RESUMO
As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.
Assuntos
Escherichia coli , Robótica , Robótica/métodos , Escherichia coli/genética , Engenharia de Proteínas/métodos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/economia , Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Polietilenotereftalatos/química , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost. RESULTS: After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4-256 µg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds. SIGNIFICANCE AND NOVELTY: Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice.
Assuntos
Caprilatos , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/economia , Humanos , Caprilatos/química , Caprilatos/sangue , Precipitação Química , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/economia , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.
Assuntos
Luciferases , Luciferases/metabolismo , Luciferases/química , Luciferases/genética , Humanos , Medições Luminescentes/métodos , Reprodutibilidade dos Testes , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/economia , Ensaios Enzimáticos/métodosRESUMO
The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing-the gold standard of SARS-CoV-2 diagnosis-is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen (LFA) tests.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Antígenos Virais/isolamento & purificação , Atletas , COVID-19/sangue , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/economia , Teste Sorológico para COVID-19/economia , Teste Sorológico para COVID-19/métodos , Redução de Custos , Ensaios de Triagem em Larga Escala/economia , Humanos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , África do Sul , Manejo de Espécimes/economia , Medicina Esportiva/economia , Medicina Esportiva/métodosRESUMO
Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.
Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , COVID-19/sangue , Teste Sorológico para COVID-19/economia , Teste em Amostras de Sangue Seco , Ensaios de Triagem em Larga Escala/economia , Humanos , Imunoensaio/economia , Imunoglobulina G/sangue , Técnicas Analíticas Microfluídicas/economia , Reprodutibilidade dos Testes , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
BACKGROUND: Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys. METHODS: Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. RESULTS: We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. CONCLUSION: We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Distrofia Miotônica/diagnóstico , Desnaturação de Ácido Nucleico , Expansão das Repetições de Trinucleotídeos , Custos e Análise de Custo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/normas , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Distrofia Miotônica/genética , Sensibilidade e EspecificidadeRESUMO
Therapeutic drug development is a long, expensive, and complex process that usually takes 12-15 years. In the early phases of drug discovery, in particular, there is a growing need for animal models that ensure the reduction in both cost and time. Caenorhabditis elegans has been traditionally used to address fundamental aspects of key biological processes, such as apoptosis, aging, and gene expression regulation. During the last decade, with the advent of large-scale platforms for screenings, this invertebrate has also emerged as an essential tool in the pharmaceutical research industry to identify novel drugs and drug targets. In this review, we discuss the reasons why C. elegans has been positioned as an outstanding cost-effective option for drug discovery, highlighting both the advantages and drawbacks of this model. Particular attention is paid to the suitability of this nematode in large-scale genetic and pharmacological screenings. High-throughput screenings in C. elegans have indeed contributed to the breakthrough of a wide variety of candidate compounds involved in extensive fields including neurodegeneration, pathogen infections and metabolic disorders. The versatility of this nematode, which enables its instrumentation as a model of human diseases, is another attribute also herein underscored. As illustrative examples, we discuss the utility of C. elegans models of both human neurodegenerative diseases and parasitic nematodes in the drug discovery industry. Summing up, this review aims to demonstrate the impact of C. elegans models on the drug discovery pipeline.
Assuntos
Caenorhabditis elegans/fisiologia , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Avaliação Pré-Clínica de Medicamentos/economia , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Animais , Especificidade da EspécieRESUMO
High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.
Assuntos
Sementes/anatomia & histologia , Zea mays/anatomia & histologia , Análise Custo-Benefício , Conjuntos de Dados como Assunto , Aprendizado Profundo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Fenótipo , Sementes/classificação , Gravação em Vídeo/métodos , Zea mays/classificaçãoRESUMO
Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal-derived foods. The current research describes for the first time the development of a semi-quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCß) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC-262536 and PF-06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD-2226, ostarine, S-1, S-6, and S-23), and 5 ng/mL (andarine, BMS-564929, LGD-4033, RAD140, and S-9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high-throughput cost-effective tool for the routine screening for a range of SARM compounds in sport and livestock animals.
Assuntos
Anabolizantes/análise , Androgênios/análise , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Anabolizantes/sangue , Androgênios/sangue , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/economia , Análise Custo-Benefício , Dopagem Esportivo , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Ensaios de Triagem em Larga Escala/economia , Cavalos , Detecção do Abuso de Substâncias/economia , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/economiaRESUMO
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 , SmartphoneRESUMO
The traditional qPCR instrument is bulky, expensive, and inconvenient to carry, so we report a portable rotary real-time fluorescent PCR (polymerase chain reaction) that completes the PCR amplification of DNA in the field, and the reaction can be observed in real-time. Through the analysis of a target gene, namely pGEM-3Zf (+), the gradient amplification and melting curves are compared to commercial devices. The results confirm the stability of our device. This is the first use of a mechanical rotary structure to achieve gradient amplification curves and melting curves comparable to commercial instruments. The average power consumption of our system is about 7.6 W, which is the lowest energy consumption for real-time fluorescence quantification in shunting PCR and enables the use of our device in the field thanks to its self-contained power supply based on a lithium battery. In addition, all of the equipment costs only about 710 dollars, which is far lower than the cost of a commercial PCR instrument because the control system through mechanical displacement replaces the traditional TEC (thermoelectric cooler) temperature control. Moreover, the equipment has a low technical barrier, which can suit the needs of non-professional settings, with strong repeatability.
Assuntos
Fontes de Energia Elétrica/economia , Fluorescência , Ensaios de Triagem em Larga Escala/economia , Lítio/economia , Reação em Cadeia da Polimerase em Tempo Real/economia , Lítio/química , TemperaturaRESUMO
More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.
Assuntos
Sistemas CRISPR-Cas/genética , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise Custo-Benefício , Células HEK293 , Ensaios de Triagem em Larga Escala/economia , Humanos , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.
Assuntos
DNA de Plantas/isolamento & purificação , Genômica , Ensaios de Triagem em Larga Escala , Genômica/economia , Genômica/métodos , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Plantas/genéticaRESUMO
Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.
Assuntos
5-Metilcitosina/análogos & derivados , Biomarcadores Tumorais/metabolismo , Epigênese Genética , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/genética , 5-Metilcitosina/metabolismo , Animais , Análise Custo-Benefício , Fluorescência , Ensaios de Triagem em Larga Escala/economia , Humanos , Camundongos , Neoplasias/classificação , Estudo de Prova de ConceitoRESUMO
Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.
Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Bioensaio/economia , Bioensaio/instrumentação , Descoberta de Drogas/economia , Descoberta de Drogas/instrumentação , Avaliação Pré-Clínica de Medicamentos/economia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/instrumentação , HumanosRESUMO
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.
Assuntos
Antígenos de Plaquetas Humanas/análise , Ensaios de Triagem em Larga Escala , Testes Sorológicos , Antígenos de Plaquetas Humanas/sangue , Antígenos de Plaquetas Humanas/genética , Estudos de Coortes , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Genótipo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Teste de Histocompatibilidade/economia , Teste de Histocompatibilidade/métodos , Humanos , Recém-Nascido , Integrina beta3 , Isoanticorpos/análise , Isoanticorpos/sangue , Países Baixos , Fenótipo , Valor Preditivo dos Testes , Gravidez , Sensibilidade e Especificidade , Testes Sorológicos/economia , Testes Sorológicos/métodos , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/diagnóstico , Trombocitopenia Neonatal Aloimune/genética , Trombocitopenia Neonatal Aloimune/imunologia , Fatores de TempoRESUMO
In this work, we propose an innovative high throughput (HT) apparatus using the bar adsorptive microextraction (BAµE) technique, which enables the simultaneous enrichment of up to 100 samples. This novel configuration was combined with microliquid desorption and high-performance liquid chromatography-diode array detection to monitor trace levels of eight benzodiazepines (diazepam, prazepam, bromazepam, oxazepam, lorazepam, alprazolam, temazepam and loflazepate) in biological samples. The proposed methodology was fully developed, optimized and validated, resulting in suitable intraday and interday precision (RSD ≤â¯15%), with recovery yields ranging from 33.0% to 104.5%. The lower limits of quantification were between 20.0 and 100.0⯵gâ¯L-1, using 1.0â¯mL of urine and 0.5â¯mL of plasma or serum samples. The application of the proposed methodology to real matrices resulted in average sample preparation time of around 2â¯min per sample, demonstrating that it is user-friendly, cost-effective and a rapid decision-making tool, whenever large number of samples are involved.
Assuntos
Benzodiazepinas/sangue , Benzodiazepinas/urina , Ensaios de Triagem em Larga Escala/economia , Microextração em Fase Líquida/economia , Adsorção , Cromatografia Líquida de Alta Pressão/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Microextração em Fase Líquida/instrumentaçãoRESUMO
Flavones are known as an inhibitor of tankyrase, a potential drug target of cancer. We here expedited the use of different computational approaches and presented a fast, easy, cost-effective and high throughput screening method to identify flavones analogs as potential tankyrase inhibitors. For this, we developed a field point based (3D-QSAR) quantitative structure-activity relationship model. The developed model showed acceptable predictive and descriptive capability as represented by standard statistical parameters r2 (0.89) and q2 (0.67). This model may help to explain SAR data and illustrated the key descriptors which were firmly related with the anticancer activity. Using the QSAR model a dataset of 8000 flavonoids were evaluated to classify the bioactivity, which resulted in the identification of 1480 compounds with the IC50 value of less than 5 µM. Further, these compounds were scrutinized through molecular docking and ADMET risk assessment. Total of 25 compounds identified which further analyzed for drug-likeness, oral bioavailability, synthetic accessibility, lead-likeness, and alerts for PAINS & Brenk. Besides, metabolites of screened compounds were also analyzed for pharmacokinetics compliance. Finally, compounds F2, F3, F8, F11, F13, F20, F21 and F25 with predicted activity (IC50) of 1.59, 1, 0.62, 0.79, 3.98, 0.79, 0.63 and 0.64, respectively were find as top hit leads. This study is offering the first example of a computationally-driven tool for prioritization and discovery of novel flavone scaffold for tankyrase receptor affinity with high therapeutic windows.
Assuntos
Flavonas/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Relação Quantitativa Estrutura-Atividade , Tanquirases/antagonistas & inibidores , Algoritmos , Domínio Catalítico , Análise Custo-Benefício , Flavonas/química , Flavonas/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Flavonoides/farmacologia , Ensaios de Triagem em Larga Escala/economia , Humanos , Modelos Teóricos , Estrutura Molecular , Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Tanquirases/química , Tanquirases/metabolismoRESUMO
The world is heading toward a dangerous post-antibiotic era where antibiotics fail to treat infections. Staphylococcus aureus is the leading cause of healthcare-associated infections worldwide, and an ever-increasing percentage of them are methicillin-resistant (MRSA). New strategies are urgently needed to combat this pathogen. Wall teichoic acids (WTA) in S. aureus are polyribitol phosphate polymers that play important roles in virulence and resistance to ß-lactam antibiotics. Here, we describe a high-throughput whole-cell screening platform for inhibitors targeting WTA biosynthesis. This platform takes advantage of the unique dispensability patterns of genes encoding WTA biosynthesis. We further describe follow-up dose-response assays to identify WTA inhibitors among the primary bioactives. WTA inhibitors offer an exciting opportunity for the development of novel antibacterial leads of unique mechanism in the fight against drug-resistant staphylococcal infections.
Assuntos
Antibacterianos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Testes de Sensibilidade Microbiana/métodos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Ácidos Teicoicos/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Ensaios de Triagem em Larga Escala/economia , Humanos , Testes de Sensibilidade Microbiana/economia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/genéticaRESUMO
Response to anticancer agents is often restricted to subsets of patients. The recognition of factors underlying this heterogeneity and the identification of biomarkers associated with response to drugs would greatly improve the efficacy of drug treatment. Platforms that can comprehensively map drug response in high-throughput ex vivo provide a unique tool to identify associated biomarkers and provide hypotheses for mechanisms underlying variable response. Such screens can be performed on cell lines and short-term cultures of primary cells to take advantage of the respective models' strength, which include, e.g., the ability to silence genes in cell lines and the "indefinite" supply of primary cells where clonal selection can be avoided. Cohorts of such samples represent the natural diversity of cancers, including rarer mutations and combinatorial patterns of mutations.We here summarize a simple and scalable method for the measurement of viability after drug exposure based on ATP measurements as a surrogate for viability, which we use to measure and understand drug response in cell lines and primary cells.