the identification of pathogenic yeast from clinical samples. METHODS: We tested 284 clinical yeast isolates from various clinical specimens using ASTA MALDI-TOF MS, and the results were compared with those using molecular sequencing of the ITS or D1-D2 regions of rDNA and biochemical assays. RESULTS: A total of 284 isolates were tested and found to be distributed across 14 species including Candida albicans (n = 100) and other yeast species (n = 184). ASTA MALDI-TOF MS correctly identified 95.1% (270/284) of the yeast species compared to molecular sequencing. Among them, 262 isolates showed acceptable MALDI-TOF MS scores (≥140), and 98.1% (257/262) isolates were identified correctly. In addition, among 22 isolates with a MALDI-TOF MS score <140, 59.1% (13/22) of the isolates showed concordance with molecular typing at the species level. Clustering analysis revealed the effectiveness of the new MALDI-TOF MS system for the identification of yeast species. CONCLUSIONS: ASTA MALDI-TOF MS showed high accuracy in the identification of yeast species; it involves facile sample preparation and extraction procedures. ASTA MALDI-TOF MS is expected to be useful for yeast identification in clinical microbiology laboratories due to its reliability and cost-effectiveness.


Assuntos
Tipagem Molecular/métodos , Micoses , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leveduras , Análise por Conglomerados , Humanos , Micoses/diagnóstico , Micoses/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Leveduras/química , Leveduras/classificação , Leveduras/isolamento & purificação
13.
Asian-Australas J Anim Sci ; 32(3): 437-441, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30145873

RESUMO

OBJECTIVE: Paraoxonase 1 (PON1), a calcium-dependent serum enzyme, has been shown to be involved in lipid metabolism. In this study, we examined the putative correlation of the serum PON1 level of Hanwoo, Korean native cattle, with gender and meat quality grade. METHODS: PON1 levels were estimated by determining the arylesterase and paraoxonase activities (AE and PO, respectively) in serum samples from Hanwoo individuals (n = 56). Serum PON1 levels were analyzed in different gender groups (female [n = 21], castrated male [n = 17], and male [n = 18]), and meat quality grades (≥1 [n = 23], 2 [n = 21], and 3 [n = 12]). RESULTS: Serum PON1 levels were similar in female (AE = 120±55 U/mL, PO = 84±43 mU/mL) and castrated male (123±44 U/mL, PO = 89±30 mU/mL), while male showed a significantly lower level (AE = 65±43 U/mL, PO = 44±34 mU/mL). Furthermore, analysis of serum PON1 levels in three different grades of meat quality showed similar levels in the grades ≥1 (AE = 118±49 U/mL, PO = 84±37 mU/mL) and 2 (AE = 116±54 U/mL, PO = 82±43 mU/mL), while the level was significantly lower in the grade 3 (AE = 58±35 U/mL, PO = 39±27 mU/mL) of lower meat quality. CONCLUSION: We discovered the gender-dependent differences in serum PON1 levels of Hanwoo and a positive association of the serum PON1 level with meat quality. Results in this study suggest that PON1 would be a useful serum marker for preliminary screening of Hanwoo individuals with high-quality meat and applicable for genetic improvement.

14.
Biotechnol Lett ; 40(5): 755-763, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29464570

RESUMO

OBJECTIVES: The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. RESULTS: Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. CONCLUSIONS: 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.


Assuntos
Butirilcolinesterase/genética , Hidrolases de Éster Carboxílico/genética , Técnicas de Cultura de Células/métodos , Hepatócitos/citologia , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Oseltamivir/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Stem Cell Res ; 26: 72-75, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29247817

RESUMO

Pregnane X receptor (PXR) is a key nuclear receptor that mediates drug metabolism and stimulates hepatocyte proliferation. However, the lack of PXR expression in human pluripotent stem cell-derived hepatocytes limits their application for drug screening and toxicity testing. Here, we generated a PXR-mCherry reporter human induced pluripotent stem cell (hiPSC) line using the CRISPR/Cas9 system. PXR-mCherry hiPSCs were pluripotent and had differentiation potential and a normal karyotype. This cell line is an important tool for identifying factors that increase PXR-mediated drug metabolism and hepatocyte proliferation.


Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Genes Reporter , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/antagonistas & inibidores
16.
Stem Cell Rev Rep ; 14(3): 438-450, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29238913

RESUMO

During liver development, nonpolarized hepatic progenitor cells differentiate into mature hepatocytes with distinct polarity. This polarity is essential for maintaining the intrinsic properties of hepatocytes. The balance between the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) plays a decisive role in differentiation of polarized hepatocytes. In this study, we found that phthalazinone pyrazole (PP), a selective inhibitor of Aurora-A kinase (Aurora-A), suppressed the EMT during the differentiation of hepatocyte-like cells (HLCs) from human embryonic stem cells. The differentiated HLCs treated with PP at the hepatoblast stage showed enhanced hepatic morphology and functions, particularly with regard to the expression of drug metabolizing enzymes. Moreover, we found that these effects were mediated though suppression of the AKT pathway, which is involved in induction of the EMT, and upregulation of hepatocyte nuclear factor 4α expression rather than Aurora-A inhibition. In conclusion, these findings provided insights into the regulatory role of the EMT on in vitro hepatic maturation, suggesting that inhibition of the EMT may drive transformation of hepatoblast cells into mature and polarized HLCs.


Assuntos
Hepatócitos/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Pirazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Hepatócitos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imuno-Histoquímica , Fígado/citologia
17.
J Korean Med Sci ; 33(53): e343, 2018 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-30595684

RESUMO

Background: Linkage of public healthcare data is useful in stroke research because patients may visit different sectors of the health system before, during, and after stroke. Therefore, we aimed to establish high-quality big data on stroke in Korea by linking acute stroke registry and national health claim databases. Methods: Acute stroke patients (n = 65,311) with claim data suitable for linkage were included in the Clinical Research Center for Stroke (CRCS) registry during 2006-2014. We linked the CRCS registry with national health claim databases in the Health Insurance Review and Assessment Service (HIRA). Linkage was performed using 6 common variables: birth date, gender, provider identification, receiving year and number, and statement serial number in the benefit claim statement. For matched records, linkage accuracy was evaluated using differences between hospital visiting date in the CRCS registry and the commencement date for health insurance care in HIRA. Results: Of 65,311 CRCS cases, 64,634 were matched to HIRA cases (match rate, 99.0%). The proportion of true matches was 94.4% (n = 61,017) in the matched data. Among true matches (mean age 66.4 years; men 58.4%), the median National Institutes of Health Stroke Scale score was 3 (interquartile range 1-7). When comparing baseline characteristics between true matches and false matches, no substantial difference was observed for any variable. Conclusion: We could establish big data on stroke by linking CRCS registry and HIRA records, using claims data without personal identifiers. We plan to conduct national stroke research and improve stroke care using the linked big database.


Assuntos
Bases de Dados Factuais , Armazenamento e Recuperação da Informação , Acidente Vascular Cerebral/patologia , Doença Aguda , Idoso , Big Data , Feminino , Humanos , Revisão da Utilização de Seguros , Masculino , Pessoa de Meia-Idade , Sistema de Registros
18.
Front Aging Neurosci ; 9: 341, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29114219

RESUMO

The endoplasmic reticulum (ER) is an indispensable cellular organelle that remains highly active in neuronal cells. The ER bears the load of maintaining protein homeostasis in the cellular network by managing the folding of incoming nascent peptides; however, the stress imposed by physiological/environmental factors can cause ER dysfunctions that lead to the activation of ER unfolded protein response (UPRER). Aging leads to deterioration of several cellular pathways and therefore weakening of the UPRER. The decline in functioning of the UPRER during aging results in accumulation of misfolded proteins that becomes intracellular inclusions in neuronal cells, resulting in toxicity manifested as neurodegenerative diseases. With ascension in cases of neurodegenerative diseases, understanding the enigma behind aging driven UPRER dysfunction may lead to possible treatments.

19.
Sci Rep ; 6: 21684, 2016 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-26899675

RESUMO

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity, their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor, CAR; and pregnane X receptor, PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs, whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore, ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition, an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus, AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics.


Assuntos
Epigênese Genética , Hepatócitos/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Adulto , Diferenciação Celular , Metilação de DNA , Hepatócitos/citologia , Humanos , Análise em Microsséries , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptor de Pregnano X , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Elementos de Resposta , Análise de Sequência de DNA , Transdução de Sinais , Ativação Transcricional
20.
Zygote ; 24(3): 442-56, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26197710

RESUMO

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine ß-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the ß-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine ß-casein gene.


Assuntos
Blastocisto/metabolismo , Sistemas CRISPR-Cas , Caseínas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Engenharia Genética/métodos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Blastocisto/citologia , Western Blotting , Caseínas/genética , Bovinos , Linhagem Celular , Células Cultivadas , Endonucleases/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Recombinação Homóloga , Humanos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...