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1.
Arch Virol ; 164(11): 2699-2706, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31435867

RESUMO

Recently, a clinical need for an improved human papilloma virus (HPV) test that covers a broad range of genotypes has emerged as a valuable primary screening tool for cervical lesions. The liquid bead microarray (LBMA) assay is a recently developed high-throughput platform covering a broad range of genotypes. Here, we compared the clinical performance of two recently developed LBMA assays, GeneFinderTM HPV Liquid Bead Microarray (GeneFinder) and CareGENETM HPV genotyping kit-O (CareGENE), in the Korean general population. A total of 3,148 cervical swabs were tested by the GeneFinder and CareGENE assays. Cases with discrepant results between the two assays were subjected to direct sequencing as a reference method for evaluating the performance of the two LBMA assays. Among all swabs tested, 12.6% showed HPV positivity, and the prevalent HPV genotypes were HPV53, 70, 16, 39, and 51, in that order. The concordance rates between the two assays for the detection of HPV and for genotyping were 96.6% (kappa = 0.836) and 94.5% (kappa = 0.779), respectively. The two LBMA assays showed comparable sensitivity and specificity for HPV detection (GeneFinder: sensitivity 94.4% and specificity 98.7%, CareGENE: sensitivity 89.8% and specificity 99.6%) and for genotyping (GeneFinder: sensitivity 91.0% and specificity 96.6%, CareGENE: sensitivity 90.2% and specificity 99.1%). This is the first demonstration that CareGENE has comparable clinical performance to GeneFinder, which has been established to show excellent performance for screening HPV in previous studies. Both LBMA platforms are thus considered to be valuable tools for HPV detection and genotyping to improve cervical screening in the general population.


Assuntos
Alphapapillomavirus/genética , Detecção Precoce de Câncer/métodos , Ensaios de Triagem em Larga Escala/métodos , Análise em Microsséries/métodos , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alphapapillomavirus/classificação , Alphapapillomavirus/isolamento & purificação , Feminino , Técnicas de Genotipagem , Humanos , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/virologia , Adulto Jovem
2.
J Agric Food Chem ; 67(31): 8599-8608, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287302

RESUMO

Because foods are perceived through combined inputs from taste and odor, which are determined by the concentration of the individual odor and taste molecules, the unified high-throughput quantitation of volatile odorants and non-volatile tastants with the very same instrumental setup has been a long-standing but yet unmet dream. The research presented here for the first time demonstrates, after only minimal sample workup, the highly accurate, rapid, and sensitive unified quantitation of odorants and tastants of key flavor molecules in apple juice on a single ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) platform over a large dynamic range of up to 6 orders of magnitude. While flavor-active aldehydes, ketones, and organic acids were analyzed after derivatization with 3-nitrophenylhydrazine, taste-active polyphenols and odor-active esters were directly analyzed by means of UHPLC-MS/MS with and without target analyte enrichment through stir-bar sorptive extraction. This "unified flavor quantitation" approach holds promise to accelerate the transition of today's labor and time-consuming, low-throughput analysis of odorants and tastants into a new era of high-performance quantitation of key flavor molecules.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Aromatizantes/química , Ensaios de Triagem em Larga Escala/métodos , Odorantes/análise , Espectrometria de Massas em Tandem/métodos , Sucos de Frutas e Vegetais/análise , Malus/química , Compostos Orgânicos Voláteis/química
3.
Plant Sci ; 286: 49-56, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300141

RESUMO

Progress in the functional biochemical analysis of plant glycosyltransferases (GTs) has been slow because plant GTs are generally membrane proteins, operate as part of larger, multimeric complexes, and utilize a vast complexity of substrate acceptors. Therefore, the field would benefit from development of adequate high throughput expression as well as product detection and characterization techniques. Here we review current approaches to tackle such obstacles and suggest a new path forward: nucleic acid programmable protein arrays (NAPPA) with liquid sample desorption ionization (LS-DESI-MS) mass spectrometry. NAPPA utilizes in vitro transcription and translation to produce epitope-tagged fusion proteins from cloned GT cDNAs. LS-DESI is a soft ionization technique that allows rapid and sensitive MS-based product characterization in situ. Coupling both approaches provides the opportunity to examine individual GT functions as well as protein-protein interactions. Furthermore, advances in automated oligosaccharide synthesis and lipid nanodisc technology should allow testing of plant GT activity in presence of numerous substrate acceptors and lipid environments in a high throughput fashion. Thus, NAPPA-DESI-MS has great potential to make headway in biochemical characterization of the large number of plant GTs.


Assuntos
Parede Celular/química , Ensaios de Triagem em Larga Escala/métodos , Plantas/química , Polissacarídeos/biossíntese , Análise Serial de Proteínas/métodos , Glicosiltransferases/análise , Espectrometria de Massas/métodos , Proteínas de Plantas/análise , Plantas/enzimologia
4.
Chem Commun (Camb) ; 55(64): 9543-9546, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31334509

RESUMO

The novel Vial@FPBA strategy was established for a large-scale pharmacokinetic study of glycosides, during which glycosides were absorbed into a boronic acid-functionalized 96-well glass plate and directly desorbed for UHPLC-MS/MS analysis. Hence, specific and high-throughput glycoside enrichment was achieved simultaneously. The LODs were reduced up to 50 times compared to the case of the methanol method. Meanwhile, sample pre-processing time was greatly saved by skipping the protein sedimentation and supernatant concentration steps.


Assuntos
Ácidos Borônicos/química , Glicosídeos/farmacocinética , Ensaios de Triagem em Larga Escala/métodos , Cromatografia Líquida de Alta Pressão/métodos , Vidro , Limite de Detecção , Espectrometria de Massas em Tandem/métodos
5.
Food Chem ; 300: 125192, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31362158

RESUMO

Dietary fiber has several benefits for humans, and the development of healthier rice with an improved dietary fiber composition has attracted increasing amounts of attention. Based on the method of AOAC 2002.02, we developed a simplified method to screen polished rice containing high total dietary fiber (TDF). Mutant cw with a high TDF content could be distinguished easily from R7954 (indica) and Nipponbare (japonica) by the digestion-resistant phenotype, which is characterized as an almost intact grain after hydrolysis by pepsin, pancreatic α-amylase and amyloglucosidase. The individuals identified from the F2 population showed digestion resistance all had TDF content higher than 5%, while those without a digestion-resistant phenotype had TDF content lower than 5%. The phenotype of digestion resistance could be a valuable index for identifying rice with higher TDF content, and the identification of this phenotype provides a simplified, economical and high throughput method for high TDF rice breeding.


Assuntos
Fibras na Dieta/análise , Análise de Alimentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Oryza/química , Culinária , Fibras na Dieta/metabolismo , Digestão , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Hidrólise , Pepsina A/química , Pepsina A/metabolismo , Sementes/química , Amido/análise , Amido/química , alfa-Amilases/química , alfa-Amilases/metabolismo
6.
Nat Commun ; 10(1): 2745, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227705

RESUMO

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.


Assuntos
Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Tiorredoxina Redutase 1/metabolismo , Animais , Produtos Biológicos/farmacologia , Misturas Complexas , Dissulfetos/química , Corantes Fluorescentes/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética
8.
J Agric Food Chem ; 67(25): 7174-7182, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31240931

RESUMO

Intake of endocrine-disrupting chemicals (EDCs) by humans could disturb the metabolism of hormones, induce cancer, and damage the liver and other organs. Phthalate acid esters (PAEs) and alkylphenols (APs) are important EDCs and environmental contaminants. With the increasing use of plastics and nonionic surfactants worldwide, PAEs and APs have entered environmental water and accumulated in edible fish, which are finally consumed by humans. In this study, a coated direct inlet probe (CDIP) based on an atmospheric solid analysis probe, which can rapidly and simultaneously extract both PAEs and APs in fish, was developed. Twelve PAEs and APs were quantified by using a stable-isotope-labeled internal standard. Standard curves of the PAEs and APs having correlation coefficients of R2 ≥ 0.9837 were obtained. The limit of detection of the PAEs and APs was distributed from 0.01 to 40 ng g-1. The relative recovery of the method was 78-120% between low, medium, and high spiked levels. Combined with principal component analysis, PAE- and AP-contaminated Carassius auratus from different habitats could be identified. Multiple sample analysis mode allowed the extraction of up to 12 samples at once, and the total analysis time (including sample pretreatment, extraction, and analysis time) was less than 10 min per sample, which indicates that CDIP is useful for rapid quantitative analysis.


Assuntos
Ésteres/análise , Carpa Dourada , Ensaios de Triagem em Larga Escala/métodos , Fenóis/análise , Ácidos Ftálicos/análise , Animais , Disruptores Endócrinos/análise , Disruptores Endócrinos/isolamento & purificação , Ésteres/isolamento & purificação , Carpa Dourada/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Limite de Detecção , Fenóis/isolamento & purificação , Ácidos Ftálicos/isolamento & purificação , Extração em Fase Sólida
9.
Nat Commun ; 10(1): 2620, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197165

RESUMO

Conventional drug screens and treatments often ignore the underlying complexity of brain network dysfunctions, resulting in suboptimal outcomes. Here we ask whether we can correct abnormal functional connectivity of the entire brain by identifying and combining multiple neuromodulators that perturb connectivity in complementary ways. Our approach avoids the combinatorial complexity of screening all drug combinations. We develop a high-speed platform capable of imaging more than 15000 neurons in 50ms to map the entire brain functional connectivity in large numbers of vertebrates under many conditions. Screening a panel of drugs in a zebrafish model of human Dravet syndrome, we show that even drugs with related mechanisms of action can modulate functional connectivity in significantly different ways. By clustering connectivity fingerprints, we algorithmically select small subsets of complementary drugs and rapidly identify combinations that are significantly more effective at correcting abnormal networks and reducing spontaneous seizures than monotherapies, while minimizing behavioral side effects. Even at low concentrations, our polytherapy performs superior to individual drugs even at highest tolerated concentrations.


Assuntos
Epilepsias Mioclônicas/tratamento farmacológico , Modelos Biológicos , Rede Nervosa/efeitos dos fármacos , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos , Neurotransmissores/farmacologia , Algoritmos , Animais , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Mapeamento Encefálico/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Confocal/métodos , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotransmissores/uso terapêutico , Peixe-Zebra
10.
Planta Med ; 85(9-10): 738-744, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31185502

RESUMO

Chronic heart failure is the terminal stage of various cardiovascular diseases. Despite the availability of several classes of drugs, there is still an unmet need for effective treatment. Based on bench work during the past two decades, we have proposed that enhancement of ß 2-adrenergic receptor signaling in combination with the presently preferred ß 1-adrenergic receptor blockade would be a promising strategy. Chinese herbal medicines have been shown to be effective in the treatment of heart failure, although the mechanisms largely remain unknown. In the present study, we screened an herbal medicine compound/extract library for ß-adrenergic receptor ligands to determine the target of certain effective botanical remedies and seek a leading compound(s) for chronic heart failure treatment. Using a high-throughput screening assay, we identified higenamine, which has a long history in chronic heart failure treatment in traditional Chinese medicine, to be a potent ß-adrenergic receptor agonist. Further experiments using specific inhibitors showed that higenamine activated both ß 1-adrenergic receptor and ß 2-adrenergic receptor. Inhibition of its action by pertussis toxin (a Gi inhibitor) indicated that it is a ß 2-adrenergic receptor Gs/Gi dual agonist. Contractility experiments demonstrated a positive inotropic effect of higenamine. In conclusion, we found an herbal compound, higenamine, to be a dual agonist for ß 1/ß 2-adrenergic receptors with no preference in stimulating the Gs and Gi pathways in ß 2-adrenergic receptor signaling. Our results elucidated not only the target of higenamine to explain its pharmacological effect in treating chronic heart failure, but also the mechanisms of its cardiac toxicity.


Assuntos
Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Alcaloides/farmacologia , Medicamentos de Ervas Chinesas/química , Ensaios de Triagem em Larga Escala/métodos , Tetra-Hidroisoquinolinas/farmacologia , Alcaloides/química , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Simulação de Acoplamento Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Tetra-Hidroisoquinolinas/química
11.
Nat Commun ; 10(1): 2163, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092822

RESUMO

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.


Assuntos
Neoplasias da Mama/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Linhagem Celular , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Camundongos , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
12.
Nat Commun ; 10(1): 2261, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113940

RESUMO

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Cristalografia por Raios X , DNA/imunologia , DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/imunologia , Interferons/metabolismo , Macrófagos , Modelos Moleculares , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/isolamento & purificação , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
13.
Nat Commun ; 10(1): 2189, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097698

RESUMO

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates <40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Desoxicitidina/análogos & derivados , Inibidores de Janus Quinases/farmacologia , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Adulto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Linhagem Celular Tumoral , Criança , Pré-Escolar , Citarabina/farmacologia , Citarabina/uso terapêutico , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Intervalo Livre de Doença , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Lactente , Inibidores de Janus Quinases/uso terapêutico , Leucemia Experimental/etiologia , Leucemia Experimental/mortalidade , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Taxoides/farmacologia , Taxoides/uso terapêutico , Irradiação Corporal Total/efeitos adversos , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
14.
Zhonghua Zhong Liu Za Zhi ; 41(5): 351-356, 2019 May 23.
Artigo em Chinês | MEDLINE | ID: mdl-31137168

RESUMO

Objective: To establish a quantitative assay of serum Golgi protein 73 (GP73) using xMAP technology and evaluate its performance. Methods: Monoclonal antibodies against GP73 were prepared and purified, and antibody pair screening was performed by double-antibody sandwich enzyme-linked immunosorbent assay. The screened antibodies were used to construct a Luminex liquid chip detection system, and the analysis performance of the detection system was evaluated. The serum levels of GP73 were detected in 90 clinical samples from healthy controls and patients with chronic hepatitis B infection (CHB) and hepatocellular carcinoma (HCC). Results: Five anti-GP73 monoclonal antibodies were prepared and purified, and 5 antibody pairs were successfully screened. The Luminex liquid chip detection system of GP73 was successfully constructed using 8F10D1 and 10B9F11 antibody pairs. The analytical performance evaluation showed that the sensitivity of this system was 0.25 ng/ml and the dynamic range was 0.25-100 ng/ml. No cross reactivity was observed. The intra- and inter-assay variation for GP73 was <8% and <11%, respectively. The recovery was 83%-92%. The linear regression equation was y=1.141x+ 6.436 (r(2)=0.998 4, P<0.001). The GP73 concentrations in the serum samples of healthy control, CHB group, and HCC group were 42.8 (38.68, 55.90) ng/ml, 61.49 (43.59, 81) ng/ml, and 122.78 (49.36 liter, 264.55) ng/ml, respectively. The levels of GP73 in HCC group were significantly higher than those in CHB group and healthy controls (P<0.05). Moreover, the levels of GP73 in CHB group were significantly higher than those in healthy controls (P<0.05). Conclusions: A liquid chip detection system of GP73 was successfully constructed. It provides a powerful tool for the clinical application of GP73 in the future.


Assuntos
Carcinoma Hepatocelular/sangue , Hepatite B Crônica/sangue , Ensaios de Triagem em Larga Escala/métodos , Neoplasias Hepáticas/sangue , Proteínas de Membrana/sangue , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala/normas , Humanos
15.
Methods Mol Biol ; 1966: 71-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041739

RESUMO

The constitutive androstane receptor (CAR; NR1I3) is a xenobiotic receptor that upregulates metabolism and detoxification mechanisms in the liver in response to chemical stimulation. Drug-induced activation of CAR may result in clinically significant drug-drug interactions and lead to complicated therapeutic outcomes. Accumulating evidence has also suggested that CAR may be a potential drug target for metabolic disorders and liver cancer by modulating cell cycle progression, energy homeostasis, and cell proliferation. Therefore, identification of CAR activators is of potential importance in both drug development and clinical practice. Notably, while CAR is localized in the nucleus and constitutively activated in immortalized cell lines, it is sequestered in the cytoplasm and translocates to the nucleus upon chemical-provoked activation in primary cultured hepatocytes. Here, we have developed a methodology that takes advantage of nuclear translocation being the first and essential step of CAR activation in human primary hepatocytes to perform high-content screening of human CAR modulators by adapting the EYFP-hCAR translocation assay to a 96-well format with automated sample dispensing and fluorescence imaging analysis.


Assuntos
Núcleo Celular/metabolismo , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Células Cultivadas , Humanos , Microscopia/métodos
16.
Methods Mol Biol ; 1966: 137-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041744

RESUMO

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may cause toxicity or even provide utility in the diagnosis or treatment of cancers that have elevated cellular PPIX levels. This chapter describes a high-throughput plate-based imaging technique for determining cellular protoporphyrin levels by using the GE Healthcare InCell 6000 confocal imaging system to detect the presence and location of PPIX in each cell and may be adapted for use with other imaging systems. Laser excitation and a scientific-grade complementary metal oxide semiconductor (CMOS) camera generate short exposure times, decreasing photobleaching in the target cells that may result in inaccurate measurements of PPIX and increasing screening throughput. Nuclear staining was detected by using a laser with 405-nm excitation and 455-nm emission wavelengths, and the presence of PPIX was measured using 405-nm excitation and 706-nm emission wavelengths. Image analysis involving top-hat segmentation on both nuclear and PPIX staining was performed by using the InCell Analyzer Workstation software. This assay may be adapted to screen for PPIX formation, degradation, and transportation effectors. Indeed, the inclusion of PPIX transport inhibition would be expected to further widen the linear range of fluorescence and improve the method.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microscopia Confocal/métodos , Protoporfirinas/análise , Espectrometria de Fluorescência/métodos , Células Hep G2 , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos
17.
J Agric Food Chem ; 67(24): 6892-6901, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31125221

RESUMO

We herein describe a high-throughput 96-well plate micro-solid phase extraction sample preparation technique based on novel sulfonated-polyaniline/polyacrylonitrile nanofiber mats (sulfonated-PANI/PAN NFMs) for multiresidue detection of fluoroquinolones (FQs) in various animal-origin food samples. Through the double-modification of polyaniline and sulfonic acid, the resulting functionalized sulfonated-PANI/PAN NFMs present high extraction efficiency for FQs. Compared with the existing methods, this approach demonstrates its advantages of being suitable for more sample matrices (milk, animal muscle, liver, kidney, and egg), lower sample amount (0.5 g), lower sorbent requirement (5.0 mg), lower volume of organic solvent (0.7 mL), shorter time (0.2 min per sample), and high sensitivity (0.012-0.06 µg·kg-1). In addition, sulfonated-PANI/PAN NFMs possess excellent reusability which could be reused 10 times without an obvious decrease in extraction efficiency. Combined with ultra performance liquid chromatography-tandem mass spectrometry, the novel sample preparation technique can be expected as an efficient method for routine trace FQ residue monitoring in animal-origin food samples.


Assuntos
Anti-Infecciosos/análise , Anti-Infecciosos/isolamento & purificação , Fluoroquinolonas/análise , Fluoroquinolonas/isolamento & purificação , Contaminação de Alimentos/análise , Ensaios de Triagem em Larga Escala/métodos , Microextração em Fase Sólida/métodos , Resinas Acrílicas/química , Compostos de Anilina/química , Animais , Bovinos , Galinhas , Ovos/análise , Ensaios de Triagem em Larga Escala/instrumentação , Rim/química , Limite de Detecção , Fígado/química , Carne/análise , Leite/química , Nanofibras/química , Microextração em Fase Sólida/instrumentação , Suínos
18.
Sci Total Environ ; 677: 362-372, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31059879

RESUMO

Organic chemicals from industrial, agricultural, and residential activities can enter surface waters through regulated and unregulated discharges, combined sewer overflows, stormwater runoff, accidental spills, and leaking septic-conveyance systems on a daily basis. The impact of point and nonpoint contaminant sources can result in adverse biological effects for organisms living in or near surface waters. Assessing the adverse or toxic effects that may result when exposure occurs is complicated by the fact that many commonly used chemicals lack toxicity information or water quality standards. To address these challenges, an exposure-activity ratio (EAR) screening approach was used to prioritize environmental chemistry data in a West Virginia watershed (Wolf Creek). Wolf Creek is a drinking water source and recreation resource with documented water quality impacts from point and nonpoint sources. The EAR screening approach uses high-throughput screening (HTS) data from ToxCast as a method of integrating environmental chemical occurrence and biological effects data. Using water quality schedule 4433, which targets 69 organic waste compounds typically found in domestic and industrial wastewater, chemicals were screened for potential adverse biological affects at multiple sites in the Wolf Creek watershed. Cumulative EAR mixture values were greatest at Sites 2 and 3, where bisphenol A (BPA) and pentachlorophenol exhibited maximum EAR values of 0.05 and 0.002, respectively. Site 2 is downstream of an unconventional oil and gas (UOG) wastewater disposal facility with documented water quality impacts. Low-level organic contaminants were found at all sample sites in Wolf Creek, except Site 10, where Wolf Creek enters the New River. The application of an EAR screening approach allowed our study to extend beyond traditional environmental monitoring methods to identify multiple sites and chemicals that warrant further investigation.


Assuntos
Disruptores Endócrinos/análise , Monitoramento Ambiental/métodos , Ensaios de Triagem em Larga Escala/métodos , Rios , Águas Residuárias/análise , Poluentes Químicos da Água/análise , Água Potável/análise , Qualidade da Água , West Virginia
20.
Philos Trans A Math Phys Eng Sci ; 377(2147): 20180422, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31030650

RESUMO

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.


Assuntos
Descoberta de Drogas/história , Síncrotrons/história , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Ensaios de Triagem em Larga Escala/história , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/tendências , História do Século XX , História do Século XXI , Humanos , Modelos Moleculares , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo
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