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1.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360558

RESUMO

Experimental screening of large sets of compounds against macromolecular targets is a key strategy to identify novel bioactivities. However, large-scale screening requires substantial experimental resources and is time-consuming and challenging. Therefore, small to medium-sized compound libraries with a high chance of producing genuine hits on an arbitrary protein of interest would be of great value to fields related to early drug discovery, in particular biochemical and cell research. Here, we present a computational approach that incorporates drug-likeness, predicted bioactivities, biological space coverage, and target novelty, to generate optimized compound libraries with maximized chances of producing genuine hits for a wide range of proteins. The computational approach evaluates drug-likeness with a set of established rules, predicts bioactivities with a validated, similarity-based approach, and optimizes the composition of small sets of compounds towards maximum target coverage and novelty. We found that, in comparison to the random selection of compounds for a library, our approach generates substantially improved compound sets. Quantified as the "fitness" of compound libraries, the calculated improvements ranged from +60% (for a library of 15,000 compounds) to +184% (for a library of 1000 compounds). The best of the optimized compound libraries prepared in this work are available for download as a dataset bundle ("BonMOLière").


Assuntos
Algoritmos , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/normas , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Humanos
2.
Sci Rep ; 11(1): 2535, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510380

RESUMO

To provide a comprehensive analysis of small molecule genotoxic potential we have developed and validated an automated, high-content, high throughput, image-based in vitro Micronucleus (IVM) assay. This assay simultaneously assesses micronuclei and multiple additional cellular markers associated with genotoxicity. Acoustic dosing (≤ 2 mg) of compound is followed by a 24-h treatment and a 24-h recovery period. Confocal images are captured [Cell Voyager CV7000 (Yokogawa, Japan)] and analysed using Columbus software (PerkinElmer). As standard the assay detects micronuclei (MN), cytotoxicity and cell-cycle profiles from Hoechst phenotypes. Mode of action information is primarily determined by kinetochore labelling in MN (aneugencity) and γH2AX foci analysis (a marker of DNA damage). Applying computational approaches and implementing machine learning models alongside Bayesian classifiers allows the identification of, with 95% accuracy, the aneugenic, clastogenic and negative compounds within the data set (Matthews correlation coefficient: 0.9), reducing analysis time by 80% whilst concurrently minimising human bias. Combining high throughput screening, multiparametric image analysis and machine learning approaches has provided the opportunity to revolutionise early Genetic Toxicology assessment within AstraZeneca. By multiplexing assay endpoints and minimising data generation and analysis time this assay enables complex genotoxicity safety assessments to be made sooner aiding the development of safer drug candidates.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Testes para Micronúcleos/métodos , Testes de Mutagenicidade , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Aprendizado de Máquina , Testes para Micronúcleos/normas , Modelos Estatísticos , Testes de Mutagenicidade/métodos , Testes de Mutagenicidade/normas , Reprodutibilidade dos Testes
3.
Toxicol Lett ; 338: 67-77, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33290830

RESUMO

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.


Assuntos
Alérgenos/toxicidade , Cromatografia Líquida de Alta Pressão , Dermatite Alérgica de Contato/etiologia , Ensaios de Triagem em Larga Escala , Peptídeos/química , Pele/efeitos dos fármacos , Espectrometria de Massas em Tandem , Testes de Toxicidade , Alérgenos/química , Alternativas aos Testes com Animais , Cromatografia Líquida de Alta Pressão/normas , Cisteína , Ensaios de Triagem em Larga Escala/normas , Humanos , Lisina , Padrões de Referência , Reprodutibilidade dos Testes , Medição de Risco , Espectrometria de Massas em Tandem/normas
4.
J Virol Methods ; 288: 114013, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33166547

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.


Assuntos
Corantes Fluorescentes , Ensaios de Triagem em Larga Escala , RNA Viral , RNA Polimerase Dependente de RNA/metabolismo , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/diagnóstico , Síndrome Respiratória Aguda Grave/genética , Antivirais/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Concentração Inibidora 50 , RNA Mensageiro/genética , Moldes Genéticos
5.
J Clin Microbiol ; 59(3)2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33303562

RESUMO

As the coronavirus disease 2019 (COVID-19) pandemic second wave is emerging, it is of the upmost importance to screen the population immunity in order to keep track of infected individuals. Consequently, immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and positive predictive values are needed to obtain an accurate epidemiological picture. As more data accumulate about the immune responses and the kinetics of neutralizing-antibody (nAb) production in SARS-CoV-2-infected individuals, new applications are forecast for serological assays such as nAb activity prediction in convalescent-phase plasma from recovered patients. This multicenter study, involving six hospital centers, determined the baseline clinical performances, reproducibility, and nAb level correlations of 10 commercially available immunoassays. In addition, three lateral-flow chromatography assays were evaluated, as these devices can be used in logistically challenged areas. All assays were evaluated using the same patient panels in duplicate, thus enabling accurate comparison of the tests. Seven immunoassays examined in this study were shown to have excellent specificity (98 to 100%) and good to excellent positive predictive values (82 to 100%) when used in a low (5%)-seroprevalence setting. We observed sensitivities as low as 74% and as high as 95% at ≥15 days after symptom onset. The determination of optimized cutoff values through receiver operating characteristic (ROC) curve analyses had a significant impact on the diagnostic resolution of several enzyme immunoassays by increasing the sensitivity significantly without a large trade-off in specificity. We found that spike-based immunoassays seem to be better correlates of nAb activity. Finally, the results reported here will add to the general knowledge of the interlaboratory reproducibility of clinical performance parameters of immunoassays and provide new evidence about nAb activity prediction.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , COVID-19/diagnóstico , Ensaios de Triagem em Larga Escala/normas , COVID-19/imunologia , Humanos , Laboratórios , Reprodutibilidade dos Testes , SARS-CoV-2 , Sensibilidade e Especificidade , Estudos Soroepidemiológicos
6.
Methods Mol Biol ; 2222: 149-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301093

RESUMO

Molecular markers provide researchers with a powerful tool for variation analysis between plant genomes. They are heritable and widely distributed across the genome and for this reason have many applications in plant taxonomy and genotyping. Over the last decade, molecular marker technology has developed rapidly and is now a crucial component for genetic linkage analysis, trait mapping, diversity analysis, and association studies. This chapter focuses on molecular marker discovery, its application, and future perspectives for plant genotyping through pangenome assemblies. Included are descriptions of automated methods for genome and sequence distance estimation, genome contaminant analysis in sequence reads, genome structural variation, and SNP discovery methods.


Assuntos
Código de Barras de DNA Taxonômico , Técnicas de Genotipagem , Ensaios de Triagem em Larga Escala , Plantas/classificação , Plantas/genética , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico/métodos , Código de Barras de DNA Taxonômico/normas , Contaminação por DNA , Evolução Molecular , Marcadores Genéticos , Genoma de Planta , Genômica/métodos , Genótipo , Ensaios de Triagem em Larga Escala/normas , Filogenia , Polimorfismo de Nucleotídeo Único
7.
Elife ; 92020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33274713

RESUMO

High-throughput testing of drugs across molecular-characterised cell lines can identify candidate treatments and discover biomarkers. However, the cells' response to a drug is typically quantified by a summary statistic from a best-fit dose-response curve, whilst neglecting the uncertainty of the curve fit and the potential variability in the raw readouts. Here, we model the experimental variance using Gaussian Processes, and subsequently, leverage uncertainty estimates to identify associated biomarkers with a new Bayesian framework. Applied to in vitro screening data on 265 compounds across 1074 cancer cell lines, our models identified 24 clinically established drug-response biomarkers, and provided evidence for six novel biomarkers by accounting for association with low uncertainty. We validated our uncertainty estimates with an additional drug screen of 26 drugs, 10 cell lines with 8 to 9 replicates. Our method is applicable to any dose-response data without replicates, and improves biomarker discovery for precision medicine.


Assuntos
Antineoplásicos , Biomarcadores Tumorais/análise , Descoberta de Drogas/métodos , Descoberta de Drogas/normas , Estatística como Assunto/métodos , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos
8.
Protist ; 171(6): 125773, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33243724

RESUMO

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.


Assuntos
Amebíase/parasitologia , Amoeba/fisiologia , Bioensaio/normas , Doenças dos Peixes/parasitologia , Ensaios de Triagem em Larga Escala/métodos , Amoeba/citologia , Animais , Organismos Aquáticos , Pesqueiros , Ensaios de Triagem em Larga Escala/normas , Viabilidade Microbiana
9.
Exp Parasitol ; 219: 108014, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33011238

RESUMO

The objective set by WHO to reach elimination of human African trypanosomiasis (HAT) as a public health problem by 2020 is being achieved. The next target is the interruption of gambiense-HAT transmission in humans by 2030. To monitor progress towards this target, in areas where specialized local HAT control capacities will disappear, is a major challenge. Test specimens should be easily collectable and safely transportable such as dried blood spots (DBS). Monitoring tests performed in regional reference centres should be reliable, cheap and allow analysis of large numbers of specimens in a high-throughput format. The aim of this study was to assess the analytical sensitivity of Loopamp, M18S quantitative real-time PCR (M18S qPCR) and TgsGP qPCR as molecular diagnostic tests for the presence of Trypanosoma brucei gambiense in DBS. The sensitivity of the Loopamp test, with a detection limit of 100 trypanosomes/mL, was in the range of parasitaemias commonly observed in HAT patients, while detection limits for M18S and TgsGP qPCR were respectively 1000 and 10,000 trypanosomes/mL. None of the tests was entirely suitable for high-throughput use and further development and implementation of sensitive high-throughput molecular tools for monitoring HAT elimination are needed.


Assuntos
Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Trypanosoma brucei gambiense/isolamento & purificação , Tripanossomíase Africana/prevenção & controle , Algoritmos , Animais , Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , DNA de Protozoário/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Trypanosoma brucei gambiense/genética , Tripanossomíase Africana/sangue , Tripanossomíase Africana/diagnóstico
10.
Appl Microbiol Biotechnol ; 104(13): 6013-6022, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32367311

RESUMO

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 µl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.


Assuntos
Evolução Molecular Direcionada/métodos , Ensaios de Triagem em Larga Escala/métodos , Papel , Catálise , Colorimetria , Evolução Molecular Direcionada/economia , Evolução Molecular Direcionada/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/normas , Hidrolases/genética , Hidrolases/metabolismo , Mutagênese , Mutação , Reprodutibilidade dos Testes , Smartphone
11.
Sci Rep ; 10(1): 4239, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144330

RESUMO

Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Consumo de Oxigênio , Smegmamorpha , Testes de Toxicidade/métodos , Animais , Cádmio/metabolismo , Relação Dose-Resposta a Droga , Exposição Ambiental , Inocuidade dos Alimentos , Substâncias Perigosas/toxicidade , Ensaios de Triagem em Larga Escala/normas , Humanos , Mitocôndrias/metabolismo , Testes de Toxicidade/normas , Fluxo de Trabalho
12.
Hum Mutat ; 41(1): 332-341, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31471937

RESUMO

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.


Assuntos
Marcadores Genéticos , Predisposição Genética para Doença , Testes Genéticos , Ensaios de Triagem em Larga Escala , Instabilidade de Microssatélites , Repetições de Microssatélites , Alelos , Biomarcadores Tumorais , Linhagem Celular , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Estudos de Associação Genética/métodos , Testes Genéticos/métodos , Testes Genéticos/normas , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Técnicas de Diagnóstico Molecular , Fosforilação , Reprodutibilidade dos Testes
13.
Eur Neuropsychopharmacol ; 31: 1-15, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866110

RESUMO

Genomic high-throughput technologies (GHTT) such as next-generation sequencing represent a fast and cost-effective tool toward a more comprehensive understanding of the molecular background of complex diseases. However, technological advances contrast with insufficient application in clinical practice. Thus, patients, physicians, and other professionals are faced with tough challenges that forestall the efficient and effective implementation. With the increasing application of genetic testing, it is of paramount importance that physicians and other professionals in healthcare recognize the restrictions and potential of GHTT, in order to understand and interpret the complex data in the context of health and disease. At the same time, the growing volume and complexity of data is forever increasing the need for sustainable infrastructure and state-of-the-art tools for efficient data management, including their analysis and integration. The large pool of sensitive information remains difficult to interpret and fundamental questions spanning from billing to legal, social, and ethical issues have still not been resolved. Here we summarize and discuss these obstacles in an interdisciplinary context and suggest ways to overcome them. Continuous discussion with clinicians, data managers, biostatisticians, systems medicine experts, ethicists, legal scholars, and patients illuminates the strengths, weakness, and current practices in the pipeline from biomaterial to sequencing and data management. This discussion also highlights the new, cross-disciplinary working collaborations to realize the wide-ranging challenges in clinical genomics including the exceptional demands placed on the staff preparing and presenting the data, as well as the question as to how to report the data and results to patients.


Assuntos
Aconselhamento Genético/ética , Testes Genéticos/ética , Genômica/ética , Ensaios de Triagem em Larga Escala/ética , Aconselhamento Genético/legislação & jurisprudência , Aconselhamento Genético/normas , Testes Genéticos/legislação & jurisprudência , Testes Genéticos/normas , Genômica/legislação & jurisprudência , Genômica/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Psicologia
14.
EBioMedicine ; 50: 14-22, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31761619

RESUMO

BACKGROUND: Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. METHODS: We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. FINDINGS: In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. INTERPRETATION: The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. FUNDING: BMBF (03VP00402).


Assuntos
Antígenos de Protozoários/genética , Ensaios de Triagem em Larga Escala/métodos , Malária Falciparum/diagnóstico , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Estudos Transversais , Eletroforese Capilar , Ensaios de Triagem em Larga Escala/normas , Humanos , Malária Falciparum/parasitologia , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
Regul Toxicol Pharmacol ; 109: 104510, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31676319

RESUMO

Synthesis of 11 steroid hormones in human adrenocortical carcinoma cells (H295R) was measured in a high-throughput steroidogenesis assay (HT-H295R) for 656 chemicals in concentration-response as part of the US Environmental Protection Agency's ToxCast program. This work extends previous analysis of the HT-H295R dataset and model by examining the utility of a novel prioritization metric based on the Mahalanobis distance that reduced these 11-dimensional data to 1-dimension via calculation of a mean Mahalanobis distance (mMd) at each chemical concentration screened for all hormone measures available. Herein, we evaluated the robustness of mMd values, and demonstrate that covariance and variance of the hormones measured appear independent of the chemicals screened and are inherent to the assay; the Type I error rate of the mMd method is less than 1%; and, absolute fold changes (up or down) of 1.5 to 2-fold have sufficient power for statistical significance. As a case study, we examined hormone responses for aromatase inhibitors in the HT-H295R assay and found high concordance with other ToxCast assays for known aromatase inhibitors. Finally, we used mMd and other ToxCast cytotoxicity data to demonstrate prioritization of the most selective and active chemicals as candidates for further in vitro or in silico screening.


Assuntos
Inibidores da Aromatase/toxicidade , Disruptores Endócrinos/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Esteroides/biossíntese , Linhagem Celular Tumoral , Interpretação Estatística de Dados , Conjuntos de Dados como Assunto , Reações Falso-Positivas , Ensaios de Triagem em Larga Escala/normas , Humanos , Reprodutibilidade dos Testes , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Estados Unidos , United States Environmental Protection Agency/normas
16.
PLoS One ; 14(11): e0221546, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31689301

RESUMO

Within 2021, Norway intends to complete implementation of HPV DNA-based primary screening for cervical cancer for women 34-69 years, while continue cytology-based screening for women 25-33 years. Over the recent years, the incidence of cervical cancer has increased by 30% among women younger than 40 years. In this subset of women, nearly 30% were diagnosed with a normal smear, as most recent smear, prior the cancer diagnosis. This observation demands quality control of normal smears. The aim of this study was to assess increase in program sensitivity of CIN2+ after follow-up of women with false negative Pap-smears testing positive for a 3-type (-16, -18, -45) HPV mRNA test in a cohort design over one screening interval. 521 women, aged 23-39 years, and no prior history of CIN1+ or HSIL, with an ASC-US or worse smear (ASC-US+) and 1444 women with normal screening cytology comprised the study cohorts. The positivity rate for the 3-type HPV mRNA was 1.9% (28/1444). Rescreening revealed 23 women with ASC-US, two women with LSIL, two women with ASC-H, and one woman with AGUS. If the HPV mRNA-positivity rate and histology findings from samples rescreened were applied to all women with normal cytology, an estimated increase in screening sensitivity of 16.4% (95% CI:15.3-17.5) for CIN2+ and 17.3% (95% CI:16.2-18.4) for CIN3+ were achieved. By rescreening less than 2% of women with normal cytology positive for a 3-type HPV mRNA test, we achieved a significant increase in screening program sensitivity.


Assuntos
Neoplasia Intraepitelial Cervical/diagnóstico , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Adulto , Neoplasia Intraepitelial Cervical/epidemiologia , Neoplasia Intraepitelial Cervical/virologia , Estudos de Coortes , Feminino , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Programas de Rastreamento/métodos , Programas de Rastreamento/normas , Programas de Rastreamento/estatística & dados numéricos , Gradação de Tumores , Noruega/epidemiologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Prevalência , Controle de Qualidade , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal/normas , Esfregaço Vaginal/estatística & dados numéricos , Adulto Jovem
17.
Curr HIV Res ; 17(6): 429-440, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31782368

RESUMO

BACKGROUND: HIV-1 protease inhibitor (PI) is one of the most potent classes of drugs in combinational antiretroviral therapies (cART). When a PI is used in combination with other anti- HIV drugs, cART can often suppress HIV-1 below detection thus prolonging the patient's lives. However, the challenge often faced by patients is the emergence of HIV-1 drug resistance. Thus, PIs with high genetic-barrier to drug-resistance are needed. OBJECTIVE: The objective of this study was to develop a novel and simple fission yeast (Schizosaccharomyces pombe) cell-based system that is suitable for high throughput screening (HTS) of small molecules against HIV-1 protease (PR). METHODS: A fission yeast RE294-GFP strain that stably expresses HIV-1 PR and green fluorescence protein (GFP) under the control of an inducible nmt1 promoter was used. Production of HIV-1 PR induces cellular growth arrest, which was used as the primary endpoint for the search of PIs and was quantified by an absorbance-based method. Levels of GFP production were used as a counter-screen control to eliminate potential transcriptional nmt1 inhibitors. RESULTS: Both the absorbance-based HIV-1 PR assay and the GFP-based fluorescence assay were miniaturized and optimized for HTS. A pilot study was performed using a small drug library mixed with known PI drugs and nmt1 inhibitors. With empirically adjusted and clearly defined double-selection criteria, we were able to correctly identify the PIs and to exclude all hidden nmt1 inhibitors. CONCLUSION: We have successfully developed and validated a fission yeast cell-based HTS platform for the future screening and testing of HIV-1 PR inhibitors.


Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Ensaios de Triagem em Larga Escala , Schizosaccharomyces/genética , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Genes Reporter , Protease de HIV/genética , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Proteínas Recombinantes de Fusão , Reprodutibilidade dos Testes , Schizosaccharomyces/metabolismo
18.
ACS Comb Sci ; 21(11): 722-725, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31566941

RESUMO

Translation of a manual process to high throughput for research and development requires special consideration. One important and often unreported aspect is the establishment of an efficient cleaning routine. This becomes significant, as precious time and, in particular, material would be lost, that is, when low-quality high-throughput experimentation is involved. We present a fully automated cleaning routine of the challenging synthesis of cadmium selenide quantum dots. Manual, semiautomated, and fully automated cleaning protocols were executed and compared in terms of spectral similarities of the synthesized colloids. Only the fully automated protocol enabled true 24/7 operation.


Assuntos
Automação/métodos , Coloides/síntese química , Ensaios de Triagem em Larga Escala/normas , Compostos de Cádmio/síntese química , Ensaios de Triagem em Larga Escala/métodos , Pontos Quânticos/química , Compostos de Selênio/síntese química
19.
BMC Cancer ; 19(1): 825, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438998

RESUMO

BACKGROUND: To develop a new 18 high-risk human papillomavirus (HR HPV) detection and genotyping assay, which is important to evaluate the risk degree of HR HPV for causing cancers. METHODS: All 18 HR HPV and ß-globin relative DNA fragments were synthesized and cloned to a plasmid pUC57 to obtain their recombinant plasmids. Based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, each of the 18 HR HPV genotypes were investigated using their constructed recombinant plasmids. The new 18 HR HPV genotyping assay was tested using 356 clinical specimens and the results were compared to ones detected by the Roche Cobas 4800 HPV assay (Cobas). The discrepant results between two assays were resolved by sequencing and genotyping methods. RESULTS: The new 18 HR HPV MALDI-TOF MS genotyping assay was developed using HPV recombination plasmids. The sensitivity was 103 to 102 copies/reaction for the all 18 HR HPV. This new developed HR HPV genotyping test was used to detect the clinical specimens. When the results on clinical samples detected by the new MALDI-TOF MS HPV test were compared with ones detected by the Roche Cobas 4800 HPV assay in terms of 14 HR HPV, the concordance was 80.1% (kappa coefficient, 0.60; 95% confidence interval [CI], 0.52-0.69). The discrepant results were resolved by sequencing and genotyping and suggests that the developed HR HPV assay is more sensitive and specific. CONCLUSIONS: The new developed 18 HR HPV detection method based on MALDI-TOF MS platform is a high-throughput assay for the all 18 HR HPV genotypes and a powerful complement to current detection methods.


Assuntos
Técnicas de Genotipagem , Ensaios de Triagem em Larga Escala , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Adulto , Idoso , DNA Viral , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Masculino , Pessoa de Meia-Idade , Plasmídeos/genética , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
20.
EBioMedicine ; 47: 365-372, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31447394

RESUMO

BACKGROUND: Islet autoantibodies (IAbs) are the most reliable biomarkers to assess risk of progression to clinical type 1 diabetes (T1D). There are four major biochemically defined IAbs currently used in clinical trials that are equally important for disease prediction. The current screening methods use a radio-binding assay (RBA) for single IAb measurement, which are laborious and inefficient for large-scale screening. More importantly, up to 40% of patients with T1D have other autoimmune conditions that can be identified through relevant autoantibody testing. Thus, there is a need to screen for T1D and other autoimmune diseases simultaneously. METHODS: Based on our well-established electrochemiluminescence (ECL) assay platform, we developed a multiplexed ECL assay that combines 7 individual autoantibody assays together in one single well to simultaneously screen T1D, and three other autoimmune diseases including celiac disease, autoimmune thyroid disease and autoimmune poly-glandular syndrome-1 (APS-1). The 7-Plex ECL assay was extensively validated against single antibody measurements including a standard RBA and single ECL assay. FINDINGS: The 7-Plex ECL assay was well correlated to each single ECL autoantibody assay and each RBA. INTERPRETATION: The multiplexed ECL assay provides high sensitivity and disease specificity, along with high throughput and a low cost for large-scale screenings of T1D and other relevant autoimmune diseases in the general population. FUND: JDRF grants 2-SRA-2015-51-Q-R, 2-SRA-2018-533-S-B, NIH grants DK32083 and DK32493. NSFC grants 81770777.


Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/imunologia , Ensaios de Triagem em Larga Escala , Adolescente , Adulto , Autoanticorpos/sangue , Doenças Autoimunes/sangue , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Feminino , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Lactente , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto Jovem
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