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1.
Gastroenterology ; 161(1): 287-300.e16, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33771553

RESUMO

BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ductos Biliares Intra-Hepáticos/patologia , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/patologia , Mutação , Proteínas de Peixe-Zebra/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose , Ductos Biliares Intra-Hepáticos/metabolismo , Estudos de Casos e Controles , Colestase Intra-Hepática/metabolismo , Doença Crônica , Feminino , Edição de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sequenciamento do Exoma , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
2.
Hepatology ; 70(6): 2107-2122, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31016744

RESUMO

The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.


Assuntos
Células Estreladas do Fígado/fisiologia , Cirrose Hepática/etiologia , Manose-6-Fosfato Isomerase/fisiologia , Manose/farmacologia , Animais , Células Cultivadas , Glicosilação , Humanos , Masculino , Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra
3.
Semin Cell Dev Biol ; 63: 68-78, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27552918

RESUMO

The liver and pancreas are the prime digestive and metabolic organs in the body. After emerging from the neighboring domains of the foregut endoderm, they turn on distinct differentiation and morphogenesis programs that are regulated by hierarchies of transcription factors. Members of SOX family of transcription factors are expressed in the liver and pancreas throughout development and act upstream of other organ-specific transcription factors. They play key roles in maintaining stem cells and progenitors. They are also master regulators of cell fate determination and tissue morphogenesis. In this review, we summarize the current understanding of SOX transcription factors in mediating liver and pancreas development. We discuss their contribution to adult organ function, homeostasis and injury responses. We also speculate how the knowledge of SOX transcription factors can be applied to improve therapies for liver diseases and diabetes.


Assuntos
Ductos Biliares/embriologia , Fígado/embriologia , Pâncreas/embriologia , Fatores de Transcrição SOX/metabolismo , Animais , Homeostase , Humanos , Modelos Biológicos
4.
Hepatology ; 67(4): 1531-1545, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29091294

RESUMO

Bile salt export pump (BSEP) adenosine triphosphate-binding cassette B11 (ABCB11) is a liver-specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9-based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. CONCLUSIONS: Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Bile/metabolismo , Colestase Intra-Hepática/genética , Hepatócitos/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Autofagia/genética , Colestase Intra-Hepática/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Lactente , Fígado/patologia , Masculino , Mutação , Sirolimo/farmacologia , Peixe-Zebra/metabolismo
5.
Development ; 140(13): 2734-45, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23757411

RESUMO

Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin ß1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffer's vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis.


Assuntos
Laminina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Cílios/metabolismo , Lateralidade Funcional/genética , Lateralidade Funcional/fisiologia , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Laminina/genética , Organogênese/genética , Organogênese/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
6.
PLoS Genet ; 8(6): e1002754, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719264

RESUMO

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.


Assuntos
Ductos Biliares Intra-Hepáticos/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Pâncreas/crescimento & desenvolvimento , Fatores de Transcrição SOX9/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Ductos Biliares Intra-Hepáticos/embriologia , Ductos Biliares Intra-Hepáticos/metabolismo , Códon sem Sentido , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Fígado/metabolismo , Morfogênese/genética , Pâncreas/embriologia , Pâncreas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
Hepatology ; 56(5): 1958-70, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22488653

RESUMO

UNLABELLED: Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development. CONCLUSION: Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior.


Assuntos
Comunicação Celular , Movimento Celular , Células Endoteliais/citologia , Células Estreladas do Fígado/citologia , Fígado/citologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzoatos/farmacologia , Contagem de Células , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Etanol/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/crescimento & desenvolvimento , Miocárdio/metabolismo , Crista Neural/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Tetra-Hidronaftalenos/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
J Orthop Surg (Hong Kong) ; 31(2): 10225536231177102, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37288764

RESUMO

BACKGROUND: Metastasis is one of the most significant prognostic factors in osteosarcoma (OS). The goal of this study was to construct a clinical prediction model for OS patients in a population cohort and to evaluate the factors influencing the occurrence of pulmonary metastasis. METHODS: We collected data from 612 patients with osteosarcoma (OS), and 103 clinical indicators were collected. After the data were filtered, the patients were randomly divided into training and validation cohorts by using random sampling. The training cohort included 191 patients with pulmonary metastasis in OS and 126 patients with non-pulmonary metastasis, and the validation cohort included 50 patients with pulmonary metastasis in OS and 57 patients with non-pulmonary metastasis. Univariate logistics regression analysis, LASSO regression analysis and multivariate logistic regression analysis were performed to identify potential risk factors for pulmonary metastasis in patients with osteosarcoma. A nomogram was developed that included risk influencing variables selected by multivariable analysis, and used the concordance index (C-index) and calibration curve to validate the model. Receiver operating characteristic curve (ROC), decision analysis curve (DCA) and clinical impact curve (CIC) were employed to assess the model. In addition, we used a predictive model on the validation cohort. RESULTS: Logistic regression analysis was used to identify independent predictors [N Stage + Alkaline phosphatase (ALP)+Thyroid stimulating hormone (TSH)+Free triiodothyronine (FT3)]. A nomogram was constructed to predict the risk of pulmonary metastasis in patients with osteosarcoma. The performance was evaluated by the concordance index (C-index) and calibration curve. The ROC curve provides the predictive power of the nomogram (AUC = 0.701 in the training cohort, AUC = 0.786 in the training cohort). Decision curve analysis (DCA) and clinical impact curve (CIC) demonstrated the clinical value of the nomogram and higher overall net benefits. CONCLUSIONS: Our study can help clinicians effectively predict the risk of lung metastases in osteosarcoma with more readily available clinical indicators, provide more personalized diagnosis and treatment guidance, and improve the prognosis of patients. MINI ABSTRACT: A new risk model was constructed to predict the pulmonary metastasis in patients with osteosarcoma based on multiple machine learning.


Assuntos
Neoplasias Ósseas , Neoplasias Pulmonares , Osteossarcoma , Humanos , Prognóstico , Modelos Estatísticos , Aprendizado de Máquina
9.
J Clin Invest ; 133(18)2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37490339

RESUMO

X-linked myotubular myopathy (XLMTM) is a fatal congenital disorder caused by mutations in the MTM1 gene. Currently, there are no approved treatments, although AAV8-mediated gene transfer therapy has shown promise in animal models and preliminarily in patients. However, 4 patients with XLMTM treated with gene therapy have died from progressive liver failure, and hepatobiliary disease has now been recognized more broadly in association with XLMTM. In an attempt to understand whether loss of MTM1 itself is associated with liver pathology, we have characterized what we believe to be a novel liver phenotype in a zebrafish model of this disease. Specifically, we found that loss-of-function mutations in mtm1 led to severe liver abnormalities including impaired bile flux, structural abnormalities of the bile canaliculus, and improper endosome-mediated trafficking of canalicular transporters. Using a reporter-tagged Mtm1 zebrafish line, we established localization of Mtm1 in the liver in association with Rab11, a marker of recycling endosomes, and canalicular transport proteins and demonstrated that hepatocyte-specific reexpression of Mtm1 could rescue the cholestatic phenotype. Last, we completed a targeted chemical screen and found that Dynasore, a dynamin-2 inhibitor, was able to partially restore bile flow and transporter localization to the canalicular membrane. In summary, we demonstrate, for the first time to our knowledge, liver abnormalities that were directly caused by MTM1 mutation in a preclinical model, thus establishing the critical framework for better understanding and comprehensive treatment of the human disease.


Assuntos
Miopatias Congênitas Estruturais , Peixe-Zebra , Animais , Humanos , Modelos Animais de Doenças , Proteínas de Membrana Transportadoras/metabolismo , Músculo Esquelético/metabolismo , Mutação , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/terapia , Miopatias Congênitas Estruturais/patologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
10.
Hepatol Commun ; 6(11): 3083-3097, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36017776

RESUMO

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.


Assuntos
Cistos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Pró-Proteína Convertases/genética , Mutação de Sentido Incorreto/genética , Tunicamicina , Actinas/genética , Modelos Animais de Doenças , Fígado/patologia , Cistos/genética , Serina-Treonina Quinases TOR/genética , Mamíferos
11.
Cell Metab ; 4(2): 155-62, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16890543

RESUMO

The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.


Assuntos
Adenosina Trifosfatases/genética , Cobre/metabolismo , Notocorda/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , ATPases Transportadoras de Cobre , Embrião não Mamífero , Dados de Sequência Molecular , Fenótipo , Peixe-Zebra/genética
12.
Front Microbiol ; 12: 753823, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34733263

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes is critical for treatment and prevention. Here, we analyzed all high-coverage complete SARS-CoV-2 sequences from GISAID database from January 1, 2020, to January 1, 2021, to mine the mutation hotspots associated with clinical outcome and developed a model to predict the clinical outcome in different epidemic strains. Exploring the cause of mutation based on RNA-dependent RNA polymerase (RdRp) and RNA-editing enzyme, mutation was more likely to occur in severe and mild cases than in asymptomatic cases, especially A > G, C > T, and G > A mutations. The mutations associated with asymptomatic outcome were mainly in open reading frame 1ab (ORF1ab) and N genes; especially R6997P and V30L mutations occurred together and were correlated with asymptomatic outcome with high prevalence. D614G, Q57H, and S194L mutations were correlated with mild and severe outcome with high prevalence. Interestingly, the single-nucleotide variant (SNV) frequency was higher with high percentage of nt14408 mutation in RdRp in severe cases. The expression of ADAR and APOBEC was associated with clinical outcome. The model has shown that the asymptomatic percentage has increased over time, while there is high symptomatic percentage in Alpha, Beta, and Gamma. These findings suggest that mutation in the SARS-CoV-2 genome may have a direct association with clinical outcomes and pandemic. Our result and model are helpful to predict the prevalence of epidemic strains and to further study the mechanism of mutation causing severe disease.

13.
J Cell Biol ; 169(5): 777-87, 2005 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-15928205

RESUMO

Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.


Assuntos
Desenvolvimento Embrionário/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Gástrula/metabolismo , Mesoderma/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Padronização Corporal/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Forma Celular/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Gástrula/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/citologia , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt
14.
Methods Mol Biol ; 1981: 273-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016661

RESUMO

Cholestasis is a condition that impairs bile flow, resulting in retention of bile fluid in the liver. It may cause significant morbidity and mortality due to pruritus, malnutrition, and complications from portal hypertension secondary to biliary cirrhosis. The zebrafish (Danio rerio) has emerged as a valuable model organism for studying cholestasis that complements with the in vitro systems and rodent models. Its main advantages include conserved mechanisms of liver development and bile formation, rapid external development, ease of monitoring hepatobiliary morphology and function in live larvae, and accessibility to genetic and chemical manipulations. In this chapter, we provide an overview of the existing zebrafish models of cholestatic liver diseases. We discuss the strengths and limitations of using zebrafish to study cholestasis. We also provide step-by-step descriptions of the methodologies for analyzing cholestatic phenotypes in zebrafish.


Assuntos
Colestase/patologia , Modelos Animais de Doenças , Hepatopatias/patologia , Peixe-Zebra , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colestase/metabolismo , Fígado/metabolismo , Fígado/patologia , Hepatopatias/metabolismo
15.
Front Immunol ; 10: 2840, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31867007

RESUMO

Hepatic macrophages are key components of the liver immunity and consist of two main populations. Liver resident macrophages, known as Kupffer cells in mammals, are crucial for maintaining normal liver homeostasis. Upon injury, they become activated to release proinflammatory cytokines and chemokines and recruit a large population of inflammatory monocyte-derived macrophages to the liver. During the progression of liver diseases, macrophages are highly plastic and have opposing functions depending on the signaling cues that they receive from the microenvironment. A comprehensive understanding of liver macrophages is essential for developing therapeutic interventions that target these cells in acute and chronic liver diseases. Mouse studies have provided the bulk of our current knowledge of liver macrophages. The emergence of various liver disease models and availability of transgenic tools to visualize and manipulate macrophages have made the teleost zebrafish (Danio rerio) an attractive new vertebrate model to study liver macrophages. In this review, we summarize the origin and behaviors of macrophages in healthy and injured livers in zebrafish. We highlight the roles of macrophages in zebrafish models of alcoholic and non-alcoholic liver diseases, hepatocellular carcinoma, and liver regeneration, and how they compare with the roles that have been described in mammals. We also discuss the advantages and challenges of using zebrafish to study liver macrophages.


Assuntos
Modelos Animais de Doenças , Hepatopatias/imunologia , Macrófagos/fisiologia , Animais , Hematopoese , Hepatopatias Alcoólicas/imunologia , Neoplasias Hepáticas/imunologia , Regeneração Hepática , Peixe-Zebra
16.
Sci Rep ; 9(1): 5807, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967584

RESUMO

In this paper, a double hollow spherical shell composite modified with layered double hydroxide (C@Ni-Al LDH HSS) was fabricated for uranium(VI) (U(VI)) adsorption. Various batch experiments were carried out to investigate the influence of pH, concentration, time and coexistence ion on extraction. The results showed that the adsorption processes of U(VI) onto C@Ni-Al LDH HSS were spontaneous and endothermic and closely followed pseudo-second-order and Langmuir isotherm models. The equilibrium time and maximum adsorption capacity of C@Ni-Al LDH HSS was 360 min and 545.9 mg g-1. FT-IR and XPS analyses proved that the adsorption behavior was primarily attributed to the strong interaction between oxygen-containing functional groups and U(VI). Moreover, the extraction of trace U(VI) (µg L-1) in artificial and natural seawater was also studied. The results showed that C@Ni-Al LDH HSS provided a promising application for the efficient extraction of U(VI) from seawater.

17.
Neuro Oncol ; 10(1): 45-51, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18182627

RESUMO

Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Histona-Lisina N-Metiltransferase/biossíntese , Proteínas Repressoras/biossíntese , Western Blotting , Proliferação de Células , Cromatografia Líquida , Humanos , Imuno-Histoquímica , Proteômica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Transfecção
18.
J Neurosurg ; 109(3): 536-46, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18759588

RESUMO

OBJECT: The lack of primary lymphoid tissue within the central nervous system (CNS) confounds our understanding of the pathogenesis of primary CNS lymphomas (PCNSLs). Comparing the protein expression of PCNSLs and sporadic systemic lymphomas (SSLs) provides a useful strategy for identifying a molecular signature that characterizes disease-associated features and provides information regarding tumor initiation and progression. METHODS: Seven diffuse large B-cell PCNSLs were selected to undergo 2D gel electrophoresis, and profiled proteomes from these PCNSLs were compared with those from 7 diffuse large B-cell SSLs. Distinguishing proteins were sequenced using mass spectrometry. RESULTS: Two-dimensional gel electrophoresis identified an average of 706 proteins from each specimen. Computerized gel analysis and manual reconfirmation revealed a 96% similarity in the proteomes of PCNSLs and SSLs. Comparative analysis identified 9 proteins significantly overexpressed (p < 0.05) and 16 proteins downregulated in PCNSLs. The proteomic findings were further validated using Western blot and immunohistochemical staining. CONCLUSIONS: The similarities in proteomic patterns between PCNSLs and SSLs suggest that these tumor types share structural similarities, acquired during differentiation. The ultimate fate of lymphomatous cells (CNS vs systemic) may be related to differentially expressed proteins, which function in homing and host processing. Elucidating the roles of these differentially expressed proteins will prove valuable in understanding the pathogenesis of PCNSL.


Assuntos
Neoplasias do Sistema Nervoso Central/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Estudos de Casos e Controles , Neoplasias do Sistema Nervoso Central/etiologia , Neoplasias do Sistema Nervoso Central/patologia , Eletroforese em Gel Bidimensional , Feminino , Humanos , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica
20.
Nat Commun ; 9(1): 1319, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615614

RESUMO

Anteroposterior (AP) axis extension during gastrulation requires embryonic patterning and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation through direct positive and negative regulation of gene expression. Although identified as a recessive enhancer of impaired axis extension in planar cell polarity (PCP) mutants, udu functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. Gon4l limits expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrinα3b, excesses of which perturb the notochord boundary via tension-dependent and -independent mechanisms, respectively. By promoting formation of this AP-aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis along the AP embryonic axis.


Assuntos
Fatores de Ligação de DNA Eritroide Específicos/genética , Fatores de Ligação de DNA Eritroide Específicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Padronização Corporal , Adesão Celular , Comunicação Celular , Cromatina/química , Cruzamentos Genéticos , Glicoproteínas de Membrana/fisiologia , Mutação , Notocorda/fisiologia , Análise de Sequência de RNA , Transdução de Sinais , Xenopus , Peixe-Zebra
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