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1.
Sci Rep ; 13(1): 15597, 2023 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-37730847

RESUMO

Prostate cancer (PCa) stands as a prominent contributor to morbidity and mortality among males on a global scale. Cancer-associated fibroblasts (CAFs) are considered to be closely connected to tumour growth, invasion, and metastasis. We explored the role and characteristics of CAFs in PCa through bioinformatics analysis and built a CAFs-based risk model to predict prognostic treatment and treatment response in PCa patients. First, we downloaded the scRNA-seq data for PCa from the GEO. We extracted bulk RNA-seq data for PCa from the TCGA and GEO and adopted "ComBat" to remove batch effects. Then, we created a Seurat object for the scRNA-seq data using the package "Seurat" in R and identified CAF clusters based on the CAF-related genes (CAFRGs). Based on CAFRGs, a prognostic model was constructed by univariate Cox, LASSO, and multivariate Cox analyses. And the model was validated internally and externally by Kaplan-Meier analysis, respectively. We further performed GO and KEGG analyses of DEGs between risk groups. Besides, we investigated differences in somatic mutations between different risk groups. We explored differences in the immune microenvironment landscape and ICG expression levels in the different groups. Finally, we predicted the response to immunotherapy and the sensitivity of antitumour drugs between the different groups. We screened 4 CAF clusters and identified 463 CAFRGs in PCa scRNA-seq. We constructed a model containing 10 prognostic CAFRGs by univariate Cox, LASSO, and multivariate Cox analysis. Somatic mutation analysis revealed that TTN and TP53 were significantly more mutated in the high-risk group. Finally, we screened 31 chemotherapeutic drugs and targeted therapeutic drugs for PCa. In conclusion, we identified four clusters based on CAFs and constructed a new CAFs-based prognostic signature that could predict PCa patient prognosis and response to immunotherapy and might suggest meaningful clinical options for the treatment of PCa.


Assuntos
Imunoterapia , Neoplasias da Próstata , Masculino , Humanos , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Sequência de Bases , RNA-Seq , Microambiente Tumoral/genética
2.
Sci Adv ; 9(26): eade6308, 2023 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-37390204

RESUMO

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.


Assuntos
Ácido N-Acetilneuramínico , Peixe-Zebra , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Glicoproteínas , Músculo Esquelético , Piruvatos , Regeneração
3.
PLoS One ; 16(11): e0260442, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34818339

RESUMO

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.


Assuntos
Precondicionamento Isquêmico/métodos , NADPH Oxidases/genética , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Plasma , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , NADPH Oxidases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Plasma/metabolismo , Regulação para Cima , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
5.
J Cell Physiol ; 236(5): 3700-3709, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33052609

RESUMO

Cardiac hypertrophy is a compensatory response to pathological stimuli, ultimately progresses to cardiomyopathy, heart failure, or sudden death. Although many signaling pathways have been reported to be involved in the hypertrophic process, it is still not fully clear about the underlying molecular mechanisms for cardiac hypertrophy. Hedgehog acyltransferase-like (Hhatl), a sarcoplasmic reticulum-resident protein, exhibits high expression in the heart and muscle. However, the biological role of Hhatl in the heart remains unknown. In this study, we first found that the expression level of Hhatl is markedly decreased in cardiac hypertrophy. We further studied the role of hhatla, homolog of Hhatl with the zebrafish model. The depletion of hhatla in zebrafish leads to cardiac defects, as well as an enhanced level of hypertrophic markers. Besides, we found that calcineurin signaling participates in hhatla depletion-induced cardiac hypertrophy. Together, these results demonstrate a critical role for hhatla in cardiac hypertrophy.


Assuntos
Aciltransferases/metabolismo , Cardiomegalia/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Aciltransferases/genética , Animais , Biomarcadores/metabolismo , Calcineurina/metabolismo , Cardiomegalia/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica , Ventrículos do Coração/patologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
6.
Commun Biol ; 3(1): 672, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188360

RESUMO

Hematopoietic adaptor containing SH3 and SAM domains-1 (HACS1) is a signaling protein with two juxtaposed protein-protein interaction domains and an intrinsically unstructured region that spans half the sequence. Here, we describe the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif (ITIM3) of the paired immunoglobulin receptor B (PIRB). From surface plasmon resonance binding assays using a mouse and human PIRB ITIM3 phosphopeptides as ligands, the HACS1 SH3 domain and SHP2 N-terminal SH2 domain demonstrated comparable affinities in the micromolar range. Since the PIRB ITIM3 sequence represents an atypical ligand for an SH3 domain, we determined the NMR structure of the HACS1 SH3 domain and performed a chemical shift mapping study. This study showed that the binding site on the HACS1 SH3 domain for PIRB shares many of the same amino acids found in a canonical binding cleft normally associated with polyproline ligands. Molecular modeling suggests that the respective binding sites in PIRB ITIM3 for the HACS1 SH3 domain and the SHP2 SH2 domain are too close to permit simultaneous binding. As a result, the HACS1-PIRB partnership has the potential to amalgamate signaling pathways that influence both immune and neuronal cell fate.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Glicoproteínas de Membrana , Receptores Imunológicos , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Sítios de Ligação , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais , Domínios de Homologia de src
7.
J Cell Mol Med ; 24(22): 13151-13162, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32989924

RESUMO

Cardiac hypertrophy is a common pathological change in patients with progressive cardiac function failure, which can be caused by hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or arterial hypertension. Despite years of study, there is still limited knowledge about the underlying molecular mechanisms for cardiac hypertrophy. NDUFA7, a subunit of NADH:ubiquinone oxidoreductase (complex I), has been reported to be a novel HCM associated gene. However, the biological role of NDUFA7 in heart remains unknown. In this study, we found that NDUFA7 exhibited high expression in the heart, and its level was significantly decreased in mice model of cardiac hypertrophy. Moreover, we demonstrated that ndufa7 knockdown in developing zebrafish embryos resulted in cardiac development and functional defects, associated with increased expression of pathological hypertrophy biomarkers nppa (ANP) and nppb (BNP). Mechanistic study demonstrated that ndufa7 depletion promoted ROS production and calcineurin signalling activation. Moreover, NDUFA7 depletion contributed to cardiac cell hypertrophy. Together, these results report for the first time that ndufa7 is implicated in pathological cardiac hypertrophy.


Assuntos
Cardiomegalia/patologia , Cardiomiopatia Hipertrófica/patologia , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Artérias/metabolismo , Biomarcadores/metabolismo , Calcineurina/metabolismo , Cardiomegalia/enzimologia , Cardiomiopatia Hipertrófica/enzimologia , Linhagem Celular , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/genética , Técnicas de Silenciamento de Genes , Genótipo , Coração/crescimento & desenvolvimento , Coração/fisiopatologia , Insuficiência Cardíaca/metabolismo , Hipertensão/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Distribuição Tecidual , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
8.
Elife ; 92020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32779569

RESUMO

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.


The human genome counts over 20,000 genes, which can be turned on and off to create the proteins required for most of life processes. Once produced, proteins need move to specific locations in the cell, where they are able to perform their jobs. Despite striking scientific advances, 90% of human genes are still under-studied; where the proteins they code for go, and what they do remains unknown. Zebrafish share many genes with humans, but they are much easier to manipulate genetically. Here, Ichino et al. used various methods in zebrafish to create a detailed 'catalogue' of previously poorly understood genes, focusing on where the proteins they coded for ended up and the biological processes they were involved with. First, a genetic tool called gene-breaking transposons (GBTs) was used to create over 1,200 strains of genetically altered fish in which a specific protein was both tagged with a luminescent marker and unable to perform its role. Further analysis of 204 of these strains revealed new insight into the role of each protein, with many having unexpected roles and localisations. For example, in one zebrafish strain, the affected gene was similar to a human gene which, when inactivated, causes severe muscle weakness. These fish swam abnormally slowly and also had muscle problems, suggesting that the GBT fish strains could 'model' the human disease. This work sheds new light on the role of many previously poorly understood genes. In the future, similar collections of GBT fish strains could help researchers to study both normal human biology and disease. They could especially be useful in cases where the genes responsible for certain conditions are still difficult to identify.


Assuntos
Técnicas de Silenciamento de Genes , Biblioteca Gênica , Genes Reporter , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
9.
Am J Hum Genet ; 106(2): 143-152, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32032513

RESUMO

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.


Assuntos
Modelos Animais de Doenças , Marcadores Genéticos , Doenças Raras/genética , Doenças Raras/terapia , Sistema de Registros/normas , Animais , Bases de Dados Factuais , Genômica , Humanos , Doenças Raras/epidemiologia
10.
PLoS One ; 14(11): e0221796, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31730619

RESUMO

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3' Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.


Assuntos
Inibidores da Angiogênese/farmacologia , Automação Laboratorial/métodos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Peixe-Zebra , Algoritmos , Animais , Animais Geneticamente Modificados , Automação Laboratorial/instrumentação , Relação Dose-Resposta a Droga , Descoberta de Drogas/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero , Células Endoteliais/efeitos dos fármacos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/instrumentação , Indóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Oximas/farmacologia
11.
Am J Hum Genet ; 105(3): 534-548, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422819

RESUMO

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.


Assuntos
Alelos , Ácido Aspártico/metabolismo , Encefalopatias/genética , Proteínas de Ligação a Ácido Graxo/genética , Malatos/metabolismo , Mutação , Animais , Criança , Pré-Escolar , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Sequenciamento do Exoma
12.
Cancers (Basel) ; 11(7)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323958

RESUMO

Identifying novel anti-cancer drugs is important for devising better cancer treatment options. In a series of studies designed to identify novel therapeutic compounds, we recently showed that a C-20 fatty acid (12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid, a furanoic acid or F-6) present in the lipid fraction of the secretions of the Arabian Gulf catfish skin (Arius bilineatus Val.; AGCS) robustly induces neutrophil extracellular trap formation. Here, we demonstrate that a lipid mix (Ft-3) extracted from AGCS and F-6, a component of Ft-3, dose dependently kill two cancer cell lines (leukemic K-562 and breast MDA MB-231). Pure F-6 is approximately 3.5 to 16 times more effective than Ft-3 in killing these cancer cells, respectively. Multiplex assays and network analyses show that F-6 promotes the activation of MAPKs such as Erk, JNK, and p38, and specifically suppresses JNK-mediated c-Jun activation necessary for AP-1-mediated cell survival pathways. In both cell lines, F-6 suppresses PI3K-Akt-mTOR pathway specific proteins, indicating that cell proliferation and Akt-mediated protection of mitochondrial stability are compromised by this treatment. Western blot analyses of cleaved caspase 3 (cCasp3) and poly ADP ribose polymerase (PARP) confirmed that F-6 dose-dependently induced apoptosis in both of these cell lines. In 14-day cell recovery experiments, cells treated with increasing doses of F-6 and Ft-3 fail to recover after subsequent drug washout. In summary, this study demonstrates that C-20 furanoic acid F-6, suppresses cancer cell proliferation and promotes apoptotic cell death in leukemic and breast cancer cells, and prevents cell recovery. Therefore, F-6 is a potential anti-cancer drug candidate.

13.
Neurotherapeutics ; 16(4): 1149-1166, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31342410

RESUMO

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder in which the neuromuscular junction progressively degenerates, leading to movement difficulties, paralysis, and eventually death. ALS is currently being treated by only two FDA-approved drugs with modest efficacy in slowing disease progression. Often, the translation of preclinical findings to bedside terminates prematurely as the evaluation of potential therapeutic compounds focuses on a single study or a single animal model. To circumscribe these issues, we screened 3,765 novel small molecule derivatives of pimozide, a recently identified repurposed neuroleptic for ALS, in Caenorhabditis elegans, confirmed the hits in zebrafish and validated the most active compounds in mouse genetic models. Out of the 27 small molecules identified from the high-throughput screen in worms, 4 were found to recover locomotor defects in C. elegans and genetic zebrafish models of ALS. TRVA242 was identified as the most potent compound as it significantly improved efficiency in rescuing locomotor, motorneuron, and neuromuscular junction synaptic deficits in a C. elegans TDP-43 model and in multiple zebrafish genetic (TDP-43, SOD1, and C9ORF72) models of ALS. The actions of TRVA242 were also conserved in a mammalian model as it also stabilized neuromuscular junction deficits in a mouse SOD1 model of ALS. Compounds such as TRVA242 therefore represent new potential therapeutics for the treatment of ALS.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Modelos Animais de Doenças , Junção Neuromuscular/genética , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Ligação a DNA/administração & dosagem , Proteínas de Ligação a DNA/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Técnicas de Cultura de Órgãos , Pimozida/administração & dosagem , Pimozida/metabolismo , Peixe-Zebra
14.
Hum Gene Ther ; 30(9): 1101-1116, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31099266

RESUMO

Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 make permanent CF transmembrane conductance regulator (CFTR) gene correction possible. However, because of safety concerns of the CRISPR/Cas9 system and challenges in in vivo delivery to inflamed CF airway, CRISPR-based gene correction strategies need to be tested in proper animal models. In this study, we aimed at creating vectors for testing CFTR gene correction in pig models. We constructed helper-dependent adenoviral (HD-Ad) vectors to deliver CRISPR/Cas9 and a donor template (a 6 kb LacZ or 8.7 kb human CFTR expression cassette) into cultured pig cells. We demonstrated precise integration of each donor into the GGTA1 safe harbor through Cas9-induced homology directed repair with 3 kb homology arms. In addition, we showed that both LacZ and hCFTR were persistently expressed in transduced cells. Furthermore, we created a CFTR-deficient cell line for testing CFTR correction. We detected hCFTR mRNA and protein expression in cells transduced with the hCFTR vector. We also demonstrated CFTR function in the CF cells transduced with the HD-Ad delivering the CRISPR-Cas9 system and hCFTR donor at late cellular passages using the membrane potential sensitive dye-based assay (FLIPR®). Combined with our previous report on gene delivery to pig airway basal cells, these data provide the feasibility of testing CRISPR/Cas9-mediated permanent human CFTR correction through HD-Ad vector delivery in pigs.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Edição de Genes , Animais , Linhagem Celular , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Galactosiltransferases/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Marcação de Genes , Técnicas de Transferência de Genes , Genes Reporter , Loci Gênicos , Terapia Genética , Vetores Genéticos/genética , Modelos Biológicos , Mutagênese Insercional , Suínos , Transdução Genética , Transgenes
15.
N Engl J Med ; 380(15): 1433-1441, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30970188

RESUMO

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Ataxia/genética , Deficiências do Desenvolvimento/genética , Glutaminase/deficiência , Glutaminase/genética , Glutamina/metabolismo , Repetições de Microssatélites , Mutação , Atrofia/genética , Cerebelo/patologia , Pré-Escolar , Feminino , Genótipo , Glutamina/análise , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Sequenciamento Completo do Genoma
16.
Antioxid Redox Signal ; 30(14): 1760-1773, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30403148

RESUMO

AIMS: Remote ischemic conditioning (RIC) protects against organ ischemia/reperfusion injury in experimental and clinical settings. We have demonstrated that RIC prevents liver and lung inflammation/injury after hemorrhagic shock/resuscitation (S/R). In this study, we used a murine model of S/R to investigate the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in mediating hepatoprotection. RESULTS: The combination of RIC with S/R caused a synergistic rise in Nrf2 and its translocation to the nucleus in the liver. Increased activation of Nrf2 by RIC augmented heme oxygenase-1 (HO-1) and autophagy and exerted hepatoprotection, concurrent with reductions in S/R-induced TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6). In Nrf2 knockout (KO) animals, RIC did not exert hepatoprotection, and it failed to upregulate HO-1 and autophagy. Further, resuscitating wildtype (WT) animals with blood from donor WT animals undergoing RIC was hepatoprotective, but not in Nrf2 KO recipient animals. Interestingly, RIC blood from Nrf2 KO donor animals was also not protective when used to resuscitate WT animals, suggesting a role for Nrf2 both in the afferent arm of RIC where protective factors are generated and also in the efferent arm where organ protection is exerted. Finally, RIC plasma prevented oxidant-induced zebrafish mortality, but not in Nrf2a morpholino knockdown fish. INNOVATION: Activation of Nrf2 is an essential mechanism underlying the hepatoprotective effects of RIC. Nrf2 appears to play a role in the afferent limb of RIC protection, as its absence precludes the generation of the protective humoral factors induced by RIC. CONCLUSION: Our studies demonstrate the critical role of Nrf2 in the ability of RIC to prevent organ injury after S/R.


Assuntos
Precondicionamento Isquêmico , Fígado/irrigação sanguínea , Fígado/metabolismo , Fator 2 Relacionado a NF-E2/genética , Choque Hemorrágico/metabolismo , Animais , Autofagia/genética , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Insuficiência Hepática/etiologia , Insuficiência Hepática/metabolismo , Insuficiência Hepática/patologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Fígado/patologia , Fígado/ultraestrutura , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Choque Hemorrágico/complicações , Choque Hemorrágico/etiologia , Transdução de Sinais
17.
JCI Insight ; 3(24)2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30568043

RESUMO

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.


Assuntos
Músculo Esquelético/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Adulto , Animais , Modelos Animais de Doenças , Edema Cardíaco/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Células HEK293 , Hexosaminas/metabolismo , Humanos , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Doenças Musculares/fisiopatologia , Mutação , Oxo-Ácido-Liases/uso terapêutico , Doença do Armazenamento de Ácido Siálico/metabolismo , Adulto Jovem , Peixe-Zebra/embriologia
18.
Respir Res ; 19(1): 190, 2018 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-30268129

RESUMO

BACKGROUND: Pneumonia is a major cause of high morbidity and mortality in critically illness, and frequently requires support with mechanical ventilation. The latter can lead to ventilator-induced lung injury characterized by neutrophil infiltration. The cationic human neutrophil peptides (HNP) stored in neutrophils can kill microorganisms, but excessive amount of HNP released during phagocytosis may contribute to inflammatory responses and worsen lung injury. Based on our previous work, we hypothesized that blocking the cell surface purinergic receptor P2Y6 will attenuate the HNP-induced inflammatory responses while maintaining their antimicrobial activity in pneumonia followed by mechanical ventilation. METHODS: Plasma HNP levels were measured in patients with pneumonia who received mechanical ventilation and in healthy volunteers. FVB littermate control and HNP transgenic (HNP+) mice were randomized to receive P. aeruginosa intranasally. The P2Y6 antagonist (MRS2578) or vehicle control was given after P. aeruginosa instillation. Additional mice underwent mechanical ventilation at either low pressure (LP) or high pressure (HP) ventilation 48 h after pneumonia, and were observed for 24 h. RESULTS: Plasma HNP concentration increased in patients with pneumonia as compared to healthy subjects. The bacterial counts in the bronchoalveolar lavage fluid (BALF) were lower in HNP+ mice than in FVB mice 72 h after P. aeruginosa instillation. However, upon receiving HP ventilation, HNP+ mice had higher levels of cytokines and chemokines in BALF than FVB mice. These inflammatory responses were attenuated by the treatment with MRS2578 that did not affect the microbial effects of HNP. CONCLUSIONS: HNP exerted dual effects by exhibiting antimicrobial activity in pneumonia alone condition while enhancing inflammatory responses in pneumonia followed by HP mechanical ventilation. Blocking P2Y6 can attenuate the inflammation without affecting the antibacterial property of HNP. The P2Y6 receptor may be a novel therapeutic target in attenuation of the leukocyte-mediated excessive host responses in inflammatory lung diseases.


Assuntos
Modelos Animais de Doenças , Isotiocianatos/uso terapêutico , Neutrófilos , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Receptores Purinérgicos P2 , Tioureia/análogos & derivados , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Idoso , Animais , Feminino , Humanos , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/metabolismo , Pneumonia Associada à Ventilação Mecânica/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Receptores Purinérgicos P2/metabolismo , Tioureia/farmacologia , Tioureia/uso terapêutico , Resultado do Tratamento , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/microbiologia
19.
Photoacoustics ; 12: 14-21, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30225194

RESUMO

With their optically transparent appearance, zebrafish larvae are readily imaged with optical-resolution photoacoustic (PA) microscopy (OR-PAM). Previous OR-PAM studies have mapped endogenous chromophores (e.g. melanin and hemoglobin) within larvae; however, anatomical features cannot be imaged with OR-PAM alone due to insufficient optical absorption. We have previously reported on the photoacoustic radiometry (PAR) technique, which can be used simultaneously with OR-PAM to generate images dependent upon the optical attenuation properties of a sample. Here we demonstrate application of the duplex PAR/PA technique for label-free imaging of the anatomy and vasculature of zebrafish larvae in vivo at 200 and 400 MHz ultrasound detection frequencies. We then use the technique to assess the effects of anti-angiogenic drugs on the development of the larval vasculature. Our results demonstrate the effectiveness of simultaneous PAR/PA for acquiring anatomical images of optically transparent samples in vivo, and its potential applications in assessing drug efficacy and embryonic development.

20.
Hum Mol Genet ; 27(17): 3029-3045, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878199

RESUMO

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.


Assuntos
Doenças do Desenvolvimento Ósseo/etiologia , Calcificação Fisiológica , Defeitos Congênitos da Glicosilação/complicações , Genômica , Glicômica , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Simportadores/genética , Adulto , Animais , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Células Cultivadas , Estudos de Coortes , Exoma , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Lactente , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Linhagem , Fenótipo , Transporte Proteico , Simportadores/metabolismo , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
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