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1.
Nihon Yakurigaku Zasshi ; 154(3): 121-127, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527361

RESUMO

Hydrogen sulfide (H2S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or -1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H2S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu2+, and Cu2+ also reacts with H2S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.


Assuntos
Corantes Fluorescentes , Enxofre/análise , Sulfurtransferases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Humanos , Sulfeto de Hidrogênio , Imagem Óptica , Transdução de Sinais
2.
Inorg Chem ; 58(16): 10611-10615, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31380629

RESUMO

Luminescent lanthanides provide a promising alternative to organic chromophores for cellular bioimaging and bioassay applications; efficacy is closely governed by their respective quantum yields. Conventionally utilized quantum-yield measurements for lanthanides are laborious and not amenable to rapid relative comparison of compound performance. Here, we introduce a high-throughput optical imaging method to determine and directly compare relative quantum yield using Cherenkov-radiation-mediated excitation of luminescent lanthanide complexes.


Assuntos
Complexos de Coordenação/química , Ensaios de Triagem em Larga Escala , Elementos da Série dos Lantanídeos/química , Teoria Quântica , Luminescência , Conformação Molecular
3.
J Enzyme Inhib Med Chem ; 34(1): 1474-1480, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414611

RESUMO

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.


Assuntos
Lipase/metabolismo , Miniaturização , Regulação Alostérica , Candida/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Limite de Detecção , Lipase/química , Reprodutibilidade dos Testes , Especificidade por Substrato , Temperatura Ambiente
4.
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
5.
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
6.
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
7.
Toxicol Lett ; 314: 27-36, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295538

RESUMO

Some cosmetic ingredients can act as a chemical hapten to induce an immune response; therefore, evaluating the sensitizing potential of cosmetic ingredients is essential. We previously developed a novel in chemico direct peptide reactivity assay involving a spectrophotometric evaluation (Spectro-DPRA) for animal skin sensitization tests (local lymph node assay; LLNA). Based on previous research, we expanded the test materials to confirm the effectiveness of the Spectro-DPRA method for predicting the animal skin sensitization potential, and further determined the feasibility of the method for estimating the human skin sensitization potential. Spectro-DPRA showed 83.1% or 89.1% accuracy compared to a conventional LLNA or prediction based on human data, respectively, with a combination model using both a cysteine peptide and lysine peptide cut-off. To identify the effect of the lipophilicity of a chemical on predicting the skin sensitization potential, we applied our prediction model to chemicals with a Log Pow range of -1 to 4. Overall predictability was increased, and the accuracy compared to the LLNA and human data was 91.5% and 94.9%, respectively, in the combination cut-off prediction model. In conclusion, Spectro-DPRA serves as an easy, rapid, and high-throughput in chemico screening method with high accuracy to predict the human skin sensitization potential of chemicals.


Assuntos
Alternativas aos Testes com Animais/métodos , Ensaios de Triagem em Larga Escala , Oligopeptídeos/química , Testes de Irritação da Pele/métodos , Pele/efeitos dos fármacos , Animais , Cisteína , Estudos de Viabilidade , Humanos , Ensaio Local de Linfonodo , Lisina , Estrutura Molecular , Reprodutibilidade dos Testes , Medição de Risco , Pele/imunologia , Espectrofotometria , Relação Estrutura-Atividade
8.
J Cancer Res Clin Oncol ; 145(9): 2383-2396, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31280346

RESUMO

PURPOSE: Breast cancer is one of the most common malignancies among females, and its prognosis is affected by a complex network of gene interactions. Weighted gene co-expression network analysis was used to construct free-scale gene co-expression networks and to identify potential biomarkers for breast cancer progression. METHODS: The gene expression profiles of GSE42568 were downloaded from the Gene Expression Omnibus database. RNA-sequencing data and clinical information of breast cancer from TCGA were used for validation. RESULTS: A total of ten modules were established by the average linkage hierarchical clustering. We identified 58 network hub genes in the significant module (R2 = 0.44) and 6 hub genes (AGO2, CDC20, CDCA5, MCM10, MYBL2, and TTK), which were significantly correlated with prognosis. Receiver-operating characteristic curve validated that the mRNA levels of these six genes exhibited excellent diagnostic efficiency in the test data set of GSE42568. RNA-sequencing data from TCGA showed that the expression levels of these six genes were higher in triple-negative tumors. One-way ANOVA suggested that these six genes were upregulated at more advanced stages. The results of independent sample t test indicated that MCM10 and TTK were associated with tumor size, and that AGO2, CDC20, CDCA5, MCM10, and MYBL2 were overexpressed in lymph-node positive breast cancer. CONCLUSIONS: AGO2, CDC20, CDCA5, MCM10, MYBL2, and TTK were identified as candidate biomarkers for further basic and clinical research on breast cancer based on co-expression analysis.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Redes Reguladoras de Genes , Transcriptoma , Biomarcadores Tumorais/análise , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Análise por Conglomerados , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Ensaios de Triagem em Larga Escala , Humanos , Análise em Microsséries , Prognóstico
9.
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
10.
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
12.
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
13.
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
14.
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
15.
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
16.
J Microbiol ; 57(6): 470-478, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054138

RESUMO

Methanogens are an important biogenic source of methane, especially in estuarine waters across a river-to-sea gradient. However, the diversity and trophic strategy of methanogens in this gradient are not clear. In this study, the diversity and trophic strategy of methanogens in sediments across the Yellow River (YR) to the Bohai Sea (BS) gradient were investigated by high-throughput sequencing based on the 16S rRNA gene. The results showed that the diversity of methanogens in sediments varied from multitrophic communities in YR samples to specific methylotrophic communities in BS samples. The methanogenic community in YR samples was dominated by Methanosarcina, while that of BS samples was dominated by methylotrophic Methanococcoides. The distinct methanogens suggested that the methanogenic community of BS sediments did not originate from YR sediment input. High-throughput sequencing of the mcrA gene revealed that active Methanococcoides dominated in the BS enrichment cultures with trimethylamine as the substrate, and methylotrophic Methanolobus dominated in the YR enrichment cultures, as detected to a limited amount in in situ sediment samples. Methanosarcina were also detected in this gradient sample. Furthermore, the same species of Methanosarcina mazei, which was widely distributed, was isolated from the area across a river-to-sea gradient by the culture-dependent method. In summary, our results showed that a distribution of diverse methanogens across a river-to-sea gradient may shed light on adaption strategies and survival mechanisms in methanogens.


Assuntos
Biodiversidade , Euryarchaeota/classificação , Euryarchaeota/fisiologia , Rios/microbiologia , Água do Mar/microbiologia , Microbiologia da Água , China , DNA Arqueal/genética , Euryarchaeota/genética , Euryarchaeota/isolamento & purificação , Genes Arqueais/genética , Ensaios de Triagem em Larga Escala , Mathanococcus , Microbiota , Filogenia , RNA Ribossômico 16S/genética , Salinidade
17.
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
18.
Nat Commun ; 10(1): 2341, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138801

RESUMO

Understanding nanoparticle uptake by biological cells is fundamentally important to wide-ranging fields from nanotoxicology to drug delivery. It is now accepted that the arrival of nanoparticles at the cell is an extremely complicated process, shaped by many factors including unique nanoparticle physico-chemical characteristics, protein-particle interactions and subsequent agglomeration, diffusion and sedimentation. Sequentially, the nanoparticle internalisation process itself is also complex, and controlled by multiple aspects of a cell's state. Despite this multitude of factors, here we demonstrate that the statistical distribution of the nanoparticle dose per endosome is independent of the initial administered dose and exposure duration. Rather, it is the number of nanoparticle containing endosomes that are dependent on these initial dosing conditions. These observations explain the heterogeneity of nanoparticle delivery at the cellular level and allow the derivation of simple, yet powerful probabilistic distributions that accurately predict the nanoparticle dose delivered to individual cells across a population.


Assuntos
Endossomos/metabolismo , Nanopartículas/metabolismo , Células A549 , Transporte Biológico , Linhagem Celular , Endossomos/ultraestrutura , Ensaios de Triagem em Larga Escala , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Nanopartículas/ultraestrutura
19.
Chem Commun (Camb) ; 55(42): 5886-5889, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31041938

RESUMO

By coupling in situ [2+3] Huisgen cycloaddition with an in vitro transcription/translation luminescence assay in a crude ribosomal extract, a robust and accurate high-throughput platform was successfully developed and applied for efficient identification of novel structural types of ribosomal inhibitors with antimicrobial activity against drug-resistant bacteria.


Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Descoberta de Drogas , Ribossomos/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Química Click , Reação de Cicloadição , Farmacorresistência Bacteriana , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Estudo de Prova de Conceito , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
20.
Eur J Med Chem ; 175: 357-372, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096156

RESUMO

Lysine-specific demethylase 1 (LSD1), demethylase against mono- and di - methylated histone3 lysine 4, has emerged as a promising target in oncology. More specifically, it has been demonstrated as a key promoter in acute myeloid leukemia (AML), and several LSD1 inhibitors have already entered into clinical trials for the treatment of AML. In this paper, a series of new indole derivatives were designed and synthesized based on a lead compound obtained by a high-throughput screening with our in-house compound library. Among the synthetic compounds, 9e was characterized as a potent LSD1 inhibitor with an IC50 of 1.230 µM and can inhibit the proliferation of THP-1 cells effectively. And most importantly, this is the first irreversible LSD1 inhibitor that is not derived from monoamine oxidase inhibitors. Hence, the discovery of 9e may serve as a proof of concept work for AML treatment.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Indóis/síntese química , Indóis/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Furanos/química , Ensaios de Triagem em Larga Escala , Histona Desmetilases/metabolismo , Humanos , Indóis/química , Indóis/metabolismo , Concentração Inibidora 50 , Leucemia Mieloide Aguda/patologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
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