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1.
iScience ; 27(2): 108968, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38327788

RESUMO

Excessive or aberrant NLRP3 inflammasome activation has been implicated in the progression and initiation of many inflammatory conditions; however, currently no NLRP3 inflammasome inhibitors have been approved for therapeutic use in the clinic. Here we have identified that the natural product brazilin effectively inhibits both priming and activation of the NLRP3 inflammasome in cultured murine macrophages, a human iPSC microglial cell line and in a mouse model of acute peritoneal inflammation. Through computational modeling, we predict that brazilin can adopt a favorable binding pose within a site of the NLRP3 protein which is essential for its conformational activation. Our results not only encourage further evaluation of brazilin as a therapeutic agent for NLRP3-related inflammatory diseases, but also introduce this small-molecule as a promising scaffold structure for the development of derivative NLRP3 inhibitor compounds.

2.
Nat Commun ; 13(1): 536, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35087090

RESUMO

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Fosfofrutoquinase-2/metabolismo , Animais , Autofagia , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Doenças por Armazenamento dos Lisossomos/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Lipofuscinoses Ceroides Neuronais/genética , Neurônios/metabolismo , Fosfofrutoquinase-2/genética , Regulação para Cima
3.
Mol Ther Methods Clin Dev ; 23: 348-358, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34729381

RESUMO

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.

4.
J Pediatr Hematol Oncol ; 43(8): e1223-e1227, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34001790

RESUMO

Malignant central nervous system (CNS) tumors in young children have a poor prognosis and pose a therapeutic challenge. We describe 11 patients with high-risk CNS tumors (6 atypical teratoid/rhabdoid tumor, 4 nonmedulloblastoma CNS embryonal tumors, and 1 glioblastoma multiforme) who received 32 consolidation cycles of myeloablative carboplatin/thiotepa followed by autologous peripheral blood stem cell rescue. All patients underwent successful stem cell harvest without significant complications. Mean time to absolute neutrophil count ≥0.5×103/µL was 10.2±1.3 days and the mean length of hospital stay was 15.7±3.0 days. There were no regimen-related deaths. Five-year event-free survival and overall survival were 45.5±15.0% and 58.4±16.3%, respectively. Tandem carboplatin/thiotepa consolidation with autologous stem cell rescue is well-tolerated in young children with nonmedulloblastoma CNS tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Sistema Nervoso Central/terapia , Agonistas Mieloablativos/administração & dosagem , Transplante de Células-Tronco de Sangue Periférico/métodos , Células-Tronco/citologia , Condicionamento Pré-Transplante/métodos , Carboplatina/administração & dosagem , Neoplasias do Sistema Nervoso Central/patologia , Pré-Escolar , Terapia Combinada , Feminino , Seguimentos , Humanos , Lactente , Masculino , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Tiotepa/administração & dosagem , Transplante Autólogo
5.
Stem Cells ; 38(10): 1292-1306, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32621788

RESUMO

Inhibition of E-cad in mouse embryonic stem cells (mESCs) leads to a switch from LIF-BMP to Activin/Nodal-dependent pluripotency, consistent with transition from a naïve to primed pluripotent phenotype. We have used both genetic ablation and steric inhibition of E-cad function in mESCs to assess alterations to phenotype using quantitative mass spectrometry analysis, network models, and functional assays. Proteomic analyses revealed that one third of detected proteins were altered in E-cad null mESCs (Ecad-/- mESCs) compared to wild type (624 proteins were downregulated and 705 were proteins upregulated). Network pathway analysis and subsequent cellular flux assays confirmed a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, specifically through mitochondrial complex III downregulation and hypoxia inducible factor 1a target upregulation. Central to this was the transcriptional coactivator EP300. E-cad is a well-known tumor suppressor, its downregulation during cancer initiation and metastasis can be linked to the metabolic switch known as Warburg effect. This study highlights a phenomena found in both primed pluripotent state and cancer stemness and links it to loss of E-cad. Data are available via ProteomeXchange with identifier PXD012679.


Assuntos
Caderinas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Ciclo Celular/genética , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glicólise , Camundongos , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Proteoma/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo
6.
Methods Mol Biol ; 2081: 161-175, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31721124

RESUMO

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice. Further, we describe whole body bioluminescence imaging of conscious mice in a noninvasive manner, thus enforcing the 3R's (replacement, reduction, and refinement) of biomedical animal research.


Assuntos
Expressão Gênica , Genes Reporter , Medições Luminescentes/métodos , Animais , Técnicas Biossensoriais , Ordem dos Genes , Vetores Genéticos/genética , Luciferases de Vaga-Lume/genética , Camundongos , Plasmídeos/genética , Transfecção , Transgenes
7.
Biochim Biophys Acta Mol Basis Dis ; 1866(9): 165559, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31655107

RESUMO

The Neuronal Ceroid Lipofuscinoses (NCL), otherwise known as Batten disease, are a group of neurodegenerative diseases caused by mutations in 13 known genes. All except one NCL is autosomal recessive in inheritance, with similar aetiology and characterised by the accumulation of autofluorescent storage material in the lysosomes of cells. Age of onset and the rate of progression vary between the NCLs. They are collectively one of the most common lysosomal storage diseases, but the enigma remains of how genetically distinct diseases result in such remarkably similar pathogenesis. Much has been learnt from cellular studies about the function of the proteins encoded by the affected genes. Such research has utilised primitive unicellular models such as yeast and amoeba containing gene orthologues, cells derived from naturally occurring (sheep) and genetically engineered (mouse) animal models or patient-derived cells. Most recently, patient-derived induced pluripotent stem cell (iPSC) lines have been differentiated into neural cell-types to study molecular pathogenesis in the cells most profoundly affected by disease. Here, we review how cell models have informed much of the biochemical understanding of the NCLs and how more complex models are being used to further this understanding and potentially act as platforms for therapeutic efficacy studies in the future.


Assuntos
Modelos Biológicos , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Modelos Animais de Doenças , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Lipofuscinoses Ceroides Neuronais/genética
8.
Stem Cells Cloning ; 11: 85-93, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30519053

RESUMO

BACKGROUND: Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. OBJECTIVE: We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. METHODS: Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. RESULTS: Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and ßIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. CONCLUSION: We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.

9.
Stem Cell Reports ; 10(6): 1766-1781, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29681545

RESUMO

Human neural development begins at embryonic day 19 and marks the beginning of organogenesis. Neural stem cells in the neural tube undergo profound functional, morphological, and metabolic changes during neural specification, coordinated by a combination of exogenous and endogenous cues. The temporal cell signaling activities that mediate this process, during development and in the postnatal brain, are incompletely understood. We have applied gene expression studies and transcription factor-activated reporter lentiviruses during in vitro neural specification of human pluripotent stem cells. We show that nuclear factor κB orchestrates a multi-faceted metabolic program necessary for the maturation of neural progenitor cells during neurogenesis.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético , NF-kappa B/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Autofagia , Biomarcadores , Ciclo Celular , Diferenciação Celular/genética , Células Cultivadas , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Imuno-Histoquímica , Modelos Biológicos , Neurogênese/genética , Fenótipo , Transdução de Sinais
10.
Methods Mol Biol ; 1651: 49-64, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28801899

RESUMO

The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo.


Assuntos
Genes Reporter , Luciferases de Vaga-Lume/análise , Substâncias Luminescentes/análise , Medições Luminescentes/métodos , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Clonagem Molecular , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Células HEK293 , Humanos , Lentivirus/genética , Luciferases de Vaga-Lume/genética , Substâncias Luminescentes/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Transdução Genética/métodos , Transgenes
11.
Cell Rep ; 14(8): 1883-91, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26904936

RESUMO

The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.


Assuntos
Reprogramação Celular , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Derme/citologia , Derme/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicólise/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Luciferases/genética , Luciferases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação Oxidativa , Via de Pentose Fosfato/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transdução Genética
12.
J Neurooncol ; 57(2): 123-6, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12125972

RESUMO

Choroid plexus carcinomas (CPC) are rare central nervous system malignancies. While attempted surgical resection is imperative, the benefit of adjuvant therapy, particularly in the setting of a gross total resection (GTR), is unclear. We reviewed all pediatric cases of CPC reported in the literature between 1985 and 2000. Seventy-five cases of CPC were identified. Mean age at the time of diagnosis was 26 months. Thirty-seven patients had a GTR and 38 patients had a subtotal resection (STR). Thirty-eight cases (51%) were alive and 37 cases (49%) were deceased at time of publication. For cases with a GTR, survival was 84% compared to an 18% survival for patients with a STR. For patients with a GTR, all forms of adjuvant therapy were statistically equivalent. Our retrospective literature review confirms the importance of GTR in the therapy of CPC, with GTR alone being the single most important predictor of survival. The prognosis is poor for any patient with a STR, with the exception of those patients for whom adjuvant therapy allowed for an eventual GTR. The small number of patients receiving a GTR and no further therapy precluded a statistical comparison of no therapy in the setting of a GTR versus any form of adjuvant therapy. However, all four of these patients are alive, raising the possibility that adjuvant therapy in the setting of a GTR may not be required.


Assuntos
Carcinoma/tratamento farmacológico , Carcinoma/cirurgia , Quimioterapia Adjuvante , Neoplasias do Plexo Corióideo/tratamento farmacológico , Neoplasias do Plexo Corióideo/cirurgia , Adolescente , Carcinoma/radioterapia , Criança , Pré-Escolar , Neoplasias do Plexo Corióideo/radioterapia , Terapia Combinada , Humanos , Lactente , Reprodutibilidade dos Testes , Estudos Retrospectivos , Resultado do Tratamento
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