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2.
Mol Cell Biol ; 42(10): e0016322, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36125265

RESUMO

Insulin and insulin-like growth factor 1 (IGF1) signaling is transduced by insulin receptor substrate 1 (IRS1) and IRS2. To elucidate physiological and redundant roles of insulin and IGF1 signaling in adult hearts, we generated mice with inducible cardiomyocyte-specific deletion of insulin and IGF1 receptors or IRS1 and IRS2. Both models developed dilated cardiomyopathy, and most mice died by 8 weeks post-gene deletion. Heart failure was characterized by cardiomyocyte loss and disarray, increased proapoptotic signaling, and increased autophagy. Suppression of autophagy by activating mTOR signaling did not prevent heart failure. Transcriptional profiling revealed reduced serum response factor (SRF) transcriptional activity and decreased mRNA levels of genes encoding sarcomere and gap junction proteins as early as 3 days post-gene deletion, in concert with ultrastructural evidence of sarcomere disruption and intercalated discs within 1 week after gene deletion. These data confirm conserved roles for constitutive insulin and IGF1 signaling in suppressing autophagic and apoptotic signaling in the adult heart. The present study also identifies an unexpected role for insulin and IGF1 signaling in regulating an SRF-mediated transcriptional program, which maintains expression of genes encoding proteins that support sarcomere integrity in the adult heart, reduction of which results in rapid development of heart failure.


Assuntos
Insuficiência Cardíaca , Fator de Crescimento Insulin-Like I , Camundongos , Animais , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Insulina/metabolismo , Fator de Resposta Sérica/metabolismo , Sarcômeros/metabolismo , Miócitos Cardíacos/metabolismo , Insuficiência Cardíaca/metabolismo , Serina-Treonina Quinases TOR/metabolismo , RNA Mensageiro/metabolismo , Conexinas/metabolismo
3.
Nat Commun ; 13(1): 4984, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-36008396

RESUMO

CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.


Assuntos
Edição de Genes , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional
4.
Elife ; 92020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32955440

RESUMO

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than a E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/instrumentação
5.
JCI Insight ; 5(6)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32213702

RESUMO

Pressure overload (PO) cardiac hypertrophy and heart failure are associated with generalized insulin resistance and hyperinsulinemia, which may exacerbate left ventricular (LV) remodeling. While PO activates insulin receptor tyrosine kinase activity that is transduced by insulin receptor substrate 1 (IRS1), the present study tested the hypothesis that IRS1 and IRS2 have divergent effects on PO-induced LV remodeling. We therefore subjected mice with cardiomyocyte-restricted deficiency of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) to PO induced by transverse aortic constriction (TAC). In WT mice, TAC-induced LV hypertrophy was associated with hyperactivation of IRS1 and Akt1, but not IRS2 and Akt2. CIRS1KO hearts were resistant to cardiac hypertrophy and heart failure in concert with attenuated Akt1 activation. In contrast, CIRS2KO hearts following TAC developed more severe LV dysfunction than WT controls, and this was prevented by haploinsufficiency of Akt1. Failing human hearts exhibited isoform-specific IRS1 and Akt1 activation, while IRS2 and Akt2 activation were unchanged. Kinomic profiling identified IRS1 as a potential regulator of cardioprotective protein kinase G-mediated signaling. In addition, gene expression profiling revealed that IRS1 signaling may promote a proinflammatory response following PO. Together, these data identify IRS1 and Akt1 as critical signaling nodes that mediate LV remodeling in both mice and humans.


Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/complicações , Humanos , Hiperinsulinismo/complicações , Resistência à Insulina/fisiologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo
6.
Nat Commun ; 11(1): 1281, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152313

RESUMO

Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Regulação da Expressão Gênica , Transcrição Gênica , Animais , Técnicas Biossensoriais , Humanos , RNA Guia de Cinetoplastídeos/genética
7.
ACS Synth Biol ; 8(4): 906-910, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30939239

RESUMO

CRISPR is a versatile technology for genomic editing and regulation, but the expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and expressed from a single transcriptional unit. We demonstrate that these gRNA arrays can stably regulate gene expression in a synergistic manner via dCas9-mediated repression. This approach expands the number of gRNAs that can be expressed in this model organism and may enable the versatile editing or transcriptional regulation of a greater number of genes in vivo.


Assuntos
RNA Guia de Cinetoplastídeos/genética , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Edição de Genes/métodos , Expressão Gênica/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética
8.
Trends Biotechnol ; 37(2): 181-197, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30497870

RESUMO

Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial consortia for diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and the development of new tools to construct synthetic consortia and program their behaviors, will vastly expand the functions that can be performed by communities of interacting microorganisms. Here, we review recent advancements in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss ongoing and emerging efforts to apply consortia for various biotechnological applications, and suggest future applications.


Assuntos
Biotecnologia/métodos , Metabolismo , Consórcios Microbianos , Biologia Sintética/métodos , Regulação da Expressão Gênica , Redes e Vias Metabólicas/genética
9.
Cardiovasc Pathol ; 27: 68-70, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28171828

RESUMO

Immunological vascular phenomena can be the initial manifestation of bacterial infection and endocarditis. Here, we report a rare case of leukocytoclastic vasculitis without immune complexes or cryoglobulinemia in a patient with infective endocarditis, congenital heart disease, and a prior mechanical valve replacement. The patient completely recovered following antibiotic therapy, and skin lesions disappeared without immune suppression, which suggested infection-mediated vasculitis. While the treatment of leukocytoclastic vasculitis typically involves immunosuppressive therapy, the treatment for infection-mediated vasculitis is eradication of the infection.


Assuntos
Endocardite Bacteriana/complicações , Transposição dos Grandes Vasos/complicações , Vasculite Leucocitoclástica Cutânea/etiologia , Adulto , Feminino , Próteses Valvulares Cardíacas , Humanos , Complicações Pós-Operatórias , Infecções Estafilocócicas/complicações
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