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Although somatic cell reprogramming to generate inducible pluripotent stem cells (iPSCs) is associated with profound epigenetic changes, the roles and mechanisms of epigenetic factors in this process remain poorly understood. Here, we identify Jmjd3 as a potent negative regulator of reprogramming. Jmjd3-deficient MEFs produced significantly more iPSC colonies than did wild-type cells, whereas ectopic expression of Jmjd3 markedly inhibited reprogramming. We show that the inhibitory effects of Jmjd3 are produced through both histone demethylase-dependent and -independent pathways. The latter pathway involves Jmjd3 targeting of PHF20 for ubiquitination and degradation via recruitment of an E3 ligase, Trim26. Importantly, PHF20-deficient MEFs could not be converted to fully reprogrammed iPSCs, even with knockdown of Jmjd3, Ink4a, or p21, indicating that PHF20 is required for reprogramming. Our findings demonstrate, to the best of our knowledge, a previously unrecognized role of Jmjd3 in cellular reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls this process.
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Reprogramação Celular , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Cinética , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Regulação para CimaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1004524.].
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Type I interferon (IFN) is critical for controlling pathogen infection; however, its regulatory mechanisms in plasmacytoid cells (pDCs) still remain unclear. Here, we have shown that nucleic acid sensors cGAS-, STING-, MDA5-, MAVS-, or transcription factor IRF3-deficient mice produced high amounts of type I IFN-α and IFN-ß (IFN-α/ß) in the serum and were resistant to lethal plasmodium yoelii YM infection. Robust IFN-α/ß production was abolished when gene encoding nucleic acid sensor TLR7, signaling adaptor MyD88, or transcription factor IRF7 was ablated or pDCs were depleted. Further, we identified SOCS1 as a key negative regulator to inhibit MyD88-dependent type I IFN signaling in pDCs. Finally, we have demonstrated that pDCs, cDCs, and macrophages were required for generating IFN-α/ß-induced subsequent protective immunity. Thus, our findings have identified a critical regulatory mechanism of type I IFN signaling in pDCs and stage-specific function of immune cells in generating potent immunity against lethal YM infection.
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Imunidade Adaptativa/imunologia , Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Malária/imunologia , Transdução de Sinais/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Knockout , Plasmodium yoelii , Reação em Cadeia da PolimeraseRESUMO
Microbial products, such as lipopolysaccharide (LPS), can elicit efficient innate immune responses against invading pathogens. However, priming with LPS can induce a form of innate immune memory, termed innate immune "tolerance", which blunts subsequent NF-κB signaling. Although epigenetic and transcriptional reprogramming has been shown to play a role in innate immune memory, the involvement of post-translational regulation remains unclear. Here, we report that ubiquitin-specific protease 3 (USP3) participates in establishing "tolerance" innate immune memory through non-transcriptional feedback. Upon NF-κB signaling activation, USP3 is stabilized and exits the nucleus. The cytoplasmic USP3 specifically removes the K63-linked polyubiquitin chains on MyD88, thus negatively regulating TLR/IL1ß-induced inflammatory signaling activation. Importantly, cytoplasmic translocation is a prerequisite step for USP3 to deubiquitinate MyD88. Additionally, LPS priming could induce cytoplasmic retention and faster and stronger cytoplasmic translocation of USP3, enabling it to quickly shut down NF-κB signaling upon the second LPS challenge. This work identifies a previously unrecognized post-translational feedback loop in the MyD88-USP3 axis, which is critical for inducing normal "tolerance" innate immune memory.
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Fator 88 de Diferenciação Mieloide , NF-kappa B , NF-kappa B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Lipopolissacarídeos/farmacologia , Transdução de Sinais , Imunidade Inata , Tolerância ImunológicaRESUMO
Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.
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Colite , Linfócitos T , Camundongos , Animais , Timo , Interleucina-17 , Transdução de SinaisRESUMO
Stringent control of the NF-kappaB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-kappaB-dependent responses by interacting with IKKalpha and IKKbeta and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-kappaB and its responsive genes, TNF-alpha and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-kappaB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity.
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Imunidade Inata , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Clonagem Molecular , RNA Helicases DEAD-box/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação , Peptídeos e Proteínas de Sinalização Intracelular/química , Ligantes , Camundongos , Fosforilação , Receptores Toll-Like/metabolismoRESUMO
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.
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Proteínas de Transporte/imunologia , Proteína DEAD-box 58/imunologia , Proteínas de Ligação a DNA/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , Complexos Multiproteicos/imunologia , Fosfoproteínas Fosfatases/imunologia , Transdução de Sinais/imunologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Fosfoproteínas Fosfatases/genética , Receptores Imunológicos , Transdução de Sinais/genética , Proteínas com Motivo Tripartido , Células Vero , Viroses/genética , Viroses/imunologia , Vírus/genética , Vírus/imunologiaRESUMO
BACKGROUND & AIMS: The aim of this study was to characterize baseline morphologic features of crypts in nondysplastic Barrett's esophagus and correlate them with DNA content abnormalities and risk of progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). METHODS: The morphologic features of nondysplastic crypts in baseline biopsy specimens from 212 BE patients (2956 biopsy specimens) were graded histologically using a 4-point scale (crypt atypia levels, 0-3). DNA content abnormalities were detected using flow cytometry. RESULTS: In patients who had dysplasia in their baseline biopsy specimens, dysplasia was associated significantly with increasing grades of crypt atypia in the background nondysplastic Barrett's esophagus (P < .001). In a subset of patients without dysplasia at baseline (N = 149), a higher grade of crypt atypia was associated with longer Barrett's esophagus segment length (5.5 vs 3.3 cm; P = .0095), and a higher percentage of cells with 4N DNA content (3.67 ± 1.27 vs 2.93 ± 1.22; P = .018). Crypt atypia was associated with the development of any neoplasia (low-grade dysplasia and HGD/EAC). Although no significant association was noted between the grade of crypt atypia and increased 4N, aneuploidy, or progression to HGD/EAC, only patients with grade 2 or 3 crypt atypia showed increased 4N, aneuploidy, or progression to HGD/EAC. CONCLUSIONS: Patients with Barrett's esophagus likely develop dysplasia via a progressive increase in the level of crypt atypia before the onset of dysplasia, and these changes may reflect some alteration of DNA content.
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Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Lesões Pré-Cancerosas , Humanos , Esôfago de Barrett/complicações , Neoplasias Esofágicas/patologia , Aneuploidia , Hiperplasia , DNA , Avaliação de Resultados em Cuidados de Saúde , Progressão da Doença , Lesões Pré-Cancerosas/patologiaRESUMO
Stringent control of the type I interferon signaling pathway is important for maintaining host immune responses and homeostasis, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. Here we report that the pattern-recognition receptor NLRP4 regulated the activation of type I interferon mediated by double-stranded RNA or DNA by targeting the kinase TBK1 for degradation. NLRP4 recruited the E3 ubiquitin ligase DTX4 to TBK1 for Lys48 (K48)-linked polyubiquitination at Lys670, which led to degradation of TBK1. Knockdown of either DTX4 or NLRP4 abrogated K48-linked ubiquitination and degradation of TBK1 and enhanced the phosphorylation of TBK1 and the transcription factor IRF3. Our results identify a previously unrecognized role for NLRP4 in the regulation of type I interferon signaling and provide molecular insight into the mechanisms by which NLRP4-DTX4 targets TBK1 for degradation.
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Interferon Tipo I/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata/imunologia , Immunoblotting , Imunoprecipitação , Interferon Tipo I/imunologia , Fosforilação , Proteínas Serina-Treonina Quinases/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/imunologia , Transfecção , Ubiquitina-Proteína Ligases/imunologia , UbiquitinaçãoRESUMO
Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.
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Yersinia pseudotuberculosis , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo , Cálcio/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Variações do Número de Cópias de DNA , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
MOTIVATION: The efficiency of CRISPR/Cas9-mediated protein knockout is determined by three factors: sequence-specific sgRNA activity, frameshift probability and the characteristics of targeted amino acids. A number of computational methods have been developed for predicting sgRNA efficiency from different perspectives. However, an integrative method that combines all three factors for rational sgRNA selection is still lacking. RESULTS: We developed GuidePro, a two-layer ensemble predictor that enables the integration of multiple factors for the prioritization of sgRNAs in protein knockouts. Tested on independent datasets, GuidePro outperforms existing methods and demonstrates consistent superior performance in predicting phenotypes caused by protein loss-of-function, suggesting its robustness for prioritizing sgRNAs in various applications of CRISPR/Cas9 knockouts. AVAILABILITY AND IMPLEMENTATION: GuidePro is available at https://github.com/MDhewei/GuidePro. A web application for prioritizing sgRNAs that target protein-coding genes in human, monkey and mouse genomes is available at https://bioinformatics.mdanderson.org/apps/GuidePro. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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BACKGROUND: Of the US population, 10% reports a penicillin allergy but more than 90% can ultimately tolerate penicillin. Confirmation of these allergies in the pediatric population may improve future health outcomes and decrease costs. Referring patients for confirmatory testing is the first step in clarifying penicillin allergies. OBJECTIVE: To increase the number of referrals of patients with listed penicillin allergies from the University of California, San Diego academic general pediatrics clinics to Rady Children's Hospital allergy clinics using an educational session and a best practice advisory (BPA) in the electronic medical record. METHODS: An educational session with attendings and 3 plan-do-study-act (PDSA) cycles were completed using a BPA alert that triggered for all patients with a documented penicillin-class drug allergy to draw attention and facilitate referral. The BPA was modified at each PDSA cycle based on physician input. RESULTS: At baseline, 1.9% of referrals to the allergy clinic were for penicillin-class drug allergies. After an attending physician educational session, the percentage increased to 13.7%. The BPA was implemented with further increase to 27.8% of all allergy referrals in the course of 3 PDSA cycles. Not all patients with penicillin-class drug allergies were referred, and the reasons were documented when the physicians dismissed the BPA. Physicians did not refer 35% of the time because of time constraints, as opposed to patient or parent disinterest, which was 8% of the time. CONCLUSION: Referrals to the allergist for confirmatory testing in patients with listed penicillin allergies increased by more than 10 fold. This study illustrates successful tools to support delabeling.
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Hipersensibilidade a Drogas , Penicilinas , Melhoria de Qualidade , Antibacterianos/efeitos adversos , Criança , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/tratamento farmacológico , Humanos , Penicilinas/efeitos adversos , Atenção Primária à Saúde , Encaminhamento e ConsultaRESUMO
Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two Vibrio cholerae strains (El Tor C6706 and Classical O395) in mucus using single-cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae can penetrate mucus using flagellar motility and that alkaline pH increases swimming speed and, consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive hemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric GMP (c-di-GMP). Overall, our results support the hypothesis that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single-cell tracking that V. cholerae can penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection, because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome will offer new strategies to fight cholera.
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Cólera/microbiologia , Muco/química , Vibrio cholerae/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cólera/metabolismo , Toxina da Cólera/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Muco/metabolismo , Muco/microbiologia , Suínos , Vibrio cholerae/genéticaRESUMO
Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the present study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer's patches and cecum during the clonal invasive phase of the infection, while the highest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues and will be readily applicable to other infection models.
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Carga Bacteriana , Variações do Número de Cópias de DNA , Plasmídeos/genética , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/fisiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Camundongos , Especificidade de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Virulência , Fatores de Virulência/genética , Infecções por Yersinia pseudotuberculosis/diagnósticoRESUMO
Angiotensin II (ANG II) is a potent vasoconstrictor and may reduce renal blood flow (RBF), causing renal hypoxia. Hypotensive hemorrhage elevates plasma ANG II levels and is associated with increased risk of acute kidney injury. We hypothesized that ANG II antagonism prevents renal vasoconstriction and hypoxia caused by hemorrhage. Pigs were anaesthetized, surgically prepared, and randomized to intravenous losartan (1.5 mg·kg-1·h-1, n = 8) or an equal volume of intravenous Ringer acetate (vehicle-treated, n = 8). Hemorrhage was induced by continuous aspiration of blood to reach and sustain mean arterial pressure of <50 mmHg for 30 min. Plasma ANG II levels, hemodynamics and oxygenation were assessed 60 min prehemorrhage, 30-min after the start of hemorrhage, and 60 min posthemorrhage. Erythropoietin mRNA was analyzed in cortical and medullary tissue sampled at the end of the experiment. Hypotensive hemorrhage increased plasma ANG II levels and decreased RBF and oxygen delivery in both groups. Losartan-treated animals recovered in RBF and oxygen delivery, whereas vehicle-treated animals had persistently reduced RBF and oxygen delivery. In accordance, renal vascular resistance increased over time post hemorrhage in vehicle-treated animals but was unchanged in losartan-treated animals. Renal oxygen extraction rate and cortical erythropoietin mRNA levels increased in the vehicle group but not in the losartan group. In conclusion, ANG II antagonism alleviates prolonged renal vasoconstriction and renal hypoxia in a large animal model of hypotensive hemorrhage.
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Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Hemorragia/patologia , Hipóxia/prevenção & controle , Rim/irrigação sanguínea , Losartan/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Hemodinâmica , Hipotensão , Masculino , Oxigênio , SuínosRESUMO
BACKGROUND AND AIMS: Estrogen is an important risk factor for cholesterol gallstone disease because women are twice as likely as men to form gallstones. The classical estrogen receptor α (ERα), but not ERß, in the liver plays a critical role in the formation of estrogen-induced gallstones in female mice. The molecular mechanisms underlying the lithogenic effect of estrogen on gallstone formation have become more complicated with the identification of G protein-coupled receptor 30 (GPR30), an estrogen receptor. APPROACH AND RESULTS: We investigated the biliary and gallstone phenotypes in ovariectomized female GPR30-/- , ERα-/- , and wild-type mice injected intramuscularly with the potent GPR30-selective agonist G-1 at 0 or 1 µg/day and fed a lithogenic diet for 8 weeks. The activation of GPR30 by G-1 enhanced cholelithogenesis by suppressing expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme for the classical pathway of bile salt synthesis. These metabolic abnormalities led to an increase in biliary cholesterol concentrations in company with hepatic hyposecretion of biliary bile salts, thereby inducing cholesterol-supersaturated gallbladder bile and accelerating cholesterol crystallization. G-1 also impairs gallbladder emptying, leading to sluggish gallbladder motility and promoting the development of biliary sludge in the early stage of gallstone formation. The prevalence rates of gallstones were 80% in wild-type and ERα-/- mice treated with G-1 compared to 10% in wild-type mice receiving no G-1. However, no gallstones were formed in GPR30-/- mice treated with G-1. CONCLUSIONS: GPR30 produces additional lithogenic actions, working independently of ERα, to increase susceptible to gallstone formation in female mice; both GPR30 and ERα are potential therapeutic targets for cholesterol gallstone disease, particularly in women and patients exposed to high levels of estrogen.
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Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Cálculos Biliares/patologia , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bile/efeitos dos fármacos , Bile/metabolismo , Ácidos e Sais Biliares/biossíntese , Colesterol/metabolismo , Modelos Animais de Doenças , Receptor alfa de Estrogênio/genética , Feminino , Vesícula Biliar/patologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ovariectomia , Quinolinas/administração & dosagem , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fatores SexuaisRESUMO
Stringent control of NF-κB and mitogen-activated protein kinase (MAPK) signaling is critical during innate immune responses. TGF-ß activated kinase-1 (TAK1) is essential for NF-κB activation in T and B cells but has precisely the opposite activity in myeloid cells. Specific deletion of TAK1 (Map3k7(ΔM/ΔM)) led to development of splenomegaly and lymphomegaly associated with neutrophilia. Compared with wild-type cells, TAK1-deficient neutrophils enhanced the phosphorylation of the kinases IKK, p38, and JNK and the production of interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) after lipopolysaccharide (LPS) stimulation. Map3k7(ΔM/ΔM) mice were significantly more susceptible to LPS-induced septic shock and produced higher amounts of IL-1ß, IL-6, and TNF-α in plasma than do wild-type mice. Specific ablation of p38 rescued the phenotype and functional properties of Map3k7(ΔM/ΔM) mice. Our findings identify a previously unrecognized role of TAK1 as a negative regulator of p38 and IKK activation in a cell type-specific manner.
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Antígeno CD11b , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Neutrófilos/enzimologia , Receptores de Quimiocinas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Antígeno CD11b/metabolismo , Proliferação de Células , Regulação para Baixo , Deleção de Genes , MAP Quinase Quinase Quinases/genética , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Modelos Imunológicos , Neutrófilos/citologia , Neutrófilos/imunologia , Fenótipo , Receptores de Quimiocinas/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. METHODS: We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. RESULTS: The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. SIGNIFICANCE: These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.
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Epilepsia , Doenças Metabólicas , Animais , Astrócitos/metabolismo , Encéfalo/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/genética , Glutamina , Humanos , Camundongos , Convulsões/patologiaRESUMO
Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERß, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERß fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17ß-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 µg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (-/-) mice treated with 6 µg/day 17ß-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.