RESUMO
BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.
RESUMO
Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.
Assuntos
Cartilagem Articular , Condrócitos , Animais , Camundongos , Condrócitos/metabolismo , Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células CultivadasRESUMO
Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.
Assuntos
Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular/genética , Regiões Promotoras Genéticas , Reprogramação Celular/genética , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteína Desglicase DJ-1/metabolismoRESUMO
Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.
Assuntos
Transição Epitelial-Mesenquimal , Células-Tronco Pluripotentes Induzidas , Animais , Reprogramação Celular/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR. Allele-specific RNA-seq of these iPSCs revealed that XCR initiates at a subset of genes clustered near the centromere region. The initial phase of XCR occurs when the cells transit through mesenchymal-epithelial transition (MET) before complete shutoff of Xist expression. Moreover, regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy. Our results identified clustered genes on the Xi that show reactivation in the initial phase of XCR during reprogramming and suggest a possible role for histone demethylation in this process.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Família Multigênica , Ativação Transcricional , Inativação do Cromossomo X/genética , Alelos , Animais , Biomarcadores , Técnicas de Reprogramação Celular , Fibroblastos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Célula Única , TranscriptomaRESUMO
We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.
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Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Ácidos Graxos/metabolismo , Incubadoras , Metabolismo dos Lipídeos , Análise Espectral Raman , Animais , Linhagem Celular , Camundongos , Imagem com Lapso de TempoRESUMO
Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Hemofilia A/genética , Hemofilia A/terapia , Vírus Sendai/genética , Animais , Testes de Coagulação Sanguínea , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Modelos Animais de Doenças , Fator VIII/genética , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Knockout , Transdução Genética , TransgenesRESUMO
BACKGROUND: Central nervous system (CNS) gliosarcoma (GSM) is a rare primary neoplasm characterized by the presence of glial and sarcomatous components. OBJECTIVE: In this report, we describe the clinical and neuroimaging aspects of three cases of GSM and correlate these aspects with pathological findings. We also provide a brief review of relevant literature. METHODS: Three patients were evaluated with magnetic resonance imaging (MRI), and biopsies confirmed the diagnosis of primary GSM, without previous radiotherapy. RESULTS: The analysis of conventional sequences (T1, T1 after contrast injection, T2, Fluid attenuation inversion recovery, SWI and DWI/ADC map) and advanced (proton 1H MR spectroscopy and perfusion) revealed an irregular, necrotic aspect of the lesion, peritumoral edema/infiltration and isointensity of the solid component on a T2-weighted image. These features were associated with irregular and peripheral contrast enhancement, lipid and lactate peaks, increased choline and creatine levels in proton spectroscopy, increased relative cerebral blood volume (rCBV) in perfusion, multifocality and drop metastasis in one of the cases. CONCLUSION: These findings are discussed in relation to the general characteristics of GSM reported in the literature.
Assuntos
Neoplasias Encefálicas , Gliossarcoma , Neoplasias Encefálicas/diagnóstico por imagem , Gliossarcoma/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
Abstract Background: Central nervous system (CNS) gliosarcoma (GSM) is a rare primary neoplasm characterized by the presence of glial and sarcomatous components. Objective: In this report, we describe the clinical and neuroimaging aspects of three cases of GSM and correlate these aspects with pathological findings. We also provide a brief review of relevant literature. Methods: Three patients were evaluated with magnetic resonance imaging (MRI), and biopsies confirmed the diagnosis of primary GSM, without previous radiotherapy. Results: The analysis of conventional sequences (T1, T1 after contrast injection, T2, Fluid attenuation inversion recovery, SWI and DWI/ADC map) and advanced (proton 1H MR spectroscopy and perfusion) revealed an irregular, necrotic aspect of the lesion, peritumoral edema/infiltration and isointensity of the solid component on a T2-weighted image. These features were associated with irregular and peripheral contrast enhancement, lipid and lactate peaks, increased choline and creatine levels in proton spectroscopy, increased relative cerebral blood volume (rCBV) in perfusion, multifocality and drop metastasis in one of the cases. Conclusion: These findings are discussed in relation to the general characteristics of GSM reported in the literature.
Resumo Introdução: O gliossarcoma (GSM) do sistema nervoso central (SNC) é uma neoplasia primária rara, caracterizada pela presença de componentes gliais e sarcomatosos. Objetivo: Nosso objetivo é descrever os aspectos clínicos e de neuroimagem de três casos com este diagnóstico e correlacioná-los com os achados patológicos. Também foi realizada uma breve revisão da literatura relevante. Métodos: Três pacientes foram avaliados por ressonância magnética (RM), e biópsias confirmaram o diagnóstico de GSM primário, sem radioterapia prévia. Resultados: Foram analisadas as sequências convencionais (T1, T1 após injeção de contraste, T2, FLAIR-fluid attenuation inversion recovery, SWI, DWI/mapa ADC) e as sequências avançadas (espectroscopia de prótons 1H e perfusão), observando-se aspecto necrótico e irregular da lesão, edema/infiltração peritumoral, isointensidade do componente sólido em T2, associada a realce irregular e periférico pelo meio de contraste, pico de lípides e de lactato e aumento dos níveis de colina e creatina na espectroscopia de prótons, aumento do volume sanguíneo cerebral relativo (rCBV) na perfusão, multifocalidade e "drop" mestástase em um dos casos. Conclusão: O presente estudo descreve características do GSM, discutindo as informações na literatura científica, ilustrando algumas particularidades desses tumores.
Assuntos
Humanos , Neoplasias Encefálicas/diagnóstico por imagem , Gliossarcoma/diagnóstico por imagem , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.
Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Retrovirus Endógenos , Inativação Gênica , Células-Tronco Embrionárias Murinas , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/virologia , Vírus Sendai/genética , Vírus Sendai/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Uncoupling protein 1 (UCP1) is a mitochondrial protein that is expressed in both brown and beige adipocytes. UCP1 uncouples the mitochondrial electron transport chain from ATP synthesis to produce heat via non-shivering thermogenesis. Due to their ability to dissipate energy as heat and ameliorate metabolic disorders, UCP1-expressing adipocytes are considered as a potential target for anti-obesity treatment. To monitor the expression of UCP1 in live mice in a non-invasive manner, we generated the Ucp1-iRFP720 knock-in (Ucp1-iRFP720 KI) mice, in which the gene encoding a near-infrared fluorescent protein iRFP720 is inserted into the Ucp1 gene locus. Using the heterozygous Ucp1-iRFP720 KI mice, we observed robust iRFP fluorescence in the interscapular region where brown adipose tissue is located. Moreover, the iRFP fluorescence was clearly observable in inguinal white adipose tissues in live mice administered with ß3-adrenergic receptor agonist CL316,243. We also found that the homozygous Ucp1-iRFP720 KI mice, which are deficient in UCP1, displayed prominent iRFP fluorescence in the inguinal regions at the standard housing temperature. Consistent with this, the mice exhibited expanded populations of beige-like adipocytes in inguinal white adipose tissue, in which the Ucp1 promoter was dramatically activated. Thus, the Ucp1-iRFP720 KI mice provide a convenient model for non-invasive in vivo imaging of UCP1 expression in both brown and beige adipocytes in live mice.
Assuntos
Expressão Gênica , Proteínas Luminescentes/genética , Imagem Molecular , Proteína Desacopladora 1/genética , Adipócitos Bege/metabolismo , Animais , Linhagem Celular , Marcação de Genes , Loci Gênicos , Genótipo , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Imagem Molecular/métodos , Espectroscopia de Luz Próxima ao Infravermelho , Proteína Desacopladora 1/metabolismo , Proteína Vermelha FluorescenteRESUMO
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold a huge promise for regenerative medicine, drug development, and disease modeling. PSCs have unique metabolic features that are akin to those of cancer cells, in which glycolysis predominates to produce energy as well as building blocks for cellular components. Recent studies indicate that the unique metabolism in PSCs is not a mere consequence of their preference for a low oxygen environment, but is an active process for maintaining self-renewal and pluripotency, possibly in preparation for rapid response to the metabolic demands of differentiation. Understanding the regulatory mechanisms of this unique metabolism in PSCs is essential for proper derivation, generation, and maintenance of PSCs. In this review, we discuss the metabolic features of PSCs and describe the current understanding of the mechanisms of the metabolic shift during reprogramming from somatic cells to iPSCs, in which the metabolism switches from oxidative phosphorylation (OxPhos) to glycolysis.
Assuntos
Reprogramação Celular , Metabolismo Energético , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/citologiaRESUMO
Causes of cervical ulceration include infection, collagen disease, malignant tumors and external stimuli. Cervical ulceration during pregnancy is rare. We present a case of cervical ulceration caused by group C streptococcal infection during pregnancy. A 36-year-old woman (gravida 1, para 0) complained of metrorrhagia, and a circular cervical ulcer of about 1.5 cm in diameter was detected on her cervix at 37 weeks' gestation. A biopsy and a cultivation test of the ulcer were performed, and pathological diagnosis was made as suppurative inflammation, and group C streptococcal infection was detected by the cultivation test. The ulcer had expanded to about 3 cm in diameter at the onset of labor at 40 weeks' gestation. An emergency cesarean section was performed because of failed induction of labor, and she was delivered of a male baby. The ulcer became gradually smaller after delivery, and completely disappeared on the 35th day after delivery.
Assuntos
Complicações Infecciosas na Gravidez/diagnóstico , Infecções Estreptocócicas/diagnóstico , Úlcera/diagnóstico , Doenças do Colo do Útero/diagnóstico , Adulto , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/microbiologia , Infecções Estreptocócicas/complicações , Infecções Estreptocócicas/microbiologia , Úlcera/etiologia , Úlcera/microbiologia , Doenças do Colo do Útero/etiologia , Doenças do Colo do Útero/microbiologiaRESUMO
We present a case of a 30-year-old man who had a 3-year history of low back pain. MRI demonstrated an infiltrative mass, affecting the vertebral body and pedicles of L4, with some extension to the vertebral canal. There was also tumor invasion in the inferior vena cava and in the left iliopsoas muscle. The anatomopathological examination of the resected L4 vertebral body was of a malignant neoplasia compatible with mesenchymal chondrosarcoma (high histological grade). About 2 months after surgery, he developed a progressive bladder incontinence, bilateral leg weakness and severe back pain. A new MRI was obtained, confirming progression of the disease. An occipital scalp lesion was detected and biopsy confirmed cutaneous metastasis. Primary malignant bone tumors are rare but should be ruled out in young patients with persistent low back pain. We present a case of a confirmed mesenchymal chondrosarcoma affecting lumbar spine, with MRI and pathological illustrations. Early diagnosis may improve the chances of local disease control and even cure.
RESUMO
Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells. The PD image and its derived PD index reflected the mitochondrial content, enabling quantitative evaluation of the degrees of somatic cell reprogramming and PSC differentiation. Moreover, the PD index allowed unbiased grouping of PSC colonies into those with high or low pluripotency without the aid of invasive methods. Finally, the PD imaging system produced three-dimensional images of PSC colonies, providing further criteria to evaluate pluripotency of PSCs. Thus, the PD imaging system may be utilized for screening of live PSCs with potentially high pluripotency prior to more rigorous quality control processes.
Assuntos
Microscopia de Fluorescência/métodos , Células-Tronco Pluripotentes/citologia , Frações Subcelulares/ultraestrutura , Animais , Diferenciação Celular , Corantes Fluorescentes , Humanos , Camundongos , Mitocôndrias/ultraestrutura , Células NIH 3T3 , Células-Tronco Pluripotentes/ultraestruturaAssuntos
Transplante de Rim/efeitos adversos , Transtornos Linfoproliferativos/etiologia , Transtornos Linfoproliferativos/patologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia , Toxoplasmose Cerebral/patologia , Adulto , Diagnóstico Diferencial , Feminino , Humanos , Transtornos Linfoproliferativos/diagnóstico por imagem , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Complicações Pós-Operatórias/diagnóstico por imagem , Toxoplasmose Cerebral/diagnóstico por imagemRESUMO
Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Cefaleia/complicações , Poliarterite Nodosa/diagnóstico , Tuberculose/diagnóstico , Transtornos da Visão/complicações , Adulto , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Infarto Encefálico/complicações , Neoplasias Encefálicas/complicações , Humanos , Imageamento por Ressonância Magnética , Masculino , Poliarterite Nodosa/complicações , Tuberculose/complicações , Acuidade VisualRESUMO
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is accompanied by morphological, functional, and metabolic alterations before acquisition of full pluripotency. Although the genome-wide effects of the reprogramming factors on gene expression are well documented, precise mechanisms by which gene expression changes evoke phenotypic responses remain to be determined. We used a Sendai virus-based system that permits reprogramming to progress in a strictly KLF4-dependent manner to screen for KLF4 target genes that are critical for the progression of reprogramming. The screening identified Tcl1 as a critical target gene that directs the metabolic shift from oxidative phosphorylation to glycolysis. KLF4-induced TCL1 employs a two-pronged mechanism, whereby TCL1 activates AKT to enhance glycolysis and counteracts PnPase to diminish oxidative phosphorylation. These regulatory mechanisms described here highlight a central role for a reprogramming factor in orchestrating the metabolic shift toward the acquisition of pluripotency during iPSC generation.
Assuntos
Reprogramação Celular , Metabolismo Energético , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Reprogramação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicólise , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: This analysis estimated the cost-effectiveness of intravitreal aflibercept injection(s) (IAI) for wet age-related macular degeneration (wAMD) compared with other treatments in Japan. METHODS: This was a cost-utility analysis based on published data. A state-transition cohort model was constructed with six health states based on best-corrected visual acuity in the better-seeing eye. The cycle time was 4 weeks, and the time horizon was 12 years. The model compared IAI 2 mg every 8 weeks (2q8) for 2 years after three initial monthly injections, ranibizumab as needed, ranibizumab 0.5 mg every 4 weeks (0.5q4), pegaptanib sodium 0.3 mg every 6 weeks, verteporfin photodynamic therapy (PDT), and best supportive care, assumed to include medical management and monitoring, but no active therapy. Costs (expressed as Japanese yen [JPY]) and quality-adjusted life years (QALYs) gained were estimated for each treatment and discounted at 2.0%. Input data were obtained from clinical studies, the Japanese drug tariff and social insurance reimbursement schedule, and expert opinion. The analysis was conducted from the societal perspective, including medical costs as well as costs of blindness. RESULTS: IAI 2q8 was dominant (i.e. more effective in terms of QALYs and less costly) to all other comparators (ranibizumab as needed, ranibizumab 0.5q4, pegaptanib sodium, PDT, and best supportive care), as shown by the incremental cost-utility ratio (i.e. cost per QALY gained). LIMITATIONS: The strengths of the analysis include the wide range of comparators evaluated and the use of Japanese-specific utility data. The limitations include the use of one eye, inclusion of published data up to 2 years only, and assumptions on disease course over 5 years. CONCLUSIONS: IAI 2q8 was more effective in terms of QALYs and less costly compared with other treatments for wAMD in Japan.