Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.903
Filtrar
1.
Nat Commun ; 11(1): 4059, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792628

RESUMO

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here, we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. The fluorescence-based neutralization assay is specific to measure COVID-19 neutralizing antibodies without cross reacting with patient specimens with other viral, bacterial, or parasitic infections. Collectively, our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.


Assuntos
Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Vacinas Virais/imunologia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Testes Sorológicos/métodos , Células Vero , Ensaio de Placa Viral
2.
Molecules ; 25(17)2020 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-32842509

RESUMO

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the Informational spectrum method applied for small molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Cisteína Endopeptidases/química , Mezlocilina/química , Inibidores de Proteases/química , Raltegravir Potássico/química , Proteínas não Estruturais Virais/química , Sítio Alostérico , Antivirais/farmacologia , Betacoronavirus/enzimologia , Betacoronavirus/patogenicidade , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Reposicionamento de Medicamentos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Mezlocilina/farmacologia , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Raltegravir Potássico/farmacologia , Termodinâmica , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Molecules ; 25(17)2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842606

RESUMO

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Catepsina L/química , Compostos Fitoquímicos/química , Inibidores de Proteases/química , Receptores Virais/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Sítios de Ligação , Catepsina L/antagonistas & inibidores , Catepsina L/genética , Catepsina L/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/virologia , Cumarínicos/química , Cumarínicos/isolamento & purificação , Cumarínicos/farmacologia , Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Índia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monossacarídeos/química , Monossacarídeos/isolamento & purificação , Monossacarídeos/farmacologia , Pandemias , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Plantas Medicinais/química , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologia , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Termodinâmica , Internalização do Vírus/efeitos dos fármacos
4.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1405-1413, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748598

RESUMO

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 µm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 µm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.


Assuntos
Emulsões , Ensaios de Triagem em Larga Escala , Microfluídica , Citometria de Fluxo , Microfluídica/métodos
6.
Nat Commun ; 11(1): 3436, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632160

RESUMO

The world is entering a new era of the COVID-19 pandemic in which there is an increasing call for reliable antibody testing. To support decision making on the deployment of serology for either population screening or diagnostics, we present a detailed comparison of serological COVID-19 assays. We show that among the selected assays there is a wide diversity in assay performance in different scenarios and when correlated to virus neutralizing antibodies. The Wantai ELISA detecting total immunoglobulins against the receptor binding domain of SARS CoV-2, has the best overall characteristics to detect functional antibodies in different stages and severity of disease, including the potential to set a cut-off indicating the presence of protective antibodies. The large variety of available serological assays requires proper assay validation before deciding on deployment of assays for specific applications.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/normas , Anticorpos Neutralizantes/sangue , Betacoronavirus , Técnicas de Laboratório Clínico , Ensaio de Imunoadsorção Enzimática , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes , Testes de Neutralização , Pandemias , Sensibilidade e Especificidade
7.
Proc Natl Acad Sci U S A ; 117(29): 16839-16847, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641515

RESUMO

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.


Assuntos
Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/classificação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Leucaférese/métodos , Campos Magnéticos , Microfluídica/instrumentação
8.
Nat Commun ; 11(1): 3714, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709852

RESUMO

The detailed understanding of the binding of small molecules to proteins is the key for the development of novel drugs or to increase the acceptance of substrates by enzymes. Nowadays, computer-aided design of protein-ligand binding is an important tool to accomplish this task. Current approaches typically rely on high-throughput docking essays or computationally expensive atomistic molecular dynamics simulations. Here, we present an approach to use the recently re-parametrized coarse-grained Martini model to perform unbiased millisecond sampling of protein-ligand interactions of small drug-like molecules. Remarkably, we achieve high accuracy without the need of any a priori knowledge of binding pockets or pathways. Our approach is applied to a range of systems from the well-characterized T4 lysozyme over members of the GPCR family and nuclear receptors to a variety of enzymes. The presented results open the way to high-throughput screening of ligand libraries or protein mutations using the coarse-grained Martini model.


Assuntos
Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas/química , Bacteriófago T4/enzimologia , Biofísica , Biologia Computacional , Ensaios de Triagem em Larga Escala , Ligantes , Simulação de Acoplamento Molecular , Muramidase/química , Conformação Proteica , Termodinâmica
9.
J Clin Microbiol ; 58(8)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513859

RESUMO

The role of serologic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in both the clinical and public health settings, will continue to evolve as we gain increasing insight into our immune response to the virus. Here, we evaluated four high-throughput serologic tests for detection of anti-SARS-CoV-2 IgG antibodies, from Abbott Laboratories (Abbott Park, IL), Epitope Diagnostics, Inc. (San Diego, CA), Euroimmun (Lubeck, Germany), and Ortho-Clinical Diagnostics (Rochester, NY), using a panel of serially collected serum samples (n = 224) from 56 patients with confirmed coronavirus disease 2019 (COVID-19), healthy donor sera from 2018, and a cross-reactivity serum panel collected in early 2020. The sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays in convalescent-phase serum samples collected more than 14 days post-symptom onset or post-initial positive reverse transcriptase PCR (RT-PCR) result were 92.9% (78/84), 88.1% (74/84), 97.6% (82/84), and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays were 97.3% (36/37), 73% (27/37), 94.6% (35/37), and 97.3% (36/37), respectively. Overall assay specificity/positive predictive values based on a 5% prevalence rate were 99.6%/92.8%, 99.6%/90.6%, 98.0%/71.2%, and 99.6%/92.5%, respectively, for the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays. In conclusion, we show high sensitivity in convalescent-phase sera and high specificity for the Abbott, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays. With the unprecedented influx of commercially available serologic tests for detection of antibodies against SARS-CoV-2, it remains imperative that laboratories thoroughly evaluate such assays for accuracy prior to implementation.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Adulto Jovem
10.
Sci Total Environ ; 729: 139028, 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32498177

RESUMO

The goal of the present study was to select a Gram-positive (Gram+) and Gram-negative (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clinical and environmental relevance. The assay sensitivity ranged from the ng/mL to µg/mL range. The applicability of the assays for toxicological characterization of aquatic environmental samples was demonstrated for hospital effluent extract. A future application includes effect-directed analysis to identify yet unknown antibiotic contaminants or their transformation products.


Assuntos
Bioensaio , Antibacterianos , Bacillus subtilis , Escherichia coli , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana
11.
Proc Natl Acad Sci U S A ; 117(24): 13261-13266, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482866

RESUMO

Modern organic reaction discovery and development relies on the rapid assessment of large arrays of hypothesis-driven experiments. The time-intensive nature of reaction analysis presents the greatest practical barrier for the execution of this iterative process that underpins the development of new bioactive agents. Toward addressing this critical bottleneck, we report herein a high-throughput analysis (HTA) method of reaction mixtures by photocapture on a 384-spot diazirine-terminated self-assembled monolayer, and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) analysis. This analytical platform has been applied to the identification of a single-electron-promoted reductive coupling of acyl azolium species.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Benzimidazóis/síntese química , Benzimidazóis/química , Diazometano/química , Oxirredução , Raios Ultravioleta
12.
Nat Protoc ; 15(7): 2203-2229, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541940

RESUMO

Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters. The method is based on dynamic self-assembly of commercially available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivatives assemble with diol-type ligands containing a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to determine the amount of each enantiomer in the mixture with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asymmetric reaction products. The procedure comprises high-throughput liquid dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asymmetric transformations and chiral drug development. The procedure takes ~4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.


Assuntos
Aminas/química , Amino Álcoois/química , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Estereoisomerismo
13.
Life Sci ; 257: 118025, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32598933

RESUMO

BACKGROUND: Glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) co-agonists have emerged as treatment options for reversing diabetes and obesity. Here, we screened the high potency receptor-biased GLP-1R agonists via a newly designed high-throughput GLP-1R extracellular domain (ECD)-based system and demonstrated its in vitro and in vivo therapeutic characters. METHODS: Twelve 9-mer peptides (named XEL1-XEL12) which were screened from a large phage-displayed peptide library were fused to the N-terminus of GIP (3-30) to generate another twelve fusion peptides, termed XEL13-24. Using the six lysine-altered XEL17 as leading sequences, eighteen fatty chain modified fusion peptides were further assessed via in vitro GLP-1R/GIPR-based cell assay. Moreover, the acute and long-acting in vivo effects of selected candidate on diabetic db/db mice and diet-induced obesity (DIO) rats were both carefully evaluated. RESULTS: XEL17 exhibited balanced activation potency on GLP-1R/GIPR in stable cell lines, and further assessment was performed to evaluate the XEL32, a fatty chain modified XEL17 derivative. Preclinical pharmacodynamic results in diabetic db/db mice demonstrated that XEL32 held outstanding insulinotropic and glucose-lowering activities. In addition, protracted antidiabetic effects of XEL32 were also proved by the hypoglycemic test and multiple oral glucose tolerance test. Furthermore, chronic treatment of XEL32 in DIO rats exhibited outstanding beneficial effects on body weight control, fat loss, food intake control, hemoglobin A1C (HbA1C) reduction as well as the glucose tolerance. CONCLUSIONS: XEL32, as a novel GLP-1/GIP dual receptor agonist, may supply efficient glycemic control and weight loss.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/metabolismo , Perda de Peso/efeitos dos fármacos , Animais , Glicemia/metabolismo , China , Diabetes Mellitus/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/efeitos dos fármacos , Teste de Tolerância a Glucose , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Obesidade/metabolismo , Ratos , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/efeitos dos fármacos
14.
Nat Commun ; 11(1): 3186, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581242

RESUMO

Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.


Assuntos
Análise de Injeção de Fluxo/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Linhagem Celular Tumoral , Análise de Injeção de Fluxo/normas , Ensaios de Triagem em Larga Escala , Humanos , Íons/química , Lipidômica/métodos , Espectrometria de Massas/normas , Metabolômica/normas , Soro/química , Soro/metabolismo
15.
Toxicon ; 184: 28-38, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32502555

RESUMO

Many organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.


Assuntos
Venenos de Crotalídeos , Fosfolipases A2/metabolismo , Venenos de Serpentes , Sequência de Aminoácidos , Animais , Bothrops , Fracionamento Químico , Cromatografia Líquida , Elapidae , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Proteínas de Répteis , Venenos de Víboras , Viperidae
16.
Prostate ; 80(11): 885-894, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32483877

RESUMO

BACKGROUND: Eradication of persistent androgen receptor (AR) activity in castration-resistant prostate cancer may be a promising strategy to overcome castration resistance. We aimed to identify novel compounds that inhibit AR activity and could be potential therapeutic agents for prostate cancer. METHODS: A high-throughput screening system involving cell lines stably expressing AR protein and AR-responsive luciferase was employed for the 1260 compound library. Molecular and antitumor effects on candidate pathways that interacted with AR signaling were examined in prostate cancer cells expressing AR. RESULTS: The high-throughput screening identified various potential compounds that interfered with AR signaling through known and novel pathways. Among them, a 5-hydroxytryptamine 5A (5-HT5A) receptor antagonist suppressed AR activity through protein kinase A signaling, which was confirmed by 5-HT5A receptor knockdown. Consistently, 5-HT5A receptor inhibitors showed cytotoxic effects toward prostate cancer cells. CONCLUSIONS: Taken together, this study identifies 5-HT5A receptor as a promising therapeutic target for prostate cancer via its interaction with AR signaling.


Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Androgênios/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo
17.
Virus Res ; 286: 198068, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32565126

RESUMO

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a renewed interest in studying the role of the spike S glycoprotein in regulating coronavirus infections in the natural host. Taking advantage of the cryo-electron microscopy structure of SARS-CoV-2 S trimer in the prefusion conformation, we performed a virtual screening simulation with the aim to identify novel molecules that could be used as fusion inhibitors. The spike glycoprotein structure has been completed using modeling techniques and its inner cavity, needful for the postfusion transition of the trimer, has been scanned for the identification of strongly interacting available drugs. Finally, the stability of the protein-drug top complexes has been tested using classical molecular dynamics simulations. The free energy of interaction of the molecules to the spike protein has been evaluated through the MM/GBSA method and per-residue decomposition analysis. Results have been critically discussed considering previous scientific knowledge concerning the selected compounds and sequence alignments have been carried out to evaluate the spike glycoprotein similarity among the betacoronavirus family members. Finally, a cocktail of drugs that may be used as SARS-CoV-2 fusion inhibitors has been suggested.


Assuntos
Antivirais/química , Betacoronavirus/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Indóis/química , Perileno/análogos & derivados , Glicoproteína da Espícula de Coronavírus/química , Sulfonamidas/química , Antivirais/farmacologia , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Indóis/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Perileno/química , Perileno/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sulfonamidas/farmacologia , Termodinâmica , Interface Usuário-Computador , Internalização do Vírus/efeitos dos fármacos
18.
APMIS ; 128(8): 497-505, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32562292

RESUMO

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.


Assuntos
Automação Laboratorial/métodos , Ensaios de Triagem em Larga Escala , Testes de DNA para Papilomavírus Humano/métodos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Fluxo de Trabalho , Feminino , Humanos , Países Baixos , Papillomaviridae/classificação , Papillomaviridae/genética , Estudos Retrospectivos , Fatores de Tempo
19.
J Clin Virol ; 129: 104474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32504946

RESUMO

BACKGROUND: High-throughput assays for the SARS-CoV-2 virus are critical to increasing test capacity and slowing the spread of COVID-19. Abbott Molecular developed and received emergency use authorization (EUA) to deploy the new RealTime SARS-CoV-2 assay, run on the automated m2000sp/rt system. OBJECTIVE: To evaluate analytical and clinical performance of the RealTime SARS-CoV-2 assay compared to the SARS-CoV-2 CDC-based laboratory developed test (LDT) in clinical use by the University of Washington Clinical Virology Laboratory (UW Virology). METHODS: RealTime SARS-CoV-2 assay limit of detection (LOD) was evaluated by testing two dilution panels of 60 replicates each. Cross-reactivity was evaluated by testing 24 clinical samples positive for various non‒SARS-CoV-2 respiratory viruses. Clinical performance was evaluated using 30 positive and 30 negative SARS-CoV-2 clinical samples previously tested using the UW Virology SARS-CoV-2 LDT. RESULTS: Exceeding the 100 copies/mL LOD reported in the RealTime SARS-CoV-2 assay EUA product insert, 19 of 20 replicates were detected at 50 copies/mL and 16 of 20 replicates were detected at 25 copies/mL. All clinical samples positive for 24 non‒SARS-CoV-2 respiratory viruses were SARS-CoV-2 negative on the RealTime SARS-CoV-2 assay. The assay had high sensitivity (93%) and specificity (100%) for detecting SARS-CoV-2 in clinical samples. Two positive samples that tested negative with the RealTime SARS-CoV-2 assay had cycle numbers of 35.94 or greater and required dilution prior to testing. One of these samples was also inconclusive on the SARS-CoV-2 LDT. CONCLUSION: The RealTime SARS-CoV-2 assay is acceptable for clinical use. With the high-throughput, fully automated m2000 system, this assay will accelerate the pace of SARS-CoV-2 testing.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Automação Laboratorial/métodos , Betacoronavirus/genética , Ensaios de Triagem em Larga Escala/métodos , Hospitais Universitários , Humanos , Pandemias , RNA Viral/genética , Sensibilidade e Especificidade , Washington
20.
J Clin Virol ; 129: 104480, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32505777

RESUMO

Serological SARS-CoV-2 assays are urgently needed for diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how recently commercially available, high-throughput immunoassays, using different recombinant SARS-CoV-2 antigens, perform with clinical samples. Focusing on IgG and total antibodies, we demonstrate the performance of four automated immunoassays (Abbott Architect™ i2000 (N protein-based)), Roche cobas™ e 411 analyzer (N protein-based, not differentiating between IgA, IgM or IgG antibodies), LIAISON®XL platform (S1 and S2 protein-based), VIRCLIA® automation system (S1 and N protein-based) in comparison to two ELISA assays (Euroimmun SARS-CoV-2 IgG (S1 protein-based) and Virotech SARS-CoV-2 IgG ELISA (N protein-based)) and an in-house developed plaque reduction neutralization test (PRNT). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. When calculating the overall sensitivity, in a time frame of 49 days after first PCR-positivity, the PRNT as gold standard, showed the highest sensitivity with 93.3% followed by the dual-target assay for the VIRCLIA® automation system with 89%. The overall sensitivity in the group of N protein-based assays ranged from 66.7 to 77.8% and in the S protein-based-assays from 71.1 to 75.6%. Five follow-up samples of three individuals were only detected in either an S and/or N protein-based assay, indicating an individual different immune response to SARS-CoV-2 and the influence of the used assay in the detection of IgG antibodies. This should be further analysed. The specificity of the examined assays was ≥ 97%. However, because of the low or unknown prevalence of SARS-CoV-2, the examined assays in this study are currently primarily eligible for epidemiological investigations, as they have limited information in individual testing.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/sangue , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Automação Laboratorial/métodos , Humanos , Pandemias , Sensibilidade e Especificidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA