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1.
STAR Protoc ; 2(4): 100842, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34585169

RESUMO

Here, we outline detailed protocols to isolate and profile murine splenic dendritic cells (DCs) through advanced flow cytometry of the myeloid compartment and single-cell transcriptomic profiling with integrated cell surface protein expression through CITE-seq. This protocol provides a general transferrable road map for different tissues and species. For complete details on the use and execution of this protocol, please refer to Lukowski et al. (2021).


Assuntos
Perfilação da Expressão Gênica , Células Mieloides , Animais , Citometria de Fluxo/métodos , Proteínas de Membrana , Camundongos , Análise em Microsséries
2.
iScience ; 24(5): 102402, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33997687

RESUMO

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

3.
J Infect Dis ; 222(5): 820-831, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32246148

RESUMO

BACKGROUND: Influenza A virus (IAV) causes a wide range of extrarespiratory complications. However, the role of host factors in these complications of influenza virus infection remains to be defined. METHODS: Here, we sought to use transcriptional profiling, virology, histology, and echocardiograms to investigate the role of a high-fat diet in IAV-associated cardiac damage. RESULTS: Transcriptional profiling showed that, compared to their low-fat counterparts (LF mice), mice fed a high-fat diet (HF mice) had impairments in inflammatory signaling in the lung and heart after IAV infection. This was associated with increased viral titers in the heart, increased left ventricular mass, and thickening of the left ventricular wall in IAV-infected HF mice compared to both IAV-infected LF mice and uninfected HF mice. Retrospective analysis of clinical data revealed that cardiac complications were more common in patients with excess weight, an association which was significant in 2 out of 4 studies. CONCLUSIONS: Together, these data provide the first evidence that a high-fat diet may be a risk factor for the development of IAV-associated cardiovascular damage and emphasizes the need for further clinical research in this area.


Assuntos
Dieta Hiperlipídica , Cardiopatias/virologia , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae/complicações , Animais , Índice de Massa Corporal , Peso Corporal , Citocinas/sangue , Citocinas/genética , Ecocardiografia , Feminino , Perfilação da Expressão Gênica , Coração/virologia , Cardiopatias/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Influenza Humana/complicações , Fator Regulador 7 de Interferon/genética , Interleucina-1beta/genética , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/virologia , RNA Viral/metabolismo , Fatores de Risco , Transdução de Sinais/genética , Ubiquitinas/genética
4.
Cell Stem Cell ; 23(4): 586-598.e8, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30290179

RESUMO

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.


Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Análise de Célula Única , Transcriptoma , Proteínas Supressoras de Tumor/genética , Animais , Células Cultivadas , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Supressoras de Tumor/metabolismo
5.
Genome Res ; 28(7): 1053-1066, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29752298

RESUMO

Heterogeneity of cell states represented in pluripotent cultures has not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC-CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method and, through this, identified four subpopulations distinguishable on the basis of their pluripotent state, including a core pluripotent population (48.3%), proliferative (47.8%), early primed for differentiation (2.8%), and late primed for differentiation (1.1%). For each subpopulation, we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four transcriptionally distinct predictor gene sets composed of 165 unique genes that denote the specific pluripotency states; using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to threefold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations and support our conclusions with results from two orthogonal pseudotime trajectory methods.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , RNA/genética , Diferenciação Celular/genética , Linhagem Celular , Análise por Conglomerados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Expressão Gênica/genética , Heterogeneidade Genética , Marcadores Genéticos/genética , Humanos , Análise de Sequência de RNA/métodos , Transcrição Gênica/genética
7.
Nature ; 543(7643): 65-71, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28199314

RESUMO

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.


Assuntos
Carcinoma Neuroendócrino/genética , Genoma Humano/genética , Genômica , Neoplasias Pancreáticas/genética , Sequência de Bases , Proteínas de Ligação a Calmodulina/genética , Montagem e Desmontagem da Cromatina/genética , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , DNA Glicosilases/genética , Análise Mutacional de DNA , Reparo do DNA/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Serina-Treonina Quinases TOR/metabolismo , Telômero/genética , Telômero/metabolismo
8.
Gastroenterology ; 152(1): 68-74.e2, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27856273

RESUMO

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/genética , Reparo de Erro de Pareamento de DNA/genética , Mutação , Neoplasias Pancreáticas/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Genoma , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Proto-Oncogênicas p21(ras)/genética
9.
Nature ; 531(7592): 47-52, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26909576

RESUMO

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.


Assuntos
Genes Neoplásicos/genética , Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias Pancreáticas/classificação , Neoplasias Pancreáticas/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Ductal Pancreático/classificação , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Histona Desmetilases/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Análise de Sobrevida , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra
10.
J Pathol ; 237(3): 363-78, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26172396

RESUMO

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Análise Mutacional de DNA , Ativação Enzimática , Amplificação de Genes , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Ligantes , Terapia de Alvo Molecular , Mutação , Fenótipo , Fosforilação , Medicina de Precisão , Valor Preditivo dos Testes , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , Microambiente Tumoral
11.
Nature ; 518(7540): 495-501, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25719666

RESUMO

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.


Assuntos
Análise Mutacional de DNA , Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Reparo do DNA/genética , Feminino , Genes BRCA1 , Genes BRCA2 , Marcadores Genéticos/genética , Instabilidade Genômica/genética , Genótipo , Humanos , Camundongos , Neoplasias Pancreáticas/classificação , Neoplasias Pancreáticas/tratamento farmacológico , Platina/farmacologia , Mutação Puntual/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Commun ; 5: 5224, 2014 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25351503

RESUMO

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinogênese/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Rearranjo Gênico/genética , Genoma Humano/genética , Carcinogênese/patologia , Quebra Cromossômica , Cromossomos Humanos/genética , Humanos , Mutação/genética
13.
Biotechniques ; 57(1): 31-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25005691

RESUMO

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.


Assuntos
Pontos de Quebra do Cromossomo , Cromossomos Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Bases , Carcinoma Ductal Pancreático/genética , Aberrações Cromossômicas , Primers do DNA , Humanos , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias Pancreáticas/genética , Reação em Cadeia da Polimerase/métodos , Fluxo de Trabalho
14.
Int J Cancer ; 135(5): 1110-8, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24500968

RESUMO

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-ß, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.


Assuntos
Carcinoma Ductal Pancreático/genética , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Adesão Celular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Integrina alfa2/genética , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Ductos Pancreáticos/patologia , Células Estreladas do Pâncreas/patologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/biossíntese , Receptores Imunológicos/genética , Análise de Sequência de DNA , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Proteínas Wnt/genética , Proteínas Roundabout
15.
PLoS One ; 8(11): e74380, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24250782

RESUMO

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.


Assuntos
Biologia Computacional , Neoplasias/genética , Mutação Puntual/genética , Software , Variações do Número de Cópias de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Neoplasias/patologia , Reação em Cadeia da Polimerase/métodos
16.
J Med Chem ; 53(15): 5536-48, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20684598

RESUMO

The hematopoietic prostaglandin D(2) synthase has a proinflammatory effect in a range of diseases, including allergic asthma, where its product prostaglandin D(2) (PGD(2)) has a role in regulating many of the hallmark disease characteristics. Here we describe the development and characterization of a novel series of hematopoietic prostaglandin D(2) synthase inhibitors with potency similar to that of known inhibitors. Compounds N-benzhydryl-5-(3-hydroxyphenyl)thiophene-2-carboxamide (compound 8) and N-(1-amino-1-oxo-3-phenylpropan-2-yl)-6-(thiophen-2-yl)nicotinamide (compound 34) demonstrated low micromolar potency in the inhibition of the purified enzyme, while only 34 reduced Toll-like receptor (TLR) inducible PGD(2) production in both mouse primary bone marrow-derived macrophages and the human megakaryocytic cell line MEG-01S. Importantly, 34 demonstrated a greater selectivity for inhibition of PGD(2) synthesis versus other eicosanoids that lie downstream of PGH(2) (PGE(2) and markers of prostacyclin (6-keto PGF(1alpha)) and thromboxane (TXB(2))) when compared to the known inhibitors HQL-79 (compound 1) and 2-phenyl-5-(1H-pyrazol-3-yl)thiazole (compound 2). Compound 34 therefore represents a selective hematopoietic prostaglandin D(2) synthase inhibitor.


Assuntos
Hematopoese , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Niacinamida/análogos & derivados , Tiofenos/síntese química , Animais , Células Cultivadas , Cristalografia por Raios X , Humanos , Oxirredutases Intramoleculares/biossíntese , Ligantes , Lipocalinas/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/enzimologia , Camundongos , Modelos Moleculares , Niacinamida/síntese química , Niacinamida/química , Niacinamida/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Receptores Toll-Like/fisiologia
17.
Eur J Med Chem ; 45(2): 447-54, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19939518

RESUMO

Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.


Assuntos
Inibidores Enzimáticos/farmacologia , Hematopoese , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutationa Transferase/antagonistas & inibidores , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Modelos Moleculares , Conformação Molecular , Nocodazol/química , Nocodazol/metabolismo , Nocodazol/farmacologia
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