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1.
Blood ; 135(1): 41-55, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

2.
Nat Metab ; 1(9): 912-926, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31572976

RESUMO

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial TGFß signaling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGFß signaling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These pro-inflammatory effects of endothelial TGFß signaling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type specific therapeutic intervention aimed at control of this disease.

3.
ACS Appl Mater Interfaces ; 11(41): 38092-38102, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31566949

RESUMO

Stretchable conductors are required for next-generation soft electronics. Achieving both high electrical conductivity and high stretchability in conductors composed of elastomers and conductive fillers, however, is challenging. Here, a generic, versatile strategy is reported for producing ultrastretchable conductors exhibiting both superior electrical conductivity (>103 S/cm) and stretchability (>1600%). This is achieved by adding small amounts of immiscible secondary fluid into silver (Ag)-filled inks. Capillary forces in these ternary systems induce the self-assembly of conductive particle networks at a low percolation threshold (6-7 vol %), cutting silver consumption by more than 2/3 compared to conventional conductive elastomers. Ag-filled polydimethylsiloxane exhibits superior cyclic durability sustaining 100% tensile strain for 1000 cycles with only a minor loss of conductivity. Ag-filled thermoplastic polyurethane displays unprecedented reversibility with nonretarded switching from conductive to nonconductive states during repeated stretching up to 200% strain. Patterned strain sensors and conductive wirings were 3D-printed to demonstrate the technical feasibility.

4.
Nat Commun ; 9(1): 2447, 2018 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-29961079

RESUMO

Signet-ring cell carcinoma (SRCC) has specific epidemiology and oncogenesis in gastric cancer, however, with no systematical investigation for prognostic genomic features. Here we report a systematic investigation conducted in 1868 Chinese gastric cancer patients indicating that signet-ring cells content was related to multiple clinical characteristics and treatment outcomes. We thus perform whole-genome sequencing on 32 pairs of SRC samples, and identify frequent CLDN18-ARHGAP26/6 fusion (25%). With 797 additional patients for validation, prevalence of CLDN18-ARHGAP26/6 fusion is noticed to be associated with signet-ring cell content, age at diagnosis, female/male ratio, and TNM stage. Importantly, patients with CLDN18-ARHGAP26/6 fusion have worse survival outcomes, and get no benefit from oxaliplatin/fluoropyrimidines-based chemotherapy, which is consistent with the fact of chemo-drug resistance acquired in CLDN18-ARHGAP26 introduced cell lines. Overall, this study provides insights into the clinical and genomic features of SRCC, and highlights the importance of frequent CLDN18-ARHGAP26/6 fusions in chemotherapy response for SRCC.


Assuntos
Carcinoma de Células em Anel de Sinete/genética , Claudinas/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Mutantes Quiméricas , Neoplasias Gástricas/genética , Antineoplásicos/uso terapêutico , Carcinoma de Células em Anel de Sinete/tratamento farmacológico , Linhagem Celular Tumoral , Claudinas/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas Ativadoras de GTPase/fisiologia , Humanos , Masculino , Oxaliplatina/uso terapêutico , Estudos Retrospectivos , Neoplasias Gástricas/tratamento farmacológico , Resultado do Tratamento , Sequenciamento Completo do Genoma
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 35(1): 1-8, 2018 Feb 10.
Artigo em Chinês | MEDLINE | ID: mdl-29419850

RESUMO

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.


Assuntos
Consenso , Testes Genéticos/métodos , Testes Genéticos/normas , Guias de Prática Clínica como Assunto , China , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
6.
Nature ; 545(7653): 224-228, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28467822

RESUMO

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicólise , Neovascularização Fisiológica , Transdução de Sinais , Animais , Movimento Celular , Proliferação de Células , Feminino , Hexoquinase/metabolismo , Linfangiogênese , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo
7.
Oncotarget ; 7(5): 5461-9, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26701727

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore,the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Janus Quinase 1/genética , Neoplasias Hepáticas/tratamento farmacológico , Mutação/genética , Pirazóis/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Apoptose , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células , Feminino , Humanos , Janus Quinase 1/antagonistas & inibidores , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Oncotarget ; 6(24): 20160-76, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26062443

RESUMO

Lack of clinically relevant tumor models dramatically hampers development of effective therapies for hepatocellular carcinoma (HCC). Establishment of patient-derived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of HCC becomes important. In this study, we first established a cohort of 65 stable PDX models of HCC from corresponding Chinese patients. Then we showed that the histology and gene expression patterns of PDX models were highly consistent between xenografts and case-matched original tumors. Genetic alterations, including mutations and DNA copy number alterations (CNAs), of the xenografts correlated well with the published data of HCC patient specimens. Furthermore, differential responses to sorafenib, the standard-of-care agent, in randomly chosen xenografts were unveiled. Finally, in the models expressing high levels of FGFR1 gene according to the genomic data, FGFR1 inhibitor lenvatinib showed greater efficacy than sorafenib. Taken together, our data indicate that PDX models resemble histopathological and genomic characteristics of clinical HCC tumors, as well as recapitulate the differential responses of HCC patients to the standard-of-care treatment. Overall, this large collection of PDX models becomes a clinically relevant platform for drug screening, biomarker discovery and translational research in preclinical setting.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Carcinoma Hepatocelular/patologia , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genômica , Humanos , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Thorac Oncol ; 9(3): 285-94, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24496003

RESUMO

INTRODUCTION: The aim of this study was to identify anaplastic lymphoma kinase (ALK) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. METHODS: Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK-rearranged non-small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. RESULTS: Two ALK-rearranged NSCLC PDX models were identified: one carried a well-known EML4-ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 (HIP1)-ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK-rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4-ALK-positive model, the HIP1-ALK-containing PDX model was sensitive to treatment with crizotinib. CONCLUSIONS: Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1-ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1-ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1-ALK-positive NSCLC may benefit from clinical applications of crizotinib.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Crizotinibe , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Fusão Oncogênica/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
10.
Anal Biochem ; 406(1): 14-8, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20599634

RESUMO

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C betaII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K(m)(ATP)), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.


Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/metabolismo , Proteína Básica da Mielina/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Concentração Inibidora 50 , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Espectrometria de Massas , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Espectrometria de Fluorescência , Fatores de Tempo
11.
Biochemistry ; 47(6): 1640-51, 2008 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-18201104

RESUMO

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.


Assuntos
Canais de Cálcio/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Calmodulina/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência
12.
Nat Chem Biol ; 2(2): 87-94, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16415859

RESUMO

Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for individual beta-galactosidase molecules by detecting one fluorescent product at a time. A molecular memory phenomenon arises at high substrate concentrations, characterized by clusters of turnover events separated by periods of low activity. Such memory lasts for decades of timescales ranging from milliseconds to seconds owing to the presence of interconverting conformers with broadly distributed lifetimes. We proved that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.


Assuntos
beta-Galactosidase/química , Catálise , Galactosídeos/química , Galactosídeos/metabolismo , Cinética , Microscopia de Fluorescência , Conformação Molecular , Oxazinas/química , Oxazinas/metabolismo , beta-Galactosidase/metabolismo
13.
Anal Chem ; 77(7): 2043-9, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15801736

RESUMO

We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.


Assuntos
Técnicas de Química Analítica/métodos , Proteínas Quinases/análise , Trifosfato de Adenosina/metabolismo , Técnicas de Química Combinatória , Microscopia Crioeletrônica , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Micelas , Fosforilação , Proteína Quinase C/análise , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Reprodutibilidade dos Testes , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Titulometria
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