RESUMO
During neurovascular development, brain endothelial cells (BECs) respond to secreted signals from the neuroectoderm that regulate CNS angiogenesis, the formation of new blood vessels in the brain, and barriergenesis, the acquisition of blood-brain barrier (BBB) properties. Wnt/ß-catenin signaling and Vegf signaling are both required for CNS angiogenesis; however, the relationship between these pathways is not understood. Furthermore, while Wnt/ß-catenin signaling is essential for barriergenesis, the role of Vegf signaling in this vital process remains unknown. Here, we provide the first direct evidence, to our knowledge, that Vegf signaling is not required for barriergenesis and that activation of Wnt/ß-catenin in BECs is independent of Vegf signaling during neurovascular development. Using double transgenic glut1b:mCherry and plvap:EGFP zebrafish (Danio rerio) to visualize the developing brain vasculature, we performed a forward genetic screen and identified a new mutant allele of kdrl, an ortholog of mammalian Vegfr2. The kdrl mutant lacks CNS angiogenesis but, unlike the Wnt/ß-catenin pathway mutant gpr124, acquires BBB properties in BECs. To examine Wnt/ß-catenin pathway activation in BECs, we chemically inhibited Vegf signaling and found robust expression of the Wnt/ß-catenin transcriptional reporter line 7xtcf-Xla.Siam:EGFP. Taken together, our results establish that Vegf signaling is essential for CNS angiogenesis but is not required for Wnt/ß-catenin-dependent barriergenesis. Given the clinical significance of either inhibiting pathological angiogenesis or stimulating neovascularization, our study provides valuable new insights that are critical for the development of effective therapies that target the vasculature in neurological disorders.
Assuntos
Barreira Hematoencefálica , beta Catenina , Animais , beta Catenina/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Mamíferos/metabolismo , Neovascularização Patológica , Fatores de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização Wnt/fisiologia , Peixe-Zebra/metabolismoRESUMO
Post-transplant diarrhea is a common complication after solid organ transplantation and is frequently attributed to the widely prescribed immunosuppressant mycophenolate mofetil (MMF). Given recent work identifying the relationship between MMF toxicity and gut bacterial ß-glucuronidase activity, we evaluated the relationship between gut microbiota composition, fecal ß-glucuronidase activity, and post-transplant diarrhea. We recruited 97 kidney transplant recipients and profiled the gut microbiota in 273 fecal specimens using 16S rRNA gene sequencing. We further characterized fecal ß-glucuronidase activity in a subset of this cohort. Kidney transplant recipients with post-transplant diarrhea had decreased gut microbial diversity and decreased relative gut abundances of 12 genera when compared to those without post-transplant diarrhea (adjusted p value < .15, Wilcoxon rank sum test). Among the kidney transplant recipients with post-transplant diarrhea, those with higher fecal ß-glucuronidase activity had a more prolonged course of diarrhea (≥7 days) compared to patients with lower fecal ß-glucuronidase activity (91% vs 40%, p = .02, Fisher's exact test). Our data reveal post-transplant diarrhea as a complex phenomenon with decreased gut microbial diversity and commensal gut organisms. This study further links commensal bacterial metabolism with an important clinical outcome measure, suggesting fecal ß-glucuronidase activity could be a novel biomarker for gastrointestinal-related MMF toxicity.
Assuntos
Microbioma Gastrointestinal , Transplante de Rim , Diarreia , Glucuronidase , Humanos , RNA Ribossômico 16SRESUMO
The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.
Assuntos
Barreira Hematoencefálica/fisiologia , Neovascularização Fisiológica , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Células Endoteliais/metabolismo , Genes Reporter , Testes Genéticos , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Regiões Promotoras Genéticas/genética , Receptores Acoplados a Proteínas G/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Oftalmopatias Hereditárias/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Miopia/metabolismo , Cegueira Noturna/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Adulto , Animais , Canais de Cálcio Tipo L/genética , Modelos Animais de Doenças , Oftalmopatias Hereditárias/embriologia , Oftalmopatias Hereditárias/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/embriologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Masculino , Miopia/embriologia , Miopia/genética , Cegueira Noturna/embriologia , Cegueira Noturna/genética , Retina/embriologia , Retina/metabolismo , Sinapses/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS(G12V)). METHODS: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated. RESULTS: We demonstrated that transient transgenic expression of KRAS(G12V) in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS(G12V) induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS(G12V) induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord. CONCLUSIONS: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/tratamento farmacológico , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica , Humanos , Imuno-Histoquímica , Queratina-5/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transgenes , Peixe-Zebra , Proteínas ras/metabolismoRESUMO
During neuroinflammation, the proinflammatory cytokine Interleukin-1ß (IL-1ß) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1ß on the in vivo development of the BBB. We generated a doxycycline-inducible transgenic zebrafish model that drives secretion of Il-1ß in the CNS. To validate the utility of our model, we showed Il-1ß dose-dependent mortality, recruitment of neutrophils, and expansion of microglia. Using live imaging, we discovered that Il-1ß causes a significant reduction in CNS angiogenesis and barriergenesis. To demonstrate specificity, we rescued the Il-1ß induced phenotypes by targeting the zebrafish il1r1 gene using CRISPR/Cas9. Mechanistically, we determined that Il-1ß disrupts BBB development by decreasing Wnt/ß-catenin transcriptional activation in brain endothelial cells. Given that several neurodevelopmental disorders are associated with inflammation, our findings support further investigation into the connections between proinflammatory cytokines, neuroinflammation, and neurovascular development.
RESUMO
During neuroinflammation, the proinflammatory cytokine interleukin-1ß (IL-1ß) impacts blood-brain barrier (BBB) function by disrupting brain endothelial tight junctions, promoting vascular permeability, and increasing transmigration of immune cells. Here, we examined the effects of Il-1ß on the in vivo initiation of BBB development. We generated doxycycline-inducible transgenic zebrafish to secrete Il-1ß in the CNS. To validate the utility of our model, we showed Il-1ß dose-dependent mortality, recruitment of neutrophils, and expansion of microglia. Using live imaging, we discovered that Il-1ß causes a significant reduction in CNS angiogenesis and barriergenesis. To demonstrate specificity, we rescued the Il-1ß induced phenotypes by targeting the zebrafish il1r1 gene using CRISPR-Cas9. Mechanistically, we determined that Il-1ß disrupts the initiation of BBB development by decreasing Wnt/ß-catenin transcriptional activation in brain endothelial cells. Given that several neurodevelopmental disorders are associated with inflammation, our findings support further investigation into the connections between proinflammatory cytokines, neuroinflammation, and neurovascular development.
RESUMO
Interleukin-1ß (IL-1ß) is a potent proinflammatory cytokine that plays a vital role in the innate immune system. To observe the innate immune response in vivo, several transgenic zebrafish lines have been developed to model IL-1ß-induced inflammation and to visualize immune cell migration and proliferation in real time. However, our understanding of the IL-1ß response in zebrafish is limited due to an incomplete genome annotation and a lack of functional data for the cytokine receptors involved in the inflammatory process. Here, we use a combination of database mining, genetic analyses, and functional assays to identify zebrafish Interleukin-1 receptor, type 1 (Il1r1). We identified putative zebrafish il1r1 candidate genes that encode proteins with predicted structures similar to human IL1R1. To examine functionality of these candidates, we designed highly effective morpholinos to disrupt gene expression in a zebrafish model of embryonic Il-1ß-induced systemic inflammation. In this double transgenic model, ubb:Gal4-EcR, uas:il1ßmat , the zebrafish ubiquitin b (ubb) promoter drives expression of the modified Gal4 transcription factor fused to the ecdysone receptor (EcR), which in turn drives the tightly-regulated expression and secretion of mature Il-1ß only in the presence of the ecdysone analog tebufenozide (Teb). Application of Teb to ubb:Gal4-EcR, uas:il1ßmat embryos causes premature death, fin degradation, substantial neutrophil expansion, and generation of reactive oxygen species (ROS). To rescue these deleterious phenotypes, we injected ubb:Gal4-EcR, uas:il1ßmat embryos with putative il1r1 morpholinos and found that knockdown of only one candidate gene prevented the adverse effects caused by Il-1ß. Mosaic knockout of il1r1 using the CRISPR/Cas9 system phenocopied these results. Taken together, our study identifies the functional zebrafish Il1r1 utilizing a genetic model of Il-1ß-induced inflammation and provides valuable new insights to study inflammatory conditions specifically driven by Il-1ß or related to Il1r1 function in zebrafish.
Assuntos
Receptores de Interleucina-1 , Peixe-Zebra , Animais , Humanos , Receptores de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Peixe-Zebra/genética , Morfolinos/metabolismo , Inflamação/induzido quimicamente , Inflamação/genéticaRESUMO
The inflammatory response is a vital defense mechanism against trauma and pathogen induced damage, but equally important is its appropriate resolution. In some instances of severe trauma or sustained infection, inappropriate and persistent activation of the immune response can occur, resulting in a dangerous systemic inflammatory response. Untreated, this systemic inflammatory response can lead to tissue damage, organ shutdown, and death. Replicating this condition in tractable model organisms can provide insight into the mechanisms involved in the induction, maintenance, and resolution of inflammation. To that end, we developed a non-invasive, inducible, and reversible model of systemic inflammation in zebrafish. Using the Gal4-EcR/UAS system activated by the ecdysone analog tebufenozide, we generated transgenic zebrafish that allow for chemically induced, ubiquitous secretion of the mature form of zebrafish interleukin-1ß (Il-1ßmat) in both larval and adult developmental stages. To ensure a robust immune response, we attached a strong signal peptide from the Gaussia princeps luciferase enzyme to promote active secretion of the cytokine. We observe a dose-dependent inflammatory response involving neutrophil expansion accompanied by tissue damage and reduced survival. Washout of tebufenozide permits inflammation resolution. We also establish the utility of this model for the identification of small molecule anti-inflammatory compounds by treatment with the immunosuppressant rapamycin. Taken together, these features make this model a valuable new tool that can aid in identifying potential new therapies while broadening our understanding of systemic inflammation, its impact on the immune system, and its resolution.
Assuntos
Inflamação , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Inflamação/genética , Síndrome de Resposta Inflamatória Sistêmica , Proteínas de Peixe-Zebra/genéticaRESUMO
A case of migration of a foreign body from the stomach to the thoracic esophagus is described. The bullet was successfully retrieved endoscopically after exploratory laparotomy was performed to address the patient's injuries. Enteral migration of bullets is a rare phenomenon that should be considered when the location of retained ballistic fragments is inconsistent with gunshot wounds and expected trajectories.
RESUMO
The aryl hydrocarbon receptor (AHR) has endogenous functions in mammalian vascular development and is necessary for mediating the toxic effects of a number of environmental contaminants. Studies in mice have demonstrated that AHR is necessary for the formation of the renal, retinal, and hepatic vasculature. In fish, exposure to the prototypic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the AHR biomarker cyp1a throughout the developing vasculature and produces vascular malformations in the head and heart. However, it is not known whether the vascular structures that are sensitive to loss of AHR function are also disrupted by aberrant AHR activation. Here, we report that TCDD-exposure in zebrafish disrupts development of 1) the subintestinal venous plexus (SIVP), which vascularizes the developing liver, kidney, gut, and pancreas, and 2) the superficial annular vessel (SAV), an essential component of the retinal vasculature. Furthermore, we determined that TCDD exposure increased the expression of bmp4, a key molecular mediator of SIVP morphogenesis. We hypothesize that the observed SIVP phenotypes contribute to one of the hallmarks of TCDD exposure in fish - the failure of the yolk sac to absorb. Together, our data describe novel TCDD-induced vascular phenotypes and provide molecular insight into critical factors producing the observed vascular malformations.
Assuntos
Dibenzodioxinas Policloradas/toxicidade , Veia Retiniana/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fígado/irrigação sanguínea , Veia Retiniana/crescimento & desenvolvimento , Veias/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
INTRODUCTION: The demand for radiation therapy services in New Zealand is growing due to an increasing and ageing population. The radiation therapist (RT) workforce is currently in a vulnerable state and this study aimed to understand RT perceptions on intent to remain in both the workplace and profession. Understanding factors that contribute to satisfaction and retention are important for the development of strategies by healthcare leaders to improve workforce sustainability. MATERIALS AND METHODS: All current practising RTs were invited via email link to complete an online survey. Multivariate regression models were used to investigate any impact of demographic, workplace and professional variables on intent to remain in the workplace and intent to leave the profession. RESULTS: Three hundred and sixty two (91% response rate) RTs completed the survey. Key findings include: a) 33% are thinking of leaving their current workplace with 31% of these intending to leave within the next 12â¯months; b) 35% intend to change careers before they retire; and c) 25% indicated they would leave the profession if they could. Workplace satisfaction, being challenged and a lack of career development opportunities were common factors that influence intention to leave both the workplace and profession. CONCLUSIONS: Strategies to ensure the sustainability of the RT workforce in New Zealand need to focus on developing a robust framework for career development including advanced practice opportunities that challenge RTs and ensuring workplaces create an environment that promote a sense of pride, camaraderie and flexibility in how they operate.
RESUMO
The spliceosome consists of accessory proteins and small nuclear ribonucleoproteins (snRNPs) that remove introns from RNA. As splicing defects are associated with degenerative conditions, a better understanding of spliceosome formation and function is essential. We provide insight into the role of a spliceosome protein U4/U6.U5 tri-snRNP-associated protein 1, or Squamous cell carcinoma antigen recognized by T-cells (Sart1). Sart1 recruits the U4.U6/U5 tri-snRNP complex to nuclear RNA. The complex then associates with U1 and U2 snRNPs to form the spliceosome. A forward genetic screen identifying defects in choroid plexus development and whole-exome sequencing (WES) identified a point mutation in exon 12 of sart1 in Danio rerio (zebrafish). This mutation caused an up-regulation of sart1. Using RNA-Seq analysis, we identified additional upregulated genes, including those involved in apoptosis. We also observed increased activated caspase 3 in the brain and eye and down-regulation of vision-related genes. Although splicing occurs in numerous cells types, sart1 expression in zebrafish was restricted to the brain. By identifying sart1 expression in the brain and cell death within the central nervous system (CNS), we provide additional insights into the role of sart1 in specific tissues. We also characterized sart1's involvement in cell death and vision-related pathways.
Assuntos
Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/metabolismo , Predisposição Genética para Doença , Mutação , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/etiologia , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , Apoptose/genética , Caspase 3/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , Modelos Animais de Doenças , Estudos de Associação Genética , Fenótipo , Análise de Sequência de RNA , Spliceossomos/metabolismo , Sequenciamento do ExomaRESUMO
The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myxoma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish.
Assuntos
Proteínas Hedgehog/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Histocitoquímica , Humanos , Proteínas de Membrana , Receptores Patched , Receptor Patched-1 , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaAssuntos
Inocuidade dos Alimentos , Doenças Transmitidas por Alimentos/prevenção & controle , Saúde Pública/legislação & jurisprudência , Surtos de Doenças/prevenção & controle , Indústria Alimentícia/legislação & jurisprudência , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Estados UnidosRESUMO
Mycophenolate mofetil (MMF) is commonly prescribed and has proven advantages over other immunosuppressive drugs. However, frequent gastrointestinal side effects through an unknown mechanism limit its use. We have found that consumption of MMF alters the composition of the gut microbiota, selecting for bacteria expressing the enzyme ß-glucuronidase (GUS) and leading to an up-regulation of GUS activity in the gut of mice and symptomatic humans. In the mouse, vancomycin eliminated GUS-expressing bacteria and prevented MMF-induced weight loss and colonic inflammation. Our work provides a mechanism for the toxicity associated with MMF and a future direction for the development of therapeutics.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Glucuronidase/metabolismo , Vancomicina/farmacologia , Animais , Proteínas de Bactérias/genética , Peso Corporal/efeitos dos fármacos , Colite/etiologia , Colite/prevenção & controle , Modelos Animais de Doenças , Feminino , Trato Gastrointestinal/microbiologia , Glucuronidase/genética , Imunossupressores/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Ácido Micofenólico/toxicidade , Regulação para Cima/efeitos dos fármacos , Vancomicina/uso terapêuticoRESUMO
The high mobility group transcription factor SOX9 is expressed in stem cells, progenitor cells, and differentiated cell-types in developing and mature organs. Exposure to a variety of toxicants including dioxin, di(2-ethylhexyl) phthalate, 6:2 chlorinated polyfluorinated ether sulfonate, and chlorpyrifos results in the downregulation of tetrapod Sox9 and/or zebrafish sox9b. Disruption of Sox9/sox9b function through environmental exposures or genetic mutations produce a wide range of phenotypes and adversely affect organ development and health. We generated a dominant-negative sox9b (dnsox9b) to inhibit sox9b target gene expression and used the Gal4/UAS system to drive dnsox9b specifically in cardiomyocytes. Cardiomyocyte-specific inhibition of sox9b function resulted in a decrease in ventricular cardiomyocytes, an increase in atrial cardiomyocytes, hypoplastic endothelial cushions, and impaired epicardial development, ultimately culminating in heart failure. Cardiomyocyte-specific dnsox9b expression significantly reduced end diastolic volume, which corresponded with a decrease in stroke volume, ejection fraction, and cardiac output. Further analysis of isolated cardiac tissue by RT-qPCR revealed cardiomyocyte-specific inhibition of sox9b function significantly decreased the expression of the critical cardiac development genes nkx2.5, nkx2.7, and myl7, as well as c-fos, an immediate early gene necessary for cardiomyocyte progenitor differentiation. Together our studies indicate sox9b transcriptional regulation is necessary for cardiomyocyte development and function.
Assuntos
Coração/embriologia , Morfogênese , Miócitos Cardíacos/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Células HEK293 , Humanos , Camundongos , Fatores de Transcrição SOX9/metabolismo , Transcrição Gênica , Peixe-ZebraRESUMO
BACKGROUND: Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has major advantages over other immunosuppressive drugs, but frequent gastrointestinal (GI) side-effects limit its use. The mechanism(s) underlying MMF-related GI toxicity have yet to be elucidated. METHODS: To investigate MMF-related GI toxicity, experimental mice were fed chow containing MMF (0.563%) and multiple indices of toxicity, including weight loss and colonic inflammation, were measured. Changes in intestinal microbial composition were detected using 16S rRNA Illumina sequencing, and downstream PICRUSt analysis was used to predict metagenomic pathways involved. Germ-free (GF) mice and mice treated with orally administered broad-spectrum antibiotics (ABX) were utilized to interrogate the importance of the microbiota in MMF-induced GI toxicity. RESULTS: Mice treated with MMF exhibited significant weight loss, related to loss of body fat and muscle, and marked colonic inflammation. MMF exposure was associated with changes in gut microbial composition, as demonstrated by a loss of overall diversity, expansion of Proteobacteria (specifically Escherichia/Shigella), and enrichment of genes involved in lipopolysaccharide (LPS) biosynthesis, which paralleled increased levels of LPS in the feces and serum. MMF-related GI toxicity was dependent on the intestinal microbiota, as MMF did not induce weight loss or colonic inflammation in GF mice. Furthermore, ABX prevented and reversed MMF-induced weight loss and colonic inflammation. CONCLUSIONS: An intact intestinal microbiota is required to initiate and sustain the GI toxicity of MMF. MMF treatment causes dynamic changes in the composition of the intestinal microbiota that may be a targetable driver of the GI side-effects of MMF.
Assuntos
Modelos Animais de Doenças , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Imunossupressores/toxicidade , Microbiota/efeitos dos fármacos , Ácido Micofenólico/toxicidade , Animais , Colo/efeitos dos fármacos , Colo/microbiologia , Vida Livre de Germes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunossupressores/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos , Microbiota/imunologia , Ácido Micofenólico/uso terapêutico , Proteobactérias , RNA Ribossômico 16S , Análise de Sequência de RNA , Redução de Peso/efeitos dos fármacosRESUMO
Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration-approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing hearing loss.
Assuntos
Cisplatino/efeitos adversos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Perda Auditiva Provocada por Ruído/induzido quimicamente , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Citoproteção/efeitos dos fármacos , Resistência a Medicamentos , Células Germinativas/metabolismo , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Indóis/farmacologia , Indóis/uso terapêutico , Sistema da Linha Lateral/efeitos dos fármacos , Sistema da Linha Lateral/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/análise , Peixe-ZebraRESUMO
PURPOSE: To identify in vivo a promoter fragment that specifically directs transgene expression in all zebrafish cone photoreceptors. This promoter subsequently enables GFP labeling of cones for facile morphologic analysis and purification and genetic rescue of achromatopsia. METHODS: Promoter fragments of the zebrafish cone transducin alpha (TalphaC) gene were subcloned upstream of EGFP and microinjected into one- to two-cell-stage embryos. Promoter activity was assessed by fluorescence microscopy in wholemounts and retinal cryosections, and cone photoreceptors were purified by flow cytometry. Visual physiology was assessed by the optokinetic response (OKR) assay. RESULTS: A 3.2-kb promoter fragment from zebrafish TalphaC specifically directed robust transgene expression in retinal cone photoreceptors and pineal photoreceptors. With this promoter, a stable transgenic line expressing EGFP in all zebrafish cone photoreceptors types was generated, and populations of cones were purified. Achromatopsia in the nof mutant was rescued using the identified promoter fragment to direct transgenic expression of wild-type cone transducin in mutant cones. CONCLUSIONS: A 3.2-kb TalphaC promoter fragment replicates the temporal and spatial pattern of endogenous TalphaC expression. The integrity of cones can be readily assessed in an EGFP transgenic line generated with this promoter, enabling downstream genetic and chemical screens for cone determinants.