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1.
PLoS One ; 15(8): e0238479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866217

RESUMO

The performances of the ImmuView Streptococcus pneumoniae (Sp) and Legionella pneumophila (Lp) urinary antigen test were compared to that of the BinaxNOW Sp and Lp assays, using frozen urine from 166 patients with Legionnaires' disease (LD) and 59 patients with pneumococcal pneumonia. Thirty Sp-positive or contrived cerebrospinal fluids (CSF) were also tested. Test specimens were collected and tested at different sites, with each site testing unique specimens by technologists blinded to expected results. No significant differences in test concordances were detected for the ImmuView and BinaxNOW assays for the Sp or Lp targets for urine from patients with pneumococcal pneumonia or LD when performance from both sites were combined. At one of two test sites the ImmuView Lp assay was more sensitive than the BinaxNOW assay, with no correlation between test performance and Lp serogroup 1 monoclonal type. Urines from six of seven patients with LD caused by Legionella spp. bacteria other than Lp serogroup 1 were negative in both assays. Both tests had equivalent performance for Sp-positive CSF. The clinical sensitivities for pneumococcal pneumonia were 88.1 and 94.4% for the ImmuView and Binax assays, and 87.6 and 84.2% for the Lp assays, respectively. Test specificities for pneumococcal pneumonia were 96.2 and 97.0% for the ImmuView and Binax assays, and 99.6 and 99.1% for the Lp assays. Both assays were highly specific for Sp in pediatric urines from children with nasopharyngeal colonization by the bacterium. ImmuView and BinaxNOW assay performance was equivalent in these studies.


Assuntos
Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/urina , Bioensaio/métodos , Líquido Cefalorraquidiano/microbiologia , Legionella pneumophila/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Urina/microbiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Testes Imunológicos/métodos , Lactente , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Doença dos Legionários/urina , Masculino , Meningite/metabolismo , Meningite/microbiologia , Meningite/urina , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/urina , Sensibilidade e Especificidade , Sorogrupo , Adulto Jovem
2.
Rev Soc Bras Med Trop ; 53: e20200314, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32997053

RESUMO

INTRODUCTION: Rapid and accurate tuberculosis detection is critical for improving patient diagnosis and decreasing tuberculosis transmission. Molecular assays can significantly increase laboratory costs; therefore, the average time and economic impact should be evaluated before implementing a new technology. The aim of this study was to evaluate the cost and average turnaround time of smear microscopy and Xpert assay at a university hospital. METHODS: The turnaround time and cost of the laboratory diagnosis of tuberculosis were calculated based on the mean cost and activity based costing (ABC). RESULTS: The average turnaround time for smear microscopy was 16.6 hours while that for Xpert was 24.1 hours. The Xpert had a mean cost of USD 17.37 with an ABC of USD 10.86, while smear microscopy had a mean cost of USD 13.31 with an ABC of USD 6.01. The sensitivity of smear microscopy was 42.9% and its specificity was 99.1%, while the Xpert assay had a sensitivity of 100% and a specificity of 96.7%. CONCLUSIONS: The Xpert assay has high accuracy; however, the turnaround time and cost of smear microscopy were lower than those of Xpert.


Assuntos
Bioensaio/economia , Patologia Molecular/economia , Tuberculose Pulmonar/diagnóstico , Bioensaio/métodos , Custos e Análise de Custo , Humanos , Microscopia , Mycobacterium tuberculosis , Patologia Molecular/métodos , Sensibilidade e Especificidade , Tuberculose , Tuberculose Pulmonar/economia
3.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716395

RESUMO

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.


Assuntos
Autofagia/fisiologia , Bioensaio/métodos , Condrócitos/patologia , Citometria de Fluxo/métodos , Osteoartrite/patologia , Linhagem Celular Transformada , Condrócitos/efeitos dos fármacos , Descoberta de Drogas/métodos , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/farmacologia , Proteínas Associadas aos Microtúbulos/uso terapêutico , Osteoartrite/tratamento farmacológico , Osteoartrite/fisiopatologia
4.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478749

RESUMO

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.


Assuntos
Bioensaio/métodos , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Automação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Reprodutibilidade dos Testes , Rotenona/farmacologia
5.
J Vis Exp ; (159)2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32478759

RESUMO

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 µM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.


Assuntos
Bioensaio/métodos , Estresse Oxidativo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Colorimetria , Células Hep G2 , Humanos , Limite de Detecção , Peroxidação de Lipídeos , Lipoproteínas LDL/metabolismo , Malondialdeído/sangue , Oxirredução , Padrões de Referência , Reprodutibilidade dos Testes
6.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510477

RESUMO

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.


Assuntos
Bioensaio/métodos , Interneurônios/citologia , Terapia a Laser , Microscopia Confocal , Regeneração/fisiologia , Peixe-Zebra/fisiologia , Anestesia , Animais , Animais Geneticamente Modificados , Corpo Celular/metabolismo , Morte Celular , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Larva/citologia , Modelos Logísticos , Peixe-Zebra/genética
7.
PLoS One ; 15(5): e0232747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374765

RESUMO

The potential effects of Bt (Bacillus thuringiensis) maize on non-target organisms must be conducted before the Bt maize is commercially planted. Folsomia candida is one of the non-target organisms of Bt maize, also as an important indicator of soil quality and environmental pollution. In this study, a 90-day F. candida feeding test were conducted to evaluate the potential effects of two Bt maize lines IE09S034 and BT799 and their non-Bt conventional isolines Zong 31 and Zheng 58. The results show that Bt maize lines had no significant effects on the survival rate, reproduction, adult body length, larval body length, and the activities of acetyl cholinesterase, catalase and superoxide dismutase on the F. candida. Namely, Bt maize had no toxic effects on the F. candida.


Assuntos
Bacillus thuringiensis/genética , Candida/metabolismo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Zea mays/genética , Acetilcolinesterase/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bioensaio/métodos , Catalase/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Larva , Reprodução/genética , Microbiologia do Solo , Superóxido Dismutase/metabolismo , Zea mays/microbiologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-32361467

RESUMO

Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area.


Assuntos
Bioensaio/métodos , Imunoconjugados/química , Espectrometria de Massas em Tandem/métodos , Calicheamicinas/química , Cromatografia Líquida de Alta Pressão , Doxorrubicina/química , Flurbiprofeno/química , Interações Hidrofóbicas e Hidrofílicas , Ibuprofeno/química , Irinotecano/química , Cetoprofeno/química , Maitansina/química , Conformação Molecular , Norfloxacino/química , Pentaclorofenol/química , Multimerização Proteica , Relação Estrutura-Atividade , Tolnaftato/química
9.
Breast Cancer Res ; 22(1): 43, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393398

RESUMO

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/patologia , Animais , Proteína BRCA2/metabolismo , Bioensaio/métodos , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/metabolismo , Sobrevivência Celular , Estudos de Coortes , Feminino , Recombinação Homóloga , Humanos , Camundongos , Síndromes Neoplásicas Hereditárias/epidemiologia , Síndromes Neoplásicas Hereditárias/metabolismo , Sri Lanka/epidemiologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Ann Biol Clin (Paris) ; 78(3): 329-342, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32420887

RESUMO

Accreditation of an in vitro diagnostic assay according to the NF/EN/ISO 15189 standard requires to analyze its technical performance before implementation for routine use, and annually when reviewing effectiveness of quality controls. Performance is evaluated through repeatability, intermediate fidelity, accuracy and uncertainty of measurement. The coefficients of variation (CV) of the intra-assay and inter-assay precision tests must be compared with those of "peers" (results from laboratories employing the same method) and also with those obtained with "all methods", i.e., results from all laboratories performing the same assay, irrespective of the method. To our best knowledge, there is currently no French or international recommendation on what the acceptable limits of performance for specific IgE and tryptase assays should be. Therefore, the AllergoBioNet network of hospital allergy laboratories set out to characterize the performance of their current methods as a basis for the development of recommendations. The results provided by 24 centers were analyzed and led to consensus recommendations for specific IgE, total IgE and tryptase assays.


Assuntos
Bioensaio/métodos , Imunoglobulina E/análise , Triptases/análise , Acreditação , Bioensaio/normas , Consenso , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , França , Humanos , Laboratórios/normas , Controle de Qualidade , Reprodutibilidade dos Testes
11.
Pharm Res ; 37(6): 99, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32435855

RESUMO

PURPOSE: To evaluate the performance of artificial membranes in in vitro lipolysis-permeation assays useful for absorption studies of drugs loaded in lipid-based formulations (LBFs). METHODS: Polycarbonate as well as PVDF filters were treated with hexadecane, or lecithin in n-dodecane solution (LiDo) to form artificial membranes. They were thereafter used as absorption membranes separating two compartments mimicking the luminal and serosal side of the intestine in vitro. Membranes were subjected to dispersions of an LBF that had been digested by porcine pancreatin and spiked with the membrane integrity marker Lucifer Yellow (LY). Three fenofibrate-loaded LBFs were used to explore the in vivo relevance of the assay. RESULTS: Of the explored artificial membranes, only LiDo applied to PVDF was compatible with lipolysis by porcine pancreatin. Formulation ranking based on mass transfer in the LiDo model exposed was the same as drug release in single-compartment lipolysis. Ranking based on observed apparent permeability coefficients of fenofibrate with different LBFs were the same as those obtained in a cell-based model. CONCLUSIONS: The LiDo membrane was able to withstand lipolysis for a sufficient assay period. However, the assay with porcine pancreatin as digestive agent did not predict the in vivo ranking of the assayed formulations better than existing methods. Comparison with a Caco-2 based assay method nonetheless indicates that the in vitro in vivo relationship of this cell-free model could be improved with alternative digestive agents.


Assuntos
Portadores de Fármacos/química , Fenofibrato/química , Lipídeos/química , Lipólise , Membranas Artificiais , Administração Oral , Adsorção , Alcanos/química , Animais , Bioensaio/métodos , Células CACO-2 , Digestão , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Excipientes/química , Fenofibrato/administração & dosagem , Humanos , Lecitinas/química , Modelos Biológicos , Pancreatina/metabolismo , Permeabilidade , Solubilidade , Suínos
12.
J Vis Exp ; (159)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32449701

RESUMO

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.


Assuntos
Bioensaio/métodos , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Células Germinativas/metabolismo , Ligação Proteica
13.
J Vis Exp ; (159)2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32449708

RESUMO

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.


Assuntos
Bioensaio/métodos , Nucleotídeos/metabolismo , Fosforilação/genética
14.
BMC Infect Dis ; 20(1): 301, 2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321418

RESUMO

BACKGROUND: In Ghana, pre-school-aged children (PSAC) are at risk of intestinal schistosomiasis and are living in need of praziquantel treatment. To better assess the infection burden within this vulnerable demographic group, we have provided a comparative assessment of the prevalence of Schistosoma mansoni in pre-school-aged children by urine circulating cathodic antigen (CCA) dipsticks, real-time PCR Taqman® faecal assays and Kato-Katz coproscopy. METHODS: In all, 190 pre-school-aged children were sampled from three endemic communities (viz. Tomefa, Torgahkope/Adakope, and Manheam) around Weija dam, Southern Ghana. Fresh stool and urine samples were collected from all participants for diagnosis. RESULTS: Among all the three communities, the urine-CCA assay recorded the highest prevalence values of 90.5% (95% CI 80.4-96.4), 87.9% (95% CI 76.7-95), and 81.2% (95% CI 69.9-89.6) in Tomefa, Torgahkope/Adakope, and Manheam respectively. Prevalence by real-time PCR was 50% (95% CI 35.5-64.5), 8% (95% CI 2.2-19.2) and 16.7% (95% CI 8.3-28.5), while by Kato-Katz was 55.6% (95% CI 42.5-68.1), 8.6% (95% CI 2.9-19) and 11.6% (95% CI 5.1-21.6) respectively. Children aged 1 year and over were found to be positive with the urine-CCA assay; by the ages of 3-4, over 50% were urine-CCA patent. The sensitivity and specificity of the POC-CCA dipsticks, when compared against the combined results of Kato-Katz/TaqMan results was found to be 84.1% (95% CI = 72.7-92.1) and 12.9% (95% CI = 6.6-22) respectively. CONCLUSIONS: We propose that the urine-CCA dipstick may be a useful rapid diagnostic tool to estimate the prevalence of intestinal schistosomiasis in PSAC, particularly in rapid identification of at-risk areas. However, our assessment has shown that it possible to record false positives when compared to combined Kato-Katz and qPCR results. To guide PSAC praziquantel treatment needs, we propose the urine CCA assay should be included in routine surveillance of intestinal schistosomiasis alongside other diagnostics such as Kato-Katz and urine filtration.


Assuntos
Antígenos de Helmintos/urina , Testes Diagnósticos de Rotina/métodos , Fezes/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Esquistossomose mansoni/diagnóstico , Urinálise/métodos , Animais , Antígenos de Helmintos/análise , Bioensaio/métodos , Líquidos Corporais/química , Líquidos Corporais/imunologia , Líquidos Corporais/parasitologia , Pré-Escolar , Fezes/química , Feminino , Gana/epidemiologia , Humanos , Lactente , Masculino , Sistemas Automatizados de Assistência Junto ao Leito , Praziquantel/uso terapêutico , Prevalência , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/urina , Sensibilidade e Especificidade
15.
Artigo em Inglês | MEDLINE | ID: mdl-32234669

RESUMO

Giardia duodenalis, the most prevalent human intestinal parasite causes the disease, giardiasis. On an annual basis G. duodenalis infects ~1 billion people, of which ~280 million develop symptomatic disease. Giardiasis can be severe and chronic, causing malnutrition, stunted growth and poor cognitive development in children. Current treatment options rely on drugs with declining efficacy and side-effects. To improve the health and well-being of millions of people world-wide, new anti-Giardia drugs with different modes of action to currently used drugs are required. The Medicines for Malaria Venture's Pathogen Box, a collection of bio-active compounds specifically chosen to stimulate infectious disease drug discovery, represents an opportunity for the discovery of new anti-Giardia agents. While the anti-Giardia activity of Pathogen Box compounds has been reported, this work failed to identify known anti-Giardia controls within the compound set. It also reported the activity of compounds previously screened and shown to be inactive by others, suggesting data may be inaccurate. Given these concerns the anti-Giardia activity of Pathogen Box compounds was re-assessed in the current study. Data from this work identified thirteen compounds with anti-Giardia IC50 values ≤2 µM. Five of these compounds were reference compounds (marketed drugs with known anti-microbial activity), or analogues of compounds with previously described anti-Giardia activity. However, eight, including MMV676358 and MMV028694, which demonstrated potent sub-µM IC50s against assemblage A, B and metronidazole resistant parasites (0.3 µM and 0.9 µM respectively), may represent new leads for future drug development. Interestingly, only four of these compounds were identified in the previously reported Pathogen Box screen highlighting the importance of assay selection and design when assessing compounds for activity against infectious agents.


Assuntos
Antiparasitários/isolamento & purificação , Antiparasitários/farmacologia , Bioensaio/métodos , Descoberta de Drogas/métodos , Giardia lamblia/efeitos dos fármacos , Giardia/efeitos dos fármacos , Descoberta de Drogas/instrumentação , Giardíase/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária , Prevalência
16.
Health Secur ; 18(2): 83-95, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324068

RESUMO

We conducted a comprehensive, multi-phase laboratory evaluation of the Tularemia BioThreat Alert® (BTA) test, a lateral flow assay (LFA) for the rapid detection of Francisella tularensis. The study, conducted at 2 sites, evaluated the limit of detection (LOD) of this assay using the virulent SchuS4 strain and the avirulent LVS strain of F. tularensis. In 6-phase evaluation (linear dynamic range and reproducibility, inclusivity, near-neighbor, environmental background, white powder, and environmental filter extract), 13 diverse strains of F. tularensis, 8 Francisella near neighbors, 61 environmental background organisms, 26 white powders, and a pooled aerosol extract were tested. In the 937 tests performed, the Tularemia BTA demonstrated an LOD of 107 to 108 cfu/mL, with a sensitivity of 100.00%, specificity of 98.08%, and accuracy of 98.84%. These performance data are important for accurate interpretation of qualitative results arising from screening suspicious white powders in the field.


Assuntos
Aerossóis/análise , Bioensaio/métodos , Francisella tularensis/isolamento & purificação , Pós/análise , Bioterrorismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
J Vis Exp ; (157)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32225144

RESUMO

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide. Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.


Assuntos
Bioensaio/métodos , Resistência à Doença , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Plântula/microbiologia , Cotilédone/fisiologia , Meios de Cultura , Ecótipo , Lycopersicon esculentum/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Fenótipo , Esterilização
18.
J Vis Exp ; (157)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32225148

RESUMO

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent ß-arrestin2 recruitment assay, the universality of ß-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango's sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.


Assuntos
Bioensaio/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestina 2/metabolismo , Análise de Dados , Células HEK293 , Humanos , Ligantes , Luminescência , Transfecção
19.
Nat Biomed Eng ; 4(5): 518-530, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32313101

RESUMO

The detection and quantification of low-abundance molecular biomarkers in biological samples is challenging. Here, we show that a plasmonic nanoscale construct serving as an 'add-on' label for a broad range of bioassays improves their signal-to-noise ratio and dynamic range without altering their workflow and readout devices. The plasmonic construct consists of a bovine serum albumin scaffold with approximately 210 IRDye 800CW fluorophores (with a fluorescence intensity approximately 6,700-fold that of a single 800CW fluorophore), a polymer-coated gold nanorod acting as a plasmonic antenna and biotin as a high-affinity biorecognition element. Its emission wavelength can be tuned over the visible and near-infrared spectral regions by modifying its size, shape and composition. It improves the limit of detection in fluorescence-linked immunosorbent assays by up to 4,750-fold and is compatible with multiplexed bead-based immunoassays, immunomicroarrays, flow cytometry and immunocytochemistry methods, and it shortens overall assay times (to 20 min) and lowers sample volumes, as shown for the detection of a pro-inflammatory cytokine in mouse interstitial fluid and of urinary biomarkers in patient samples.


Assuntos
Bioensaio/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Animais , Células da Medula Óssea/citologia , Linhagem Celular Tumoral , Coloides/química , Células Dendríticas/citologia , Feminino , Citometria de Fluxo , Fluorescência , Humanos , Imunoensaio , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Microesferas , Proteômica , Padrões de Referência
20.
PLoS One ; 15(3): e0227094, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126066

RESUMO

CWD is an emergent prion disease that now affects cervid species on three continents. CWD is efficiently spread in wild and captive populations, likely through both direct animal contact and environmental contamination. Here, by longitudinally assaying in feces of CWD-exposed white-tailed deer by RT-QuIC, we demonstrate fecal shedding of prion seeding activity months before onset of clinical symptoms and continuing throughout the disease course. We also examine the impact of simulated environmental conditions such as repeated freeze-thaw cycles and desiccation on fecal prion seeding activity. We found that while multiple (n = 7) freeze-thaw cycles substantially decreased fecal seeding activity, desiccation had little to no effect on seeding activity. Finally, we examined whether RT-QuIC testing of landscape fecal deposits could distinguish two premises with substantial known CWD prevalence from one in which no CWD-infected animals had been detected. In the above pilot study, this distinction was possible. We conclude that fecal shedding of CWD prions occurs over much of the disease course, that environmental factors influence prion seeding activity, and that it is feasible to detect fecal prion contamination using RT-QuIC.


Assuntos
Bioensaio/métodos , Cervos , Fezes/química , Príons/análise , Doença de Emaciação Crônica/diagnóstico , Animais , Exposição Ambiental/efeitos adversos , Estudos de Viabilidade , Prevalência , Doença de Emaciação Crônica/epidemiologia , Doença de Emaciação Crônica/transmissão
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