RESUMO
Over 80% of CTSA programs have a community advisory board (CAB). Little is known about how research discussed with CABs aligns with community priorities (bidirectionality). This program evaluation assessed researcher presentations from 2014 to 2018 to the CABs linked to our CTSA at all three sites (Minnesota, Arizona, and Florida) for relevance to local community needs identified in 2013 and/or 2016. From content analysis, of 65 presentations total, 41 (63%) addressed ≥1 local health needs (47% Minnesota, 60% Florida, and 80% Arizona). Cross-cutting topics were cancer/cancer prevention (physical activity/obesity/nutrition) and mental health. Results could help to prioritize health outcomes of community-engaged research efforts.
RESUMO
The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
Assuntos
Desoxirribonucleases/metabolismo , Marcação de Genes/métodos , Engenharia Genética/métodos , Genoma/genética , Peixe-Zebra/genética , Alelos , Animais , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Cromossomos/genética , Quebras de DNA , DNA de Cadeia Simples/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genômica/métodos , Genótipo , Mutação em Linhagem Germinativa/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Reparo de DNA por Recombinação/genéticaRESUMO
TGF-ß modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-ß. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-ß treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-ß-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-ß and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Quinase 1 de Adesão Focal/genética , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Smad , Células Swiss 3T3 , Fator de Crescimento Transformador beta/genéticaRESUMO
Transforming growth factor (TGF)-ß family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-ß signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-ß receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-ß receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-ß, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-ß receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-ß receptor recycling as well as Smad2 phosphorylation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endocitose , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Smad2/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.
Assuntos
Proteínas de Transporte/metabolismo , Neurofibromina 2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Quinases Ativadas por p21/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Biológicos , Neurofibromina 2/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
BACKGROUND: Halothane inhibits airway smooth muscle contraction in part by inhibiting the functional coupling between muscarinic receptors and one of its cognate heterotrimeric G proteins, Galphaq. Based on previous studies indicating a more potent effect of halothane and sevoflurane on airway smooth muscle contraction compared with isoflurane, the current study hypothesized that at anesthetic concentrations of 2 minimum alveolar concentration (MAC) or less, halothane and sevoflurane but not isoflurane inhibit acetylcholine-promoted Galphaq guanosine nucleotide exchange. METHODS: Galphaq guanosine nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the human M3 muscarinic receptor and human Galphaq. A radioactive, nonhydrolyzable analog of guanosine-5'-triphosphate, [35S]GTPgammaS, was used as a reporter for nucleotide exchange at Galphaq. RESULTS: Acetylcholine caused a concentration-dependent increase in Galphaq [35S]GTPgammaS-GDP exchange. Neither anesthetic affected constitutive Galphaq [35S]GTPgammaS-GDP exchange in the absence of acetylcholine. Conversely, each anesthetic caused a concentration-dependent and reversible inhibition of Galphaq [35S]GTPgammaS-GDP exchange when promoted by acetylcholine. At concentrations of 3 MAC or less, the effect of halothane and sevoflurane were significantly greater than that of isoflurane, with only a minimal inhibition by isoflurane observed at 2 MAC. CONCLUSION: The differential effects of volatile anesthetics on acetylcholine-promoted guanosine nucleotide exchange at Galphaq are consistent with the apparent more potent direct effect of halothane and sevoflurane compared with isoflurane on muscarinic receptor-mediated contraction of isolated airway smooth muscle. These differential effects also suggest a mode of anesthetic action that could be due to anesthetic-protein interactions and not simply anesthetic accumulation in the lipid membrane.
Assuntos
Anestésicos Inalatórios/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/efeitos dos fármacos , Receptor Muscarínico M3/efeitos dos fármacos , Acetilcolina/farmacologia , Algoritmos , Animais , Células COS , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citoplasma/metabolismo , Interpretação Estatística de Dados , Nucleotídeos de Guanina/metabolismo , Halotano/farmacologia , Hexanóis/farmacologia , Humanos , Immunoblotting , Isoflurano/farmacologia , Contração Isométrica , Éteres Metílicos/farmacologia , SevofluranoRESUMO
BACKGROUND: This study investigated whether halothane affects the functional coupling between the beta2 adrenergic receptor and the alpha subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (Galphas). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at Galphas and hence would not affect isoproterenol-induced relaxation of airway smooth muscle. METHODS: Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. Galphas nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human beta1 or beta2 receptor each with human short Galphas. A radioactive, nonhydrolyzable analog of GTP, [S]GTPgammaS, was used as the reporter for nucleotide exchange at Galphas. RESULTS: Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in Galphas nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal Galphas nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted Galphas nucleotide exchange in both the beta1-Galphas and beta2-Galphas expressing membranes. However, the effect was significantly greater on beta1-Galphas coupling compared with beta2-Galphas coupling, with no effect on beta2-Galphas coupling at 2.8 MAC halothane. CONCLUSION: Halothane does not inhibit the biochemical coupling between the beta2 receptor and Galphas and hence does not affect the inhibition of calcium sensitivity induced by isoproterenol. Therefore, halothane should not affect the efficacy of beta2 agonists, as suggested by studies of in vivo animal models of asthma.
Assuntos
Anestésicos Inalatórios/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/efeitos dos fármacos , Halotano/farmacologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Isoproterenol/farmacologia , Receptores Adrenérgicos beta 2/fisiologia , SuínosRESUMO
BACKGROUND: Halothane is an effective bronchodilator and inhibits airway smooth muscle contraction in part by inhibiting intracellular signaling pathways activated by the M2 muscarinic receptor and its cognate inhibitory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G protein), Gi. This study hypothesized that halothane inhibits nucleotide exchange at the alpha isoform-3 subunit of Gi (Galphai-3), but only when regulated by the M2 muscarinic receptor. METHODS: GTP hydrolysis by Galphai-3 and the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [gammaPi]-labeled GTP. Anesthetic binding to Galphai-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. Galphai-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and Galphai-3. A radioactive analog of GTP, [S]GTPgammaS, was used as a reporter for Galphai-3 nucleotide exchange. RESULTS: Although spectroscopy demonstrated halothane binding to Galphai-3, this binding had no effect on [gammaPi]-labeled GTP hydrolysis by the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal Galphai-3 nucleotide exchange measured in crude membranes when the muscarinic receptor agonist acetylcholine was omitted from the assay. Conversely, halothane caused a concentration-dependent inhibition of Galphai-3 nucleotide exchange with acetylcholine included in the assay. CONCLUSION: These data indicate that despite halothane binding to Galphai-3, halothane has no direct inhibitory effect on the intrinsic activity of the Galphai-3beta1gamma2HF heterotrimer but inhibits M2 muscarinic receptor regulation of the heterotrimer. This novel effect is consistent with the ability of halothane to inhibit airway smooth muscle contraction and bronchoconstriction induced by acetylcholine.
Assuntos
Anestésicos Inalatórios/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Halotano/farmacologia , Receptor Muscarínico M2/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Baculoviridae/metabolismo , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Nucleotídeos de Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Receptor Muscarínico M2/biossíntese , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Members of the transforming growth factor beta (TGF-beta) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-beta signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-beta receptor (TGF-beta R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-beta R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-beta-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-beta R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-beta R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.