20180312 - Evaluating the applicability of read-across tools and high throughput screening data for food relevant chemicals (SOT)

EPA Science Inventory

Alternative toxicity assessment methods to characterize the hazards of chemical substances have been proposed to reduce animal testing and screen thousands of chemicals in an efficient manner. Resources to accomplish these goals include utilizing large in vitro chemical screening...

Evaluating the Impact of Uncertainties in Clearance and Exposure When Prioritizing Chemicals Screened in High-Throughput Assays

EPA Science Inventory

The toxicity-testing paradigm has evolved to include high-throughput (HT) methods for addressing the increasing need to screen hundreds to thousands of chemicals rapidly. Approaches that involve in vitro screening assays, in silico predictions of exposure concentrations, and phar...

An in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of AChE inhibitors from roots of Coptis chinensis Franch.

PubMed

Zhao, Hengqiang; Zhou, Siduo; Zhang, Minmin; Feng, Jinhong; Wang, Shanshan; Wang, Daijie; Geng, Yanling; Wang, Xiao

2016-02-20

In this study, an in vitro acetylcholinesterase (AChE) inhibition assay based on microplate reader combined with ultrafiltration high performance liquid chromatography-electrospray quadrupole time of flight mass (UF-HPLC-ESI-Q-TOF/MS) was developed for the rapid screening and identification of acetylcholinesterase inhibitors (AChEI) from roots of Coptis chinensis Franch. Incubation conditions such as enzyme concentration, incubation time, incubation temperature and co-solvent was optimized so as to get better screening results. Five alkaloids including columbamine, jatrorrhizine, coptisine, palmatine and berberine were found with AChE inhibition activity in the 80% ethanol extract of C. chinensis Franch. The screened compounds were identified by HPLC-DAD-ESI-Q-TOF/MS compared with the reference stands and literatures. The screened results were verified by in vitro AChE inhibition assays, palmatine showed the best AChE inhibitory activities with IC50 values of 36.6μM among the five compounds. Results of the present study indicated that the combinative method using in vitro AChE inhibition assay and UF-HPLC-ESI-Q-TOF/MS could be widely applied for rapid screening and identification of AChEI from complex TCM extract. Copyright © 2015 Elsevier B.V. All rights reserved.

Possible strategies for EDC testing in the future: exploring roles of pathway-based in silico, in vitro and in vivo methods

EPA Science Inventory

Current methods for screening, testing and monitoring endocrine-disrupting chemicals (EDCs) rely relatively substantially upon moderate- to long-term assays that can, in some instances, require significant numbers of animals. Recent developments in the areas of in vitro testing...

Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.

PubMed

Dolado, Ignacio; Nieto, Joan; Saraiva, Maria João M; Arsequell, Gemma; Valencia, Gregori; Planas, Antoni

2005-01-01

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Evaluating the Value of Augmenting In Vitro Hazard Assessment with Exposure and Pharmacokinetics Considerations for Chemical Prioritization

EPA Science Inventory

Over time, toxicity-testing paradigms have progressed from low-throughput in vivo animal studies for limited numbers of chemicals to high-throughput (HT) in vitro screening assays for thousands of chemicals. Such HT in vitro methods, along with HT in silico predictions of popula...

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen].

PubMed

Flachner, Beáta; Hajdú, István; Dobi, Krisztina; Lorincz, Zsolt; Cseh, Sándor; Dormán, György

2013-01-01

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.

Using in Vitro Evolution and Whole Genome Analysis To Discover Next Generation Targets for Antimalarial Drug Discovery

PubMed Central

2018-01-01

Although many new anti-infectives have been discovered and developed solely using phenotypic cellular screening and assay optimization, most researchers recognize that structure-guided drug design is more practical and less costly. In addition, a greater chemical space can be interrogated with structure-guided drug design. The practicality of structure-guided drug design has launched a search for the targets of compounds discovered in phenotypic screens. One method that has been used extensively in malaria parasites for target discovery and chemical validation is in vitro evolution and whole genome analysis (IVIEWGA). Here, small molecules from phenotypic screens with demonstrated antiparasitic activity are used in genome-based target discovery methods. In this Review, we discuss the newest, most promising druggable targets discovered or further validated by evolution-based methods, as well as some exceptions. PMID:29451780

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

PubMed Central

Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

2016-01-01

ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing. PMID:27371585

Biomarkers of Acute Respiratory Allergen Exposure: Screening For Sensitization Potential

EPA Science Inventory

Rationale: An in vitro assay to identify respiratory sensitizers will provide a rapid screen and reduce animal use. The study goal was to identify biomarkers that differentiate allergen versus non-allergen responses following an acute exposure. Methods: Female BALB/c mice rec...

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

PubMed

Burra, D D; Mühlenbock, P; Andreasson, E

2015-09-01

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

A novel and discriminative method of in vitro disintegration time for preparation and optimization of taste-masked orally disintegrating tablets of carbinoxamine maleate.

PubMed

Liu, Yali; Li, Peng; Qian, Rong; Sun, Tianyu; Fang, Fangzhi; Wang, Zonghua; Ke, Xue; Xu, Bohui

2018-08-01

The primary objective of this study was to mask bitter taste and decrease the disintegration time of carbinoxamine maleate (CAM) orally disintegrating tablets (ODTs). In order to screen the prescription of ODTs, a novel modified in vitro disintegration method (MIVDM) was developed to measure the in vitro disintegration time. In this method, different concentrations of ethanol served as disintegration medium in order to delay the in vitro water absorption and disintegration process of tablets. The MIVDM demonstrated good in vitro and in vivo correlation and proved more precise and discriminative than other reported methods. In this research, ion exchange resins (IERs) were used to mask bitter taste for improving mouthfeel. The drug-resin ratio and reaction temperature were investigated to obtain the optimum carbinoxamine resin complexes (CRCs). The characterization of CRCs revealed an amorphous state. ODTs were prepared by direct compression. Superdisintegrants and diluents of ODTs were screened first. Further optimization was carried out by using Box-Behnken design. The effect of (X 1 ) mannitol/microcrystalline cellulose ratio, (X 2 ) the amount of low-substituted hydroxypropylcellulose and (X 3 ) the hardness was investigated for achieving the lowest (Y) in vitro disintegration time. Technological characterization, wetting time, water absorption ratio, and roughness degree were evaluated. The CRCs and ODTs proved successful taste-masking efficiency. The end product improved patients' compliance. The developed MIVDM was practical for commercial use.

Antioxidant activity, total phenolics and flavonoids contents: Should we ban in vitro screening methods?

PubMed

Granato, Daniel; Shahidi, Fereidoon; Wrolstad, Ronald; Kilmartin, Paul; Melton, Laurence D; Hidalgo, Francisco J; Miyashita, Kazuo; Camp, John van; Alasalvar, Cesarettin; Ismail, Amin B; Elmore, Stephen; Birch, Gordon G; Charalampopoulos, Dimitris; Astley, Sian B; Pegg, Ronald; Zhou, Peng; Finglas, Paul

2018-10-30

As many studies are exploring the association between ingestion of bioactive compounds and decreased risk of non-communicable diseases, the scientific community continues to show considerable interest in these compounds. In addition, as many non-nutrients with putative health benefits are reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators, measurement of antioxidant activity using in vitro assays has become very popular over recent decades. Measuring concentrations of total phenolics, flavonoids, and other compound (sub)classes using UV/Vis spectrophotometry offers a rapid chemical index, but chromatographic techniques are necessary to establish structure-activity. For bioactive purposes, in vivo models are required or, at the very least, methods that employ distinct mechanisms of action (i.e., single electron transfer, transition metal chelating ability, and hydrogen atom transfer). In this regard, better understanding and application of in vitro screening methods should help design of future research studies on 'bioactive compounds'. Copyright © 2018 Elsevier Ltd. All rights reserved.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taxvig, Camilla, E-mail: camta@food.dtu.dk; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenousmore » metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.« less

(125)I-Tetrazines and Inverse-Electron-Demand Diels-Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies.

PubMed

Albu, Silvia A; Al-Karmi, Salma A; Vito, Alyssa; Dzandzi, James P K; Zlitni, Aimen; Beckford-Vera, Denis; Blacker, Megan; Janzen, Nancy; Patel, Ramesh M; Capretta, Alfredo; Valliant, John F

2016-01-20

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels-Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with trans-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics.

Toxicity Screening of Volatile Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System

EPA Science Inventory

Traditional in vitro dosing methods require, for example, the addition of particulate matter (PM), PM extracts, or chemicals in dimethyl sulfoxide (DMSO) or water into cell culture medium. However, about 10% of chemicals nominated for study in the U.S Environmental Protection Age...

A Workflow to Investigate Exposure and Pharmacokinetic Influences on High-Throughput in Vitro Chemical Screening Based on Adverse Outcome Pathways

EPA Science Inventory

Background: Adverse outcome pathways (AOPs) link adverse effects in individuals or populations to a molecular initiating event (MIE) that can be quantified using in vitro methods. Practical application of AOPs in chemical-specific risk assessment requires incorporation of knowled...

Toxicokinetic and Dosimetry Modeling Tools for Exposure Reconstruction: US EPA's Rapid Exposure and Dosimetry (RED) Project

EPA Science Inventory

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide ...

ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS

EPA Science Inventory

There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures ...

IN VITRO SCREENING OF DEVELOPMENTAL NEUROTOXICANTS IN RAT PRIMARY CORTICAL NEURONS USING HIGH CONTENT IMAGE

EPA Science Inventory

There is a need for more efficient and cost-effective methods for identifying, characterizing and prioritizing chemicals which may result in developmental neurotoxicity. One approach is to utilize in vitro test systems which recapitulate the critical processes of nervous system d...

Gas Phase Probe Molecules for Assessing In vitro Metabolism to Infer an In vivo Response

EPA Science Inventory

Efficient and accurate in vitro high-throughput screening (HTS) methods use cellular and molecular based adverse outcome pathways (AOPs) as central elements for exposure assessment and chemical prioritization. However, not all AOPs are based on human or animal systems biology, bu...

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

EPA Science Inventory

AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Model Applications to Screen Environmental Hazards.

EPA Science Inventory

This presentation discusses methods used to extrapolate from in vitro high-throughput screening (HTS) toxicity data for an endocrine pathway to in vivo for early life stages in humans, and the use of a life stage PBPK model to address rapidly changing physiological parameters. A...

In Vitro Screening for Cytotoxic Activity of Herbal Extracts

PubMed Central

Lombardi, Valter R. M.; Cacabelos, Ramón

2017-01-01

Experimental studies have shown that a variety of chemopreventive plant components affect tumor initiation, promotion, and progression and the main difference, between botanical medicines and synthetic drugs, resides in the presence of complex metabolite mixtures shown by botanical medicine which in turn exert their action on different levels and via different mechanisms. In the present study, we performed an in vitro screening of ethanol extracts from commercial plants in order to investigate potential antitumor activity against human tumor cell lines. Experimental results obtained through a variety of methods and techniques indicated that extracts of I. verum, G. glabra, R. Frangula, and L. usitatissimum present significant reduction in in vitro tumor cell proliferation, suggesting these extracts as possible chemotherapeutical adjuvants for different cancer treatments. PMID:28386288

Species-specific predictive models of developmental toxicity using the ToxCast chemical library

EPA Science Inventory

EPA’s ToxCastTM project is profiling the in vitro bioactivity of chemicals to generate predictive models that correlate with observed in vivo toxicity. In vitro profiling methods are based on ToxCast data, consisting of over 600 high-throughput screening (HTS) and high-content sc...

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay.

PubMed

Taxvig, Camilla; Olesen, Pelle Thonning; Nellemann, Christine

2011-02-01

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects. Copyright © 2010 Elsevier Inc. All rights reserved.

Evaluation of Microelectrode Array Data using Bayesian Modeling as an Approach to Screening and Prioritization for Neurotoxicity Testing*

EPA Science Inventory

The need to assess large numbers of chemicals for their potential toxicities has resulted in increased emphasis on medium- and high-throughput in vitro screening approaches. For such approaches to be useful, efficient and reliable data analysis and hit detection methods are also ...

In Vitro Toxicity Assessment Technique for Volatile ...

EPA Pesticide Factsheets

The U.S. Environmental Protection Agency is tasked with evaluating the human health, environmental, and wildlife effects of over 80,000 chemicals registered for use in the environment and commerce. The challenge is that sparse chemical data exists; traditional toxicity testing methods are slow, costly, involve animal studies, and cannot keep up with a chemical registry that typically grows by at least 1000 chemicals every year. In recent years, High Throughput Screening (HTS) has been used in order to prioritize chemicals for traditional toxicity screening or to complement traditional toxicity studies. HTS is an in vitro approach of rapidly assaying a large number of chemicals for biochemical activity using robotics and automation. However, no method currently exists for screening volatile chemicals such as air pollutants in a HTS fashion. Additionally, significant uncertainty regarding in vitro to in in vivo extrapolation (IVIVE) remains. An approach to bridge the IVIVE gap and the current lack of ability to screen volatile chemicals in a HTS fashion is by using a probe molecule (PrM) technique. The proposed technique uses chemicals with empirical human pharmacokinetic data as PrMs to study toxicity of molecules with no known data for gas-phase analysis. We are currently studying the xenobiotic-metabolizing enzyme CYP2A6 using transfected BEAS-2B bronchial epithelial cell line. The CYP2A6 pathway activity is studied by the formation of cotinine from nicot

In vitro screening of Fe2+-chelating effect by a Fenton's reaction-luminol chemiluminescence system.

PubMed

Wada, Mitsuhiro; Komatsu, Hiroaki; Ikeda, Rie; Aburjai, Talal A; Alkhalil, Suleiman M; Kuroda, Naotaka; Nakashima, Kenichiro

2014-11-01

In vitro screening of a Fe(2+) -chelating effect using a Fenton's reaction-luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe(2+) using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10-phenanthroline were examined. EC50 values for these compounds (except 1,10-phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe(2+)-chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.

A workflow to investigate exposure and pharmacokinetic influences on high-throughput in vitro chemical screening based on adverse outcome pathways, OpenTox USA 2015 Poster

EPA Science Inventory

Adverse outcome pathways (AOP) link known population outcomes to a molecular initiating event (MIE) that can be quantified using high-throughput in vitro methods. Practical application of AOPs in chemical-specific risk assessment requires consideration of exposure and absorption,...

Antimalarial natural products drug discovery in Panama.

PubMed

Calderón, Angela I; Simithy-Williams, Johayra; Gupta, Mahabir P

2012-01-01

Malaria is still a major public health problem. The biodiversity of the tropics is extremely rich and represents an invaluable source of novel bioactive molecules. For screening of this diversity more sensitive and economical in vitro methods are needed, Flora of Panama has been studied based on ethnomedical uses for discovering antimalarial compounds. This review aims to provide an overview of in vitro screening methodologies for antimalarial drug discovery and to present results of this effort in Panama during the last quarter century. A literature search in SciFinder and PubMed and original publications of Panamanian scientists was performed to gather all the information on antimalarial drug discovery from the Panamanian flora and in vitro screening methods. A variety of colorimetric, staining, fluorometric, and mass spectrometry and radioactivity-based methods have been provided. The advantages and limitations of these methods are also discussed. Plants used in ethnomedicine for symptoms of malaria by three native Panamanian groups of Amerindians, Kuna, Ngöbe Buglé and Teribes are provided. Seven most active plants with IC(50) values < 10 μg/mL were identified Talisia nervosa Radlk. (Sapindaceae), Topobea parasitica Aubl.(Melastomataceae), Monochaetum myrtoideum Naudin (Melastomataceae), Bourreria spathulata (Miers) Hemsl.(Boraginaceae), Polygonum acuminatum Kunth (Polygonaceae), Clematis campestris A. St.-Hil. (Ranunculaceae) and Terminalia triflora (Griseb.) Lillo (Combretaceae). Thirty bioactive compounds belonging to a variety of chemical classes such as spermine and isoquinoline alkaloids, glycosylflavones, phenylethanoid glycosides, ecdysteroids, quercetin arabinofuranosides, clerodane-type diterpenoids, sipandinolid, galloylquercetin derivatives, gallates, oleamide and mangiferin derivatives.

An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

PubMed

Peng, Lan; Fan, Jialong; Tong, Chunyi; Xie, Zhenhua; Zhao, Chuan; Liu, Xuanming; Zhu, Yonghua; Liu, Bin

2016-09-15

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Method for in vitro screening of aquatic fungicides

USGS Publications Warehouse

Bailey, T.A.

1983-01-01

Methods were developed for in vitro screening of candidate aquatic fungicides for efficacy against Achlya fiagellata, A. racemosa, Saprolegnia hypogyna and S. megasperma. Agar plugs containing fungal hyphae, removed from the edge of actively growing colonies, were placed in the depressions of spot plates containing 1a??0, 10a??0 and 100 mg/I of the candidate compounds for 15 or 60 min. After exposure, the plugs were transferred on to filter papers (0a??45-A?m pore) in a holder, rinsed, and then placed on cornmeal agar medium in tri-petri dishes. The plates were checked for mycelial growth after 48, 96 and 168 h of incubation in a lighted (400-800 A?m) environmental control chamber at 20A?2A?C. Criteria for the acceptance or rejection of candidate aquatic fungicides for further study were based on the antifungal spectrum index (ASI) comparisons between respective compounds and malachite green after 48 h and the concentration level producing complete growth inhibition. Candidate compounds whose ASI was less than 50% that of malachite green after 48 h or did not inhibit growth at levels less than 100 mg/l were rejected. This method provides a base from which in vivo and definitive test regimens can be developed. Preliminary in vitro screening of candidate fungicides reduces the need for costly in vivo tests on compounds that have low antifungal activity.

In Vitro Androgen Bioassays as a Detection Method for Designer Androgens

PubMed Central

Cooper, Elliot R.; McGrath, Kristine C. Y.; Heather, Alison K.

2013-01-01

Androgens are the class of sex steroids responsible for male sexual characteristics, including increased muscle mass and decreased fat mass. Illicit use of androgen doping can be an attractive option for those looking to enhance sporting performance and/or physical appearance. The use of in vitro bioassays to detect androgens, especially designer or proandrogens, is becoming increasingly important in combating androgen doping associated with nutritional supplements. The nutritional sports supplement market has grown rapidly throughout the past decade. Many of these supplements contain androgens, designer androgens or proandrogens. Many designer or proandrogens cannot be detected by the standard highly-sensitive screening methods such as gas chromatography-mass spectrometry because their chemical structure is unknown. However, in vitro androgen bioassays can detect designer and proandrogens as these assays are not reliant on knowing the chemical structure but instead are based on androgen receptor activation. For these reasons, it may be advantageous to use routine androgen bioassay screening of nutraceutical samples to help curb the increasing problem of androgen doping. PMID:23389345

Characterization of ToxCast Phase II compounds disruption of spontaneous network activity in cortical networks grown on multi-well microelectrode array (mwMEA) plates.

EPA Science Inventory

The development of multi-well microelectrode array (mwMEA) systems has increased in vitro screening throughput making them an effective method to screen and prioritize large sets of compounds for potential neurotoxicity. In the present experiments, a multiplexed approach was used...

In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays

EPA Science Inventory

Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 Ce02 (5- 1288 nm), and 4 Ti02 (6-142 nm) NPs and microparticles (M...

In vitro avarol does affect the growth of Candida sp.

PubMed

Pejin, Boris; Ciric, Ana; Markovic, Dejan; Tommonaro, Giuseppina; Sokovic, Marina

2016-09-01

This work extends in vitro screening of antimicrobial activity of avarol, the marine natural product firstly isolated from the Mediterranean sponge Dysidea avara. Its anticandidial activity was evaluated by microdilution method against eight Candida strains, two ATCC and six clinical ones. At a different extent this compound was proven to be active against all the strains tested (MIC 0.8-6.0 μg/mL and MFC 1.6-12.0 μg/mL, respectively). According to the best of our knowledge, this is the first report on avarol activity towards any yeast strain which may be of relevance for Alzheimer's disease. Indeed, avarol derivatives showing moderate AChE activity should be screened for anticandidial activity both in vitro and in vivo.

Using In Vitro High-Throughput Screening Data for Predicting Benzo[k]Fluoranthene Human Health Hazards.

PubMed

Burgoon, Lyle D; Druwe, Ingrid L; Painter, Kyle; Yost, Erin E

2017-02-01

Today there are more than 80,000 chemicals in commerce and the environment. The potential human health risks are unknown for the vast majority of these chemicals as they lack human health risk assessments, toxicity reference values, and risk screening values. We aim to use computational toxicology and quantitative high-throughput screening (qHTS) technologies to fill these data gaps, and begin to prioritize these chemicals for additional assessment. In this pilot, we demonstrate how we were able to identify that benzo[k]fluoranthene may induce DNA damage and steatosis using qHTS data and two separate adverse outcome pathways (AOPs). We also demonstrate how bootstrap natural spline-based meta-regression can be used to integrate data across multiple assay replicates to generate a concentration-response curve. We used this analysis to calculate an in vitro point of departure of 0.751 μM and risk-specific in vitro concentrations of 0.29 μM and 0.28 μM for 1:1,000 and 1:10,000 risk, respectively, for DNA damage. Based on the available evidence, and considering that only a single HSD17B4 assay is available, we have low overall confidence in the steatosis hazard identification. This case study suggests that coupling qHTS assays with AOPs and ontologies will facilitate hazard identification. Combining this with quantitative evidence integration methods, such as bootstrap meta-regression, may allow risk assessors to identify points of departure and risk-specific internal/in vitro concentrations. These results are sufficient to prioritize the chemicals; however, in the longer term we will need to estimate external doses for risk screening purposes, such as through margin of exposure methods. © 2016 Society for Risk Analysis.

Identification of candidate reference chemicals for in vitro steroidogenesis assays.

PubMed

Pinto, Caroline Lucia; Markey, Kristan; Dix, David; Browne, Patience

2018-03-01

The Endocrine Disruptor Screening Program (EDSP) is transitioning from traditional testing methods to integrating ToxCast/Tox21 in vitro high-throughput screening assays for identifying chemicals with endocrine bioactivity. The ToxCast high-throughput H295R steroidogenesis assay may potentially replace the low-throughput assays currently used in the EDSP Tier 1 battery to detect chemicals that alter the synthesis of androgens and estrogens. Herein, we describe an approach for identifying in vitro candidate reference chemicals that affect the production of androgens and estrogens in models of steroidogenesis. Candidate reference chemicals were identified from a review of H295R and gonad-derived in vitro assays used in methods validation and published in the scientific literature. A total of 29 chemicals affecting androgen and estrogen levels satisfied all criteria for positive reference chemicals, while an additional set of 21 and 15 chemicals partially fulfilled criteria for positive reference chemicals for androgens and estrogens, respectively. The identified chemicals included pesticides, pharmaceuticals, industrial and naturally-occurring chemicals with the capability to increase or decrease the levels of the sex hormones in vitro. Additionally, 14 and 15 compounds were identified as potential negative reference chemicals for effects on androgens and estrogens, respectively. These candidate reference chemicals will be informative for performance-based validation of in vitro steroidogenesis models. Copyright © 2017. Published by Elsevier Ltd.

In vitro OECD test methods applied to screen the estrogenic effect of chemicals, used in Korea.

PubMed

Lee, Hee-Seok; Park, Eun-Jung; Han, Songyi; Oh, Gyeong-Yong; Kim, Min-Hee; Kang, Hui-Seung; Suh, Jin-Hyang; Oh, Jae-Ho; Lee, Kwang-Soo; Hwang, Myung-Sil; Moon, Guiim; Hong, Jin-Hwan; Hwang, In-Gyun

2016-09-01

In this study, 27 chemicals found in household products, which became an issue in Korea were screened for the agonistoc and antagonistic effects against human estrogen receptor using official Organization for Economic Cooperation and Development (OECD) in vitro assays, STTA assay using ERα-HeLa-9903 cell line and BG1Luc ER TA assay. In the case of human ER agonist screening by two assays, all tested chemicals did not show agonist effect against ER. In ER antagonist test by BG1Luc ER TA assay, five surfactants α-dodecyl-ω-hydroxypoly(oxyethylene), alcohols C16-18 ethoxylated, nonylphenol, ethoxylated, 3,6,9,12,15,18,21-heptaoxatritriacontan-1-ol, and α-dodecyl-ω-hydroxypoly(oxy-1,2-ethanediyl)) were found to exhibit weak antagonistic activities. The agonist/antagonist effects against human estrogen receptor of various chemicals, used in Korea by OECD test guideline are reported in this study. These results indicated that two OECD in vitro assays will can be applied in Korea by screening of agonistic/antagonistic effects against human ER of various chemicals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of the Ortho-Clinical Diagnostics Vitros ECi Anti-HCV test: comparison with three other methods.

PubMed

Watterson, Jeannette M; Stallcup, Paulina; Escamilla, David; Chernay, Patrick; Reyes, Alfred; Trevino, Sylvia C

2007-01-01

After observing a high incidence of low positive hepatitis C virus (HCV) antibody screens by the Ortho-Clinical Vitros ECi test (Orthoclinical Diagnostics, Raritan, NJ), we compared results against those obtained using another chemiluminescent analyzer, as well as two U.S. Food and Drug Administration (FDA)-approved confirmatory methodologies. To ascertain the true anti-HCV status of samples deemed low-positive by the Ortho-Clinical Vitros ECi test, we tested samples using the ADVIA Centaur HCV screen test (Siemens Medical Solutions Diagnostics), the Chiron recombinant immunoblot assay (RIBA) test (Chiron Corp., Emeryville, CA), and the Roche COBAS Amplicor HCV qualitative test (Roche Diagnostics, Indianapolis, IN) in a series of studies. Of 94 specimens positive by Vitros ECi, 19% were observed to be negative by Centaur. A separate study of 91 samples with signal-to-cutoff (s/co) values less than 8.0 showed that all but one was negative for HCV ribonucleic acid (RNA). In comparison with RIBA, 100% (77) samples positive by the Vitros ECi test with s/co values less than 12.0 were negative or indeterminate by RIBA. A final study comparing all four methods side-by-side showed 63% disagreement by Centaur for Vitros ECi low-positive samples, 75% disagreement by RIBA, and 97% disagreement by polymerase chain reaction (PCR). In conclusion, the Ortho-Clinical Vitros ECi Anti-HCV test yields a high rate of false-positive results in the low s/co range in our patient population. (c) 2007 Wiley-Liss, Inc.

The FLIGHT Drosophila RNAi database

PubMed Central

Bursteinas, Borisas; Jain, Ekta; Gao, Qiong; Baum, Buzz; Zvelebil, Marketa

2010-01-01

FLIGHT (http://flight.icr.ac.uk/) is an online resource compiling data from high-throughput Drosophila in vivo and in vitro RNAi screens. FLIGHT includes details of RNAi reagents and their predicted off-target effects, alongside RNAi screen hits, scores and phenotypes, including images from high-content screens. The latest release of FLIGHT is designed to enable users to upload, analyze, integrate and share their own RNAi screens. Users can perform multiple normalizations, view quality control plots, detect and assign screen hits and compare hits from multiple screens using a variety of methods including hierarchical clustering. FLIGHT integrates RNAi screen data with microarray gene expression as well as genomic annotations and genetic/physical interaction datasets to provide a single interface for RNAi screen analysis and datamining in Drosophila. PMID:20855970

Development and Validation of a Computational Model for Androgen Receptor Activity

PubMed Central

2016-01-01

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 μM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity. PMID:27933809

Cross-species extrapolation of toxicity information using the ...

EPA Pesticide Factsheets

In the United States, the Endocrine Disruptor Screening Program (EDSP) was established to identify chemicals that may lead to adverse effects via perturbation of the endocrine system (i.e., estrogen, androgen, and thyroid hormone systems). In the mid-1990s the EDSP adopted a two tiered approach for screening chemicals that applied standardized in vitro and in vivo toxicity tests. The Tier 1 screening assays were designed to identify substances that have the potential of interacting with the endocrine system and Tier 2 testing was developed to identify adverse effects caused by the chemical, with documentation of dose-response relationships. While this tiered approach was effective in identifying possible endocrine disrupting chemicals, the cost and time to screen a single chemical was significant. Therefore, in 2012 the EDSP proposed a transition to make greater use of computational approaches (in silico) and high-throughput screening (HTS; in vitro) assays to more rapidly and cost-efficiently screen chemicals for endocrine activity. This transition from resource intensive, primarily in vivo, screening methods to more pathway-based approaches aligns with the simultaneously occurring transformation in toxicity testing termed “Toxicity Testing in the 21st Century” which shifts the focus to the disturbance of the biological pathway predictive of the observable toxic effects. An example of such screening tools include the US Environmental Protection Agency’s

Maize Genotype and Food Matrix Affect the Provitamin A Carotenoid Bioefficacy from Staple and Carrot-fortified Feeds in Mongolian Gerbils (Meriones unguiculatus)

PubMed Central

Schmaelzle, Samantha; Gannon, Bryan; Crawford, Serra; Arscott, Sara A.; Goltz, Shellen; Palacios-Rojas, Natalia; Pixley, Kevin V.; Simon, Philipp W.; Tanumihardjo, Sherry A.

2014-01-01

Biofortification to increase provitamin A carotenoids is an agronomic approach to alleviate vitamin A deficiency. Two studies compared biofortified foods using in vitro and in vivo methods. Study 1 screened maize genotypes (n = 44) using in vitro analysis, which demonstrated decreasing micellarization with increasing provitamin A. Thereafter, seven 50% biofortified maize feeds that hypothesized a one-to-one equivalency between β-cryptoxanthin and β-carotene were fed to Mongolian gerbils. Total liver retinol differed among the maize groups (P = 0.0043). Study 2 assessed provitamin A bioefficacy from 0.5% high-carotene carrots added to 60% staple-food feeds, followed by in vitro screening. Liver retinol was highest in the potato and banana groups, maize group retinol did not differ from baseline, and all treatments differed from control (P < 0.0001). In conclusion, β-cryptoxanthin and β-carotene have similar bioefficacy; meal matrix effects influence provitamin A absorption from carrot; and in vitro micellarization does not predict bioefficacy. PMID:24341827

Screening of Lactobacillus isolates from gastrointestinal tract of guinea fowl for probiotic qualities using in vitro tests to select species-specific probiotic candidates.

PubMed

Vineetha, P G; Tomar, S; Saxena, V K; Susan, C; Sandeep, S; Adil, K; Mukesh, K

2016-08-01

A total of 32 Lactobacillus isolates, 8 each from the crop (LGFCP1-LGFCP8), proventriculus (LGFP9-LGFP16), ileum (LGFI17-LGFI24) and caeca (LGFCM25-LGFCM32) were isolated from 25 adult guinea fowl (Pearl variety), 22-28 weeks of age, and characterised morphologically, physiologically, biochemically and by molecular methods. Isolates were screened for their probiotic quality using range of in vitro tests: aggregation test, cell surface hydrophobicity, resistance to bile salts and acidic conditions, enzymatic tests and coaggregation and antagonistic test. Based on in vitro test results and a novel scoring method, the two best isolates were selected and partial 16S rRNA sequencing was done. BLAST (Basic Local Alignment Search Tool) analysis of sequence of isolate LGFCP4 showed 99% genetic identity with Lactobacillus plantarum and LGFP16 with Lactobacillus reuteri. The study shows that these two microbial agents may be suitable as potential probiotic candidates in guinea fowl, as well as in a feed supplement for other poultry species.

In Vitro Toxicity Screening Technique for Volatile Substances ...

EPA Pesticide Factsheets

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for high throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules (e.g., signaling molecules nitric oxide (NO) and carbon monoxide (CO) in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are

ASSESSMENT OF PC12 CELL DIFFERENTIATION AND NEURITE GROWTH: A COMPARISON OF MORPHOLOGICAL AND NEUROCHEMICAL MEASURES.

EPA Science Inventory

In order to screen large numbers of chemicals for their potential to produce developmental neurotoxicity new, in vitro methods are needed. One approach is to develop methods based on the biologic processes which underlie brain development including the growth and maturation of ce...

Identification and Prioritization of Chemical Mixtures from Environmental Residue Data

EPA Science Inventory

High throughput toxicity testing has greatly improved the speed at which single chemicals can be screened using in vitro methods. However, people are not exposed to a single chemical at a time, rather to a mixture of chemicals. Even with the increased speed of these methods, te...

Effect of a triclosan/PVM/MA copolymer/fluoride dentifrice on volatile sulfur compounds in vitro.

PubMed

Pilch, S; Williams, M I; Cummins, D

2005-01-01

The objective of the investigation was to document the in vitro efficacy of a triclosan/PVM/MA copolymer/fluoride (TCF) dentifrice against the formation of volatile sulfur compounds (VSC) as well as the growth of H2S-producing bacteria. Clinical studies using organoleptic judges, gas chromatography, or a portable sulfide monitor have generally been employed in the assessment of treatments for the control of oral malodor. However, these studies are not appropriate for screening purposes because of the expense and time required. An in vitro method was developed for the purpose of screening new compounds, agents or formulations for their ability to control VSC formation and for determining bio-equivalence of efficacy when implementing changes in existing formulations. The method combines basic microbiological methods, dynamic flow cell techniques and head space analysis. The in vitro VSC method was validated by comparing the efficacy of two dentifrices containing TCF with a control fluoride dentifrice as the TCF products have been clinically proven to control oral malodor. In the validation studies, the TCF-containing dentifrices were significantly better (P < 0.05) than the control dentifrice in inhibiting VSC formation and reducing H(2)S-producing bacteria. For example, when compared with baseline, the TCF dentifrices reduced VSC formation between 42 and 49% compared with the control dentifrice which reduced VSC formation 3%. There was no significant difference (P > 0.05) between the two TCF dentifrice formulations. Using an in vitro breath VSC model, it has been demonstrated that two variants of a dentifrice containing triclosan, PVM/MA copolymer and fluoride have efficacy that is significantly better than a control fluoridated dentifrice and that there is no significant difference between the triclosan/PVM/MA copolymer/fluoride dentifrice variants.

Bayesian models trained with HTS data for predicting β-haematin inhibition and in vitro antimalarial activity.

PubMed

Wicht, Kathryn J; Combrinck, Jill M; Smith, Peter J; Egan, Timothy J

2015-08-15

A large quantity of high throughput screening (HTS) data for antimalarial activity has become available in recent years. This includes both phenotypic and target-based activity. Realising the maximum value of these data remains a challenge. In this respect, methods that allow such data to be used for virtual screening maximise efficiency and reduce costs. In this study both in vitro antimalarial activity and inhibitory data for β-haematin formation, largely obtained from publically available sources, has been used to develop Bayesian models for inhibitors of β-haematin formation and in vitro antimalarial activity. These models were used to screen two in silico compound libraries. In the first, the 1510 U.S. Food and Drug Administration approved drugs available on PubChem were ranked from highest to lowest Bayesian score based on a training set of β-haematin inhibiting compounds active against Plasmodium falciparum that did not include any of the clinical antimalarials or close analogues. The six known clinical antimalarials that inhibit β-haematin formation were ranked in the top 2.1% of compounds. Furthermore, the in vitro antimalarial hit-rate for this prioritised set of compounds was found to be 81% in the case of the subset where activity data are available in PubChem. In the second, a library of about 5000 commercially available compounds (Aldrich(CPR)) was virtually screened for ability to inhibit β-haematin formation and then for in vitro antimalarial activity. A selection of 34 compounds was purchased and tested, of which 24 were predicted to be β-haematin inhibitors. The hit rate for inhibition of β-haematin formation was found to be 25% and a third of these were active against P. falciparum, corresponding to enrichments estimated at about 25- and 140-fold relative to random screening, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease.

PubMed

Frikha-Gargouri, Olfa; Ben Abdallah, Dorra; Bhar, Ilhem; Tounsi, Slim

2017-01-01

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes ( sfp, ituC and bmyB ) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight ( p = 0.000) and the antibacterial activity in vitro ( p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol ( p = 0.004) and the reduction in gall weight ( p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo . Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens . These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains.

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease

PubMed Central

Frikha-Gargouri, Olfa; Ben Abdallah, Dorra; Bhar, Ilhem; Tounsi, Slim

2017-01-01

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains. PMID:28855909

Cytotoxicity Testing: Cell Experiments

NASA Astrophysics Data System (ADS)

Grünert, Renate; Westendorf, Aron; Buczkowska, Magdalena; Hänsch, Mareike; Grüunert, Sybil; Bednarski, Patrick J.

Screening for new anticancer agents has traditionally been done with in vitro cell culture methods. Even in the genomic era of target-driven drug design, screening for cytotoxic activity is still a standard tool in the search for new anticancer agents, especially if the mode of action of a substance is not yet known. A wide variety of cell culture methods with unique end-points are available for testing the anticancer potential of a substance. Each has its advantages and disadvantages, which must be weighed in the decision to use a particular method. Often several complementary methods are used to gain information on the mode of action of a substance.

Computational Sensing and in vitro Classification of GMOs and Biomolecular Events

DTIC Science & Technology

2008-12-01

COMPUTATIONAL SENSING AND IN VITRO CLASSIFICATION OF GMOs AND BIOMOLECULAR EVENTS Elebeoba May1∗, Miler T. Lee2†, Patricia Dolan1, Paul Crozier1...modified organisms ( GMOs ) in the pres- ence of non-lethal agents. Using an information and coding- theoretic framework we develop a de novo method for...high through- put screening, distinguishing genetically modified organisms ( GMOs ), molecular computing, differentiating biological mark- ers

Comparison of some aspects of the in situ and in vitro methods in evaluation of neutral detergent fiber digestion.

PubMed

Krizsan, S J; Jančík, F; Ramin, M; Huhtanen, P

2013-02-01

The objective of the present study was to compare digestion rates (kd) of NDF for different feeds estimated with the in situ method or derived from an automated gas in vitro system. A meta-analysis was conducted to evaluate how in situ derived kd of NDF related to in vivo digestibility of NDF. Furthermore, in vitro true digestibility of the feed samples incubated within filter bags or dispersed in the medium was compared, and kd for insoluble and soluble components of those feeds were estimated. Four different concentrates and 4 forages were used in this study. Two lactating Swedish Red cows fed a diet of 60% grass silage and 40% concentrate on DM basis were used for in situ incubations and for collection of rumen fluid. The feed samples were ground through a 2.0-mm screen before the in situ incubations and a 1.0-mm screen before the in vitro gas incubations. In situ nylon bags were introduced into the rumen for determination of kd of NDF. Additional kinetic data were produced from isolated NDF and intact samples subjected to in vitro incubations in which gas production was recorded for 72 h. Samples were weighed in the bottles or within filter bags (for fiber and in vitro studies) that were placed in the bottles. The interaction between feed and method was significant (P < 0.01); kd of NDF for grass hay tended (P = 0.06) to be less whereas kd of NDF for alfalfa, barley grain, canola meal, and dried sugar beet pulp were greater (P < 0.01) when estimated with the in situ method than from gas production recordings. The meta-analysis suggested that in situ derived kd of NDF were biased and underestimated in vivo digestibility of NDF. Digestion rates of the intact samples were lower for all feeds, except for the hay, when incubated within the bags compared with dispersed in the medium (P < 0.01). Less OM and NDF were digested for all feeds when incubated within bags than dispersed in the medium (P < 0.01). It is concluded from the in vitro study that microbial activity within the bags is less than in the medium. Significant interactions between method (in situ vs. in vitro) and feed suggest that one or both methods result in biased estimates of digestion kinetics.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Comparison of Two Static in Vitro Digestion Methods for Screening the Bioaccessibility of Carotenoids in Fruits, Vegetables, and Animal Products.

PubMed

Rodrigues, Daniele B; Chitchumroonchokchai, Chureeporn; Mariutti, Lilian R B; Mercadante, Adriana Z; Failla, Mark L

2017-12-27

In vitro digestion methods are routinely used to assess the bioaccessibility of carotenoids and other dietary lipophilic compounds. Here, we compared the recovery of carotenoids and their efficiency of micellarization in digested fruits, vegetables, egg yolk, and salmon and also in mixed-vegetable salads with and without either egg yolk or salmon using the static INFOGEST method22 and the procedure of Failla et al.16 Carotenoid stability during the simulated digestion was ≥70%. The efficiencies of the partitioning of carotenoids into mixed micelles were similar when individual plant foods and salad meals were digested using the two static methods. Furthermore, the addition of cooked egg or salmon to vegetable salads increased the bioaccessibility of some carotenoids. Our findings showed that the two methods of in vitro digestion generated similar estimates of carotenoid retention and bioaccessibility for diverse foods.

Integrative data mining of high-throughput in vitro screens, in vivo data, and disease information to identify Adverse Outcome Pathway (AOP) signatures:ToxCast high-throughput screening data and Comparative Toxicogenomics Database (CTD) as a case study.

EPA Science Inventory

The Adverse Outcome Pathway (AOP) framework provides a systematic way to describe linkages between molecular and cellular processes and organism or population level effects. The current AOP assembly methods however, are inefficient. Our goal is to generate computationally-pr...

Comparative phytochemical analysis and antibacterial efficacy of in vitro and in vivo extracts from East Indian sandalwood tree (Santalum album L.).

PubMed

Misra, B B; Dey, S

2012-12-01

Sandalwood oil has been found in numerous therapeutic applications in traditional medicines such as Chinese traditional medicine and Ayurveda. However, there are no comparative accounts available in the literature that focused on in vitro and in vivo tree sample-derived extracts. Combined dichloromethane and methanol extracts were obtained from in vitro samples, that is, callus, somatic embryo and seedlings, and in vivo from leaves of non-oil-yielding young and oil-yielding matured trees. Phytochemical evaluation of the extracts reveals that the tree is rich in terpenoids, saponin, phenolics and tannins. The antibacterial properties of the five extracts were compared with sandalwood oil by screening against nine Gram-negative and five Gram-positive bacterial strains by disc diffusion, agar spot and TLC bioautography methods. Minimum inhibitory concentration (MIC) for sandalwood oil was determined to be in the range of 0·078-5 μg ml(-1) for most of the test micro-organisms screened. Bioautography results indicated the presence of potential antimicrobial constituents in somatic embryo extracts and sandalwood oil. Among the extracts screened, the somatic embryo extracts showed the strongest antibacterial activity comparable only with sandalwood oil and matured tree leaves' extract. The findings presented here also suggest that apart from sandalwood oil, other parts of this tree across developmental stages are also enriched with antibacterial principles. This study constitutes the first systematic investigation on phytochemical composition and antimicrobial efficacy of sandalwood tree across in vitro and in vivo developmental stages screened against thirteen bacterial strains by four methods. Using a battery of antimicrobial assay techniques, it is possible to follow the differential bioactive metabolic richness of plant parts, to decipher, for example comparable efficacy of somatic embryo extracts and sandalwood oil. © 2012 The Society for Applied Microbiology.

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide mechanistic insights on chemical action. However, extrapolating from in vitro chemical concentrations to target tissue or blood concentrations in vivo is fraught with uncertainties, and modeling is dependent upon pharmacokinetic variables not measured in in vitro assays. To address this need, new tools have been created for characterizing, simulating, and evaluating chemical toxicokinetics. Physiologically-based pharmacokinetic (PBPK) models provide estimates of chemical exposures that produce potentially hazardous tissue concentrations, while tissue microdosimetry PK models relate whole-body chemical exposures to cell-scale concentrations. These tools rely on high-throughput in vitro measurements, and successful methods exist for pharmaceutical compounds that determine PK from limited in vitro measurements and chemical structure-derived property predictions. These high throughput (HT) methods provide a more rapid and less resource–intensive alternative to traditional PK model development. We have augmented these in vitro data with chemical structure-based descriptors and mechanistic tissue partitioning models to construct HTPBPK models for over three hundred environmental and pharmace

Whole Organism High-Content Screening by Label-Free, Image-Based Bayesian Classification for Parasitic Diseases

PubMed Central

Paveley, Ross A.; Mansour, Nuha R.; Hallyburton, Irene; Bleicher, Leo S.; Benn, Alex E.; Mikic, Ivana; Guidi, Alessandra; Gilbert, Ian H.; Hopkins, Andrew L.; Bickle, Quentin D.

2012-01-01

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping. PMID:22860151

In vitro chemical screening assays to identify thyroid hormone disruptors.

EPA Science Inventory

Identification of chemicals with potential to impact thyroid hormone function is a priority of the US EPA’s Endocrine Disruptor Screening Program (EDSP). In vitro screening assays can be used to significantly reduce the number of chemicals that need to be considered for tes...

Accurate and noninvasive embryos screening during in vitro fertilization (IVF) assisted by Raman analysis of embryos culture medium Accurate and noninvasive embryos screening during IVF

NASA Astrophysics Data System (ADS)

Shen, A. G.; Peng, J.; Zhao, Q. H.; Su, L.; Wang, X. H.; Hu, J. M.; Yang, J.

2012-04-01

In combination with morphological evaluation tests, we employ Raman spectroscopy to select higher potential reproductive embryos during in vitro fertilization (IVF) based on chemical composition of embryos culture medium. In this study, 57 Raman spectra are acquired from both higher and lower quality embryos culture medium (ECM) from 10 patients which have been preliminarily confirmed by clinical assay. Data are fit by using a linear combination model of least squares method in which 12 basis spectra represent the chemical features of ECM. The final fitting coefficients provide insight into the chemical compositions of culture medium samples and are subsequently used as criterion to evaluate the quality of embryos. The relative fitting coefficients ratios of sodium pyruvate/albumin and phenylalanine/albumin seem act as key roles in the embryo screening, attaining 85.7% accuracy in comparison with clinical pregnancy. The good results demonstrate that Raman spectroscopy therefore is an important candidate for an accurate and noninvasive screening of higher quality embryos, which potentially decrease the time-consuming clinical trials during IVF.

Predictive In Vitro Screening of Environmental Chemicals – The ToxCast Project

EPA Science Inventory

ToxCast, the United States Environmental Protection Agency’s chemical prioritization research program, is developing methods for utilizing computational chemistry and bioactivity profiling to predict potential for toxicity and prioritize limited testing resources (www.epa.gov/toc...

In Vitro Pulmonary Toxicity of Metal Oxide Nanoparticles

EPA Science Inventory

Nanomaterials (NMs) encompass a diversity of materials with unique physicochemical characteristics which raise concerns about their potential risk to human health. Rapid predictive testing methods are needed to characterize NMs health effects as well as to screen and prioritize N...

Quantitative Assessment of Neurite Outgrowth in PC12 Cells

EPA Science Inventory

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes such as neuronal differenti...

Perspectives on pathway perturbation: Focused research to enhance 3R objectives

EPA Science Inventory

In vitro high-throughput screening (HTS) and in silico technologies are emerging as 21st century tools for hazard identification. Computational methods that strategically examine cross-species conservation of protein sequence/structural information for chemical molecular targets ...

In-vitro propagation and antimycotic potential of extracts and essential oil of roots of Aristolochia bracteolata Linn. (Aristolochiaceae).

PubMed

Gbadamosi, I T; Egunyomi, A

2012-01-01

In spite of the therapeutic importance of Aristolochia bracteolata Linn. in Nigerian ethnomedicine, it is largely collected from the wild. Owing to the acclaimed potency of the plant and the difficulty in treating candidiasis, the anticandidal activity and in vitro propagation of the plant were investigated. Phytochemical screening and preparation of extracts of the roots were done using standard procedures. Clinical isolates of Candida albicans were screened against extracts and essential oil of Aristolochia bracteolata root using agar-well diffusion method. Minimum Inhibitory Concentration (MIC) of the ethanol extract was determined using broth dilution method. The nodal cuttings of A. bracteolata were cultured on Murashige and Skoog (MS) basal media. A. bracteolata contained alkaloids, saponins and cardenolides. The water extract was inactive on all isolates. The ethanol extract (500 mg/ml) and essential oil (undiluted) exhibited anticandidal activity on 9 out of 10 isolates at 10(1) - 10(6) cfu/ml inoculums concentration. Green growth and callus formation were observed in explants cultured on MS basal media after 30 days. A. bracteolata could be a source of anticandidal phytomedicine and the in vitro propagation confirmed its sustainability as anticandidal agent.

SkinEthic Laboratories, a company devoted to develop and produce in vitro alternative methods to animal use.

PubMed

de Brugerolle, Anne

2007-01-01

SkinEthic Laboratories is a France-based biotechnology company recognised as the world leader in tissue engineering. SkinEthic is devoted to develop and produce reliable and robust in vitro alternative methods to animal use in cosmetic, chemical and pharmaceutical industries. SkinEthic models provide relevant tools for efficacy and safety screening tests in order to support an integrated decision-making during research and development phases. Some screening tests are referenced and validated as alternatives to animal use (Episkin), others are in the process of validation under ECVAM and OECD guidelines. SkinEthic laboratories provide a unique and joined experience of more than 20 years from Episkin SNC and SkinEthic SA. Their unique cell culture process allows in vitro reconstructed human tissues with well characterized histology, functionality and ultrastructure features to be mass produced. Our product line includes skin models: a reconstructed human epidermis with a collagen layer, Episkin, reconstructed human epidermis without or with melanocytes (with a tanning degree from phototype II to VI) and a reconstructed human epithelium, i.e. cornea, and other mucosa, i.e. oral, gingival, oesophageal and vaginal. Our philosophy is based on 3 main commitments: to support our customers by providing robust and reliable models, to ensure training and education in using validated protocols, allowing a large array of raw materials, active ingredients and finished products in solid, liquid, powder, cream or gel form to be screened, and, to provide a dedicated service to our partners.

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles

PubMed Central

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

2016-01-01

This article addresses the in silico–in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy. PMID:27920524

Monte Carlo simulations guided by imaging to predict the in vitro ranking of radiosensitizing nanoparticles.

PubMed

Retif, Paul; Reinhard, Aurélie; Paquot, Héna; Jouan-Hureaux, Valérie; Chateau, Alicia; Sancey, Lucie; Barberi-Heyob, Muriel; Pinel, Sophie; Bastogne, Thierry

This article addresses the in silico-in vitro prediction issue of organometallic nanoparticles (NPs)-based radiosensitization enhancement. The goal was to carry out computational experiments to quickly identify efficient nanostructures and then to preferentially select the most promising ones for the subsequent in vivo studies. To this aim, this interdisciplinary article introduces a new theoretical Monte Carlo computational ranking method and tests it using 3 different organometallic NPs in terms of size and composition. While the ranking predicted in a classical theoretical scenario did not fit the reference results at all, in contrast, we showed for the first time how our accelerated in silico virtual screening method, based on basic in vitro experimental data (which takes into account the NPs cell biodistribution), was able to predict a relevant ranking in accordance with in vitro clonogenic efficiency. This corroborates the pertinence of such a prior ranking method that could speed up the preclinical development of NPs in radiation therapy.

Development and Validation of a Computational Model for Androgen Receptor Activity

EPA Science Inventory

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can mo...

Occurrence and in vitro bioactivity of estrogen, androgen, and glucocorticoid compounds in a nationwide screen of United States stream waters

EPA Science Inventory

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a suite of chemical a...

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

PubMed

Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

2016-03-01

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

PubMed

Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K

2017-01-01

Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

Methods and options in vitro dialyzability; benefits and limitations.

PubMed

Sandberg, Ann-Sofie

2005-11-01

In vitro dialyzability methods involve a two-step digestion process simulating the gastric and intestinal phase, and dialysis through a semi-permeable membrane with a selected molecular weight cut-off. Dialyzable iron/zinc is used as an estimation of available mineral. Final pH adjustment and use of a strict time schedule were found to be critical factors for standardization. In addition the selected cut-off of the dialysis membrane and the method used for iron and zinc determination influence the results. For screening purposes, simple solubility or dialyzability methods seem preferable to the more sophisticated computer-controlled gastrointestinal model. This is likely more valuable in studies of different transit times and sites of dialyzability. In vitro solubility/dialyzability methods correlate in most cases with human absorption studies in ranking iron and zinc availability from different meals. Exceptions may be that effects of milk, certain proteins, tea, and organic acids cannot be predicted. The dialyzability methods exclude iron bound to large molecules, which in some cases is available and include iron bound to small molecules, which is not always available. In vitro experiments based on solubility/dialyzability are tools to understand factors that may affect subsequent mineral absorption.

Susceptibility Testing of Medically Important Parasites.

PubMed

Genetu Bayih, Abebe; Debnath, Anjan; Mitre, Edward; Huston, Christopher D; Laleu, Benoît; Leroy, Didier; Blasco, Benjamin; Campo, Brice; Wells, Timothy N C; Willis, Paul A; Sjö, Peter; Van Voorhis, Wesley C; Pillai, Dylan R

2017-07-01

In the last 2 decades, renewed attention to neglected tropical diseases (NTDs) has spurred the development of antiparasitic agents, especially in light of emerging drug resistance. The need for new drugs has required in vitro screening methods using parasite culture. Furthermore, clinical laboratories sought to correlate in vitro susceptibility methods with treatment outcomes, most notably with malaria. Parasites with their various life cycles present greater complexity than bacteria, for which standardized susceptibility methods exist. This review catalogs the state-of-the-art methodologies used to evaluate the effects of drugs on key human parasites from the point of view of drug discovery as well as the need for laboratory methods that correlate with clinical outcomes. Copyright © 2017 American Society for Microbiology.

In vitro screening of 50 highly prescribed drugs for thiol adduct formation--comparison of potential for drug-induced toxicity and extent of adduct formation.

PubMed

Gan, Jinping; Ruan, Qian; He, Bing; Zhu, Mingshe; Shyu, Wen C; Humphreys, W Griffith

2009-04-01

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, rapid, cell based toxicity testing methods relevant to human health. Recent advances in robotics, automation, and miniaturization have been used to address these problems. However, one challenge is that ma...

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System##

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, rapid, cell based toxicity testing methods relevant to human health. Recent advances in robotics, automation, and miniaturization have been used to address this challenge. However, one drawback to currentl...

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Complementing in vitro screening assays with in silico molecular chemistry tools to examine potential in vivo metabolite-mediated effects

EPA Science Inventory

High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive un...

New(ish) tools in the toolbox: Using in vitro bioassays for cumulative assessment of steroid hormone receptor active compounds in environmental samples

EPA Science Inventory

In vitro assays are currently a high priority tool within USEPA for screening chemicals and samples for biological activity. The work included in this abstract describes the usage and expertise in our research group for using in vitro assays to screen environmental samples for s...

The Design, Synthesis and Screening of Potential Pyridinium Oxime Prodrugs

DTIC Science & Technology

1984-02-01

In vitro and in vivo ( mice ) screening of these compounds indicates some promise as AChE regenerators, prompting the preparation of gram quantities of...PAM 6 (E-I) have been synthesized and characterized. In vitro (immobilized-AChE) and in vivo ( mice ) screening of these compounds showed promising...screening assay. An ED5 0 determination (diisopropylfluorophosphate [DFPJ challenge/ mice ) showed 5-I-2-PAM (5) to be significantly more effective than 2

Risk-Based High-Throughput Chemical Screening and Prioritization using Exposure Models and in Vitro Bioactivity Assays.

PubMed

Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon A; Wetmore, Barbara A; Csiszar, Susan A; Fantke, Peter; Zhang, Xianming; McKone, Thomas E; Jolliet, Olivier; Bennett, Deborah H

2015-06-02

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.

Translating Computational Toxicology Data Through ...

EPA Pesticide Factsheets

US EPA has been using in vitro testing methods in an effort to accelerate the pace of chemical evaluations and address the significant lack of health and environmental data on the thousands of chemicals found in commonly used products. Since 2005, EPA’s researchers have generated hazard data using in vitro methods for thousands chemicals, designed innovative chemical exposure prediction models, and created a repository of thousands of high quality chemical structure data. Recently, EPA's ToxCast research effort, released high-throughput screening data on thousands of chemicals. These chemicals were screened for potential health effects in over 700 high-throughput screening assay endpoints. As part of EPA’s commitment to transparency, all data is accessible through the Chemical Safety for Sustainability Dashboard (iCSS). Policy makers and stakeholders can analyze and use this data to help inform decisions they make about chemicals. Use of these new datasets in risk decisions will require changing a regulatory paradigm that has been used for decades. EPA recognized early in the ToxCast effort that a communications and outreach strategy was needed to parallel the research and aid with the development and use of these new data sources. The goal is to use communications and outreach to increase awareness, interest and usage of analyzing and using these new chemical evaluation methods. To accomplish this, EPA employs numerous communication and outreach including t

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

PubMed Central

Isherwood, Beverley; Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I; Canel, Marta; Serrels, Alan; Brunton, Valerie G; Carragher, Neil O

2011-01-01

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates. PMID:24310493

Screening-level assays for potentially human-infectious environmental Legionella spp.

EPA Science Inventory

In spite of the fact that Legionella species can be isolated from nonclinical settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. In this study, an in vivo murine model of pneumonia and three in vitro proliferation as...

SeqAPASS: Sequence alignment to predict across-species susceptibility

EPA Science Inventory

Efforts to shift the toxicity testing paradigm from whole organism studies to those focused on the initiation of toxicity and relevant pathways have led to increased utilization of in vitro and in silico methods. Hence the emergence of high through-put screening (HTS) programs, s...

Human health and the environment: Predicting plasma protein binding and metabolic clearance rates of environmentally relevant chemicals.

EPA Science Inventory

In silico methods provide a rapid, inexpensive means of screening a wide array of environmentally relevant pollutants, pesticides, fungicides and consumer products for further toxicity testing. Physiologically based pharmacokinetic (PBPK) models bridge the gap between in vitro as...

The role of in vitro methods as alternatives to animals in toxicity testing.

PubMed

Anadón, Arturo; Martínez, María Aranzazu; Castellano, Victor; Martínez-Larrañaga, María Rosa

2014-01-01

It is accepted that animal testing should be reduced, refined or replaced as far as it is practicably possible. There are also a wide variety of in vitro models, which are used as screening studies and mechanistic investigations. The ability of an in vitro assay to be reliable, biomedically, is essential in pharmaceutical development. Furthermore, it is necessary that cells used in in vitro testing mimic the phenotype of cells within the human target tissue. The focus of this review article is to identify the key points of in vitro assays. In doing so, the authors take into account the chemical agents that are assessed and the integrated in vitro testing strategies. There is a transfer of toxicological data from primary in vivo animal studies to in vitro assays. The key element for designing an integrated in vitro testing strategy is summarized as follows: exposure modeling of chemical agents for in vitro testing; data gathering, sharing and read-across for testing a class of chemical; a battery of tests to assemble a broad spectrum of data on different mechanisms of action to predict toxic effects; and applicability of the test and the integrated in vitro testing strategies and flexibility to adjust the integrated in vitro testing strategies to test substance. While these methods will be invaluable if effective, more studies must be done to ensure reliability and suitability of these tests for humans.

Screening Applications to Test Cellular Fitness in Transwell® Models After Nanoparticle Treatment.

PubMed

Christ, Bastian; Fey, Christina; Cubukova, Alevtina; Walles, Heike; Dembski, Sofia; Metzger, Marco

2017-01-01

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs.

Diagnostic tool for early detection of ovarian cancers using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Lieber, Chad A.; Molpus, Kelly; Brader, Kevin; Mahadevan-Jansen, Anita

2000-05-01

With an overall survival rate of about 35 percent, ovarian cancer claims more than 13,000 women in the US each year. It is estimated that roughly 1 in 70 women will develop ovarian cancer. Current screening techniques are challenged due to cost-effectiveness, variable false-positive results, and the asymptomatic nature of the early stages of ovarian cancer. The predominant screening method for ovarian cancers is transvaginal sonography (TVS). TVS is fairly accomplished at ovarian cancer detection, however it is inefficient in distinguishing between benign and malignant masses. Accurate diagnosis of the ovarian tumor relies on exploratory laparotomy, thus increasing the cost and hazard of false- positive screening methods. Raman spectroscopy has been sued successfully as a diagnostic tool in several organ systems in vitro. These studies have shown that Raman spectroscopy can be used to provide diagnosis of subtle changes in tissue pathology with high accuracy. Based on this success, we have developed a Raman spectroscopic system for application in the ovary. Using this system, the Raman signatures of normal and various types of non-normal human ovarian tissues were characterized in vitro. Raman spectra are being analyzed, and empirical as well as multivariate discriminatory algorithms developed. Based on the result of this study, a strategy for in vivo trials will be planned.

A new cadmium(II) complex with bridging dithiolate ligand: Synthesis, crystal structure and antifungal activity study

NASA Astrophysics Data System (ADS)

Singh, Mahesh Kumar; Sutradhar, Sanjit; Paul, Bijaya; Adhikari, Suman; Laskar, Folguni; Butcher, Raymond J.; Acharya, Sandeep; Das, Arijit

2017-07-01

A new polymeric complex of Cd(II) with 1,1-dicyanoethylene- 2,2-dithiolate [ i-MNT2- = {S2C:C(CN)2}2- ] as a bridging ligand has been synthesized and characterized on the basis of spectroscopy and single-crystal X-ray diffraction analysis. Single crystal X-ray diffraction analysis reveals that the Cadmium (II) complex is six coordinated 1D polymeric in nature. Biological screening effects in vitro of the synthesized polymeric complex has been tested against five fungi Synchitrium endobioticum, Pyricularia oryzae, Helminthosporium oryzae, Candida albicans(ATCC10231), Trichophyton mentagrophytes by the disc diffusion method. In vitro antifungal screening indicates that the complex exhibits fungistatic and fungicidal antifungal activity whereas K2i-MNT.H2O became silent on Synchitrium endobioticum, Pyricularia oryzae, Helminthosporium oryzae, Candida albicans (ATCC10231), Trichophyton mentagrophytes.

NCCT ToxCast Program for Nanomaterial Prioritization: High-Throughput Screening, Consideration of Exposure, and Bioactivity Profiling/Modeling

EPA Science Inventory

Find relationships between bioactivities and NM characteristics or testing conditions. Recommend a dose metric for NMs in vitro studies. Establish associations to in vivo toxicity or pathways identified from testing of conventional chemicals with ToxCast HTS methods. May be abl...

A gene expression biomarker identifies in vitro and in vivo ERα modulators in a human gene expression compendium

EPA Science Inventory

We propose the use of gene expression profiling to complement the chemical characterization currently based on HTS assay data and present a case study relevant to the Endocrine Disruptor Screening Program. We have developed computational methods to identify estrogen receptor &alp...

Identifying Breast Tumor Suppressors Using in Vitro and in Vivo RNAi Screens

DTIC Science & Technology

2011-10-01

vivo RNA interference screen, breast cancer , tumor suppressor, leukemia inhibitory factor receptor (LIFR) 16. SECURITY CLASSIFICATION OF: 17...The identification of these genes will improve the understanding of the causes of breast cancer , which may lead to therapeutic advancements for... breast cancer prevention and treatment. BODY Objective 1: Identification of breast tumor suppressors using in vitro and in vivo RNAi screens

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

PubMed

Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

2016-08-01

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola).

PubMed

Gavamukulya, Yahaya; Abou-Elella, Faten; Wamunyokoli, Fred; AEl-Shemy, Hany

2014-09-01

To determine the phytochemical composition, antioxidant and anticancer activities of ethanolic and water leaves extracts of Annona muricata (A. muricata) from the Eastern Uganda. Phytochemical screening was conducted using standard qualitative methods and a Chi-square goodness of fit test was used to assign the relative abundance of the different phytochemicals. The antioxidant activity was determined using the 2, 2-diphenyl-2-picrylhydrazyl and reducing power methods whereas the in vitro anticancer activity was determined using three different cell lines. Phytochemical screening of the extracts revealed that they were rich in secondary class metabolite compounds such as alkaloids, saponins, terpenoids, flavonoids, coumarins and lactones, anthraquinones, tannins, cardiac glycosides, phenols and phytosterols. Total phenolics in the water extract were (683.69±0.09) μg/mL gallic acid equivalents (GAE) while it was (372.92±0.15) μg/mL GAE in the ethanolic extract. The reducing power was 216.41 μg/mL in the water extract and 470.51 μg/mL GAE in the ethanolic extract. In vitro antioxidant activity IC50 was 2.0456 mg/mL and 0.9077 mg/mL for ethanolic and water leaves extracts of A. muricata respectively. The ethanolic leaves extract was found to be selectively cytotoxic in vitro to tumor cell lines (EACC, MDA and SKBR3) with IC50 values of 335.85 μg/mL, 248.77 μg/mL, 202.33 μg/mL respectively, while it had no cytotoxic effect on normal spleen cells. The data also showed that water leaves extract of A. muricata had no anticancer effect at all tested concentrations. The results showed that A. muricata was a promising new antioxidant and anticancer agent. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing.

PubMed

Jomaa, Barae; Aarts, Jac M M J G; de Haan, Laura H J; Peijnenburg, Ad A C M; Bovee, Toine F H; Murk, Albertinka J; Rietjens, Ivonne M C M

2013-01-01

This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

EPA Science Inventory

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multip...

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances.

PubMed

Navas, José M; Segner, Helmut

2006-10-25

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Advancing Predictive Hepatotoxicity at the Intersection of Experimental, in Silico, and Artificial Intelligence Technologies.

PubMed

Fraser, Keith; Bruckner, Dylan M; Dordick, Jonathan S

2018-06-18

Adverse drug reactions, particularly those that result in drug-induced liver injury (DILI), are a major cause of drug failure in clinical trials and drug withdrawals. Hepatotoxicity-mediated drug attrition occurs despite substantial investments of time and money in developing cellular assays, animal models, and computational models to predict its occurrence in humans. Underperformance in predicting hepatotoxicity associated with drugs and drug candidates has been attributed to existing gaps in our understanding of the mechanisms involved in driving hepatic injury after these compounds perfuse and are metabolized by the liver. Herein we assess in vitro, in vivo (animal), and in silico strategies used to develop predictive DILI models. We address the effectiveness of several two- and three-dimensional in vitro cellular methods that are frequently employed in hepatotoxicity screens and how they can be used to predict DILI in humans. We also explore how humanized animal models can recapitulate human drug metabolic profiles and associated liver injury. Finally, we highlight the maturation of computational methods for predicting hepatotoxicity, the untapped potential of artificial intelligence for improving in silico DILI screens, and how knowledge acquired from these predictions can shape the refinement of experimental methods.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

PubMed Central

Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

2015-01-01

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

PubMed

Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

2015-01-01

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

PubMed

Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

2018-06-26

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

PubMed

Misra, Ashish; Green, Michael R

2017-01-01

Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

DOE PAGES

Shin, Hyeong -Moo; Ernstoff, Alexi; Arnot, Jon A.; ...

2015-05-01

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate dailymore » intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.« less

Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

PubMed

Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

2004-12-01

The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

Developmental neurotoxicity testing in vitro: Models for assessing chemical effects on neurite outgrowth

EPA Science Inventory

In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to resul...

IN VITRO ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: USE OF MICROELECTRODE ARRAYS TO MEASURE FUNCTIONAL CHANGES IN NEURONAL NETWORK ONTOGENY

EPA Science Inventory

Because the Developmental Neurotoxicity Testing Battery requires large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical,...

In Vitro Assessment of Developmental Neurotoxicity: Use of Microelectrode Arrays to Measure Functional Changes in Neuronal Network Ontogeny*

EPA Science Inventory

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemica...

Optimization of DNA barcode method to assess altered chemical toxicity due to CYP-mediated metabolism

EPA Science Inventory

A drawback of current in vitro chemical testing is that many commonly used cell lines lack chemical metabolism. This hinders the use and relevance of cell culture in high throughput chemical toxicity screening. To address this challenge, we engineered HEK293T cells to overexpress...

A Road Map Towards High pH Adaptability: Phenomic and Genomic Approaches to Azalea Breeding (Rhododendron sp.)

USDA-ARS?s Scientific Manuscript database

A research grant from the Azalea Society of America has enabled us to collect and begin evaluating diverse Rhododendron viscosum germplasm to identify genetic and phenotypic variation for pH adaptability. During the Spring of 2014, we developed novel, in vitro screening methods for Rhododendron to ...

Application of the ToxMiner Database: Network Analysis Linking the ToxCast Chemicals to Known Disease-Gene Associations

EPA Science Inventory

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effecti...

A Novel Two-Step Hierarchial Quantitative Structure-Activity Relationship Modeling Workflow for Predicting Acute Toxicity of Chemicals in Rodents

EPA Science Inventory

Background: Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public–private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening. Methods and results: A database co...

ASSESSMENT OF CHEMICAL EFFECTS ON NEURONAL DIFFERENTIATION USING THE ARRAYSCAN HIGH CONTENT SCREENING SYSTEM

EPA Science Inventory

The development of alternative methods for toxicity testing is driven by the need for scientifically valid data that can be obtained in a rapid and cost-efficient manner. In vitro systems provide a model in which chemical effects on cellular events can be examined using technique...

Informatics approach using metabolic reactivity classifiers to link in vitro to in vivo data in application to the ToxCast Phase I dataset

EPA Science Inventory

Strategic combinations and tiered application of alternative testing methods to replace or minimize the use of animal models is attracting much attention. With the advancement of high throughput screening (HTS) assays and legacy databases providing in vivo testing results, suffic...

An in vitro approach for lipolysis measurement using high-resolution mass spectrometry and partial least squares based analysis.

PubMed

Chang, Wen-Qi; Zhou, Jian-Liang; Li, Yi; Shi, Zi-Qi; Wang, Li; Yang, Jie; Li, Ping; Liu, Li-Fang; Xin, Gui-Zhong

2017-01-15

The elevation of free fatty acids (FFAs) has been regarded as a universal metabolic signature of excessive adipocyte lipolysis. Nowadays, in vitro lipolysis assay is generally essential for drug screening prior to the animal study. Here, we present a novel in vitro approach for lipolysis measurement combining UHPLC-Orbitrap and partial least squares (PLS) based analysis. Firstly, the calibration matrix was constructed by serial proportions of mixed samples (blended with control and model samples). Then, lipidome profiling was performed by UHPLC-Orbitrap, and 403 variables were extracted and aligned as dataset. Owing to the high resolution of Orbitrap analyzer and open source lipid identification software, 28 FFAs were further screened and identified. Based on the relative intensity of the screened FFAs, PLS regression model was constructed for lipolysis measurement. After leave-one-out cross-validation, ten principal components have been designated to build the final PLS model with excellent performances (RMSECV, 0.0268; RMSEC, 0.0173; R 2 , 0.9977). In addition, the high predictive accuracy (R 2  = 0.9907 and RMSEP = 0.0345) of the trained PLS model was also demonstrated using test samples. Finally, taking curcumin as a model compound, its antilipolytic effect on palmitic acid-induced lipolysis was successfully predicted as 31.78% by the proposed approach. Besides, supplementary evidences of curcumin induced modification in FFAs compositions as well as lipidome were given by PLS extended methods. Different from general biological assays, high resolution MS-based method provide more sophisticated information included in biological events. Thus, the novel biological evaluation model proposed here showed promising perspectives for drug evaluation or disease diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Fostering efficacy and toxicity evaluation of traditional Chinese medicine and natural products: Chick embryo as a high throughput model bridging in vitro and in vivo studies.

PubMed

Wu, Tong; Yu, Gui-Yuan; Xiao, Jia; Yan, Chang; Kurihara, Hiroshi; Li, Yi-Fang; So, Kwok-Fai; He, Rong-Rong

2018-04-19

Efficacy and safety assessments are essential thresholds for drug candidates from preclinical to clinical research. Conventional mammalian in vivo models cannot offer rapid pharmacological and toxicological screening, whereas cell-based or cell-free in vitro systems often lead to inaccurate results because of the lack of physiological environment. Within the avian species, gallus gallus is the first bird to have its genome sequencing. Meantime, chick embryo is an easily operating, relatively transparent and extensively accessible model, whose physiological and pathological alterations can be visualized by egg candler, staining and image technologies. These features facilitate chick embryo as a high-throughput screening platform bridging in vivo and in vitro gaps in the pharmaceutical research. Due to the complicated ingredients and multiple-targets natures of traditional Chinese medicine (TCM), testing the efficacy and safety of TCM by in vitro methods are laborious and inaccurate, while testing in mammalian models consume massive cost and time. As such, the productive living organism chick embryo serves as an ideal biological system for pharmacodynamics studies of TCM. Herein, we comprehensively update recent progresses on the specialty of chick embryo in evaluation of efficacy and toxicity of drugs, with special concerns of TCM. Copyright © 2018 Elsevier Ltd. All rights reserved.

A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish

PubMed Central

Xie, Shao-Lin; Bian, Wan-Ping; Wang, Chao; Junaid, Muhammad; Zou, Ji-Xing; Pei, De-Sheng

2016-01-01

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system. PMID:27680290

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

PubMed

Sørli, Jorid B; Huang, Yishi; Da Silva, Emilie; Hansen, Jitka S; Zuo, Yi Y; Frederiksen, Marie; Nørgaard, Asger W; Ebbehøj, Niels E; Larsen, Søren T; Hougaard, Karin S

2018-01-01

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

Development and Application of In Vitro Models for Screening Drugs and Environmental Chemicals that Predict Toxicity in Animals and Humans

EPA Pesticide Factsheets

Development and Application of In Vitro Models for Screening Drugs and Environmental Chemicals that Predict Toxicity in Animals and Humans (Presented by James McKim, Ph.D., DABT, Founder and Chief Science Officer, CeeTox) (5/25/2012)

In silico study of in vitro GPCR assays by QSAR modeling

EPA Science Inventory

The U.S. EPA is screening thousands of chemicals of environmental interest in hundreds of in vitro high-throughput screening (HTS) assays (the ToxCast program). One goal is to prioritize chemicals for more detailed analyses based on activity in molecular initiating events (MIE) o...

A Workflow for Identifying Metabolically Active Chemicals to Complement in vitro Toxicity Screening

EPA Science Inventory

The new paradigm of toxicity testing approaches involves rapid screening of thousands of chemicals across hundreds of biological targets through use of in vitro assays. Such assays may lead to false negatives when the complex metabolic processes that render a chemical bioactive i...

Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

EPA Science Inventory

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

Formulation characteristics and in vitro release testing of cyclosporine ophthalmic ointments.

PubMed

Dong, Yixuan; Qu, Haiou; Pavurala, Naresh; Wang, Jiang; Sekar, Vasanthakumar; Martinez, Marilyn N; Fahmy, Raafat; Ashraf, Muhammad; Cruz, Celia N; Xu, Xiaoming

2018-06-10

The aim of the present study was to investigate the relationship between formulation/process variables versus the critical quality attributes (CQAs) of cyclosporine ophthalmic ointments and to explore the feasibility of using an in vitro approach to assess product sameness. A definitive screening design (DSD) was used to evaluate the impact of formulation and process variables. The formulation variables included drug percentage, percentage of corn oil and lanolin alcohol. The process variables studied were mixing temperature, mixing time and the method of mixing. The quality and performance attributes examined included drug assay, content uniformity, image analysis, rheology (storage modulus, shear viscosity) and in vitro drug release. Of the formulation variables evaluated, the percentage of the drug substance and the percentage of corn oil in the matrix were the most influential factors with respect to in vitro drug release. Conversely, the process parameters tested were observed to have minimal impact. An evaluation of the release mechanism of cyclosporine from the ointment revealed an interplay between formulation (e.g. physicochemical properties of the drug and ointment matrix type) and the release medium. These data provide a scientific basis to guide method development for in vitro drug release testing of ointment dosage forms. These results demonstrate that the in vitro methods used in this investigation were fit-for-purpose for detecting formulation and process changes and therefore amenable to assessment of product sameness. Published by Elsevier B.V.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Screening for small molecule inhibitors of Toxoplasma gondii.

PubMed

Kortagere, Sandhya

2012-12-01

Toxoplasma gondii, the agent that causes toxoplasmosis, is an opportunistic parasite that infects many mammalian species. It is an obligate intracellular parasite that causes severe congenital neurological and ocular disease mostly in immunocompromised humans. The current regimen of therapy includes only a few medications that often lead to hypersensitivity and toxicity. In addition, there are no vaccines available to prevent the transmission of this agent. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. The author presents in silico and in vitro strategies that are currently used to screen for novel targets and unique chemotypes against T. gondii. Furthermore, this review highlights the screening technologies and characterization of some novel targets and new chemical entities that could be developed into highly efficacious treatments for toxoplasmosis. A number of diverse methods are being used to design inhibitors against T. gondii. These include ligand-based methods, in which drugs that have been shown to be efficacious against other Apicomplexa parasites can be repurposed to identify lead molecules against T. gondii. In addition, structure-based methods use currently available repertoire of structural information in various databases to rationally design small-molecule inhibitors of T. gondii. Whereas the screening methods have their advantages and limitations, a combination of methods is ideally suited to design small-molecule inhibitors of complex parasites such as T. gondii.

Analysis Of Volatile Fingerprints: A Rapid Screening Method For Antifungal Agents For Efficacy Against Dermatophytes

NASA Astrophysics Data System (ADS)

Naraghi, Kamran; Sahgal, Natasha; Adriaans, Beverley; Barr, Hugh; Magan, Naresh

2009-05-01

The potential of using an electronic nose (E. nose) for rapid screening dermatophytes to antifungal agents was studied. In vitro, the 50 and 90% effective concentration (EC) values of five antifungal agents for T. rubrum and T. mentagrophytes were obtained by mycelial growth assays. Then, the qualitative volatile production patterns of the growth responses of these fungi to these values were incorporated into solid medium were analysed after 96-120 hrs incubation at 25° C using headspace analyses. Overall, results, using PCA and CA demonstrated that it is possible to differentiate between various treatments within 96-120 hrs. This study showed that potential exists for using qualitative volatile patterns as a rapid screening method for antifungal agents for microorganism. This approach could also facilitate the monitoring of antimicrobial drug activities and infection control programmes and perhaps drug resistance build up in microbial species.

Screening for Natural Inhibitors of Topoisomerases I from Rhamnus davurica by Affinity Ultrafiltration and High-Performance Liquid Chromatography–Mass Spectrometry

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Topoisomerase I (Topo I) catalyzes topological interconversion of duplex DNA during DNA replication and transcription, and has been deemed as important antineoplastic targets. In this study, the fraction R.d-60 from ethyl acetate extracts of Rhamnus davurica showed higher inhibitory rates against SGC-7901 and HT-29 compared with the R.d-30 fraction in vitro. However, the specific active components of R.d-60 fraction remain elusive. To this end, a method based on bio-affinity ultrafiltration and high performance liquid chromatography/electrospray mass spectrometry (HPLC- ESI-MS/MS) was developed to rapidly screen and identify the Topo I inhibitors in this fraction. The enrichment factors (EFs) were calculated to evaluate the binding affinities between the bioactive constituents and Topo I. As a result, eight ligands were identified and six of which with higher EFs showed more potential antitumor activity. Furthermore, antiproliferative assays in vitro (IC50 values) with two representative candidates (apigenin, quercetin) against SGC-7901, HT-29 and Hep G2 cells were conducted and further validated. Finally, the structure-activity relationships revealed that flavones contain a C2-C3 double bond of C ring exhibited higher bio-affinities to Topo I than those without it. This integrated method combining Topo I ultrafiltration with HPLC-MS/MS proved to be very efficient in rapid screening and identification of potential Topo I inhibitors from the complex extracts of medicinal plants, and could be further explored as a valuable high-throughput screening platform in the early drug discovery stage. PMID:28919906

Microfluidics-assisted in vitro drug screening and carrier production

PubMed Central

Tsui, Jonathan H.; Lee, Woohyuk; Pun, Suzie H.; Kim, Jungkyu; Kim, Deok-Ho

2013-01-01

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development. PMID:23856409

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

PubMed Central

Creek, Darren J.; Nijagal, Brunda; Kim, Dong-Hyun; Rojas, Federico; Matthews, Keith R.

2013-01-01

In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening. PMID:23571546

Physiologically-based Pharmacokinetic(PBPK) Models Application to Screen Environmental Hazards Related to Adverse Outcome Pathways(AOPs)

EPA Science Inventory

PBPK models are useful in estimating exposure levels based on in vitro to in vivo extrapolation (IVIVE) calculations. Linkage of large sets of chemically screened vitro signature effects to in vivo adverse outcomes using IVIVE is central to the concepts of toxicology in the 21st ...

On Selecting a Minimal Set of In Vitro Assays to Reliably Determine Estrogen Agonist Activity

EPA Science Inventory

The US EPA is charged with screening chemicals for their ability to be endocrine disruptors through interaction with the estrogen, androgen and thyroid axes. The agency is starting to explore the use of high-throughput in vitro assays to use in the Endocrine Disruptor Screening P...

Application of the ToxMiner Database: Network Analysis of Linkage between ToxCast Phase I Chemicals and Thyroid Related Disease Outcomes

EPA Science Inventory

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effecti...

Maize genotype and food matrix affect the provitamin A carotenoid bioefficacy from staple and carrot-fortified feeds in Mongolian gerbils (Meriones unguiculatus)

USDA-ARS?s Scientific Manuscript database

Biofortification of staple crops to increase provitamin A carotenoids is a promising technique to alleviate vitamin A deficiency. ß-Carotene (ßC) and ß-cryptoxanthin (ßCX) are low in typical maize but higher in biofortified varieties. In vitro screening methods evaluate bioaccessibility of carotenoi...

The comet assay: assessment of in vitro and in vivo DNA damage.

PubMed

Bajpayee, Mahima; Kumar, Ashutosh; Dhawan, Alok

2013-01-01

Rapid industrialization and pursuance of a better life have led to an increase in the amount of chemicals in the environment, which are deleterious to human health. Pesticides, automobile exhausts, and new chemical entities all add to air pollution and have an adverse effect on all living organisms including humans. Sensitive test systems are thus required for accurate hazard identification and risk assessment. The Comet assay has been used widely as a simple, rapid, and sensitive tool for assessment of DNA damage in single cells from both in vitro and in vivo sources as well as in humans. Already, the in vivo comet assay has gained importance as the preferred test for assessing DNA damage in animals for some international regulatory guidelines. The advantages of the in vivo comet assay are its ability to detect DNA damage in any tissue, despite having non-proliferating cells, and its sensitivity to detect genotoxicity. The recommendations from the international workshops held for the comet assay have resulted in establishment of guidelines. The in vitro comet assay conducted in cultured cells and cell lines can be used for screening large number of compounds and at very low concentrations. The in vitro assay has also been automated to provide a high-throughput screening method for new chemical entities, as well as environmental samples. This chapter details the in vitro comet assay using the 96-well plate and in vivo comet assay in multiple organs of the mouse.

[Evaluation on a fast weight reduction model in vitro].

PubMed

Li, Songtao; Li, Ying; Wen, Ying; Sun, Changhao

2010-03-01

To establish a fast and effective model in vitro for screening weight-reducing drugs and taking preliminary evaluation of the model. Mature adipocytes of SD rat induced by oleic acid were used to establish a obesity model in vitro. Isoprel, genistein, caffeine were selected as positive agents and curcumine as negative agent to evaluate the obesity model. Lipolysis of adipocytes was stimulated significantly by isoprel, genistein and caffeine rather than curcumine. This model could be used efficiently for screening weight-losing drugs.

FAITH – Fast Assembly Inhibitor Test for HIV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadravová, Romana; Rumlová, Michaela, E-mail: michaela.rumlova@vscht.cz; Department of Biotechnology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague

Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification ofmore » the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors. - Highlights: • Allows screening of assembly inhibitors of both mature and immature HIV-1 particles. • Based on Gag-derived proteins with CA in mature or immature conformation. • Simple and sensitive method suitable for high-throughput screening of inhibitors. • Unlike in other HIV assembly methods, works under physiological conditions. • No washing steps are necessary.« less

Method For Determining And Modifying Protein/Peptide Solubilty

DOEpatents

Waldo, Geoffrey S.

2005-03-15

A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum.

PubMed

Srivastava, Shivani; Cahill, David M; Conlan, Xavier A; Adholeya, Alok

2014-10-15

Plants are an important source for medicinal compounds. Chemical screening and selection is critical for identification of compounds of interest. Ocimum basilicum (Basil) is a rich source of polyphenolics and exhibits high diversity, therefore bioprospecting of a suitable cultivar is a necessity. This study reports on the development of a true to type novel "in vitro system" and its comparison with a conventional system for screening and selection of cultivars for high total phenolics, individual polyphenolics, and antioxidant content. We have shown for the first time using online acidic potassium permanganate chemiluminescence that extracts from Ocimum basilicum showed antioxidant potential. The current study identified the cultivar specific composition of polyphenolics and their antioxidant properties. Further, a distinct relationship between plant morphotype and polyphenolic content was also found. Of the 15 cultivars examined, "Holy Green", "Red Rubin", and "Basil Genovese" were identified as high polyphenolic producing cultivars while "Subja" was determined to be a low producer. The "in vitro system" enabled differentiation of the cultivars in their morphology, polyphenolic content, and antioxidant activity and is a cheap and efficient method for bioprospecting studies.

Engineering cancer microenvironments for in vitro 3-D tumor models

PubMed Central

Asghar, Waseem; El Assal, Rami; Shafiee, Hadi; Pitteri, Sharon; Paulmurugan, Ramasamy; Demirci, Utkan

2017-01-01

The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell–cell, cell–matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing. PMID:28458612

A preliminary study for the development and optimization by experimental design of an in vitro method for prediction of drug buccal absorption.

PubMed

Mura, Paola; Orlandini, Serena; Cirri, Marzia; Maestrelli, Francesca; Mennini, Natascia; Casella, Giada; Furlanetto, Sandra

2018-06-15

The work was aimed at developing an in vitro method able to provide rapid and reliable evaluation of drug absorption through buccal mucosa. Absorption simulator apparatus endowed with an artificial membrane was purposely developed by experimental design. The apparent permeation coefficient (P app ) through excised porcine buccal mucosa of naproxen, selected as model drug, was the target value to obtain with the artificial membrane. The multivariate approach enabled systematic evaluation of the effect on the response (P app ) of simultaneous variations of the variables (kind of lipid components for support impregnation and relative amounts). A screening phase followed by a response-surface study allowed optimization of the lipid-mixture composition to obtain the desired P app value, and definition of a design space where all mixture components combinations fulfilled the desired target at a fixed probability level. The method offers a useful tool for a quick screening in the early stages of drug discovery and/or in preformulation studies, improving efficiency and chance of success in the development of buccal delivery systems. Further studies with other model drugs are planned to confirm the buccal absorption predictive capacity of the developed membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro steroid profiling system for the evaluation of endocrine disruptors.

PubMed

Nakano, Yosuke; Yamashita, Toshiyuki; Okuno, Masashi; Fukusaki, Eiichiro; Bamba, Takeshi

2016-09-01

Endocrine disruptors (ED) are chemicals that affect various aspects of the endocrine system, often leading to the inhibition of steroidogenesis. Current chemical safety policies that restrict human exposure to such chemicals describe often time-consuming and costly methods for the evaluation of ED effects. We aimed to develop an effective tool for accurate phenotypic chemical toxicology studies. We developed an in vitro ED evaluation system using gas chromatography/mass spectrometry (GC/MS/MS) methods for metabolomic analysis of multi-marker profiles. Accounting for sample preparation and GC/MS/MS conditions, we established a screening method that allowed the simultaneous analysis of 17 steroids with good reproducibility and a linear calibration curve. Moreover, we applied the developed system to H295R human adrenocortical cells exposed to forskolin and prochloraz in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines and observed dose-dependent variations in steroid profiles. While the OECD guidelines include only testosterone and 17β-estradiol, our system enabled a comprehensive and highly sensitive analysis of steroid profile alteration due to ED exposure. The application of our ED evaluation screen could be economical and provide novel insights into the hazards of ED exposure to the endocrine system. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

PubMed

Rajkumar, M; Lee, Wang Hyu; Lee, Kui Jae

2005-01-01

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici. ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Pilot Screening to Determine Antimicrobial Synergies in a Multidrug-Resistant Bacterial Strain Library

PubMed Central

Kim, Si-Hyun; Park, Chulmin; Chun, Hye-Sun; Choi, Jae-Ki; Lee, Hyo-Jin; Cho, Sung-Yeon; Park, Sun Hee; Choi, Su-Mi; Choi, Jung-Hyun; Yoo, Jin-Hong

2016-01-01

With the rise in multidrug-resistant (MDR) bacterial infections, there has been increasing interest in combinations of ≥2 antimicrobial agents with synergistic effects. We established an MDR bacterial strain library to screen for in vitro antimicrobial synergy by using a broth microdilution checkerboard method and high-throughput luciferase-based bacterial cell viability assay. In total, 39 MDR bacterial strains, including 23 carbapenem-resistant gram-negative bacteria, 9 vancomycin-intermediate Staphylococcus aureus, and 7 vancomycin-resistant Enterococcus faecalis, were used to screen for potential antimicrobial synergies. Synergies were more frequently identified with combinations of imipenem plus trimethoprim–sulfamethoxazole for carbapenem-resistant Acinetobacter baumannii in the library. To verify this finding, we tested 34 A. baumannii clinical isolates resistant to both imipenem and trimethoprim–sulfamethoxazole by the checkerboard method. The imipenem plus trimethoprim–sulfamethoxazole combination showed synergy in the treatment of 21 (62%) of the clinical isolates. The results indicate that pilot screening for antimicrobial synergy in the MDR bacterial strain library could be valuable in the selection of combination therapeutic regimens to treat MDR bacterial infections. Further studies are warranted to determine whether this screening system can be useful to screen for the combined effects of conventional antimicrobials and new-generation antimicrobials or nonantimicrobials. PMID:26974861

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

PubMed

Ye, Lidan; Yang, Chengcheng; Yu, Hongwei

2018-01-01

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

An investigation into western white pine partial resistance against the rust pathogen Cronartium ribicola using in vitro screening method

Treesearch

D. Noshad; J.N. King

2012-01-01

Cronartium ribicola is one of the most destructive forest pathogens of North American white pines. The pathogen infects pine trees through their stomata, colonizes the stem, and produces stem cankers the following growing season. In this research, we collected samples from different white pine populations across Canada and the United States to...

QSAR Classification of ToxCast and Tox21 Chemicals on the Basis of Estrogen Receptor Assays (FutureToxII)

EPA Science Inventory

The ToxCast and Tox21 programs have tested ~8,200 chemicals in a broad screening panel of in vitro high-throughput screening (HTS) assays for estrogen receptor (ER) agonist and antagonist activity. The present work uses this large in vitro data set to develop in silico QSAR model...

In Vitro Screening of 1877 Industrial and Consumer Chemicals, Pesticides and Pharmaceuticals in up to 782 Assays: ToxCast Phase I and II (SOT)

EPA Science Inventory

In Phase II of the ToxCast program, the U.S. EPA and Tox21 partners screened 1,877 chemicals, including pesticides; food, cosmetics and personal care ingredients; pharmaceuticals; and industrial chemicals. Testing used a 782 in vitro assays across 7 technologies and multiple bi...

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

PubMed

Hashimoto, Junko; Watanabe, Taku; Seki, Tatsuya; Karasawa, Satoshi; Izumikawa, Miho; Seki, Tomoe; Iemura, Shun-Ichiro; Natsume, Tohru; Nomura, Nobuo; Goshima, Naoki; Miyawaki, Atsushi; Takagi, Motoki; Shin-Ya, Kazuo

2009-09-01

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

PubMed

Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

2015-05-01

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

PubMed

Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

1984-10-01

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

UHPLC-Q-TOF-MS/MS Method Based on Four-Step Strategy for Metabolism Study of Fisetin in Vitro and in Vivo.

PubMed

Zhang, Xia; Yin, Jintuo; Liang, Caijuan; Sun, Yupeng; Zhang, Lantong

2017-12-20

Fisetin has been identified as an anticancer agent with antiangiogenic properties in mice. However, its metabolism in vitro (rat liver microsomes) and in vivo (rats) is presently not characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was employed for data acquiring, and a four-step analytical strategy was developed to screen and identify metabolites. First, full-scan was applied, which was dependent on a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS). Then PeakView 1.2 and Metabolitepilot 1.5 software were used to load data to seek possible metabolites. Finally, metabolites were identified according to mass measurement and retention time. Moreover, isomers were distinguished based on Clog P parameter. Based on the proposed method, 53 metabolites in vivo and 14 metabolites in vitro were characterized. Moreover, metabolic pathways mainly included oxidation, reduction, hydrogenation, methylation, sulfation, and glucuronidation.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

Rapid screening, identification, and purification of neuraminidase inhibitors from Lithospermum erythrorhizon Sieb.et Zucc. by ultrafiltration with HPLC-ESI-TOF-MS combined with semipreparative HPLC.

PubMed

Zhang, Minmin; Zhao, Hengqiang; Zhao, Zhiguo; Yan, Huijiao; Lv, Ruimin; Cui, Li; Yuan, Jinpeng; Wang, Daijie; Geng, Yanling; Liu, Daicheng; Wang, Xiao

2016-06-01

We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply to the discovery of anti-influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti-influenza components from Zicao. Semipreparative high-performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high-performance liquid chromatography with the purity over 98% for all of them by high-performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β-dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti-influenza active ingredients from complex Chinese herbal medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid Screening and Characterization of Acetylcholinesterase Inhibitors from Yinhuang Oral Liquid Using Ultrafiltration-liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

PubMed Central

Zhang, Haomin; Guo, Yinan; Meng, Lingwen; Sun, Hui; Yang, Yinping; Gao, Ying; Sun, Jiaming

2018-01-01

Background: At present, approximately 17–25 million people in the world suffer from Alzheimer's disease (AD). The most efficacious and acceptable therapeutic drug clinically are the acetylcholinesterase inhibitors (AChEIs). Yinhuang oral liquid is a Chinese medicine preparation which contains AChEIs according to the literatures. However, no strategy has been presented for rapid screening and identification of AChEIs from Yinhuang oral liquid. Objective: To develop a method for rapid screening and identification of AChEIs from Yinhuang oral liquid using ultrafiltration–liquid chromatography–electrospray ionization tandem mass spectrometry (UF-LC-ESI-MS/MS). Materials and Methods: In this study, UF incubation conditions such as enzyme concentration, incubation time, and incubation temperature were optimized so as to get better screening results. The AChEIs from Yinhuang oral liquid were identified by high-performance liquid chromatography-ESI-MS and the improved Ellman method was used for the AChE inhibitory activity test in vitro. Results: The results showed that Yinhuang oral liquid can inhibit the activity of AChE. We screened and identified seven compounds with potential AChE inhibitory activity from Yinhuang oral liquid, which provided experimental basis for the treatment and prevention of AD. Conclusion: The current technique was used to directly screen the active ingredients with acetylcholinesterase inhibition from complex traditional Chinese medicine, which was simple, rapid, accurate, and suitable for high-throughput screening of AChEI from complex systems. SUMMARY A UF-LC-ESI-MS/MS method for rapid screening and identification of AChEIs from Yinhuang oral liquid was developedSeven compounds were screened and identified with potential AChE inhibitory activity from Yinhuang oral liquidIt provided experimental basis of Yinhuang oral liquid for the treating and preventing AD. Abbreviations used: (AD): Alzheimer's disease; (UF-LC-ESI-MS/MS): ultrafiltration–liquid chromatography–electrospray ionization tandem mass spectrometry; (AChEIs): acetylcholinesterase inhibitors. PMID:29720840

Intersection of toxicogenomics and high throughput screening in the Tox21 program: an NIEHS perspective.

PubMed

Merrick, B Alex; Paules, Richard S; Tice, Raymond R

Humans are exposed to thousands of chemicals with inadequate toxicological data. Advances in computational toxicology, robotic high throughput screening (HTS), and genome-wide expression have been integrated into the Tox21 program to better predict the toxicological effects of chemicals. Tox21 is a collaboration among US government agencies initiated in 2008 that aims to shift chemical hazard assessment from traditional animal toxicology to target-specific, mechanism-based, biological observations using in vitro assays and lower organism models. HTS uses biocomputational methods for probing thousands of chemicals in in vitro assays for gene-pathway response patterns predictive of adverse human health outcomes. In 1999, NIEHS began exploring the application of toxicogenomics to toxicology and recent advances in NextGen sequencing should greatly enhance the biological content obtained from HTS platforms. We foresee an intersection of new technologies in toxicogenomics and HTS as an innovative development in Tox21. Tox21 goals, priorities, progress, and challenges will be reviewed.

Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Targeting-Free Cancer Cell Screening and Killing.

PubMed

Chen, Limei; Li, Haijuan; He, Haili; Wu, Haoxi; Jin, Yongdong

2015-07-07

Fast and accurate identification of cancer cells from healthy normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic Au nanoparticles (NPs), have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct localized surface plasmon resonance (LSPR) responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

Discrete Fourier Transform-Based Multivariate Image Analysis: Application to Modeling of Aromatase Inhibitory Activity.

PubMed

Barigye, Stephen J; Freitas, Matheus P; Ausina, Priscila; Zancan, Patricia; Sola-Penna, Mauro; Castillo-Garit, Juan A

2018-02-12

We recently generalized the formerly alignment-dependent multivariate image analysis applied to quantitative structure-activity relationships (MIA-QSAR) method through the application of the discrete Fourier transform (DFT), allowing for its application to noncongruent and structurally diverse chemical compound data sets. Here we report the first practical application of this method in the screening of molecular entities of therapeutic interest, with human aromatase inhibitory activity as the case study. We developed an ensemble classification model based on the two-dimensional (2D) DFT MIA-QSAR descriptors, with which we screened the NCI Diversity Set V (1593 compounds) and obtained 34 chemical compounds with possible aromatase inhibitory activity. These compounds were docked into the aromatase active site, and the 10 most promising compounds were selected for in vitro experimental validation. Of these compounds, 7419 (nonsteroidal) and 89 201 (steroidal) demonstrated satisfactory antiproliferative and aromatase inhibitory activities. The obtained results suggest that the 2D-DFT MIA-QSAR method may be useful in ligand-based virtual screening of new molecular entities of therapeutic utility.

A method for evaluating nanoparticle transport through the blood-brain barrier in vitro.

PubMed

Guarnieri, Daniela; Muscetti, Ornella; Netti, Paolo A

2014-01-01

Blood-brain barrier (BBB) represents a formidable barrier for many therapeutic drugs to enter the brain tissue. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous system (CNS) diseases. In this context, nanoparticles are an emerging class of drug delivery systems that can be easily tailored to deliver drugs to various compartments of the body, including the brain. To identify, characterize, and validate novel nanoparticles applicable to brain delivery, in vitro BBB model systems have been developed. In this work, we describe a method to screen nanoparticles with variable size and surface functionalization in order to define the physicochemical characteristics underlying the design of nanoparticles that are able to efficiently cross the BBB.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Virtual medicinal chemistry: in silico pre-docking functional group transformation for discovery of novel inhibitors of botulinum toxin serotype A light chain.

PubMed

O'Malley, Sean; Sareth, Sina; Jiao, Guan-Sheng; Kim, Seongjin; Thai, April; Cregar-Hernandez, Lynne; McKasson, Linda; Margosiak, Stephen A; Johnson, Alan T

2013-05-01

A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.

PubMed

de-Souza-Silva, Calliandra Maria; Guilhelmelli, Fernanda; Zamith-Miranda, Daniel; de Oliveira, Marco Antônio; Nosanchuk, Joshua Daniel; Silva-Pereira, Ildinete; Albuquerque, Patrícia

2018-02-14

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs.

PubMed

Kim, Wooseong; Hendricks, Gabriel Lambert; Lee, Kiho; Mylonakis, Eleftherios

2017-06-01

The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doherty, Kimberly R., E-mail: kimberly.doherty@quintiles.com; Talbert, Dominique R.; Trusk, Patricia B.

Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks.more » Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability. - Highlights: • 24 drugs were tested for cardiac liability using an in vitro multi-parameter screen. • Changes in beating activity were the most sensitive in predicting cardiac risk. • Structural effects add in-depth insight towards mechanism of cardiac toxicity. • Testing functional and structural endpoints enhances early cardiac risk assessment.« less

High throughput screening technologies for ion channels

PubMed Central

Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

2016-01-01

Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

Discovery of Novel MDR-Mycobacterium tuberculosis Inhibitor by New FRIGATE Computational Screen

PubMed Central

Vértessy, Beáta; Pütter, Vera; Grolmusz, Vince; Schade, Markus

2011-01-01

With 1.6 million casualties annually and 2 billion people being infected, tuberculosis is still one of the most pressing healthcare challenges. Here we report on the new computational docking algorithm FRIGATE which unites continuous local optimization techniques (conjugate gradient method) with an inherently discrete computational approach in forcefield computation, resulting in equal or better scoring accuracies than several benchmark docking programs. By utilizing FRIGATE for a virtual screen of the ZINC library against the Mycobacterium tuberculosis (Mtb) enzyme antigen 85C, we identified novel small molecule inhibitors of multiple drug-resistant Mtb, which bind in vitro to the catalytic site of antigen 85C. PMID:22164290

Evaluation of OASIS QSAR Models Using ToxCast™ in Vitro Estrogen and Androgen Receptor Binding Data and Application in an Integrated Endocrine Screening Approach

PubMed Central

Bhhatarai, Barun; Wilson, Daniel M.; Price, Paul S.; Marty, Sue; Parks, Amanda K.; Carney, Edward

2016-01-01

Background: Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed. Objectives: We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 μM), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay. Methods: NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined. Results: The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92–100%) and specificity (70–81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 μM, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 μM were active in the uterotrophic assay. Conclusions: We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had high sensitivity and specificity when compounds were in-domain of the models. Based on this research, we recommend a tiered screening approach wherein a) QSAR is used to identify compounds in-domain of the ER or AR binding models and predicted to bind; b) those compounds are screened in vitro to assess binding potency; and c) the stronger binders (AC50 < 1 μM) are screened in vivo. This scheme prioritizes compounds for integrative testing and risk assessment. Importantly, compounds that are not in-domain, that are predicted either not to bind or to bind weakly, that are not active in in vitro, that require metabolism to manifest activity, or for which in vivo AR testing is in order, need to be assessed differently. Citation: Bhhatarai B, Wilson DM, Price PS, Marty S, Parks AK, Carney E. 2016. Evaluation of OASIS QSAR models using ToxCast™ in vitro estrogen and androgen receptor binding data and application in an integrated endocrine screening approach. Environ Health Perspect 124:1453–1461; http://dx.doi.org/10.1289/EHP184 PMID:27152837

A redox proteomics approach to investigate the mode of action of nanomaterials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riebeling, Christian; Wiemann, Martin; Schnekenburger, Jürgen

2016-05-15

Numbers of engineered nanomaterials (ENMs) are steadily increasing. Therefore, alternative testing approaches with reduced costs and high predictivity suitable for high throughput screening and prioritization are urgently needed to ensure a fast and effective development of safe products. In parallel, extensive research efforts are targeted to understanding modes of action of ENMs, which may also support the development of new predictive assays. Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of ENMs. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Fluorescent dye basedmore » read-outs are most frequently used for cell testing in vitro but may be limited due to possible interference of the ENMs. Recently, other assays have been put forward such as acellular determination of ROS production potential using methods like electron spin resonance, antioxidant quantification or the use of specific sensors. In addition, Omics based approaches have gained increasing attention. In particular, redox proteomics can combine the assessment of oxidative stress with the advantage of getting more detailed mechanistic information. Here we propose a comprehensive testing strategy for assessing the oxidative stress potential of ENMs, which combines acellular methods and fast in vitro screening approaches, as well as a more involved detailed redox proteomics approach. This allows for screening and prioritization in a first tier and, if required, also for unraveling mechanistic details down to compromised signaling pathways. - Highlights: • Oxidative stress is a general paradigm for nanomaterial hazard mechanism of action. • Reactive oxygen species generation can be predicted using acellular assays. • Cellular assays based on fluorescence suffer from interference by nanomaterials. • Protein carbonylation is an irreversible and predictive mark of oxidative stress. • Proteomics of carbonylation indicates affected pathways and mechanism of action.« less

Endocrine Profiling and Prioritization of Environmental Chemicals Using ToxCast Data

PubMed Central

Reif, David M.; Martin, Matthew T.; Tan, Shirlee W.; Houck, Keith A.; Judson, Richard S.; Richard, Ann M.; Knudsen, Thomas B.; Dix, David J.; Kavlock, Robert J.

2010-01-01

Background The prioritization of chemicals for toxicity testing is a primary goal of the U.S. Environmental Protection Agency (EPA) ToxCast™ program. Phase I of ToxCast used a battery of 467 in vitro, high-throughput screening assays to assess 309 environmental chemicals. One important mode of action leading to toxicity is endocrine disruption, and the U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) has been charged with screening pesticide chemicals and environmental contaminants for their potential to affect the endocrine systems of humans and wildlife. Objective The goal of this study was to develop a flexible method to facilitate the rational prioritization of chemicals for further evaluation and demonstrate its application as a candidate decision-support tool for EDSP. Methods Focusing on estrogen, androgen, and thyroid pathways, we defined putative endocrine profiles and derived a relative rank or score for the entire ToxCast library of 309 unique chemicals. Effects on other nuclear receptors and xenobiotic metabolizing enzymes were also considered, as were pertinent chemical descriptors and pathways relevant to endocrine-mediated signaling. Results Combining multiple data sources into an overall, weight-of-evidence Toxicological Priority Index (ToxPi) score for prioritizing further chemical testing resulted in more robust conclusions than any single data source taken alone. Conclusions Incorporating data from in vitro assays, chemical descriptors, and biological pathways in this prioritization schema provided a flexible, comprehensive visualization and ranking of each chemical’s potential endocrine activity. Importantly, ToxPi profiles provide a transparent visualization of the relative contribution of all information sources to an overall priority ranking. The method developed here is readily adaptable to diverse chemical prioritization tasks. PMID:20826373

IN VITRO SCREENING BATTERIES FOR NEUROTOXICANTS

EPA Science Inventory

The need to develop, validate and utilize in vitro models to test chemicals for neurotoxic potential is widely appreciated. his lecture discusses the major advantages and disadvantages of using cell and tissue culture, the carious in vitro models amenable for neurotoxicity studie...

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Use of in vivo/in vitro unscheduled DNA synthesis for identification of organ-specific carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furihata, C.; Matsushima, T.

1987-01-01

There are still only a few in vivo short-term assay methods for predicting potential organ-specific carcinogens and mutagens in mammals, although such methods are required for evaluating the in vivo effects of in vitro mutagens. In the in vivo/in vitro UDS assay methods described here, chemicals are given to experimental animals and induction of UDS in target organs is determined by in vitro organ culture or primary cell culture in the presence of (/sup 3/H)dThd. Incorporation of (/sup 3/H)dThd into DNA is measured with a liquid scintillation counter or by autoradiography. These methods have now been applied to the glandularmore » stomach, forestomach, colon, liver, kidney, pancreas, tracheal epithelium, nasal epithelium, and spermatocytes. With minor modifications, they may also be applied to other organs. The present review shows that induction of UDS in various organs correlated well with the induction of cancer in these organs. The present authors have used the present methods to identify some potential organ-specific mutagens and carcinogens in mammals. The present authors found that three dicarbonyl compounds, glyoxal, methylglyoxal, and diacetyl, induced apparent UDS and TDS in the glandular stomach, and other groups found that 2-NT, MA6BT, and CNEt6BT induced UDS in the liver. These in vivo/in vitro UDS assays are better than in vitro UDS assay for identification of potential organ-specific mutagens and carcinogens in mammals and are especially useful for identifying potential mutagens and carcinogens that are specific for certain organs, such as the stomach, liver, and kidney. They are also useful for examining the potential mutagenicities and carcinogenicities of carcinogen analogs. However, these methods are not suitable for general in vivo screening because they are not yet available for all organs. 113 references.« less

In Vitro Wound Healing Potential and Identification of Bioactive Compounds from Moringa oleifera Lam

PubMed Central

Muhammad, Abubakar Amali; Pauzi, Nur Aimi Syarina; Arulselvan, Palanisamy; Abas, Faridah; Fakurazi, Sharida

2013-01-01

Moringa oleifera Lam. (M. oleifera) from the monogeneric family Moringaceae is found in tropical and subtropical countries. The present study was aimed at exploring the in vitro wound healing potential of M. oleifera and identification of active compounds that may be responsible for its wound healing action. The study included cell viability, proliferation, and wound scratch test assays. Different solvent crude extracts were screened, and the most active crude extract was further subjected to differential bioguided fractionation. Fractions were also screened and most active aqueous fraction was finally obtained for further investigation. HPLC and LC-MS/MS analysis were used for identification and confirmation of bioactive compounds. The results of our study demonstrated that aqueous fraction of M. oleifera significantly enhanced proliferation and viability as well as migration of human dermal fibroblast (HDF) cells compared to the untreated control and other fractions. The HPLC and LC-MS/MS studies revealed kaempferol and quercetin compounds in the crude methanolic extract and a major bioactive compound Vicenin-2 was identified in the bioactive aqueous fraction which was confirmed with standard Vicenin-2 using HPLC and UV spectroscopic methods. These findings suggest that bioactive fraction of M. oleifera containing Vicenin-2 compound may enhance faster wound healing in vitro. PMID:24490175

Screening of wheat endophytes as biological control agents against Fusarium head blight using two different in vitro tests.

PubMed

Comby, Morgane; Gacoin, Marie; Robineau, Mathilde; Rabenoelina, Fanja; Ptas, Sébastien; Dupont, Joëlle; Profizi, Camille; Baillieul, Fabienne

2017-09-01

In order to find biological control agents (BCAs) for the management of Fusarium head blight (FHB), a major disease on wheat crops worldwide, 86 microorganisms isolated from inner tissues of wheat plants were discriminated for their ability to inhibit the growth of Fusarium graminearum and Fusarium culmorum by in vitro dual culture assays. A group of 22 strains appeared very effective to inhibit F. graminearum (inhibition of 30-51%) and they were also globally effective in controlling F. culmorum (inhibition of 15-53%). Further evaluation of a subselection of strains by screening on detached spikelets in vitro confirmed three species, namely Phoma glomerata, Aureobasidium proteae and Sarocladium kiliense, that have not yet been reported for their efficacy against Fusarium spp., indicating that looking for BCAs toward FHB among wheat endophytes proved to be promising. The efficacy of some strains turned out different between both in vitro screening approaches, raising the importance of finding the most appropriate screening approach for the search of BCAs. This study pointed out the interest of the test on detached wheat spikelets that provided information about a potential pathogenicity, the growth capacity and efficacy of the endophyte strains on the targeted plant, before testing them on whole plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Engineering an in vitro air-blood barrier by 3D bioprinting

PubMed Central

Horváth, Lenke; Umehara, Yuki; Jud, Corinne; Blank, Fabian; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing. PMID:25609567

An integrated microfludic device for culturing and screening of Giardia lamblia.

PubMed

Zheng, Guo-Xia; Zhang, Xue-Mei; Yang, Yu-Suo; Zeng, Shu-Rui; Wei, Jun-Feng; Wang, Yun-Hua; Li, Ya-Jie

2014-02-01

In vitro culturing of trophozoites was important for research of Giardia lamblia (G. lamblia), especially in discovery of anti-Giardia agents. The current culture methods mainly suffer from lab-intension or the obstacle in standardizing the gas condition. Thus, it could benefit from a more streamlined and integrated approach. Microfluidics offers a way to accomplish this goal. Here we presented an integrated microfluidic device for culturing and screening of G. lamblia. The device consisted of a polydimethylsiloxane (PDMS) microchip with an aerobic culture system. In the microchip, the functionality of integrated concentration gradient generator (CGG) with micro-scale cell culture enables dose-response experiment to be performed in a simple and reagent-saving way. The diffusion-based culture chambers allowed growing G. lamblia at the in vivo like environment. It notable that the highly air permeable material of parallel chambers maintain uniform anaerobic environment in different chambers easily. Using this device, G. lamblia were successfully cultured and stressed on-chip. In all cases, a dose-related inhibitory response was detected. The application of this device for these purposes represents the first step in developing a completely integrated microfluidic platform for high-throughput screening and might be expanded to other assays based on in vitro culture of G. lamblia with further tests. Copyright © 2013 Elsevier Inc. All rights reserved.

Prospective performance evaluation of selected common virtual screening tools. Case study: Cyclooxygenase (COX) 1 and 2.

PubMed

Kaserer, Teresa; Temml, Veronika; Kutil, Zsofia; Vanek, Tomas; Landa, Premysl; Schuster, Daniela

2015-01-01

Computational methods can be applied in drug development for the identification of novel lead candidates, but also for the prediction of pharmacokinetic properties and potential adverse effects, thereby aiding to prioritize and identify the most promising compounds. In principle, several techniques are available for this purpose, however, which one is the most suitable for a specific research objective still requires further investigation. Within this study, the performance of several programs, representing common virtual screening methods, was compared in a prospective manner. First, we selected top-ranked virtual screening hits from the three methods pharmacophore modeling, shape-based modeling, and docking. For comparison, these hits were then additionally predicted by external pharmacophore- and 2D similarity-based bioactivity profiling tools. Subsequently, the biological activities of the selected hits were assessed in vitro, which allowed for evaluating and comparing the prospective performance of the applied tools. Although all methods performed well, considerable differences were observed concerning hit rates, true positive and true negative hits, and hitlist composition. Our results suggest that a rational selection of the applied method represents a powerful strategy to maximize the success of a research project, tightly linked to its aims. We employed cyclooxygenase as application example, however, the focus of this study lied on highlighting the differences in the virtual screening tool performances and not in the identification of novel COX-inhibitors. Copyright © 2015 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

[Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique].

PubMed

Zhang, Lu; Xu, Jinhao; Ma, Jinbiao

2016-07-25

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

PubMed Central

Tan, Kaeling; Roberts, Anthony J.; Chonofsky, Mark; Egan, Martin J.; Reck-Peterson, Samara L.

2014-01-01

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. PMID:24403603

Molecular modeling of drug-pathophysiological Mtb protein targets: Synthesis of some 2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-tubercular agents

NASA Astrophysics Data System (ADS)

Noorulla, K. M.; Suresh, Ayyadurai Jerad; Devaraji, Vinod; Mathew, Bijo; Umesh, Devi

2017-11-01

Twenty novel 2-thioxo-1, 3-thiazolidin-4-one derivatives (5a-5t) were synthesized and evaluated for their antitubercular activity. The structure of the compounds was confirmed by IR, NMR and Mass Spectroscopy methods. In addition, single-crystal X-ray diffraction was performed for compound 5a. All the synthesized compounds were screened for their in-vitro antimycobacterial activity against MTB (H37RV, ATCC No: 27294) by Alamar Blue assay method. Compounds 5r, 5k, 5t displayed most potent in-vitro activity with MICs of 0.05, 0.1, 0.2 μg/ml concentrations respectively which are comparatively potent than the standards. Molecular docking and dynamics simulations were performed to find out the plausible mechanism of the titled compounds.

20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

EPA Science Inventory

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

Buffer-regulated biocorrosion of pure magnesium.

PubMed

Kirkland, Nicholas T; Waterman, Jay; Birbilis, Nick; Dias, George; Woodfield, Tim B F; Hartshorn, Richard M; Staiger, Mark P

2012-02-01

Magnesium (Mg) alloys are being actively investigated as potential load-bearing orthopaedic implant materials due to their biodegradability in vivo. With Mg biomaterials at an early stage in their development, the screening of alloy compositions for their biodegradation rate, and hence biocompatibility, is reliant on cost-effective in vitro methods. The use of a buffer to control pH during in vitro biodegradation is recognised as critically important as this seeks to mimic pH control as it occurs naturally in vivo. The two different types of in vitro buffer system available are based on either (i) zwitterionic organic compounds or (ii) carbonate buffers within a partial-CO(2) atmosphere. This study investigated the influence of the buffering system itself on the in vitro corrosion of Mg. It was found that the less realistic zwitterion-based buffer did not form the same corrosion layers as the carbonate buffer, and was potentially affecting the behaviour of the hydrated oxide layer that forms on Mg in all aqueous environments. Consequently it was recommended that Mg in vitro experiments use the more biorealistic carbonate buffering system when possible.

In vitro fertilisation treatment and factors affecting success.

PubMed

Huang, Jack Yu Jen; Rosenwaks, Zev

2012-12-01

The efficacy of assisted reproductive technologies has improved significantly over the past decades. The main indications for in vitro fertilisation include tubal obstruction, severe male-factor infertility, severe endometriosis, ovulatory dysfunction, diminished ovarian reserve, and infertility of unexplained cause. In vitro fertilisation has also become an effective treatment option for couples wishing to undergo pre-implantation genetic diagnosis or screening, and for those wishing to cryopreserve their oocytes or embryos for preservation of fertility. The management of women in late reproductive age poses a major challenge; the optimum in vitro fertilisation treatment for poor responders remains elusive. The success of in vitro fertilisation treatment can be optimised by taking an individualised, patient-centered approach to controlled ovarian hyperstimulation. Key components involve selection of an appropriate controlled ovarian protocol, close-cycle monitoring, adjustment of gonadotropin dosage to avoid hyper-response, and individualised timing of human chorionic gonadotropin injection. Future directions of assisted reproductive technologies include development of non-invasive embryo selection methods, use of transcriptomics, proteomics, metabolomics, and time-lapse imaging technologies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com; Cromwell, Evan F., E-mail: evan.cromwell@moldev.com; Crittenden, Carole

2013-12-15

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. Amore » number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development. - Highlights: • Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models. • We tested if evaluation of cardiotoxicity is possible in a high-throughput format. • The assay shows benefits of automated data integration across multiple parameters. • Quantitative assessment of concentration–response is possible using iPSCs. • Multi-parametric screening allows for cardiotoxicity risk assessment.« less

In Vitro Metabolic Studies of REV-ERB Agonists SR9009 and SR9011.

PubMed

Geldof, Lore; Deventer, Koen; Roels, Kris; Tudela, Eva; Van Eeno, Peter

2016-10-03

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC-HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended.

In Vitro Metabolic Studies of REV-ERB Agonists SR9009 and SR9011

PubMed Central

Geldof, Lore; Deventer, Koen; Roels, Kris; Tudela, Eva; Van Eenoo, Peter

2016-01-01

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography–high resolution mass spectrometry (LC–HRMS). Structure elucidation was performed for all metabolites by LC–HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC–HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended. PMID:27706103

A rapid generation of adenovirus vector with a genetic modification in hexon protein.

PubMed

Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

2012-02-10

The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

In Vivo Tumor Cell Targeting with “Click” Nanoparticles

PubMed Central

von Maltzahn, Geoffrey; Ren, Yin; Park, Ji-Ho; Min, Dal-Hee; Kotamraju, Venkata Ramana; Jayakumar, Jayanthi; Fogel, Valentina; Sailor, Michael J.; Ruoslahti, Erkki; Bhatia, Sangeeta N.

2008-01-01

The in vivo fate of nanomaterials strongly determines their biomedical efficacy. Accordingly, much effort has been invested into the development of library screening methods to select targeting ligands for a diversity of sites in vivo. Still, broad application of chemical and biological screens to the in vivo targeting of nanomaterials requires ligand attachment chemistries that are generalizable, efficient, covalent, orthogonal to diverse biochemical libraries, applicable under aqueous conditions, and stable in in vivo environments. To date, the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition or “click” reaction has shown considerable promise as a method for developing targeted nanomaterials in vitro. Here, we investigate the utility of “click” chemistry for the in vivo targeting of inorganic nanoparticles to tumors. We find that “click” chemistry allows cyclic LyP-1 targeting peptides to be specifically linked to azido-nanoparticles and to direct their binding to p32-expressing tumor cells in vitro. Moreover, “click” nanoparticles are able to stably circulate for hours in vivo following intravenous administration (>5h circulation time), extravasate into tumors, and penetrate the tumor interstitium to specifically bind p32-expressing cells in tumors. In the future, in vivo use of “click” nanomaterials should expedite the progression from ligand discovery to in vivo evaluation and diversify approaches toward multifunctional nanoparticle development. PMID:18611045

[Study thought of material basis of secondary development of major traditional Chinese medicine varieties on basis of combination of in vivo and in vitro experiments].

PubMed

Cheng, Xu-Dong; Jia, Xiao-Bin; Feng, Liang; Jiang, Jun

2013-12-01

The secondary development of major traditional Chinese medicine varieties is one of important links during the modernization, scientification and standardization of traditional Chinese medicines. How to accurately and effectively identify the pharmacodynamic material basis of original formulae becomes the primary problem in the secondary development, as well as the bottleneck in the modernization development of traditional Chinese medicines. On the basis of the existing experimental methods, and according to the study thought that the multi-component and complex effects of traditional Chinese medicine components need to combine multi-disciplinary methods and technologies, we propose the study thought of the material basis of secondary development of major traditional Chinese medicine varieties based on the combination of in vivo and in vitro experiments. It is believed that studies on material basis needs three links, namely identification, screening and verification, and in vivo and in vitro study method corresponding to each link is mutually complemented and verified. Finally, the accurate and reliable material basis is selected. This thought provides reference for the secondary development of major traditional Chinese medicine varieties and studies on compound material basis.

Evaluation of the GADD45α-GFP GreenScreen HC assay for rapid and reliable in vitro early genotoxicity screening.

PubMed

Luzy, Anne-Pascale; Orsini, Nicolas; Linget, Jean-Michel; Bouvier, Guy

2013-11-01

Twenty-two of Galderma's proprietary compounds were tested in the GADD45α-GFP 'GreenScreen HC' assay (GS), the SOS-ChromoTest and the Mini-Ames to evaluate GSs performance for early genotoxicity screening purposes. Forty more characterized compounds were also tested, including antibiotics: metronidazole, clindamycin, tetracycline, lymecycline and neomycin; and catecholamines: resorcinol mequinol, hydroquinone, one aneugen carbendazim, one corticoid dexamethasone, one peroxisome proliferator-activated receptor rosiglitazone, one pesticide carbaryl and two further proprietary molecules with in vitro genotoxicity data. With proprietary molecules, this study concluded that the GS renders the SOS-ChromoTest obsolete for in vitro screening. The GS confirmed all results of the Mini-Ames test (100% concordance). Compared with the micronucleus test, the GS showed a concordance of 82%. With known compounds, the GS ranked the potency of positive results for catecholamines in accordance with other genotoxicity tests and showed very reproducible results. It confirmed positive results for carbendazim, for tetracycline antibiotics and for carbaryl. The GS produced negative results for metronidazole, a nitroreduction-specific bacterial mutagen, for dexamethasone (a non-genotoxic apoptosis inducer), for rosiglitazone (a GADD45γ promoter inducer) and for clindamycin and neomycin (inhibitors of macromolecular synthesis in bacteria). As such, the GS appears to be a reproducible, robust, specific and sensitive test for genotoxicity screening. Copyright © 2012 John Wiley & Sons, Ltd.

In vitro bioactivity and phytochemical screening of selected spices used in Mauritian foods

PubMed Central

Tacouri, Diksa Devi; Ramful-Baboolall, Deena; Puchooa, Daneshwar

2013-01-01

Objective To investigate the in vitro antioxidant and antimicrobial activities along with phytochemical screening of organic and aqueous extracts of spices used in Mauritian foods. Methods Antioxidant activity of the crude extracts was evaluated in terms of total antioxidant capacity, total phenol content and total flavonoid content. The antimicrobial activity of the spices was determined by the agar well diffusion method against a gram positive and a gram negative bacteria. The qualitative and quantitative phytochemical screening were carried out by standard biochemical assays. Results All six spices were found to possess alkaloids, coumarins, flavonoids, saponins, steroids, tannins and phenols. Total phenolic content of the extracts varied between 177 and 1 890 mg GAE/g DW while the total flavonoid content varied between 2.8 and 37.6 mg QE/g DW. All six spices were found to possess strong antioxidant properties as well. Highest value was obtained for cinnamon [(24.930±0.198) µmol Fe2+/g DW] whilst turmeric showed the lowest antioxidant activity [(5.980±0.313) µmol Fe2+/g DW] (P<0.05). All extracts showed promising activity against Staphylococcus aureus and Escherichia coli. The size of the inhibition zones ranged between (11.20±0.23) mm to (26.10±2.09) mm (P<0.05) with turmeric and cinnamon being the most effective against Staphylococcus aureus while garlic was least effective against both E. coli and S. aureus. Conclusions The present study reveals the presence of potential antioxidant and antimicrobial properties in the extracts of the spices which could be further exploited.

A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

PubMed

Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

2013-02-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

Yeast as a potential vehicle for neglected tropical disease drug discovery.

PubMed

Denny, P W; Steel, P G

2015-01-01

High-throughput screening (HTS) efforts for neglected tropical disease (NTD) drug discovery have recently received increased attention because several initiatives have begun to attempt to reduce the deficit in new and clinically acceptable therapies for this spectrum of infectious diseases. HTS primarily uses two basic approaches, cell-based and in vitro target-directed screening. Both of these approaches have problems; for example, cell-based screening does not reveal the target or targets that are hit, whereas in vitro methodologies lack a cellular context. Furthermore, both can be technically challenging, expensive, and difficult to miniaturize for ultra-HTS [(u)HTS]. The application of yeast-based systems may overcome some of these problems and offer a cost-effective platform for target-directed screening within a eukaryotic cell context. Here, we review the advantages and limitations of the technologies that may be used in yeast cell-based, target-directed screening protocols, and we discuss how these are beginning to be used in NTD drug discovery. © 2014 Society for Laboratory Automation and Screening.

In vitro assessment of estrogen, androgen, and glucocorticoid bioactivity in a nationwide screen of United States stream waters

EPA Science Inventory

In vitro bioassays can identify environmental samples contaminated with bioactive chemicals that interact with steroid receptors and provide a cumulative, effect-based measurement of contamination. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR...

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

PubMed Central

Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

2015-01-01

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

Overview of a workshop on screening methods for detecting potential (anti-) estrogenic/androgenic chemicals in wildlife

USGS Publications Warehouse

Ankley, Gerald T.; Mihaich, Ellen; Stahl, Ralph G.; Tillitt, Donald E.; Colborn, Theo; McMaster, Suzzanne; Miller, Ron; Bantle, John; Campbell, Pamela; Denslow, Nancy; Dickerson, Richard L.; Folmar, Leroy C.; Fry, Michael; Giesy, John P.; Gray, L. Earl; Guiney, Patrick; Hutchinson, Thomas; Kennedy, Sean W.; Kramer, Vincent; LeBlanc, Gerald A.; Mayes, Monte; Nimrod, Alison; Patino, Reynaldo; Peterson, Richard; Purdy, Richard; Ringer, Robert; Thomas, Peter C.; Touart, Les; Van Der Kraak, Glen; Zacharewski, Tim

1998-01-01

The U.S. Congress has passed legislation requiring the U.S. Environmental Protection Agency (U.S. EPA) to develop, validate, and implement screening tests for identifying potential endocrine-disrupting chemicals within 3 years. To aid in the identification of methods suitable for this purpose, the U.S. EPA, the Chemical Manufacturers Association, and the World Wildlife Fund sponsored several workshops, including the present one, which dealt with wildlife species. This workshop was convened with 30 international scientists representing multiple disciplines in March 1997 in Kansas City, Missouri, USA. Participants at the meeting identified methods in terms of their ability to indicate (anti-) estrogenic/androgenic effects, particularly in the context of developmental and reproductive processes. Data derived from structure-activity relationship models and in vitro test systems, although useful in certain contexts, cannot at present replace in vivo tests as the sole basis for screening. A consensus was reached that existing mammalian test methods (e.g., with rats or mice) generally are suitable as screens for assessing potential (anti-) estrogenic/ androgenic effects in mammalian wildlife. However, due to factors such as among-class variation in receptor structure and endocrine function, it is uncertain if these mammalian assays would be of broad utility as screens for other classes of vertebrate wildlife. Existing full and partial life-cycle tests with some avian and fish species could successfully identify chemicals causing endocrine disruption; however, these long-term tests are not suitable for routine screening. However, a number of short-term tests with species from these two classes exist that could serve as effective screening tools for chemicals inducing (anti-) estrogenic/androgenic effects. Existing methods suitable for identifying chemicals with these mechanisms of action in reptiles and amphibians are limited, but in the future, tests with species from these classes may prove highly effective as screens. In the case of invertebrate species, too little is known at present about the biological role of estrogens and androgens in reproduction and development to recommend specific assays.

Assessing the Relevance of in vitro Studies in Nanotoxicology by Examining Correlations between in vitro and in vivo Data

PubMed Central

Han, Xianglu; Corson, Nancy; Wade-Mercer, Pamela; Gelein, Robert; Jiang, Jingkun; Sahu, Manoranjan; Biswas, Pratim; Finkelstein, Jacob N.; Elder, Alison; Oberdörster, Günter

2012-01-01

There is an urgent need for in vitro screening assays to evaluate nanoparticle (NP) toxicity. However, the relevance of in vitro assays is still disputable. We administered doses of TiO2 NPs of different sizes to alveolar epithelial cells in vitro and the same NPs by intratracheal instillation in rats in vivo to examine the correlation between in vitro and in vivo responses. The correlations were based on toxicity rankings of NPs after adopting NP surface area as dose metric, and response per unit surface area as response metric. Sizes of the anatase TiO2 NPs ranged from 3 to 100 nm. A cell-free assay for measuring reactive oxygen species (ROS) was used, and lactate dehydrogenase (LDH) release, and protein oxidation induction were the in vitro cellular assays using a rat lung Type I epithelial cell line (R3/1) following 24 hr incubation. The in vivo endpoint was number of PMNs in bronchoalveolar lavage fluid (BALF) after exposure of rats to the NPs via intratracheal instillation. Slope analyses of the dose response curves shows that the in vivo and in vitro responses were well correlated. We conclude that using the approach of steepest slope analysis offers a superior method to correlate in vitro with in vivo results of NP toxicity and for ranking their toxic potency. PMID:22487507

A General Method for Discovering Inhibitors of Protein–DNA Interactions Using Photonic Crystal Biosensors

PubMed Central

Chan, Leo L.; Pineda, Maria; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2009-01-01

Protein–DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein–DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein–DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein–DNA binding. The PC technology is used to detect binding between protein–DNA interactions that are DNA-sequence-dependent (the bacterial toxin–antitoxin system MazEF) and those that are DNA-sequence-independent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF–DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein–DNA binding. PMID:18582039

Screening and Characterization of Lactic Acid Bacteria Strains with Anti-inflammatory Activities through in vitro and Caenorhabditis elegans Model Testing

PubMed Central

Park, Mi Ri; Kim, Younghoon; Lee, Myung-Ki

2015-01-01

The present study was conducted to screen candidate probiotic strains for anti-inflammatory activity. Initially, a nitric oxide (NO) assay was used to test selected candidate probiotic strains for anti-inflammatory activity in cultures of the murine macrophage cell line, RAW 264.7. Then, the in vitro probiotic properties of the strains, including bile tolerance, acid resistance, and growth in skim milk media, were investigated. We also performed an in vitro hydrophobicity test and an intestinal adhesion assay using Caenorhabditis elegans as a surrogate in vivo model. From our screening, we obtained 4 probiotic candidate lactic acid bacteria (LAB) strains based on their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell cultures and the results of the in vitro and in vivo probiotic property assessments. Molecular characterization using 16S rDNA sequencing analysis identified the 4 LAB strains as Lactobacillus plantarum. The selected L. plantarum strains (CAU1054, CAU1055, CAU1064, and CAU1106) were found to possess desirable in vitro and in vivo probiotic properties, and these strains are good candidates for further investigations in animal models and human clinical studies to elucidate the mechanisms underlying their anti-inflammatory activities. PMID:26761805

Phytochemical screening and in vitro bioactivities of the extracts of aerial part of Boerhavia diffusa Linn.

PubMed Central

Apu, Apurba Sarker; Liza, Mahmuda Sultana; Jamaluddin, A.T.M.; Howlader, Md. Amran; Saha, Repon Kumer; Rizwan, Farhana; Nasrin, Nishat

2012-01-01

Objective To investigate the bioactivities of crude n-hexane, ethyl acetate and methanol extracts of aerial part of Boerhavia diffusa Linn. (B. diffusa) and its phytochemical analysis. Methods The identification of phytoconstituents and assay of antioxidant, thrombolytic, cytotoxic, antimicrobial activities were conducted using specific standard in vitro procedures. Results The results showed that the plant extracts were a rich source of phytoconstituents. Methanol extract showed higher antioxidant, thrombolytic activity and less cytotoxic activity than those of n-hexane and ethyl acetate extracts of B. diffusa. Among the bioactivities, antioxidant activity was the most notable compared to the positive control and thus could be a potential rich source of natural antioxidant. In case of antimicrobial screening, crude extracts of the plant showed remarkable antibacterial activity against tested microorganisms. All the extracts showed significant inhibitory activity against Candida albicuns, at a concentration of 1000 µg/disc. Conclusions The present findings suggest that, the plant widely available in Bangladesh, could be a prominent source of medicinally important natural compounds. PMID:23569993

Inhibitors of Helicobacter pylori Protease HtrA Found by ‘Virtual Ligand’ Screening Combat Bacterial Invasion of Epithelia

PubMed Central

Schneider, Petra; Hoy, Benjamin; Wessler, Silja; Schneider, Gisbert

2011-01-01

Background The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection. PMID:21483848

76 FR 27331 - Draft Guidance for Industry and Food and Drug Administration Staff; Establishing the Performance...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-11

... Characteristics of In Vitro Diagnostic Devices for Chlamydia Trachomatis and/or Neisseria Gonorrhoeae: Screening... entitled ``Establishing the Performance Characteristics of In Vitro Diagnostic Devices for Chlamydia... clinical performance of in vitro diagnostic devices (IVDs) intended for C. trachomatis and/or N...

Opportunities and Challenges in Employing In Vitro-In Vivo Extrapolation (IVIVE) to the Tox21 Dataset

EPA Science Inventory

In vitro-in vivo extrapolation (IVIVE), or the process of using in vitro data to predict in vivo phenomena, provides key opportunities to bridge the disconnect between high-throughput screening data and real-world human exposures and potential health effects. Strategies utilizing...

In Vitro Bioactivity in ToxCast Assays for Fruit and Vegetable Juices (TDS)

EPA Science Inventory

The ToxCast and Tox21 programs have generated in vitro screening data for over 1000 chemicals to aid in hazard identification and setting chemical testing priorities. These data, together with in vitro pharmacokinetic data, are used to infer possible toxic responses and external ...

Discovery of novel mGluR1 antagonists: a multistep virtual screening approach based on an SVM model and a pharmacophore hypothesis significantly increases the hit rate and enrichment factor.

PubMed

Li, Guo-Bo; Yang, Ling-Ling; Feng, Shan; Zhou, Jian-Ping; Huang, Qi; Xie, Huan-Zhang; Li, Lin-Li; Yang, Sheng-Yong

2011-03-15

Development of glutamate non-competitive antagonists of mGluR1 (Metabotropic glutamate receptor subtype 1) has increasingly attracted much attention in recent years due to their potential therapeutic application for various nervous disorders. Since there is no crystal structure reported for mGluR1, ligand-based virtual screening (VS) methods, typically pharmacophore-based VS (PB-VS), are often used for the discovery of mGluR1 antagonists. Nevertheless, PB-VS usually suffers a lower hit rate and enrichment factor. In this investigation, we established a multistep ligand-based VS approach that is based on a support vector machine (SVM) classification model and a pharmacophore model. Performance evaluation of these methods in virtual screening against a large independent test set, M-MDDR, show that the multistep VS approach significantly increases the hit rate and enrichment factor compared with the individual SB-VS and PB-VS methods. The multistep VS approach was then used to screen several large chemical libraries including PubChem, Specs, and Enamine. Finally a total of 20 compounds were selected from the top ranking compounds, and shifted to the subsequent in vitro and in vivo studies, which results will be reported in the near future. Copyright © 2011 Elsevier Ltd. All rights reserved.

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors.

PubMed

Villar-Piqué, Anna; Espargaró, Alba; Sabaté, Raimon; de Groot, Natalia S; Ventura, Salvador

2012-05-03

The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides.

Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database.

PubMed

Wang, Jing; Qiao, Chunxia; Xiao, He; Lin, Zhou; Li, Yan; Zhang, Jiyan; Shen, Beifen; Fu, Tinghuan; Feng, Jiannan

2016-01-01

According to the three-dimensional (3D) complex structure of (hIL-6⋅hIL-6R⋅gp 130) 2 and the binding orientation of hIL-6, three compounds with high affinity to hIL-6R and bioactivity to block hIL-6 in vitro were screened theoretically from the chemical databases, including 3D-Available Chemicals Directory (ACD) and MDL Drug Data Report (MDDR), by means of the computer-guided virtual screening method. Using distance geometry, molecular modeling and molecular dynamics trajectory analysis methods, the binding mode and binding energy of the three compounds were evaluated theoretically. Enzyme-linked immunosorbent assay analysis demonstrated that all the three compounds could block IL-6 binding to IL-6R specifically. However, only compound 1 could effectively antagonize the function of hIL-6 and inhibit the proliferation of XG-7 cells in a dose-dependent manner, whereas it showed no cytotoxicity to SP2/0 or L929 cells. These data demonstrated that the compound 1 could be a promising candidate of hIL-6 antagonist.

Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics.

PubMed

Merzoug, Amina; Chikhi, Abdelouahab; Bensegueni, Abderrahmane; Boucherit, Hanane; Okay, Sezer

2018-03-01

The increasing resistance of bacteria to antibacterial therapy poses an enormous health problem, it renders the development of new antibacterial agents with novel mechanism of action an urgent need. Peptide deformylase, a metalloenzyme which catalytically removes N-formyl group from N-terminal methionine of newly synthesized polypeptides, is an important target in antibacterial drug discovery. In this study, we report the structure-based virtual screening of ZINC database in order to discover potential hits as bacterial peptide deformylase enzyme inhibitors with more affinity as compared to GSK1322322, previously known inhibitor. After virtual screening, fifteen compounds of the top hits predicted were purchased and evaluated in vitro for their antibacterial activities against one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella. pneumoniae) bacteria in different concentrations by disc diffusion method. Out of these, three compounds, ZINC00039650, ZINC03872971 and ZINC00126407, exhibited significant zone of inhibition. The results obtained were confirmed using the dilution method. Thus, these proposed compounds may aid the development of more efficient antibacterial agents. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

EPA Science Inventory

This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

The ToxCast Pathway Database for Identifying Toxicity Signatures and Potential Modes of Action from Chemical Screening Data

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA), through its ToxCast program, is developing predictive toxicity approaches that will use in vitro high-throughput screening (HTS), high-content screening (HCS) and toxicogenomic data to predict in vivo toxicity phenotypes. There are ...

Are In Vitro Methods for the Detection of Endocrine Potentials in the Aquatic Environment Predictive for In Vivo Effects? Outcomes of the Projects SchussenAktiv and SchussenAktivplus in the Lake Constance Area, Germany

PubMed Central

Henneberg, Anja; Bender, Katrin; Blaha, Ludek; Giebner, Sabrina; Kuch, Bertram; Köhler, Heinz-R.; Maier, Diana; Oehlmann, Jörg; Richter, Doreen; Scheurer, Marco; Schulte-Oehlmann, Ulrike; Sieratowicz, Agnes; Ziebart, Simone; Triebskorn, Rita

2014-01-01

Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption observed in snails and field-exposed fish, respectively. PMID:24901835

AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

PubMed Central

Godugu, Chandraiah; Patel, Apurva R.; Desai, Utkarsh; Andey, Terrick; Sams, Alexandria; Singh, Mandip

2013-01-01

Background Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. Methods Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids. Results IC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Conclusion The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations. PMID:23349734

A review of the influence of growth factors and cytokines in in vitro human keratinocyte migration.

PubMed

Peplow, Philip V; Chatterjee, Marissa P

2013-04-01

Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cutaneous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play an important role, and several in vitro assays have been performed to elucidate this. The purposes of this study were to review in vitro human studies on keratinocyte migration to identify those growth factors or cytokines that stimulate keratinocyte migration and whether these assays might serve as a screening procedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo. Research papers investigating effect of growth factors and cytokines on human keratinocyte migration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, and searches of relevant journals. Fourteen different growth factors and cytokines enhanced migration in scratch wound assay and HGF together with TGF-β, and IGF-1 with EGF, were more stimulatory than either growth factor alone. HGF with TGF-β1 had a greater chemokinetic effect than either growth factor alone in transmigration assay. TGF-β1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-α, IL-1α, IGF and MGSA enhanced cell migration on ECM proteins. Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certain combinations of growth factors were more stimulatory than either alone. These and other combinations that stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repair in vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening method for testing individual or combinations of growth factors or cytokines, or growth factors combined with other modalities such as laser irradiation, prior to performing wound healing studies with laboratory animals. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ecosystem screening approach for pathogen-associated microorganisms affecting host disease.

PubMed

Galiana, Eric; Marais, Antoine; Mura, Catherine; Industri, Benoît; Arbiol, Gilles; Ponchet, Michel

2011-09-01

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen in vitro and host disease. This approach was applied to a soilborne plant pathogen, Phytophthora parasitica, structured in a biofilm, for screening the microbial community from the rhizosphere of Nicotiana tabacum (the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A Vorticella species acted through a mutualistic interaction with P. parasitica, disseminating pathogenic material by leaving the biofilm. A Phoma species established an amensal interaction with P. parasitica, strongly suppressing disease by inhibiting P. parasitica germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.

Dysfunction Screening in Experimental Arteriovenous Grafts for Hemodialysis Using Fractional-Order Extractor and Color Relation Analysis.

PubMed

Wu, Ming-Jui; Chen, Wei-Ling; Kan, Chung-Dann; Yu, Fan-Ming; Wang, Su-Chin; Lin, Hsiu-Hui; Lin, Chia-Hung

2015-12-01

In physical examinations, hemodialysis access stenosis leading to dysfunction occurs at the venous anastomosis site or the outflow vein. Information from the inflow stenosis, such as blood pressure, pressure drop, and flow resistance increases, allows dysfunction screening from the stage of early clots and thrombosis to the progression of outflow stenosis. Therefore, this study proposes dysfunction screening model in experimental arteriovenous grafts (AVGs) using the fractional-order extractor (FOE) and the color relation analysis (CRA). A Sprott system was designed using an FOE to quantify the differences in transverse vibration pressures between the inflow and outflow sites of an AVG. Experimental analysis revealed that the degree of stenosis (DOS) correlated with an increase in fractional-order dynamic errors (FODEs). Exponential regression was used to fit a non-linear curve and can be used to quantify the relationship between the FODEs and DOS (R (2) = 0.8064). The specific ranges were used to evaluate the stenosis degree, such as DOS: <50, 50-80, and >80%. A CRA-based screening method was derived from the hue angle-saturation-value color model, which describes perceptual color relationships for the DOS. It has a flexibility inference manner with color visualization to represent the different stenosis degrees, which has average accuracy >90% superior to the traditional methods. This in vitro experimental study demonstrated that the proposed model can be used for dysfunction screening in stenotic AVGs.

Insights and Perspectives on Emerging Inputs to Weight of Evidence Determinations for Food Safety: Workshop Proceedings

PubMed Central

Bialk, Heidi; Llewellyn, Craig; Kretser, Alison; Canady, Richard; Lane, Richard; Barach, Jeffrey

2013-01-01

This workshop aimed to elucidate the contribution of computational and emerging in vitro methods to the weight of evidence used by risk assessors in food safety assessments. The following issues were discussed: using in silico and high-throughput screening (HTS) data to confirm the safety of approved food ingredients, applying in silico and HTS data in the process of assessing the safety of a new food ingredient, and utilizing in silico and HTS data in communicating the safety of food ingredients while enhancing the public’s trust in the food supply. Perspectives on integrating computational modeling and HTS assays as well as recommendations for optimizing predictive methods for risk assessment were also provided. Given the need to act quickly or proceed cautiously as new data emerge, this workshop also focused on effectively identifying a path forward in communicating in silico and in vitro data. PMID:24296863

Triazole-based Zn²⁺-specific molecular marker for fluorescence bioimaging.

PubMed

Sinha, Sougata; Mukherjee, Trinetra; Mathew, Jomon; Mukhopadhyay, Subhra K; Ghosh, Subrata

2014-04-25

Fluorescence bioimaging potential, both in vitro and in vivo, of a yellow emissive triazole-based molecular marker has been investigated and demonstrated. Three different kinds of cells, viz Bacillus thuringiensis, Candida albicans, and Techoma stans pollen grains were used to investigate the intracellular zinc imaging potential of 1 (in vitro studies). Fluorescence imaging of translocation of zinc through the stem of small herb, Peperomia pellucida, having transparent stem proved in vivo bioimaging capability of 1. This approach will enable in screening cell permeability and biostability of a newly developed probe. Similarly, the current method for detection and localization of zinc in Gram seed sprouts could be an easy and potential alternative of the existing analytical methods to investigate the efficiency of various strategies applied for increasing zinc-content in cereal crops. The probe-zinc ensemble has efficiently been applied for detecting phosphate-based biomolecules. Copyright © 2014 Elsevier B.V. All rights reserved.

A Specific Screening Strategy to Reduce Prostate Cancer Mortality

DTIC Science & Technology

2013-09-01

Vashaw Scientific, Norcross, GA ). Filter, camera, and image processing from the in vitro imaging methods were used in coordination with the...Cancer Facts and Figures 2011. (2011). American Cancer Society. Atlanta, GA . 2. Jemal, A, Siegel, R, Ward, E, Murray, T, Xu, J, Smigal, C, et al. (2006...overexpression in prostate cancer: relevance to tumor differentiation. Pathology oncology research : POR 15: 91-96. 11. Ouyang, XS , Wang, X, Lee, DT, Tsao, SW

Studies on the antioxidant activities of natural vanilla extract and its constituent compounds through in vitro models.

PubMed

Shyamala, B N; Naidu, M Madhava; Sulochanamma, G; Srinivas, P

2007-09-19

Vanilla extract was prepared by extraction of cured vanilla beans with aqueous ethyl alcohol (60%). The extract was profiled by HPLC, wherein major compounds, viz., vanillic acid, 4-hydroxybenzyl alcohol, 4-hydroxy-3-methoxybenzyl alcohol, 4-hydroxybenzaldehyde and vanillin, could be identified and separated. Extract and pure standard compounds were screened for antioxidant activity using beta-carotene-linoleate and DPPH in vitro model systems. At a concentration of 200 ppm, the extract showed 26% and 43% of antioxidant activity by beta-carotene-linoleate and DPPH methods, respectively, in comparison to corresponding values of 93% and 92% for BHA. Interestingly, 4-hydroxy-3-methoxybenzyl alcohol and 4-hydroxybenzyl alcohol exhibited antioxidant activity of 65% and 45% by beta-carotene-linoleate method and 90% and 50% by DPPH methods, respectively. In contrast, pure vanillin exhibited much lower antioxidant activity. The present study points toward the potential use of vanilla extract components as antioxidants for food preservation and in health supplements as nutraceuticals.

High-throughput image analysis of tumor spheroids: a user-friendly software application to measure the size of spheroids automatically and accurately.

PubMed

Chen, Wenjin; Wong, Chung; Vosburgh, Evan; Levine, Arnold J; Foran, David J; Xu, Eugenia Y

2014-07-08

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application - SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary "Manual Initialize" and "Hand Draw" tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

Toxicokinetic Triage for Environmental Chemicals | Science ...

EPA Pesticide Factsheets

Toxicokinetic (TK) models are essential for linking administered doses to blood and tissue concentrations. In vitro-to-in vivo extrapolation (IVIVE) methods have been developed to determine TK from limited in vitro measurements and chemical structure-based property predictions, providing a less resource–intensive alternative to traditional in vivo TK approaches. High throughput TK (HTTK) methods use IVIVE to estimate doses that produce steady-state plasma concentrations equivalent to those producing biological activity in in vitro screening studies (e.g., ToxCast). In this study, the domain of applicability and assumptions of HTTK approaches were evaluated using both in vivo data and simulation analysis. Based on in vivo data for 87 chemicals, specific properties (e.g., in vitro HTTK data, physico-chemical descriptors, chemical structure, and predicted transporter affinities) were identified that correlate with poor HTTK predictive ability. For 350 xenobiotics with literature HTTK data, we then differentiated those xenobiotics for which HTTK approaches are likely to be sufficient, from those that may require additional data. For 272 chemicals we also developed a HT physiologically-based TK (HTPBTK) model that requires somewhat greater information than a steady-state model, but allows non-steady state dynamics and can predict chemical concentration time-courses for a variety of exposure scenarios, tissues, and species. We used this HTPBTK model to show that the

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

In vitro SCREENING ANTIBACTERIAL ACTIVITY OF Bidens pilosa LINNÉ AND Annona crassiflora MART. AGAINST OXACILLIN RESISTANT Staphylococcus aureus (ORSA) FROM THE AERIAL ENVIRONMENT AT THE DENTAL CLINIC

PubMed Central

da Silva, Jeferson; Cerdeira, Cláudio Daniel; Chavasco, Juliana Moscardini; Cintra, Ana Beatriz Pugina; da Silva, Carla Brigagão Pacheco; de Mendonça, Andreia Natan; Ishikawa, Tati; Boriollo, Marcelo Fabiano Gomes; Chavasco, Jorge Kleber

2014-01-01

Currently multiresistant Staphylococcus aureus is one common cause of infections with high rates of morbidity and mortality worldwide, which directs scientific endeavors in search for novel antimicrobials. In this study, nine extracts from Bidens pilosa (root, stem, flower and leaves) and Annona crassiflora (rind fruit, stem, leaves, seed and pulp) were obtained with ethanol: water (7:3, v/v) and their in vitro antibacterial activity evaluated through both the agar diffusion and broth microdilution methods against 60 Oxacillin Resistant S. aureus (ORSA) strains and against S. aureus ATCC6538. The extracts from B. pilosa and A. crassiflora inhibited the growth of the ORSA isolates in both methods. Leaves of B. pilosa presented mean of the inhibition zone diameters significantly higher than chlorexidine 0.12% against ORSA, and the extracts were more active against S. aureus ATCC (p < 0.05). Parallel, toxicity testing by using MTT method and phytochemical screening were assessed, and three extracts (B. pilosa, root and leaf, and A. crassiflora, seed) did not evidence toxicity. On the other hand, the cytotoxic concentrations (CC50 and CC90) for other extracts ranged from 2.06 to 10.77 mg/mL. The presence of variable alkaloids, flavonoids, tannins and saponins was observed, even though there was a total absence of anthraquinones. Thus, the extracts from the leaves of B. pilosa revealed good anti-ORSA activity and did not exhibit toxicity. PMID:25076435

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

PubMed Central

Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

2017-01-01

Summary The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

PubMed

Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

2016-01-01

The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

PubMed Central

Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

2012-01-01

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

Research on major antitumor active components in Zi-Cao-Cheng-Qi decoction based on hollow fiber cell fishing with high performance liquid chromatography.

PubMed

Li, Miaomiao; Hu, Shuang; Chen, Xuan; Wang, Runqin; Bai, Xiaohong

2018-02-05

Hollow fiber cell fishing (HFCF) based on hepatoma HepG-2 cells, human renal tubular ACHN cells or human cervical carcinoma HeLa cells, coupled with high-performance liquid chromatography (HPLC), was developed and employed to research the major active components in Zi-Cao-Cheng-Qi decoction both in vitro and in vivo. The research showed that the active components, such as hesperidin, magnolol, honokiol, shikonin, emodin and β,β'-dimethylacrylshikonin were screened out by HFCF based on the cancer cells in vitro, furthermore they can be absorbed into blood and reach in the target organ, and some of the active components can be fished by the cells and maintain effective concentrations. Before application of HFCF with HPLC, cell growth state, cell survival rate, positive effect on screening results binding between active centers on the fiber and target components, repeatability of retention times and relative peak areas of the target analytes were analysed and investigated. In short, HFCF with HPLC is a simple, inexpensive, effective, and reliable method that can be used in researching active components from traditional Chinese medicine (TCM) and its formula both in vitro and in vivo, elucidating preliminarily the TCM characteristics of multiple components and multiple targets, laying a foundation for expounding the antitumor efficacy material basis in TCM. Copyright © 2017 Elsevier B.V. All rights reserved.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

PubMed

Broce, Sean; Hensley, Lisa; Sato, Tomoharu; Lehrer-Graiwer, Joshua; Essrich, Christian; Edwards, Katie J; Pajda, Jacqueline; Davis, Christopher J; Bhadresh, Rami; Hurt, Clarence R; Freeman, Beverly; Lingappa, Vishwanath R; Kelleher, Colm A; Karpuj, Marcela V

2016-05-14

Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

In vitro bioassays for detecting dioxin-like activity--application potentials and limits of detection, a review.

PubMed

Eichbaum, Kathrin; Brinkmann, Markus; Buchinger, Sebastian; Reifferscheid, Georg; Hecker, Markus; Giesy, John P; Engwall, Magnus; van Bavel, Bert; Hollert, Henner

2014-07-15

Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology. While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs. This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of chick embryo in screening for teratogenicity.

PubMed

Kotwani, A

1998-04-01

A teratology screening system would detect agents hazardous to the conceptus before they can perturb embryonic development in humans. The back log of untested chemicals and the rate at which new substances enter the market exceed the developmental effects testing by standard in vivo method. Thus, cheaper, quicker in vitro systems afford a unique opportunity for investigating the direct interaction of substances with developing morphogenetic system (MGSs), since maternal influences are excluded. As a carrier of a complete set of MGSs, the chick embryo in ovo manifests an advantage over those in vitro systems that employ isolated embryos or embryonic tissues that have only limited survival. Under controlled experimental conditions including standardization of subjects, administration technique and mode of evaluation, according to the basic principles of teratology, the chick embryo test is demonstrated to be reliable and to afford quantifiable end points for evaluation. Individual compounds, mixtures of compounds and against and antagonist can easily be administered and tested. The chick embryo possesses its own basic enzyme-catalyzed drug-transformation capacity and moreover, it can be used for screening specific human metabolites. Different newer techniques e.g. chick embryotoxicity screening test (CHEST), Chick embryo blastoderm model etc are described in detail. Chick embryo fulfills all the criteria which a test should have at a lower level of tier system in teratological studies i.e. modest laboratory equipment, moderate skill, minimal expenditure of time and money, ease of accessibility of embryo, known embryological development, possibility of experimenting on a large scale for statistically valid results and whole animals are also not required.

A biomaterial screening approach reveals microenvironmental mechanisms of drug resistance.

PubMed

Schwartz, Alyssa D; Barney, Lauren E; Jansen, Lauren E; Nguyen, Thuy V; Hall, Christopher L; Meyer, Aaron S; Peyton, Shelly R

2017-12-11

Traditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell-cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments.

In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay##

EPA Science Inventory

Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms of...

In vitro-to-in vivo extrapolation of xenoestrogens using an estrogen responsive in vitro transcriptional activation assay and the in vivo uterotrophic assay

EPA Science Inventory

Widespread contamination of waters with both natural and synthetic estrogens is a concern for potential adverse ecological and human health effects. In vitro assays are valuable screening tools for identifying contaminated environmental samples and chemical specific mechanisms o...

A tiered approach for integrating exposure and dosimetry with in vitro dose-response data in the modern risk assessment paradigm

EPA Science Inventory

High-throughput (HT) risk screening approaches apply in vitro dose-response data to estimate potential health risks that arise from exposure to chemicals. However, much uncertainty is inherent in relating bioactivities observed in an in vitro system to the perturbations of biolog...

Application of the ToxMiner Database: Network Analysis of ...

EPA Pesticide Factsheets

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effective alternatives to animal testing. The chemicals selected for Phase I are composed largely by a diverse set of pesticide active ingredients, which had sufficient supporting in vivo data included as part of their registration process with the EPA. Other miscellaneous chemicals of environmental concern were also included. Application of HTS to environmental toxicants is a novel approach to predictive toxicology and health risk assessment, and differs from what is required for drug efficacy screening in that biochemical interaction of environmental chemicals are sometimes weaker than that seen with drugs and their intended targets. Additionally, the chemical space covered by environmental chemicals is much broader compared to that of pharmaceuticals. The ToxMiner database has been created and added to the EPA’s ACToR (Aggregated Computational Toxicology Resource) chemical database. One purpose of the ToxMiner database is to link biological, metabolic and cellular pathway data to genes and in vitro assay data for the initial subset of chemicals screened in the ToxCast Phase I HTS assays. Also included in ToxMiner is human disease information, which correlates with ToxCast assays that tar

A gene expression biomarker accurately predicts estrogen ...

EPA Pesticide Factsheets

The EPA’s vision for the Endocrine Disruptor Screening Program (EDSP) in the 21st Century (EDSP21) includes utilization of high-throughput screening (HTS) assays coupled with computational modeling to prioritize chemicals with the goal of eventually replacing current Tier 1 screening tests. The ToxCast program currently includes 18 HTS in vitro assays that evaluate the ability of chemicals to modulate estrogen receptor α (ERα), an important endocrine target. We propose microarray-based gene expression profiling as a complementary approach to predict ERα modulation and have developed computational methods to identify ERα modulators in an existing database of whole-genome microarray data. The ERα biomarker consisted of 46 ERα-regulated genes with consistent expression patterns across 7 known ER agonists and 3 known ER antagonists. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments in MCF-7 cells. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% or 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) OECD ER reference chemicals including “very weak” agonists and replicated predictions based on 18 in vitro ER-associated HTS assays. For 114 chemicals present in both the HTS data and the MCF-7 c

Application of the ToxMiner Database: Network Analysis ...

EPA Pesticide Factsheets

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effective alternatives to animal testing. The chemicals selected for Phase I are composed largely by a diverse set of pesticide active ingredients, which had sufficient supporting in vivo data included as part of their registration process with the EPA. Other miscellaneous chemicals of environmental concern were also included. Application of HTS to environmental toxicants is a novel approach to predictive toxicology and health risk assessment, and differs from what is required for drug efficacy screening in that biochemical interaction of environmental chemicals are sometimes weaker than that seen with drugs and their intended targets. Additionally, the chemical space covered by environmental chemicals is much broader compared to that of pharmaceuticals. The ToxMiner database has been created and added to the EPA’s ACToR (Aggregated Computational Toxicology Resource) chemical database. One purpose of the ToxMiner database is to link biological, metabolic, and cellular pathway data to genes and in vitro assay data for the initial subset of chemicals screened in the ToxCast Phase I HTS assays. Also included in ToxMiner is human disease information, which correlates with ToxCast assays that ta

Evolving the EPA Endocrine Disruptor Screening Program: The case for and against using high-throughput screening assays in EDSP Tier 1

EPA Science Inventory

Testing has begun as part of the EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 battery of 11 in vitro and in vivo tests. A recognized issue with the EDSP is that the current Tier 1 screening battery is highly resource intensive in terms of cost, time and animal usage fo...

Discovery of novel Pim-1 kinase inhibitors by a hierarchical multistage virtual screening approach based on SVM model, pharmacophore, and molecular docking.

PubMed

Ren, Ji-Xia; Li, Lin-Li; Zheng, Ren-Lin; Xie, Huan-Zhang; Cao, Zhi-Xing; Feng, Shan; Pan, You-Li; Chen, Xin; Wei, Yu-Quan; Yang, Sheng-Yong

2011-06-27

In this investigation, we describe the discovery of novel potent Pim-1 inhibitors by employing a proposed hierarchical multistage virtual screening (VS) approach, which is based on support vector machine-based (SVM-based VS or SB-VS), pharmacophore-based VS (PB-VS), and docking-based VS (DB-VS) methods. In this approach, the three VS methods are applied in an increasing order of complexity so that the first filter (SB-VS) is fast and simple, while successive ones (PB-VS and DB-VS) are more time-consuming but are applied only to a small subset of the entire database. Evaluation of this approach indicates that it can be used to screen a large chemical library rapidly with a high hit rate and a high enrichment factor. This approach was then applied to screen several large chemical libraries, including PubChem, Specs, and Enamine as well as an in-house database. From the final hits, 47 compounds were selected for further in vitro Pim-1 inhibitory assay, and 15 compounds show nanomolar level or low micromolar inhibition potency against Pim-1. In particular, four of them were found to have new scaffolds which have potential for the chemical development of Pim-1 inhibitors.

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

PubMed

Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

2005-01-01

This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

Promotion of anagen, increased hair density and reduction of hair fall in a clinical setting following identification of FGF5-inhibiting compounds via a novel 2-stage process

PubMed Central

Burg, Dominic; Yamamoto, Masakuni; Namekata, Masato; Haklani, Joseph; Koike, Koichiro; Halasz, Maria

2017-01-01

Background There are very few effective, scientifically validated treatments with known mechanisms of action for treatment of hair loss in both men and women. Fibroblast growth factor 5 (FGF5) is an important factor in the irreversible transition from anagen to catagen, and inhibition of FGF5 prolongs anagen phase and reduces hair loss. Objective We aimed to screen botanically derived molecules for FGF5 inhibitory activity in vitro and assess efficacy in a clinical setting. Methods We screened for FGF5 inhibitory efficacy via a novel 2-step in vitro pipeline consisting of an engineered FGF5 responsive cell line, followed by an activated dermal papillae (DP) cell method. Efficacy in a clinical setting was assessed in a randomized, single-blind, placebo-controlled trial against early- to mid-stage pattern hair loss in men and women. Results We observed FGF5 inhibitory activity for a number of compounds from the monoterpenoid family, many showing greater inhibitory efficacy than our previously reported crude plant extracts. Evaluation of a lead candidate in a clinical study over 112 days showed a significant improvement in anagen:telogen (AT) ratio (p = 0.002), reduced hair fall (p = 0.007) and improved visual grading (p = 0.004). Scientifically matched photography on a subgroup of randomly chosen participants highlighted significant improvement in hair density, with increases evident in all tested participants compared to baseline. Conclusion Isolates from the monoterpenoid family displayed efficacy in FGF5 inhibition in vitro. A topical formulation containing a leading isolate significantly improved AT ratio, reduced hair fall and increased apparent hair density in the tested population of men and women. PMID:28280377

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

PubMed

Wu, Jian-Hui; Sun, Zu-Yue

2013-06-01

To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

Survey of ecotoxicologically-relevant reproductive endpoint coverage within the ECOTOX database across ToxCast ER agonists (ASCCT)

EPA Science Inventory

The U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) has been charged with screening thousands of chemicals for their potential to affect the endocrine systems of humans and wildlife. In vitro high throughput screening (HTS) assays have been proposed as a way to prioritize...

The US EPA's Endocrine Disruptor Screening Program: In VItro and In Vivo Mammalian Tier 1 Screening Assays

EPA Science Inventory

In response to emerging concerns that environmental chemicals may have adverse effects on human health by altering the function of the endocrine system, the Food Quality Protection Act mandated that the U.S. EPA develop and implement an endocrine disruptor screening program (EDSP...

Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents.

PubMed

Cho, Choi-Fong; Wolfe, Justin M; Fadzen, Colin M; Calligaris, David; Hornburg, Kalvis; Chiocca, E Antonio; Agar, Nathalie Y R; Pentelute, Bradley L; Lawler, Sean E

2017-06-06

Culture-based blood-brain barrier (BBB) models are crucial tools to enable rapid screening of brain-penetrating drugs. However, reproducibility of in vitro barrier properties and permeability remain as major challenges. Here, we report that self-assembling multicellular BBB spheroids display reproducible BBB features and functions. The spheroid core is comprised mainly of astrocytes, while brain endothelial cells and pericytes encase the surface, acting as a barrier that regulates transport of molecules. The spheroid surface exhibits high expression of tight junction proteins, VEGF-dependent permeability, efflux pump activity and receptor-mediated transcytosis of angiopep-2. In contrast, the transwell co-culture system displays comparatively low levels of BBB regulatory proteins, and is unable to discriminate between the transport of angiopep-2 and a control peptide. Finally, we have utilized the BBB spheroids to screen and identify BBB-penetrant cell-penetrating peptides (CPPs). This robust in vitro BBB model could serve as a valuable next-generation platform for expediting the development of CNS therapeutics.

Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

PubMed

Saraswathi, M S; Kannan, G; Uma, S; Thangavelu, R; Backiyarani, S

2016-05-01

Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards

PubMed Central

Mordwinkin, Nicholas M.; Burridge, Paul W.; Wu, Joseph C.

2013-01-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach. PMID:23229562

Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls.

PubMed

Möller, Ines; Wintermeyer, Annette; Bender, Katja; Jübner, Martin; Thomas, Andreas; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

2011-09-01

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine. Copyright © 2010 John Wiley & Sons, Ltd.

In vitro antioxidant potential of dicliptera roxburghiana

PubMed Central

2013-01-01

Background Stress caused by free radicals accumulation result into many hazardous diseases. A number of investigations are focusing to find out the plant oriented natural antioxidant moieties. The basic aim of this research was to investigate the antioxidant potential, total Phenolic and flavonoids contents and photochemical screening of the crude methanol extract and its derived various fractions Dicliptera roxburghiana of Acanthaceae family. Methods Crude methanol extract of aerial parts of Dicliptera roxburghiana (DRME) was partitioned in to n-hexane (DRHF), chloroform (DRCF), ethyl acetate (DREF), n-butanol (DRBF) and the remaining soluble portion as residual aqueous fraction (DRAF). We evaluated the antioxidant activities of the extract and various fractions through different analytical methods such as DPPH, superoxide anion, ABTS, H2O2, hydroxyl radical and phosphomolybdate radical inhibition. In vitro lipid peroxidation and reducing power of the plant was also analyzed. Total flavonoid and phenolic contents of the extract and all fractions were also quantified. Plant was also subjected for preliminary phytochemical screening to confirm the presence or absence of various constituents in the plant. Results Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, alkaloids, saponins, terpenoids and coumarines. Quantitative analysis revealed the maximum amount of total phenolic and flavonoid contents in DRME while lowest in DRHF. Methanol extract, DREF, DRCF and DRBF exhibited promising antioxidant potential for DPPH, ABTS, H2O2, phosphomolybdate, superoxide anion and hydroxyl radical scavenging capabilities, while these were not appreciable for DRHF and DRAF. All fractions except DRHF and DRAF possess strong reducing power ability and showed appreciable lipid peroxidation inhibition. Conclusion These research investigations revealed that Dicliptera roxburghiana is a potent source of natural antioxidants. Hence the plant can be used for management of different stress and anxiety related ailments. PMID:23777321

Development of a central nervous system axonal myelination assay for high throughput screening.

PubMed

Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

2016-04-22

Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

Screening Active Components from Yu-Ping-Feng-San for Regulating Initiative Key Factors in Allergic Sensitization

PubMed Central

Zhu, Zhijie; Yu, Xi; Liu, Hailiang; Wang, Huizhu; Fan, Hongwei; Wang, Dawei; Jiang, Guorong; Hong, Min

2014-01-01

Yu-ping-feng-san (YPFS) is a Chinese medical formula that is used clinically for allergic diseases and characterized by reducing allergy relapse. Our previous studies demonstrated that YPFS efficiently inhibited T helper 2 cytokines in allergic inflammation. The underlying mechanisms of action of YPFS and its effective components remain unclear. In this study, it was shown that YPFS significantly inhibited production of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived initiative factor in allergic inflammation, in vitro and in vivo. A method of human bronchial epithelial cell (16HBE) binding combined with HPLC-MS (named 16HBE-HPLC-MS) was established to explore potential active components of YPFS. The following five components bound to 16HBE cells: calycosin-7-glucoside, ononin, claycosin, sec-o-glucosylhamaudol and formononetin. Serum from YPFS-treated mice was analyzed and three major components were detected claycosin, formononetin and cimifugin. Among these, claycosin and formononetin were detected by 16HBE-HPLC-MS and in the serum of YPFS-treated mice. Claycosin and formononetin decreased the level of TSLP markedly at the initial stage of allergic inflammation in vivo. Nuclear factor (NF)-κB, a key transcription factor in TSLP production, was also inhibited by claycosin and formononetin, either in terms of transcriptional activation or its nuclear translocation in vitro. Allergic inflammation was reduced by claycosin and formononetin when they are administered only at the initial stage in a murine model of atopic contact dermatitis. Thus, epithelial cell binding combined with HPLC-MS is a valid method for screening active components from complex mixtures of Chinese medicine. It was demonstrated that the compounds screened from YPFS significantly attenuated allergic inflammation probably by reducing TSLP production via regulating NF-κB activation. PMID:25198676

A demonstration of the uncertainty in predicting the estrogenic activity of individual chemicals and mixtures from an in vitro estrogen receptor transcriptional activation assay (T47D-KBluc) to the in vivo uterotrophic assay using oral exposure

EPA Science Inventory

In vitro estrogen receptor assays are valuable screening tools for identifying environmental samples and chemicals that display estrogenic activity. However, in vitro potency cannot necessarily be extrapolated to estimates of in vivo potency because in vitro assays are currently...

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Evaluating In Vitro-In Vivo Extrapolation of Toxicokinetics

PubMed Central

MacMillan, Denise K; Ford, Jermaine; Fennell, Timothy R; Black, Sherry R; Snyder, Rodney W; Sipes, Nisha S; Westerhout, Joost; Setzer, R Woodrow; Pearce, Robert G; Simmons, Jane Ellen; Thomas, Russell S

2018-01-01

Abstract Prioritizing the risk posed by thousands of chemicals potentially present in the environment requires exposure, toxicity, and toxicokinetic (TK) data, which are often unavailable. Relatively high throughput, in vitro TK (HTTK) assays and in vitro-to-in vivo extrapolation (IVIVE) methods have been developed to predict TK, but most of the in vivo TK data available to benchmark these methods are from pharmaceuticals. Here we report on new, in vivo rat TK experiments for 26 non-pharmaceutical chemicals with environmental relevance. Both intravenous and oral dosing were used to calculate bioavailability. These chemicals, and an additional 19 chemicals (including some pharmaceuticals) from previously published in vivo rat studies, were systematically analyzed to estimate in vivo TK parameters (e.g., volume of distribution [Vd], elimination rate). For each of the chemicals, rat-specific HTTK data were available and key TK predictions were examined: oral bioavailability, clearance, Vd, and uncertainty. For the non-pharmaceutical chemicals, predictions for bioavailability were not effective. While no pharmaceutical was absorbed at less than 10%, the fraction bioavailable for non-pharmaceutical chemicals was as low as 0.3%. Total clearance was generally more under-estimated for nonpharmaceuticals and Vd methods calibrated to pharmaceuticals may not be appropriate for other chemicals. However, the steady-state, peak, and time-integrated plasma concentrations of nonpharmaceuticals were predicted with reasonable accuracy. The plasma concentration predictions improved when experimental measurements of bioavailability were incorporated. In summary, HTTK and IVIVE methods are adequately robust to be applied to high throughput in vitro toxicity screening data of environmentally relevant chemicals for prioritizing based on human health risks. PMID:29385628

In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus

PubMed Central

Sathish, Kumar SR; Kokati, Venkata Bhaskara Rao

2012-01-01

Objective To investigate the antibacterial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus (MDRSA). Methods Fifty one actinobacterial strains were isolated from salt pans soil, costal area in Kothapattanam, Ongole, Andhra Pradesh. Primary screening was done using cross-streak method against MDRSA. The bioactive compounds are extracted from efficient actinobacteria using solvent extraction. The antimicrobial activity of crude and solvent extracts was performed using Kirby-Bauer method. MIC for ethyl acetate extract was determined by modified agar well diffusion method. The potent actinobacteria are identified using Nonomura key, Shirling and Gottlieb 1966 with Bergey's manual of determinative bacteriology. Results Among the fifty one isolates screened for antibacterial activity, SRB25 were found efficient against MDRSA. The ethyl acetate extracts showed high inhibition against test organism. MIC test was performed with the ethyl acetate extract against MDRSA and found to be 1 000 µg/mL. The isolated actinobacteria are identified as Streptomyces sp with the help of Nonomura key. Conclusions The current investigation reveals that the marine actinobacteria from salt pan environment can be able to produce new drug molecules against drug resistant microorganisms. PMID:23569848

In silico study of in vitro GPCR assays by QSAR modeling ...

EPA Pesticide Factsheets

The U.S. EPA is screening thousands of chemicals of environmental interest in hundreds of in vitro high-throughput screening (HTS) assays (the ToxCast program). One goal is to prioritize chemicals for more detailed analyses based on activity in molecular initiating events (MIE) of adverse outcome pathways (AOPs). However, the chemical space of interest for environmental exposure is much wider than this set of chemicals. Thus, there is a need to fill data gaps with in silico methods, and quantitative structure-activity relationships (QSARs) are a proven and cost effective approach to predict biological activity. ToxCast in turn provides relatively large datasets that are ideal for training and testing QSAR models. The overall goal of the study described here was to develop QSAR models to fill the data gaps in a larger environmental database of ~32k structures. The specific aim of the current work was to build QSAR models for 18 G-Protein Coupled Receptor (GPCR) assays, part of the aminergic category. Two QSAR modeling strategies were adopted: classification models were developed to separate chemicals into active/non-active classes, and then regression models were built to predict the potency values of the bioassays for the active chemicals. Multiple software programs were used to calculate constitutional, topological and substructural molecular descriptors from two-dimensional (2D) chemical structures. Model-fitting methods included PLSDA (partial least squares d

Importance of supply integrity for in vitro fertilization and embryo culture.

PubMed

Morbeck, Dean E

2012-06-01

The quality of in vitro culture conditions is a key component of a successful clinical embryology laboratory. Many, but not all, supplies used in the embryology laboratory are screened by the supplier with a bioassay. Embryology laboratories use a variety of approaches to verify the quality of mineral oil, protein, and disposables before clinical use; however, a best practice has not been determined. Some laboratories test every supply, even those already screened by the supplier, whereas other laboratories perform as little testing as possible. Despite screening by the supplier, recent reports of embryo toxicity, specifically with mineral oil, highlight that the integrity of the supply system has gaps. This review describes current bioassay quality control testing and discusses how it applies to screening of products with documented lot-to-lot variation. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S.

1991-01-01

Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

Identifying Novel Molecular Structures for Advanced Melanoma by Ligand-Based Virtual Screening

PubMed Central

Wang, Zhao; Lu, Yan; Seibel, William; Miller, Duane D.; Li, Wei

2009-01-01

We recently discovered a new class of thiazole analogs that are highly potent against melanoma cells. To expand the structure-activity relationship study and to explore potential new molecular scaffolds, we performed extensive ligand-based virtual screening against a compound library containing 342,910 small molecules. Two different approaches of virtual screening were carried out using the structure of our lead molecule: 1) connectivity-based search using Scitegic Pipeline Pilot from Accelerys and 2) molecular shape similarity search using Schrodinger software. Using a testing compound library, both approaches can rank similar compounds very high and rank dissimilar compounds very low, thus validating our screening methods. Structures identified from these searches were analyzed, and selected compounds were tested in vitro to assess their activity against melanoma cancer cell lines. Several molecules showed good anticancer activity. While none of the identified compounds showed better activity than our lead compound, they provided important insight into structural modifications for our lead compound and also provided novel platforms on which we can optimize new classes of anticancer compounds. One of the newly synthesized analogs based on this virtual screening has improved potency and selectivity against melanoma. PMID:19445498

Predictive Models for Carcinogenicity and Mutagenicity ...

EPA Pesticide Factsheets

Mutagenicity and carcinogenicity are endpoints of major environmental and regulatory concern. These endpoints are also important targets for development of alternative methods for screening and prediction due to the large number of chemicals of potential concern and the tremendous cost (in time, money, animals) of rodent carcinogenicity bioassays. Both mutagenicity and carcinogenicity involve complex, cellular processes that are only partially understood. Advances in technologies and generation of new data will permit a much deeper understanding. In silico methods for predicting mutagenicity and rodent carcinogenicity based on chemical structural features, along with current mutagenicity and carcinogenicity data sets, have performed well for local prediction (i.e., within specific chemical classes), but are less successful for global prediction (i.e., for a broad range of chemicals). The predictivity of in silico methods can be improved by improving the quality of the data base and endpoints used for modelling. In particular, in vitro assays for clastogenicity need to be improved to reduce false positives (relative to rodent carcinogenicity) and to detect compounds that do not interact directly with DNA or have epigenetic activities. New assays emerging to complement or replace some of the standard assays include VitotoxTM, GreenScreenGC, and RadarScreen. The needs of industry and regulators to assess thousands of compounds necessitate the development of high-t

A Non-Biological Method for Screening Active Components against Influenza Virus from Traditional Chinese Medicine by Coupling a LC Column with Oseltamivir Molecularly Imprinted Polymers

PubMed Central

Yang, Ya-Jun; Li, Jian-Yong; Liu, Xi-Wang; Zhang, Ji-Yu; Liu, Yu-Rong; Li, Bing

2013-01-01

To develop a non-biological method for screening active components against influenza virus from traditional Chinese medicine (TCM) extraction, a liquid chromatography (LC) column prepared with oseltamivir molecularly imprinted polymer (OSMIP) was employed with LC-mass spectrometry (LC-MS). From chloroform extracts of compound TCM liquid preparation, we observed an affinitive component m/z 249, which was identified to be matrine following analysis of phytochemical literatures, OSMIP-LC column on-line of control compounds and MS/MS off-line. The results showed that matrine had similar bioactivities with OS against avian influenza virus H9N2 in vitro for both alleviating cytopathic effect and hemagglutination inhibition and that the stereostructures of these two compounds are similar while their two-dimensional structures were different. In addition, our results suggested that the bioactivities of those affinitive compounds were correlated with their chromatographic behaviors, in which less difference of the chromatographic behaviors might have more similar bioactivities. This indicates that matrine is a potential candidate drug to prevent or cure influenza for human or animal. In conclusion, the present study showed that molecularly imprinted polymers can be used as a non-biological method for screening active components against influenza virus from TCM. PMID:24386385

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening

PubMed Central

Smith, Alec S.T.; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A.; Kim, Deok-Ho

2016-01-01

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. PMID:28007615

Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cline, Gary W., E-mail: gary.cline@yale.edu; Zhao, Xiaojian; Jakowski, Amy B.

2011-09-02

Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity tomore » islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution characteristics suggest several GPCRs as potential targets for PET imaging of pancreatic BCM.« less

Environmental Impact on Vascular Development Predicted by High-Throughput Screening

PubMed Central

Judson, Richard S.; Reif, David M.; Sipes, Nisha S.; Singh, Amar V.; Chandler, Kelly J.; DeWoskin, Rob; Dix, David J.; Kavlock, Robert J.; Knudsen, Thomas B.

2011-01-01

Background: Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High-throughput screening (HTS) in the U.S. Environmental Protection Agency (EPA) ToxCast™ project provides vast data on an expanding chemical library currently consisting of > 1,000 unique compounds across > 500 in vitro assays in phase I (complete) and Phase II (under way). This public data set can be used to evaluate concentration-dependent effects on many diverse biological targets and build predictive models of prototypical toxicity pathways that can aid decision making for assessments of human developmental health and disease. Objective: We mined the ToxCast phase I data set to identify signatures for potential chemical disruption of blood vessel formation and remodeling. Methods: ToxCast phase I screened 309 chemicals using 467 HTS assays across nine assay technology platforms. The assays measured direct interactions between chemicals and molecular targets (receptors, enzymes), as well as downstream effects on reporter gene activity or cellular consequences. We ranked the chemicals according to individual vascular bioactivity score and visualized the ranking using ToxPi (Toxicological Priority Index) profiles. Results: Targets in inflammatory chemokine signaling, the vascular endothelial growth factor pathway, and the plasminogen-activating system were strongly perturbed by some chemicals, and we found positive correlations with developmental effects from the U.S. EPA ToxRefDB (Toxicological Reference Database) in vivo database containing prenatal rat and rabbit guideline studies. We observed distinctly different correlative patterns for chemicals with effects in rabbits versus rats, despite derivation of in vitro signatures based on human cells and cell-free biochemical targets, implying conservation but potentially differential contributions of developmental pathways among species. Follow-up analysis with antiangiogenic thalidomide analogs and additional in vitro vascular targets showed in vitro activity consistent with the most active environmental chemicals tested here. Conclusions: We predicted that blood vessel development is a target for environmental chemicals acting as putative vascular disruptor compounds (pVDCs) and identified potential species differences in sensitive vascular developmental pathways. PMID:21788198

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

PubMed

Arutla, Viswanath; Leal, Joseph; Liu, Xiaowei; Sokalingam, Sriram; Raleigh, Michael; Adaralegbe, Adejimi; Liu, Li; Pentel, Paul R; Hecht, Sidney M; Chang, Yung

2017-05-08

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC 50 ) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

PubMed

Fernando, Ruani N; Chaudhari, Umesh; Escher, Sylvia E; Hengstler, Jan G; Hescheler, Jürgen; Jennings, Paul; Keun, Hector C; Kleinjans, Jos C S; Kolde, Raivo; Kollipara, Laxmikanth; Kopp-Schneider, Annette; Limonciel, Alice; Nemade, Harshal; Nguemo, Filomain; Peterson, Hedi; Prieto, Pilar; Rodrigues, Robim M; Sachinidis, Agapios; Schäfer, Christoph; Sickmann, Albert; Spitkovsky, Dimitry; Stöber, Regina; van Breda, Simone G J; van de Water, Bob; Vivier, Manon; Zahedi, René P; Vinken, Mathieu; Rogiers, Vera

2016-06-01

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

“Watching the Detectives” Report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November, 2015

PubMed Central

Fernando, Ruani N.; Chaudhari, Umesh; Escher, Sylvia E.; Hengstler, Jan G.; Hescheler, Jürgen; Jennings, Paul; Keun, Hector C.; Kleinjans, Jos C. S.; Kolde, Raivo; Kollipara, Laxmikanth; Kopp-Schneider, Annette; Limonciel, Alice; Nemade, Harshal; Nguemo, Filomain; Peterson, Hedi; Prieto, Pilar; Rodrigues, Robim M.; Sachinidis, Agapios; Schäfer, Christoph; Sickmann, Albert; Spitkovsky, Dimitry; Stöber, Regina; van Breda, Simone G.J.; van de Water, Bob; Vivier, Manon; Zahedi, René P.

2017-01-01

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro and in silico based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and “-omics” technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitutes a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich “-omics” databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project. PMID:27129694

Comparison of estrogen mixtures in vitro vs. in vivo

EPA Science Inventory

Numerous sources contribute to widespread contamination of drinking water sources with both natural and synthetic estrogens, which isa concern for potential ecological and human health effects. In vitro screening assays are valuable tools for identifying mechanisms of toxicity bu...

Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

NASA Astrophysics Data System (ADS)

Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

2016-09-01

We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening.

PubMed

Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y; Wei, Jie; Glicksman, Marcie A; Cuny, Gregory D; Stein, Ross L; Cohen, David E

2008-12-01

Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

Discovery of Dual ETA/ETB Receptor Antagonists from Traditional Chinese Herbs through in Silico and in Vitro Screening

PubMed Central

Wang, Xing; Zhang, Yuxin; Liu, Qing; Ai, Zhixin; Zhang, Yanling; Xiang, Yuhong; Qiao, Yanjiang

2016-01-01

Endothelin-1 receptors (ETAR and ETBR) act as a pivotal regulator in the biological effects of ET-1 and represent a potential drug target for the treatment of multiple cardiovascular diseases. The purpose of the study is to discover dual ETA/ETB receptor antagonists from traditional Chinese herbs. Ligand- and structure-based virtual screening was performed to screen an in-house database of traditional Chinese herbs, followed by a series of in vitro bioassay evaluation. Aristolochic acid A (AAA) was first confirmed to be a dual ETA/ETB receptor antagonist based intracellular calcium influx assay and impedance-based assay. Dose-response curves showed that AAA can block both ETAR and ETBR with IC50 of 7.91 and 7.40 μM, respectively. Target specificity and cytotoxicity bioassay proved that AAA is a selective dual ETA/ETB receptor antagonist and has no significant cytotoxicity on HEK293/ETAR and HEK293/ETBR cells within 24 h. It is a feasible and effective approach to discover bioactive compounds from traditional Chinese herbs using in silico screening combined with in vitro bioassay evaluation. The structural characteristic of AAA for its activity was especially interpreted, which could provide valuable reference for the further structural modification of AAA. PMID:26999111

Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

PubMed Central

Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

2016-01-01

We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD. PMID:27653664

The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening: Special focus on IK1.

PubMed

Goversen, Birgit; van der Heyden, Marcel A G; van Veen, Toon A B; de Boer, Teun P

2018-03-01

Preclinical drug screens are not based on human physiology, possibly complicating predictions on cardiotoxicity. Drug screening can be humanised with in vitro assays using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, in contrast to adult ventricular cardiomyocytes, iPSC-CMs beat spontaneously due to presence of the pacemaking current I f and reduced densities of the hyperpolarising current I K1 . In adult cardiomyocytes, I K1 finalises repolarisation by stabilising the resting membrane potential while also maintaining excitability. The reduced I K1 density contributes to proarrhythmic traits in iPSC-CMs, which leads to an electrophysiological phenotype that might bias drug responses. The proarrhythmic traits can be suppressed by increasing I K1 in a balanced manner. We systematically evaluated all studies that report strategies to mature iPSC-CMs and found that only few studies report I K1 current densities. Furthermore, these studies did not succeed in establishing sufficient I K1 levels as they either added too little or too much I K1 . We conclude that reduced densities of I K1 remain a major flaw in iPSC-CMs, which hampers their use for in vitro drug screening. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Identification and screening of rare actinomycetes isolated from Neesia altissima Bl.

NASA Astrophysics Data System (ADS)

Pratiwi, R. H.; Hidayat, I.; Hanafi, M.; Mangunwardoyo, W.

2017-07-01

Actinomycetes is the main source of antibiotics and endophytic actinomycetes from medicinal plants has considerable potential as like the host. The aim of this research is to identify rare actinomycetes isolated from Neesia altissima and to screen their antagonistic activity against diarrhea-causing bacteria in order to find new potential secondary metabolites. Samples of N. altissima were collected from mount Halimun-Salak National Park. Endophytic actinomycetes were isolated from roots of N. altissima by surface sterilization method. Screening of antagonistic activity was conducted against five diarrhea-causing bacteria such as Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 25241, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 by using diffusion disc methods. The endophytic actinomycete showed in vitro antibacterial activity against four diarrhea-causing bacteria, except the B. cereus ATCC 10876. The phylogenetic tree generated from 16S rRNA sequence showed that sequence of endophytic actinomycetes isolates nested in the clade belonging to the genus Nonomuraea. Sequence of UICC B-94 formed a monophyletic clade with N. jabiensis strain A4036 and N. rubra strain AC 615. Therefore, it is named as Nonomuraea sp. strain UICC B-94.

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions.

PubMed

De Diego, Nuria; Fürst, Tomáš; Humplík, Jan F; Ugena, Lydia; Podlešáková, Kateřina; Spíchal, Lukáš

2017-01-01

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidops is as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance.

Optically Based Rapid Screening Method for Proven Optimal Treatment Strategies Before Treatment Begins

DTIC Science & Technology

2016-08-01

are currently re- evaluating our IHC analysis to provide more refined response data (i.e. proliferation, percent tumor, percent fibrosis, etc.) to test...vitro results (in Fig. 4). We attempted to include only tumor cells in our image analysis, by evaluation of the cellmorphologywith respect to...tomography for evaluation of the activity of lapatinib, a dual inhibitor of the ErbB1 andErbB2 tyrosine kinases, in patients with advanced tumors. Jpn J

Synthesis of analogues of Eunicea gamma-cembranolides containing cyclic ethers via saponification.

PubMed

Rodríguez, A D; Piña, I C; Acosta, A L; Ramírez, C; Soto, J J

2001-02-09

A method for the synthesis of derivatives of the lead structures euniolide (1), 12,13-bisepieupalmerin (2), and eupalmerin acetate (3) containing tetrahydrofuran and tetrahydropyran ring systems was developed on the basis of alkali-induced intramolecular oxacyclizations. Representatives of the new analogues were submitted to the in vitro antitumor cell-line-screening program of the National Cancer Institute (NCI). While it was shown that a variety of structural modifications are possible, these transformations led typically to nontoxic synthetic cembranoids.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

PubMed Central

Miller, Tyler E.; Liau, Brian B.; Wallace, Lisa C.; Morton, Andrew R.; Xie, Qi; Dixit, Deobrat; Factor, Daniel C.; Kim, Leo J. Y.; Morrow, James J.; Wu, Qiulian; Mack, Stephen C.; Hubert, Christopher G.; Gillespie, Shawn M.; Flavahan, William A.; Hoffmann, Thomas; Thummalapalli, Rohit; Hemann, Michael T.; Paddison, Patrick J.; Horbinski, Craig M.; Zuber, Johannes; Scacheri, Peter C.; Bernstein, Bradley E.; Tesar, Paul J.; Rich, Jeremy N.

2017-01-01

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months1. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology2–4 for use in orthotopic patient-derived xenograft (PDX) models, creating a high-throughput negative selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators necessary for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, supporting targeting the transcription elongation machinery as a therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, which could supply untapped opportunities for therapeutic intervention. PMID:28678782

Diaquachloro-tris(ethyl-p-Aminobenzoate)copper(II) Chloride: Synthesis, Characterization and In Vitro Investigation of Antibacterial Activity

NASA Astrophysics Data System (ADS)

Rahardjo, S. B.; Lestari, W. W.; Syaima, H.

2017-07-01

The new complex of [Cu(benz)3(H2O)2Cl]Cl has been synthesized in 1:4 mole ratio of CuCl2·2H2O and ethyl-p-aminobenzoate (benz) in ethanol at room temperature. The complex was characterized by UV-Vis and infrared spectroscopy, Atomic Absorption Spectroscopy (AAS), thermal analysis, magnetic measurement and molar conductivity. Infrared spectra indicated that benz was coordinated to the metal ion through nitrogen of primary amine group. Water molecules and chloride ion were also coordinated to Cu(II). CuCl2·2H2O, benz, and Cu(II) complex were screened for investigating in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli using a modified Kirby-Bauer method.

Optical diagnostics of osteoblast cells and osteogenic drug screening

NASA Astrophysics Data System (ADS)

Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

2016-02-01

Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

In vitro screening techniques for reactive metabolites for minimizing bioactivation potential in drug discovery.

PubMed

Prakash, Chandra; Sharma, Raman; Gleave, Michelle; Nedderman, Angus

2008-11-01

Drug induced toxicity remains one of the major reasons for failures of new pharmaceuticals, and for the withdrawal of approved drugs from the market. Efforts are being made to reduce attrition of drug candidates, and to minimize their bioactivation potential in the early stages of drug discovery in order to bring safer compounds to the market. Therefore, in addition to potency and selectivity; drug candidates are now selected on the basis of acceptable metabolism/toxicology profiles in preclinical species. To support this, new approaches have been developed, which include extensive in vitro methods using human and animal hepatic cellular and subcellular systems, recombinant human drug metabolizing enzymes, increased automation for higher-throughput screens, sensitive analytical technologies and in silico computational models to assess the metabolism aspects of the new chemical entities. By using these approaches many compounds that might have serious adverse reactions associated with them are effectively eliminated before reaching clinical trials, however some toxicities such as those caused by idiosyncratic responses, are not detected until a drug is in late stages of clinical trials or has become available to the market. One of the proposed mechanisms for the development of idiosyncratic drug toxicity is the bioactivation of drugs to form reactive metabolites by drug metabolizing enzymes. This review discusses the different approaches to, and benefits of using existing in vitro techniques, for the detection of reactive intermediates in order to minimize bioactivation potential in drug discovery.

Generation of stable PDX derived cell lines using conditional reprogramming.

PubMed

Borodovsky, Alexandra; McQuiston, Travis J; Stetson, Daniel; Ahmed, Ambar; Whitston, David; Zhang, Jingwen; Grondine, Michael; Lawson, Deborah; Challberg, Sharon S; Zinda, Michael; Pollok, Brian A; Dougherty, Brian A; D'Cruz, Celina M

2017-12-06

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

The Combined Application of the Caco-2 Cell Bioassay Coupled with In Vivo (Gallus gallus) Feeding Trial Represents an Effective Approach to Predicting Fe Bioavailability in Humans

PubMed Central

Tako, Elad; Bar, Haim; Glahn, Raymond P.

2016-01-01

Research methods that predict Fe bioavailability for humans can be extremely useful in evaluating food fortification strategies, developing Fe-biofortified enhanced staple food crops and assessing the Fe bioavailability of meal plans that include such crops. In this review, research from four recent poultry (Gallus gallus) feeding trials coupled with in vitro analyses of Fe-biofortified crops will be compared to the parallel human efficacy studies which used the same varieties and harvests of the Fe-biofortified crops. Similar to the human studies, these trials were aimed to assess the potential effects of regular consumption of these enhanced staple crops on maintenance or improvement of iron status. The results demonstrate a strong agreement between the in vitro/in vivo screening approach and the parallel human studies. These observations therefore indicate that the in vitro/Caco-2 cell and Gallus gallus models can be integral tools to develop varieties of staple food crops and predict their effect on iron status in humans. The cost-effectiveness of this approach also means that it can be used to monitor the nutritional stability of the Fe-biofortified crop once a variety has released and integrated into the food system. These screening tools therefore represent a significant advancement to the field for crop development and can be applied to ensure the sustainability of the biofortification approach. PMID:27869705

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

An Accelerated Release Method of Risperidone Loaded PLGA Microspheres with Good IVIVC.

PubMed

Hu, Xiaoqin; Zhang, Jianwei; Tang, Xuemei; Li, Mingyuan; Ma, Siyu; Liu, Cheng; Gao, Yue; Zhang, Yue; Liu, Yan; Yu, Fanglin; Yang, Yang; Guo, Jia; Li, Zhiping; Mei, Xingguo

2018-01-01

A long release period lasting several days or several weeks is always needed and thereby it is tedious and time consuming to screen formulations of such microspheres with so long release period and evaluate their release profiles in vitro with conventional long-term or "real-time" release method. So, an accelerated release testing of such system is necessary for formulation design as well as quality control purpose. The purpose of this study is to obtain an accelerated release method of risperidone loaded poly(lactic-co-glycolic acid) (PLGA) microspheres with good in vitro/in vivo correlation (IVIVC). Two formulations of risperidone loaded PLGA microspheres used for evaluating IVIVC were prepared by O/W method. The accelerated release condition was optimized by investigating the effect of pH, osmotic pressure, temperature and ethanol concentration on the release of risperidone from microspheres and the in vitro accelerated release profiles of risperidone from PLGA microspheres were obtained under this optimized accelerated release condition. The plasma concentration of risperidone were also detected after subcutaneous injection of risperidone loaded microspheres to rats. The in vivo cumulative absorption profiles were then calculated using Wagner-Nelson model, Loo- Riegelman model and numerical convolution model, respectively. The correlation between in vitro accelerated release and in vivo cumulative absorption were finally evaluated with Least Square Method. It was shown that temperature and ethanol concentration significantly affected the release of risperidone from the microspheres while pH and osmotic pressure of release media slightly affected the release behavior of risperidone. The in vitro release of risperidone from microspheres were finally undergone in PBS (pH7.0, 300mosm) with 20% (V/V) ethanol at 45°C. The sustained and complete release of risperidone was observed in both formulations under the accelerated release condition although these two release profiles were dissimilar. The correlation coefficients (R2) of IVIVC were all above 0.95 and the slopes were all between 0.9564 and 1.1868 in spite of fitted model and microsphere formulation. An in vitro accelerated release method of risperidone microspheres with good IVIVC was established in this paper and this accelerated release method was supposed to have great potential in both in vivo performance prediction and quality control for risperidone loaded PLGA microspheres. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Computational toxicology and in silico modeling of embryogenesis

EPA Science Inventory

High-throughput screening (HTS) is providing a rich source of in vitro data for predictive toxicology. ToxCast™ HTS data presently covers 1060 broad-use chemicals and captures >650 in vitro features for diverse biochemical and receptor binding activities, multiplexed reporter gen...

Cross species comparisons of chemical interaction with recombinant steroid receptors in vitro.

EPA Science Inventory

Typically, mammalian receptors are used for in vitro hazard identification and screening for endocrine disrupting compounds. There is concern, however, that differences may exist in the affinities of environmental compounds for steroid receptors from other vertebrate classes. S...

Identification of sumoylation activating enzyme 1 inhibitors by structure-based virtual screening.

PubMed

Kumar, Ashutosh; Ito, Akihiro; Hirohama, Mikako; Yoshida, Minoru; Zhang, Kam Y J

2013-04-22

SUMO activating enzyme 1 (SUMO E1) is responsible for the activation of SUMO in the first step of the sumoylation cascade. SUMO E1 is linked to many human diseases including cancer, thus making it a potential therapeutic target. There are few reported SUMO E1 inhibitors including several natural products. To identify small molecule inhibitors of SUMO E1 with better drug-like properties for potential therapeutic studies, we have used structure-based virtual screening to identify hits from the Maybridge small molecule library for biological assay. Our virtual screening protocol involves fast docking of the entire small molecule library with rigid protein and ligands followed by redocking of top hits using a method that incorporates both ligand and protein flexibility. Subsequently, the top-ranking compounds were prioritized using the molecular dynamics simulation-based binding free energy calculation. Out of 24 compounds that were acquired and tested using in vitro sumoylation assay, four of them showed more than 85% inhibition of sumoylation with the most active compound showing an IC50 of 14.4 μM. A similarity search with the most active compound in the ZINC database has identified three more compounds with improved potency. These compounds share a common phenyl urea scaffold and have been confirmed to inhibit SUMO E1 by in vitro SUMO-1 thioester bond formation assay. Our study suggests that these phenyl urea compounds could be used as a starting point for the development of novel therapeutic agents.

Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

PubMed Central

Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

1997-01-01

mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

High-Throughput and Rapid Screening of Novel ACE Inhibitory Peptides from Sericin Source and Inhibition Mechanism by Using in Silico and in Vitro Prescriptions.

PubMed

Sun, Huaju; Chang, Qing; Liu, Long; Chai, Kungang; Lin, Guangyan; Huo, Qingling; Zhao, Zhenxia; Zhao, Zhongxing

2017-11-22

Several novel peptides with high ACE-I inhibitory activity were successfully screened from sericin hydrolysate (SH) by coupling in silico and in vitro approaches for the first time. Most screening processes for ACE-I inhibitory peptides were achieved through high-throughput in silico simulation followed by in vitro verification. QSAR model based predicted results indicated that the ACE-I inhibitory activity of these SH peptides and six chosen peptides exhibited moderate high ACE-I inhibitory activities (log IC 50 values: 1.63-2.34). Moreover, two tripeptides among the chosen six peptides were selected for ACE-I inhibition mechanism analysis which based on Lineweaver-Burk plots indicated that they behave as competitive ACE-I inhibitors. The C-terminal residues of short-chain peptides that contain more H-bond acceptor groups could easily form hydrogen bonds with ACE-I and have higher ACE-I inhibitory activity. Overall, sericin protein as a strong ACE-I inhibition source could be deemed a promising agent for antihypertension applications.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

PubMed

Szaszkó, Mária; Hajdú, István; Flachner, Beáta; Dobi, Krisztina; Magyar, Csaba; Simon, István; Lőrincz, Zsolt; Kapui, Zoltán; Pázmány, Tamás; Cseh, Sándor; Dormán, György

2017-02-01

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

PubMed Central

Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

2013-01-01

Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

*Biomarkers of acute respiratory allergen exposure: Screening for sensitization potential

EPA Science Inventory

Effective hazard screening will require the development of high-throughput or in vitro assays for the identification of potential sensitizers. The goal of this preliminary study was to identify potential biomarkers that differentiate the response to allergens vs non-allergens fol...

Screening for Developmental Neurotoxicants using In Vitro "Brain on a Chip" Cultures

EPA Science Inventory

Currently there are thousands of chemicals in the environment that have not been screened for their potential to cause developmental neurotoxicity (DNT). The use of microelectrode array (MEA) technology allows for simultaneous extracellular measurement of action potential (spike)...

Pathway Profiling and Tissue Modeling Using ToxCast HTS Data

EPA Science Inventory

High-throughput screening (HTS) and high-content screening (HCS) assays are providing data-rich studies to probe and profile the direct cellular effects of thousands of chemical compounds in commerce or potentially entering the environment. In vitro profiling may compare unknown ...

Picking Cell Lines for High-Throughput Transcriptomic Toxicity Screening (SOT)

EPA Science Inventory

High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captu...

Cell cultures in drug discovery and development: The need of reliable in vitro-in vivo extrapolation for pharmacodynamics and pharmacokinetics assessment.

PubMed

Jaroch, Karol; Jaroch, Alina; Bojko, Barbara

2018-01-05

For ethical and cost-related reasons, use of animals for the assessment of mode of action, metabolism and/or toxicity of new drug candidates has been increasingly scrutinized in research and industrial applications. Implementation of the 3 "Rs" 1 ; rule (Reduction, Replacement, Refinement) through development of in silico or in vitro assays has become an essential element of risk assessment. Physiologically based pharmacokinetic (PBPK 2 ) modeling is the most potent in silico tool used for extrapolation of pharmacokinetic parameters to animal or human models from results obtained in vitro. Although, many types of in vitro assays are conducted during drug development, use of cell cultures is the most reliable one. Two-dimensional (2D) cell cultures have been a part of drug development for many years. Nowadays, their role is decreasing in favor of three-dimensional (3D) cell cultures and co-cultures. 3D cultures exhibit protein expression patterns and intercellular junctions that are closer to in vivo states in comparison to classical monolayer cultures. Co-cultures allow for examinations of the mutual influence of different cell lines. However, the complexity and high costs of co-cultures and 3D equipment exclude such methods from high-throughput screening (HTS). 3 In vitro absorption, distribution, metabolism, and excretion assessment, as well as drug-drug interaction (DDI), are usually performed with the use of various cell culture based assays. Progress in in silico and in vitro methods can lead to better in vitro-in vivo extrapolation (IVIVE 4 ) outcomes and have a potential to contribute towards a significant reduction in the number of laboratory animals needed for drug research. As such, concentrated efforts need to be spent towards the development of an HTS in vitro platform with satisfactory IVIVE features. Copyright © 2017 Elsevier B.V. All rights reserved.

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity--proof of concept.

PubMed

Becker, Richard A; Friedman, Katie Paul; Simon, Ted W; Marty, M Sue; Patlewicz, Grace; Rowlands, J Craig

2015-04-01

Rapid high throughput in vitro screening (HTS) assays are now available for characterizing dose-responses in assays that have been selected for their sensitivity in detecting estrogen-related endpoints. For example, EPA's ToxCast™ program recently released endocrine assay results for more than 1800 substances and the interagency Tox21 consortium is in the process of releasing data for approximately 10,000 chemicals. But such activity measurements alone fall short for the purposes of priority setting or screening because the relevant exposure context is not considered. Here, we extend the method of exposure:activity profiling by calculating the exposure:activity ratios (EARs) using human exposure estimates and AC50 values for a range of chemicals tested in a suite of seven estrogenic assays in ToxCast™ and Tox21. To provide additional context, relative estrogenic exposure:activity quotients (REEAQ) were derived by comparing chemical-specific EARs to the EAR of the ubiquitous dietary phytoestrogen, genistein (GEN). Although the activity of a substance in HTS-endocrine assays is not a measure of health hazard or risk, understanding how such a dose compares to human exposures provides a valuable additional metric that can be used in decision-making; substances with small EARs and REEAQs would indicate low priority for further endocrine screening or testing. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells.

PubMed

Wahlin, Karl J; Maruotti, Julien A; Sripathi, Srinivasa R; Ball, John; Angueyra, Juan M; Kim, Catherine; Grebe, Rhonda; Li, Wei; Jones, Bryan W; Zack, Donald J

2017-04-10

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus.

PubMed

Sathish, Kumar S R; Kokati, Venkata Bhaskara Rao

2012-10-01

To investigate the antibacterial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus (MDRSA). Fifty one actinobacterial strains were isolated from salt pans soil, costal area in Kothapattanam, Ongole, Andhra Pradesh. Primary screening was done using cross-streak method against MDRSA. The bioactive compounds are extracted from efficient actinobacteria using solvent extraction. The antimicrobial activity of crude and solvent extracts was performed using Kirby-Bauer method. MIC for ethyl acetate extract was determined by modified agar well diffusion method. The potent actinobacteria are identified using Nonomura key, Shirling and Gottlieb 1966 with Bergey's manual of determinative bacteriology. Among the fifty one isolates screened for antibacterial activity, SRB25 were found efficient against MDRSA. The ethyl acetate extracts showed high inhibition against test organism. MIC test was performed with the ethyl acetate extract against MDRSA and found to be 1 000 µg/mL. The isolated actinobacteria are identified as Streptomyces sp with the help of Nonomura key. The current investigation reveals that the marine actinobacteria from salt pan environment can be able to produce new drug molecules against drug resistant microorganisms.

Predicting hepatotoxicity using ToxCast in vitro bioactivity and chemical structure

EPA Science Inventory

Background: The U.S. EPA ToxCastTM program is screening thousands of environmental chemicals for bioactivity using hundreds of high-throughput in vitro assays to build predictive models of toxicity. We represented chemicals based on bioactivity and chemical structure descriptors ...

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

Evaluating ToxCast Assays for their Ability to Detect Genotoxicity

EPA Science Inventory

It has become evident over the past several decades, that though the standard battery of genotoxicity tests including bacterial and in vitro mammalian mutagenesis and in vitro and in vivo clastogenicity assays have been quite useful in screening out potent genotoxicants during th...

In vitro Perturbations of Targets in Cancer Hallmark Processes Predict Rodent Chemical Carcinogenesis

EPA Science Inventory

Thousands of untested chemicals in the environment require efficient characterization of carcinogenic potential in humans. A proposed solution is rapid testing of chemicals using in vitro high-throughput screening (HTS) assays for targets in pathways linked to disease processes ...

Neuronal models for evaluation of proliferation in vitro using high content screening.

PubMed

Mundy, William R; Radio, Nicholas M; Freudenrich, Theresa M

2010-04-11

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation. Published by Elsevier Ireland Ltd.

Plants used in Guatemala for the treatment of protozoal infections. I. Screening of activity to bacteria, fungi and American trypanosomes of 13 native plants.

PubMed

Cáceres, A; López, B; González, S; Berger, I; Tada, I; Maki, J

1998-10-01

Extracts were prepared from 13 native plants used for the treatment of protozoal infections. Activity against bacteria and fungi was demonstrated by dilution procedures; Trypanosoma cruzi was evaluated in vitro against epimastigote and trypomastigotes and in vivo against trypomastigotes. In active extracts, toxicity was evaluated by Artemia salina nauplii, oral acute toxicity (1-5 g/kg) and oral and intraperitoneal subacute toxicity in mice (500 mg/kg). From the plants screened, six showed activity (< or = 2 mg/ml) against bacteria, three against yeasts, five against Microsporum gypseum and five against T. cruzi in vitro and/or in vivo. In vitro and in vivo activity was demonstrated by Neurolaena lobata and Solanum americanum; in vitro or in vivo activity was shown by Acalypha guatemalensis, Petiveria alliacea and Tridax procumbens. Toxicity studies showed that extracts from S. americanum are toxic to A. salina (aqueous, 160 ppm). None showed acute or oral toxicity to mice; S. americanum showed intraperitoneal subacute toxicity.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays.

PubMed

Martins, Flaviano S; Miranda, Ieso C; Rosa, Carlos A; Nicoli, Jacques R; Neves, Maria J

2008-01-01

Probiotics are viable defined microorganisms (bacteria or yeasts) that exert a beneficial effect on the health of the host when ingested in adequate amounts. Screening for such biotherapeutic agents is commonly performed by in vitro assays simulating gastrointestinal environment to determine the ability to survive in the digestive tract. In the present study, the possibility of extrapolation of data obtained in in vitro assays to in vivo conditions was studied using five Saccharomyces cerevisiae strains isolated from Brazilian Atlantic rain forest. Trehalose contents and survival after exposure to a combination of physiological stresses generally found in the gastrointestinal tract of humans were determined for the five yeasts and compared to the behavior of Saccharomyces boulardii, a well-known probiotic. The results were completed with the colonization capacity of the gastrointestinal tract of gnotobiotic mice by these yeast strains. Some results obtained by in vitro assays are not confirmed by in vivo experiments, indicating that the extrapolation cannot be always done.

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test.

PubMed

Alves, Eloísa Nunes; Presgrave, Rosaura de Farias; Presgrave, Octávio Augusto França; Sabagh, Fernanda Peres; de Freitas, João Carlos Borges Rolim; Corrado, Alexandre P

2008-07-01

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

Review of Angiotensin-converting Enzyme Inhibitory Assay: Rapid Method in Drug Discovery of Herbal Plants

PubMed Central

Ahmad, Islamudin; Yanuar, Arry; Mulia, Kamarza; Mun’im, Abdul

2017-01-01

The renin-angiotensin-aldosterone system is a signaling pathway which responsible in the blood pressure regulation. Angiotensin-converting enzyme (ACE) is one of the key elements responsible for the hypertensive mechanism. It converts angiotensin-I to angiotensin-II. The discovery history of the ACE inhibitory activity assay method has been through a long stage for decades and development continues until today. The ACE inhibitory activity has become an effective screening method in the search for new antihypertensive agents from herbal plants. Some of in vitro assay methods were used to examine the activity of ACE inhibitors based on the substrate usage, such as; Cushman and Cheung Method using a substrate hippuryl-histidyl-leucine (HHL), Holmquist method using a substrate furanacryloyl-tripeptide, Elbl and Wagner method using a substrate benzoil-[l-14C] glicyl-L-histidine-L-leucine, Carmel and Yaron method using a substrate o-aminobenzoylglycyl-p-nitrophenylalanilproline, and Lam method using 3-hydroxybutyrylglycyl-glycyl-glycine as substrate. Several different methods to measure the results of enzymatic reactions or separating substrate with products, including spectrophotometric, fluorometric, high-performance liquid chromatography, electrophoresis, and radiochemistry. Application of the test method for screening the ACE inhibitors activity and investigation of active compounds from natural products can be done easily with this method, it is very helpful in research because the results obtained are simple, accurate, and rapid. PMID:28503045

Aseptic Culture Systems of Radopholus similis for In Vitro Assays on Musa spp. and Arabidopsis thaliana

PubMed Central

Elsen, A.; Lens, K.; Nguyet, D. T. M.; Broos, S.; Stoffelen, R.; De Waele, D.

2001-01-01

Radopholus similis is one of the most damaging nematodes in bananas. Chemical control is currently the most-used method, but nematode control through genetic improvement is widely encouraged. The objective of this study was to establish an aseptic culture system for R. similis and determine whether R. similis can infect and reproduce on in vitro banana plantlets and in vitro Arabidopsis thaliana. In the study's first part, a suitable aseptic culture system was developed using alfalfa callus. Radopholus similis could penetrate and reproduce in the callus. Six weeks after inoculation with 25 females, the reproduction ratio was 26.3 and all vermiform stages were present. The reproduction ratio increased to 223.2 after 12 weeks. Results of a greenhouse test showed that R. similis did not lose its pathogenicity after culturing on alfalfa callus. In the study's second part, the infection and reproduction of the nematodes cultured on the callus were studied on both in vitro banana plantlets and A. thaliana. Radopholus similis infected and reproduced on both banana and A. thaliana. Furthermore, nematode damage was observed in the root systems of both hosts. These successful infections open new perspectives for rapid in vitro screening for resistance in banana cultivars and anti-nematode proteins expressed in A. thaliana. PMID:19266012

Advances in In Vitro and In Silico Tools for Toxicokinetic Dose ...

EPA Pesticide Factsheets

Recent advances in vitro assays, in silico tools, and systems biology approaches provide opportunities for refined mechanistic understanding for chemical safety assessment that will ultimately lead to reduced reliance on animal-based methods. With the U.S. commercial chemical landscape encompassing thousands of chemicals with limited data, safety assessment strategies that reliably predict in vivo systemic exposures and subsequent in vivo effects efficiently are a priority. Quantitative in vitro-in vivo extrapolation (QIVIVE) is a methodology that facilitates the explicit and quantitative application of in vitro experimental data and in silico modeling to predict in vivo system behaviors and can be applied to predict chemical toxicokinetics, toxicodynamics and also population variability. Tiered strategies that incorporate sufficient information to reliably inform the relevant decision context will facilitate acceptance of these alternative data streams for safety assessments. This abstract does not necessarily reflect U.S. EPA policy. This talk will provide an update to an international audience on the state of science being conducted within the EPA’s Office of Research and Development to develop and refine approaches that estimate internal chemical concentrations following a given exposure, known as toxicokinetics. Toxicokinetic approaches hold great potential in their ability to link in vitro activities or toxicities identified during high-throughput screen

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

Arisugacins A and B, novel and selective acetylcholinesterase inhibitors from Penicillium sp. FO-4259. I. Screening, taxonomy, fermentation, isolation and biological activity.

PubMed

Kuno, F; Otoguro, K; Shiomi, K; Iwai, Y; Omura, S

1996-08-01

An in vitro screening method for selective acetylcholinesterase (AChE) inhibitors was established. Inhibitory activity of AChE and butyrylcholinesterase (BuChE) was measured and the culture broths of microorganisms that showed selective inhibition against AChE were characterized. By using this method, a strain producing the novel and selective inhibitors of AChE, arisugacins A and B, was picked out among over seven thousand microorganisms tested. Arisugacins were obtained as white powders from the culture broth together with three known compounds, territrems B and C and cyclopenin that also showed selective inhibition against AChE. Arisugacins and territrems are members of the meroterpenoid compounds. They showed potent inhibitory activities against AChE with IC50 values in range of 1.0 approximately 25.8 nM. Furthermore, they showed greater than 2,000-fold more potent inhibition against AChE than BuChE.

Advances in Antibody Design

PubMed Central

Tiller, Kathryn E.; Tessier, Peter M.

2017-01-01

The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody–drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human

EPA Science Inventory

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however...

Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

EPA Science Inventory

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, c...

Population-Based in Vitro Hazard and Concentration–Response Assessment of Chemicals: The 1000 Genomes High-Throughput Screening Study

PubMed Central

Abdo, Nour; Xia, Menghang; Brown, Chad C.; Kosyk, Oksana; Huang, Ruili; Sakamuru, Srilatha; Zhou, Yi-Hui; Jack, John R.; Gallins, Paul; Xia, Kai; Li, Yun; Chiu, Weihsueh A.; Motsinger-Reif, Alison A.; Austin, Christopher P.; Tice, Raymond R.

2015-01-01

Background: Understanding of human variation in toxicity to environmental chemicals remains limited, so human health risk assessments still largely rely on a generic 10-fold factor (10½ each for toxicokinetics and toxicodynamics) to account for sensitive individuals or subpopulations. Objectives: We tested a hypothesis that population-wide in vitro cytotoxicity screening can rapidly inform both the magnitude of and molecular causes for interindividual toxicodynamic variability. Methods: We used 1,086 lymphoblastoid cell lines from the 1000 Genomes Project, representing nine populations from five continents, to assess variation in cytotoxic response to 179 chemicals. Analysis included assessments of population variation and heritability, and genome-wide association mapping, with attention to phenotypic relevance to human exposures. Results: For about half the tested compounds, cytotoxic response in the 1% most “sensitive” individual occurred at concentrations within a factor of 10½ (i.e., approximately 3) of that in the median individual; however, for some compounds, this factor was > 10. Genetic mapping suggested important roles for variation in membrane and transmembrane genes, with a number of chemicals showing association with SNP rs13120371 in the solute carrier SLC7A11, previously implicated in chemoresistance. Conclusions: This experimental approach fills critical gaps unaddressed by recent large-scale toxicity testing programs, providing quantitative, experimentally based estimates of human toxicodynamic variability, and also testable hypotheses about mechanisms contributing to interindividual variation. Citation: Abdo N, Xia M, Brown CC, Kosyk O, Huang R, Sakamuru S, Zhou YH, Jack JR, Gallins P, Xia K, Li Y, Chiu WA, Motsinger-Reif AA, Austin CP, Tice RR, Rusyn I, Wright FA. 2015. Population-based in vitro hazard and concentration–response assessment of chemicals: the 1000 Genomes high-throughput screening study. Environ Health Perspect 123:458–466; http://dx.doi.org/10.1289/ehp.1408775 PMID:25622337

Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations.

PubMed

Ge, Z; Taylor, D E

1997-09-01

Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains. The copP gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing. The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination. The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe. Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted. The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.

Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration.

PubMed

Ma, Hui-li; Jiang, Qiao; Han, Siyuan; Wu, Yan; Cui Tomshine, Jin; Wang, Dongliang; Gan, Yaling; Zou, Guozhang; Liang, Xing-Jie

2012-01-01

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Screening method for inhibitors against formosan subterranean termite beta-glucosidases in vivo.

PubMed

Zhu, Betty C R; Henderson, Gregg; Laine, Roger A

2005-02-01

Cellulose, a main structural constituent of plants, is the major nutritional component for wood-feeding termites. Enzymatic hydrolysis of cellulose to glucose occurs by the action of cellulases, a mixture of the three major classes of enzymes including endo-1,4-beta-glucanases, exo-1,4-beta-glucanases, and beta-glucosidase. Lower termites, such as the Formosan subterranean termite, Coptotermes formosanus Shiraki, require cellulolytic protozoa to efficiently digest cellulose for survival. Inhibitors developed against any of these cellulase system enzymes would be a potential termite treatment avenue. Our effort was to develop a screening system to determine whether termites could be controlled by administration of cellulase system inhibitors. Some reported compounds such as gluconolactone, conduritol B epoxide, and 1-deoxynojirimycin are potential beta-glucosidase inhibitors, but they have only been tested in vitro. We describe an in vivo method to test the inhibitory ability of the designated chemicals to act on beta-1,4-glucosidases, one member of the cellulase system that is the key step that releases glucose for use as an energy and carbon source for termites. Inhibition in releasing glucose from cellooligosaccharides might be sufficient to starve termites. Fluorescein di-beta-D-glucopyranoside was used as the artificial enzyme substrate and the fluorescent intensity of the reaction product (fluorescein) quantified with an automated fluorescence plate reader. Several known in vitro beta-1,4-glucosidase inhibitors were tested in vivo, and their inhibitory potential was determined. Endogenous and protozoan cellulase activities are both assumed to play a role.

Micronuclei versus Chromosomal Aberrations Induced by X-Ray in Radiosensitive Mammalian Cells

PubMed Central

PLAMADEALA, Cristina; WOJCIK, Andrzej; CREANGA, Dorina

2015-01-01

Background: An experimental study was accomplished to compare estimation methods of ionizing radiations genotoxicity in mammalian cell cultures by means of two cytogenetic parameters with focus on aberrant cells characterized by multiple chromosomal damages. Methods: In vitro study was carried out on the genotoxicity of low-medium doses of 190 kV X-rays absorbed in Chinese hamster ovary cell cultures. Micronuclei and ten types of chromosomal aberrations were identified with Giemsa dying and optical microscope screening. Results: The first parameter consisting in micronuclei relative frequency has led to higher linear correlation coefficient than the second one consistent with chromosomal aberrations relative frequency. However, the latter parameter estimated as the sum of all chromosomal aberrations appeared to be more sensitive to radiation dose increasing in the studied dose range, from 0 to 3 Gy. The number of micronuclei occurring simultaneously in a single cell was not higher than 3, while the number of chromosomal aberrations observed in the same cell reached the value of 5 for doses over 1 Gy. Conclusion: Polynomial dose-response curves were evidenced for cells with Ni micronuclei (i=1,3) while non-monotonic curves were evidenced through detailed analysis of aberrant cells with Ni chromosomal changes (i=(1,5)¯) - in concordance with in vitro studies from literature. The investigation could be important for public health issues where micronucleus screening is routinely applied but also for research purposes where various chromosomal aberrations could be of particular interest. PMID:25905075

Changing the Paradigm of Toxicity Testing From Observational to Predictive: An Update on Two Global In Vitro Screening Initiatives

EPA Science Inventory

This is a transcript of a one hour roundtable hosted by the Editor-in-Chief of the journal Applied In Vitro Toxicology. This transcript was published as a roundtable discussion and was not peer reviewed.

Follow-up actions from positive results of in vitro genetic toxicity testing

EPA Science Inventory

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in...

ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

EPA Science Inventory

In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

COMPARISON OF THE ESTROGENIC POTENCIES OF ESTRADIOL, ETHYNYLESTRADIOL, DIETHYLSTILBESTROL, NONYLPHENOL AND METHOXYCHLOR IN VIVO AND IN VITRO

EPA Science Inventory

Five natural, pharmaceutical, or xenobiotic chemicals (17b-estradiol, ethynylestradiol, diethystilbestrol, nonylphenol, methoxychlor) were tested in two in vitro (MCF-7 breast tumor cell proliferation [E-screen], yeast estrogen system [YES]), and one in vivo (male sheepshead min...

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

PubMed

Katt, Moriah E; Placone, Amanda L; Wong, Andrew D; Xu, Zinnia S; Searson, Peter C

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

PubMed Central

Katt, Moriah E.; Placone, Amanda L.; Wong, Andrew D.; Xu, Zinnia S.; Searson, Peter C.

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment. PMID:26904541

Effect of training data size and noise level on support vector machines virtual screening of genotoxic compounds from large compound libraries.

PubMed

Kumar, Pankaj; Ma, Xiaohua; Liu, Xianghui; Jia, Jia; Bucong, Han; Xue, Ying; Li, Ze Rong; Yang, Sheng Yong; Wei, Yu Quan; Chen, Yu Zong

2011-05-01

Various in vitro and in-silico methods have been used for drug genotoxicity tests, which show limited genotoxicity (GT+) and non-genotoxicity (GT-) identification rates. New methods and combinatorial approaches have been explored for enhanced collective identification capability. The rates of in-silco methods may be further improved by significantly diversified training data enriched by the large number of recently reported GT+ and GT- compounds, but a major concern is the increased noise levels arising from high false-positive rates of in vitro data. In this work, we evaluated the effect of training data size and noise level on the performance of support vector machines (SVM) method known to tolerate high noise levels in training data. Two SVMs of different diversity/noise levels were developed and tested. H-SVM trained by higher diversity higher noise data (GT+ in any in vivo or in vitro test) outperforms L-SVM trained by lower noise lower diversity data (GT+ in in vivo or Ames test only). H-SVM trained by 4,763 GT+ compounds reported before 2008 and 8,232 GT- compounds excluding clinical trial drugs correctly identified 81.6% of the 38 GT+ compounds reported since 2008, predicted 83.1% of the 2,008 clinical trial drugs as GT-, and 23.96% of 168 K MDDR and 27.23% of 17.86M PubChem compounds as GT+. These are comparable to the 43.1-51.9% GT+ and 75-93% GT- rates of existing in-silico methods, 58.8% GT+ and 79% GT- rates of Ames method, and the estimated percentages of 23% in vivo and 31-33% in vitro GT+ compounds in the "universe of chemicals". There is a substantial level of agreement between H-SVM and L-SVM predicted GT+ and GT- MDDR compounds and the prediction from TOPKAT. SVM showed good potential in identifying GT+ compounds from large compound libraries based on higher diversity and higher noise training data.

Comparison of Clobetasol Propionate Generics Using Simplified in Vitro Bioequivalence Method for Topical Drug Products.

PubMed

Soares, Kelen Carine Costa; de Souza, Weidson Carlos; de Souza Texeira, Leonardo; da Cunha-Filho, Marcilio Sergio Soares; Gelfuso, Guilherme Martins; Gratieri, Tais

2017-11-20

The aim of this paper is to propose a simple in vitro skin penetration experiment in which the drug is extracted from the whole skin piece as a test valid for formulation screening and optimization during development process, equivalence assessment during quality control or post-approval after changes to the product. Twelve clobetasol propionate (CP) formulations (six creams and six ointments) from the local market were used as a model to challenge the proposed methodology in comparison to in vitro skin penetration following tape-stripping for drug extraction. To support the results, physicochemical tests for pH, viscosity, density and assay, as well as in vitro release were performed. Both protocols, extracting the drug from the skin using the tape-stripping technique or extracting from the full skin were capable of differentiating CP formulations. Only one formulation did not present statistical difference from the reference drug product in penetration tests and only other two oitments presented equivalent release to the reference. The proposed protocol is straightforward and reproducible. Results suggest the bioinequavalence of tested CP formulations reinforcing the necessity of such evaluations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening

NASA Astrophysics Data System (ADS)

García-Gutiérrez, P.; Landa-Piedra, A.; Rodríguez-Romero, A.; Parra-Unda, R.; Rojo-Domínguez, A.

2011-12-01

We describe in this work a successful virtual screening and experimental testing aimed to the identification of novel inhibitors of superoxide dismutase of the worm Taenia solium ( TsCu/Zn-SOD), a human parasite. Conformers from LeadQuest® database of drug-like compounds were selected and then docked on the surface of TsCu/Zn-SOD. Results were screened looking for ligand contacts with receptor side-chains not conserved in the human homologue, with a subsequent development of a score optimization by a set of energy minimization steps, aimed to identify lead compounds for in vitro experiments. Six out of fifty experimentally tested compounds showed μM inhibitory activity toward TsCu/Zn-SOD. Two of them showed species selectivity since did not inhibit the homologous human enzyme when assayed in vitro.

Bayesian Models Leveraging Bioactivity and Cytotoxicity Information for Drug Discovery

PubMed Central

Ekins, Sean; Reynolds, Robert C.; Kim, Hiyun; Koo, Mi-Sun; Ekonomidis, Marilyn; Talaue, Meliza; Paget, Steve D.; Woolhiser, Lisa K.; Lenaerts, Anne J.; Bunin, Barry A.; Connell, Nancy; Freundlich, Joel S.

2013-01-01

SUMMARY Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data, to experimentally validate virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screen a commercial library and experimentally confirm actives with hit rates exceeding typical HTS results by 1-2 orders of magnitude. The first dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery. PMID:23521795

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

PubMed Central

2017-01-01

ABSTRACT The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. PMID:29327641

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid screening of bioactive compounds from natural products by integrating 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays.

PubMed

Zhang, Yufeng; Xiao, Shun; Sun, Lijuan; Ge, Zhiwei; Fang, Fengkai; Zhang, Wen; Wang, Yi; Cheng, Yiyu

2013-05-13

A high throughput method was developed for rapid screening and identification of bioactive compounds from traditional Chinese medicine, marine products and other natural products. The system, integrated with five-channel chromatographic separation and dual UV-MS detection, is compatible with in vitro 96-well microplate based bioassays. The stability and applicability of the proposed method was validated by testing radical scavenging capability of a mixture of seven known compounds (rutin, dihydroquercetin, salvianolic acid A, salvianolic acid B, glycyrrhizic acid, rubescensin A and tangeretin). Moreover, the proposed method was successfully applied to the crude extracts of traditional Chinese medicine and a marine sponge from which 12 bioactive compounds were screened and characterized based on their anti-oxidative or anti-tumor activities. In particular, two diterpenoid derivatives, agelasine B and (-)-agelasine D, were identified for the first time as anti-tumor compounds from the sponge Agelas mauritiana, showing a considerable activity toward MCF-7 cells (IC50 values of 7.84±0.65 and 10.48±0.84 μM, respectively). Our findings suggested that the integrated system of 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays can be a versatile and high efficient approach for the discovery of active compounds from natural products. Copyright © 2013 Elsevier B.V. All rights reserved.

Virtual Liver: Quantitative Dose-Response Using Systems Biology

EPA Science Inventory

The U.S. EPA’s ToxCast™ program uses hundreds of high-throughput, in vitro assays to screen chemicals in order to rapidly identify signatures of toxicity. These assays measure the in vitro concentrations at which cellular pathways are perturbed by chemicals. The U.S. EPA’s Virtu...

Incorporating High-Throughput Exposure Predictions with Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

EPA Science Inventory

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast™ HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compare...

Comparison of L1000 and Affymetrix Microarray for In Vitro Concentration-Response Gene Expression Profiling (SOT)

EPA Science Inventory

Advances in high-throughput screening technologies and in vitro systems have opened doors for cost-efficient evaluation of chemical effects on a diversity of biological endpoints. However, toxicogenomics platforms remain too costly to evaluate large libraries of chemicals in conc...

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

High-throughput screening (HTS) generates in vitro data for characterizing potential chemical hazard. TK models are needed to allow in vitro to in vivo extrapolation (IVIVE) to real world situations. The U.S. EPA has created a public tool (R package “httk” for high throughput tox...

Post-thaw motility of frozen boar sperm does not predict success with in vitro fertilization

USDA-ARS?s Scientific Manuscript database

Using cryopreserved boar sperm rather than liquid semen for in vitro fertilization (IVF) allows improved IVF consistency. However, cryopreservation of boar sperm results in reduced post-thaw motility, fertilization and embryo development. Boars are often screened on an individual basis prior to use ...

PRELIMINARY ASSESSMENTS OF IN VITRO PHARMACOKINETIC DATA AND EXPOSURE INFORMATION FOR THE TOXCAST PHASE II CHEMICALS

EPA Science Inventory

Momentum has been growing in Toxicology to assess the utility of high-throughput screening (HTS) assays in the determination of chemical testing priorities. However, in vitro potencies determined in these assays do not consider in vivo bioavailability, clearance or exposure estim...

Toxicity testing in the 21st Century: Extrapolation from in vitro to in vivo using xenoestrogens as a model

EPA Science Inventory

Slides associated with the following abstract: Numerous sources contribute to widespread contamination of drinking water sources with both natural and synthetic estrogens, which is a concern for potential ecological and human health effects. In vitro screening assays are valuabl...

Complementing in vitro hazard assessment with exposure and pharmacokinetics considerations for chemical prioritization

EPA Science Inventory

Traditional toxicity testing involves a large investment in resources, often using low-throughput in vivo animal studies for limited numbers of chemicals. An alternative strategy is the emergence of high-throughput (HT) in vitro assays as a rapid, cost-efficient means to screen t...

In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles

EPA Science Inventory

As in vitro assays are increasingly used to screen chemicals for their potential to produce endocrine disrupting adverse effects, it is important to understand their predictive capacity. The potential for a set of six benzothiazoles to affect endpoints related to thyroid hormone ...

Predictive Endocrine Testing in the 21st Century Using In Vitro Assays of Estrogen Receptor Signaling Responses

EPA Science Inventory

Thousands of environmental chemicals are subject to regulatory review for their potential to be endocrine disruptors (ED). In vitro high-throughput screening (HTS) assays have emerged as a potential tool for prioritizing chemicals for ED-related whole-animal tests. In this study,...

Screening larch in vitro for resistance to Mycosphaerella laricina

Treesearch

M.E. Ostry; Paula M. Pijut; D.D. Skilling

1991-01-01

Needle blight of larch caused by Mycosphaerella laricina seriously limits the productivity of susceptible trees in the north central and northeastern United States. Adventitious shoots, derived from cotyledon tissue culture, of selected European larch (Larix decidua) and a hybrid larch were inoculated in vitro with three isolates...

UPLC-ESI-MS/MS and HPTLC Method for Quantitative Estimation of Cytotoxic Glycosides and Aglycone in Bioactivity Guided Fractions of Solanum nigrum L.

PubMed Central

Chester, Karishma; Paliwal, Sarvesh; Khan, Washim; Ahmad, Sayeed

2017-01-01

Solanum nigrum L., is traditionally used for the management of the various liver disorders. Investigating the effect of polarity based fractionation of S. nigrum for its hepatoprotective effect on Hep G2 cells in vitro to provide base of its activity by quantifying in steroidal glycosides responsible for hepatoprotective potential. A new UPLC-ESI-MS/MS method following a high performance thin layer chromatography (HPTLC) has been developed and validated for quantification of steroidal glycosides and aglycone (solasonine, solamargine, and solasodine, respectively). The in vitro antioxidant potential, total phenolics, and flavonoid content were also determined in different fractions. The newly developed UPLC-ESI-MS/MS and HPTLC methods were linear (r2 ≥ 0.99), precise, accurate, and showing recovery more than 97%. The n-butanol enriched fraction of S. nigrum berries was found to be the most potent hepatoprotective fraction against all other fractions as it showed significantly (p < 0.01) better in vitro anti-oxidant potential than other fractions. Quantification by both methods revealed that, content of steroidal glycosides and aglycones are more than 20% in n-butanol fraction as compared to other fractions. The screened steroidal glycoside n-butanol enriched fraction underwent bioefficacy studies against D-galactosamine and H2O2 induced toxicity in HepG2 cell line showing significant (p < 0.05) liver protection. However, developed method can be used for the quality control analysis with respect to targeted metabolites and it can be explored for the pharmacokinetic and pharmacodynamic analysis in future. PMID:28729835

Impact of preimplantation genetic screening on donor oocyte-recipient cycles in the United States.

PubMed

Barad, David H; Darmon, Sarah K; Kushnir, Vitaly A; Albertini, David F; Gleicher, Norbert

2017-11-01

Our objective was to estimate the contribution of preimplantation genetic screening to in vitro fertilization pregnancy outcomes in donor oocyte-recipient cycles. This was a retrospective cross-sectional study of US national data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System between 2005 and 2013. Society for Assisted Reproductive Technology Clinic Outcome Reporting relies on voluntarily annual reports by more than 90% of US in vitro fertilization centers. We evaluated pregnancy and live birth rates in donor oocyte-recipient cycles after the first embryo transfer with day 5/6 embryos. Statistical models, adjusted for patient and donor ages, number of embryos transferred, race, infertility diagnosis, and cycle year were created to compare live birth rates in 392 preimplantation genetic screening and 20,616 control cycles. Overall, pregnancy and live birth rates were significantly lower in preimplantation genetic screening cycles than in control cycles. Adjusted odds of live birth for preimplantation genetic screening cycles were reduced by 35% (odds ratio, 0.65, 95% confidence interval, 0.53-0.80; P < .001). Preimplantation genetic screening, as practiced in donor oocyte-recipient cycles over the past 9 years, has not been associated with improved odds of live birth or reduction in miscarriage rates. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of the Caco-2 permeability assay to screen drug compounds for intestinal absorption and efflux.

PubMed

Press, Barry

2011-01-01

In vitro permeability assays are a valuable tool for scientists during lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, correlation of in vitro permeability data to in vivo absorption results is critical for understanding the structural-physicochemical relationship (SPR) of drugs exhibiting low levels of absorption. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for both intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization. This chapter provides technical information for performing and optimizing the Caco-2 assay. In addition, techniques are discussed for dealing with some of the most pressing issues surrounding in vitro permeability assays (i.e., low aqueous solubility of test compounds and low postassay recovery). Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data.

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

PubMed

Coady, Katherine K; Kan, H Lynn; Schisler, Melissa R; Gollapudi, B Bhaskar; Neal, Barbara; Williams, Amy; LeBaron, Matthew J

2014-08-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reflective lens-free imaging on high-density silicon microelectrode arrays for monitoring and evaluation of in vitro cardiac contractility

PubMed Central

Pauwelyn, Thomas; Stahl, Richard; Mayo, Lakyn; Zheng, Xuan; Lambrechts, Andy; Janssens, Stefan; Lagae, Liesbet; Reumers, Veerle; Braeken, Dries

2018-01-01

The high rate of drug attrition caused by cardiotoxicity is a major challenge for drug development. Here, we developed a reflective lens-free imaging (RLFI) approach to non-invasively record in vitro cell deformation in cardiac monolayers with high temporal (169 fps) and non-reconstructed spatial resolution (352 µm) over a field-of-view of maximally 57 mm2. The method is compatible with opaque surfaces and silicon-based devices. Further, we demonstrated that the system can detect the impairment of both contractility and fast excitation waves in cardiac monolayers. Additionally, the RLFI device was implemented on a CMOS-based microelectrode array to retrieve multi-parametric information of cardiac cells, thereby offering more in-depth analysis of drug-induced (cardiomyopathic) effects for preclinical cardiotoxicity screening applications. PMID:29675322

The design principles of axilla deodorant fragrances.

PubMed

McGee, T; Rankin, K M; Baydar, A

1998-11-30

There are a number of ways that deodorant products control malodor: a) by suppressing sweat, b) by inhibiting bacterial activity, and c) by covering malodor. The paper focuses on the Givaudan Roure methodology used to develop fragrances that effectively cover malodor. Several steps are involved in the development of a successful deodorant fragrance. First, we test for substantivity of the deodorant fragrance material in the axilla, using odor value technology. Second, using an in vitro test with reconstituted axilla odor, we determine the effectiveness of the substantive fragrance material with carefully screened panelists. Third, using a multichannel olfactive blender, the perfumer creates a fragrance heart with effective deodorant fragrance materials that cover malodor in the vapor phase. Finally, the hedonically pleasing heart is used to create the final fragrance, which is then optimized using our in vitro test method.

Patient-derived Models of Human Breast Cancer: Protocols for In vitro and In vivo Applications in Tumor Biology and Translational Medicine

PubMed Central

DeRose, Yoko S.; Gligorich, Keith M.; Wang, Guoying; Georgelas, Ann; Bowman, Paulette; Courdy, Samir J.; Welm, Alana L.; Welm, Bryan E.

2013-01-01

Research models that replicate the diverse genetic and molecular landscape of breast cancer are critical for developing the next generation therapeutic entities that can target specific cancer subtypes. Patient-derived tumorgrafts, generated by transplanting primary human tumor samples into immune-compromised mice, are a valuable method to model the clinical diversity of breast cancer in mice, and are a potential resource in personalized medicine. Primary tumorgrafts also enable in vivo testing of therapeutics and make possible the use of patient cancer tissue for in vitro screens. Described in this unit are a variety of protocols including tissue collection, biospecimen tracking, tissue processing, transplantation, and 3-dimensional culturing of xenografted tissue, that enable use of bona fide uncultured human tissue in designing and validating cancer therapies. PMID:23456611

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

PubMed

Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

Application of Physiologically-Based Pharmacokinetic/Pharmacodynamic Model for Interpretation of High-throughput Screening Assay for Thyroperoxidase Inhibition

EPA Science Inventory

In vitro based assays are used to identify potential endocrine disrupting chemicals. Thyroperoxidase (TPO), an enzyme essential for thyroid hormone (TH) synthesis, is a target site for disruption of the thyroid axis for which a high-throughput screening (HTPS) assay has recently ...

GENOMIC AND PROTEOMIC BASIS FOR INTERSPECIES EXTRAPOLATIONS BASED UPON ESTROGEN AND ANDROGEN RECEPTORSTRUCTURE AND FUNCTION AMONG ANIMALS

EPA Science Inventory

Most in vitro hazard assessments for the screening and identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSP Tier 1 Screening (T1S) protocols, use mammalian steroid hormone receptors. There is uncertainty, however, concerning differences th...

The Use of Purified Rat Leydig Cells Complements the H295R Screen to Detect Chemical Induced Alterations in Testosterone Production

EPA Science Inventory

Exposure to endocrine disrupting contaminants can compromise testosterone production and lead to abnormal male reproductive development and altered spermatogenesis. In vitro high throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the mai...

Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

EPA Science Inventory

Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...

The Use of Purified Rat Leydig Cells Complements the H295R Screen to Detect Chemical-Induced Alterations in Testosterone Production

EPA Science Inventory

Exposure to endocrine disrupting contaminants can compromise testosterone production and lead to abnormal male reproductive development and altered spermatogenesis. In vitro high throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the mai...

Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

EPA Science Inventory

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

Neural Progenitor Cells as Models for High-Throughput Screens of Developmental Neurotoxicity: State of the Science

EPA Science Inventory

In vitro, high-throughput approaches have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramificat...

Comparison of Chemical-induced Changes in Proliferation and Apoptosis in Human and Mouse Neuroprogenitor Cells.***

EPA Science Inventory

There is a need to develop rapid and efficient models to screen chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present study...

COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

EPA Science Inventory

A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

Extrapolating toxicity data across species using U.S. EPA SeqAPASS tool

EPA Science Inventory

In vitro high-throughput screening (HTS) and in silico technologies have emerged as 21st century tools for chemical hazard identification. In 2007 the U.S. Environmental Protection Agency (EPA) launched the ToxCast Program, which has screened thousands of chemicals in hundreds of...

COMPARISON OF CHEMICAL-INDUCED CHANGES IN PROLIFERATION AND APOPTOSIS IN HUMAN AND MOUSE NEUROPROGENITOR CELLS.

EPA Science Inventory

There is a need to develop rapid and efficient models for screening chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present s...

Recent Developments in Toxico-Cheminformatics and Progress Towards a New Paradigm for Predictive Toxicology (2)

EPA Science Inventory

EPAs National Center for Computational Toxicology is building capabilities to support a new paradigm for toxicity screening and prediction through harnessing of legacy toxicity data, creation of data linkages, and generation of new in vitro screening data. In association with EPA...

Recent Developments in Toxico-Cheminformatics and Progress Towards a New Paradigm for Predictive Toxicology

EPA Science Inventory

EPA’s Computational Toxicology Center is building capabilities to support a new paradigm for toxicity screening and prediction through harnessing of legacy toxicity data, creation of data linkages, and generation of new in vitro screening data. In association with EPA’s ToxCastTM...

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Old Story in the Parallel Synthesis World: An Approach to Hydantoin Libraries.

PubMed

Bogolubsky, Andrey V; Moroz, Yurii S; Savych, Olena; Pipko, Sergey; Konovets, Angelika; Platonov, Maxim O; Vasylchenko, Oleksandr V; Hurmach, Vasyl V; Grygorenko, Oleksandr O

2018-01-08

An approach to the parallel synthesis of hydantoin libraries by reaction of in situ generated 2,2,2-trifluoroethylcarbamates and α-amino esters was developed. To demonstrate utility of the method, a library of 1158 hydantoins designed according to the lead-likeness criteria (MW 200-350, cLogP 1-3) was prepared. The success rate of the method was analyzed as a function of physicochemical parameters of the products, and it was found that the method can be considered as a tool for lead-oriented synthesis. A hydantoin-bearing submicromolar primary hit acting as an Aurora kinase A inhibitor was discovered with a combination of rational design, parallel synthesis using the procedures developed, in silico and in vitro screenings.

An enzyme complex increases in vitro dry matter digestibility of corn and wheat in pigs.

PubMed

Park, Kyu Ree; Park, Chan Sol; Kim, Beob Gyun

2016-01-01

Two experiments were conducted to determine the effects of enzyme complex on in vitro dry matter (DM) digestibility for feed ingredients. The objective of experiment 1 was to screen feed ingredients that can be effective substrates for an enzyme complex, mainly consisted of β-pentosanase, β-glucanase and α-amylase, using in vitro digestibility methods. In experiment 1, the test ingredients were three grain sources (barley, corn and wheat) and six protein supplements (canola meal, copra expellers, cottonseed meal, distillers dried grains with solubles, palm kernel expellers and soybean meal). In vitro ileal and total tract digestibility (IVID and IVTTD, respectively) of DM for test ingredients were determined. In vitro digestibility methods consisted of two- or three-step procedure simulating in vivo digestion in the pig gastrointestinal tracts with or without enzyme complex. As the enzyme complex added, the IVID of DM for corn and wheat increased (p < 0.05) by 5.0 and 2.6 percentage unit, respectively. The IVTTD of DM for corn increased (p < 0.05) by 3.1 percentage unit with enzyme complex addition. As the effect of enzyme complex was the greatest in corn digestibility, corn grains were selected to determine the in vitro digestibility of the fractions (starch, germ, hull and gluten) that maximally respond to the enzyme complex in experiment 2. The IVID of DM for corn starch, germ and hull increased (p < 0.05) by 16.0, 2.8 and 1.2 percentage unit, respectively. The IVTTD of DM for corn starch and hull also increased (p < 0.05) by 8.6 and 0.9 percentage unit, respectively, with enzyme complex addition. In conclusion, the enzyme complex increases in vitro DM digestibility of corn and wheat, and the digestibility increments of corn are mainly attributed to the increased digestibility of corn starch.

In-vitro evidence for efficacy of antimicrobial mouthrinses

PubMed Central

Pan, Pauline C.; Harper, Scott; Ricci-Nittel, Danette; Lux, Renate; Shi, Wenyuan

2010-01-01

SUMMARY Objectives The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro. Methods Nine mouthrinses were tested in a recirculating flow-through biofilm model (RFTB) with viability assessment by ATP bioluminescence. In addition, five mouthrinses were evaluated in a batch chamber slide biofilm (BCSB) model, using live- dead staining and confocal laser scanning microscopy. Results In the RFTB model, essential oil (EO) and chlorhexidine (CHX)-containing rinses showed equivalent antimicrobial activity and were more effective than a range of cetyl pyridinium chloride (CPC1) formulations. In the BCSB model, twice-daily mouthrinse exposure demonstrated that the EO rinse was significantly more effective than rinses containing amine and stannous fluorides, a combination of CPC/CHX and CPC2. EO showed biofilm kill comparable to the CHX rinse. Conclusions The present studies have shown that mouthrinses vary significantly in their capability to kill plaque biofilm bacteria in BCSB and RFTB models. The EO mouthrinse demonstrated superior antiplaque biofilm activity to AFSF, CPC/CHX, and CPC rinses and comparable activity to CHX. The methods tested may be of value for the in-vitro screening of antiseptic rinses with different modes of antimicrobial action. PMID:20621239

Novel approach to integrated DNA adductomics for the assessment of in vitro and in vivo environmental exposures.

PubMed

Chang, Yuan-Jhe; Cooke, Marcus S; Hu, Chiung-Wen; Chao, Mu-Rong

2018-06-25

Adductomics is expected to be useful in the characterization of the exposome, which is a new paradigm for studying the sum of environmental causes of diseases. DNA adductomics is emerging as a powerful method for detecting DNA adducts, but reliable assays for its widespread, routine use are currently lacking. We propose a novel integrated strategy for the establishment of a DNA adductomic approach, using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS), operating in constant neutral loss scan mode, screening for both known and unknown DNA adducts in a single injection. The LC-QqQ-MS/MS was optimized using a representative sample of 23 modified 2'-deoxyribonucleosides reflecting a range of biologically relevant DNA lesions. Six internal standards (ISTDs) were evaluated for their ability to normalize, and hence correct, possible variation in peak intensities arising from matrix effects, and the quantities of DNA injected. The results revealed that, with appropriate ISTDs adjustment, any bias can be dramatically reduced from 370 to 8.4%. Identification of the informative DNA adducts was achieved by triggering fragmentation spectra of target ions. The LC-QqQ-MS/MS method was successfully applied to in vitro and in vivo studies to screen for DNA adducts formed following representative environmental exposures: methyl methanesulfonate (MMS) and five N-nitrosamines. Interestingly, five new DNA adducts, induced by MMS, were discovered using our adductomic approach-an added strength. The proposed integrated strategy provides a path forward for DNA adductomics to become a standard method to discover differences in DNA adduct fingerprints between populations exposed to genotoxins, and facilitate the field of exposomics.

In vitro porcine blood-brain barrier model for permeability studies: pCEL-X software pKa(FLUX) method for aqueous boundary layer correction and detailed data analysis.

PubMed

Yusof, Siti R; Avdeef, Alex; Abbott, N Joan

2014-12-18

In vitro blood-brain barrier (BBB) models from primary brain endothelial cells can closely resemble the in vivo BBB, offering valuable models to assay BBB functions and to screen potential central nervous system drugs. We have recently developed an in vitro BBB model using primary porcine brain endothelial cells. The model shows expression of tight junction proteins and high transendothelial electrical resistance, evidence for a restrictive paracellular pathway. Validation studies using small drug-like compounds demonstrated functional uptake and efflux transporters, showing the suitability of the model to assay drug permeability. However, one limitation of in vitro model permeability measurement is the presence of the aqueous boundary layer (ABL) resulting from inefficient stirring during the permeability assay. The ABL can be a rate-limiting step in permeation, particularly for lipophilic compounds, causing underestimation of the permeability. If the ABL effect is ignored, the permeability measured in vitro will not reflect the permeability in vivo. To address the issue, we explored the combination of in vitro permeability measurement using our porcine model with the pKa(FLUX) method in pCEL-X software to correct for the ABL effect and allow a detailed analysis of in vitro (transendothelial) permeability data, Papp. Published Papp using porcine models generated by our group and other groups are also analyzed. From the Papp, intrinsic transcellular permeability (P0) is derived by simultaneous refinement using a weighted nonlinear regression, taking into account permeability through the ABL, paracellular permeability and filter restrictions on permeation. The in vitro P0 derived for 22 compounds (35 measurements) showed good correlation with P0 derived from in situ brain perfusion data (r(2)=0.61). The analysis also gave evidence for carrier-mediated uptake of naloxone, propranolol and vinblastine. The combination of the in vitro porcine model and the software analysis provides a useful tool to better predict BBB permeability in vivo and gain better mechanistic information about BBB permeation. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Integrated in vivo genetic and pharmacologic screening identifies co-inhibition of EGRF and ROCK as a potential treatment regimen for triple-negative breast cancer.

PubMed

Iskit, Sedef; Lieftink, Cor; Halonen, Pasi; Shahrabi, Aida; Possik, Patricia A; Beijersbergen, Roderick L; Peeper, Daniel S

2016-07-12

Breast cancer is the second most common cause of cancer-related deaths worldwide among women. Despite several therapeutic options, 15% of breast cancer patients succumb to the disease owing to tumor relapse and acquired therapy resistance. Particularly in triple-negative breast cancer (TNBC), developing effective treatments remains challenging owing to the lack of a common vulnerability that can be exploited by targeted approaches. We have previously shown that tumor cells have different requirements for growth in vivo than in vitro. Therefore, to discover novel drug targets for TNBC, we performed parallel in vivo and in vitro genetic shRNA dropout screens. We identified several potential drug targets that were required for tumor growth in vivo to a greater extent than in vitro. By combining pharmacologic inhibitors acting on a subset of these candidates, we identified a synergistic interaction between EGFR and ROCK inhibitors. This combination effectively reduced TNBC cell growth by inducing cell cycle arrest. These results illustrate the power of in vivo genetic screens and warrant further validation of EGFR and ROCK as combined pharmacologic targets for breast cancer.

Utilization of in vitro Caco-2 permeability and liver microsomal half-life screens in discovering BMS-488043, a novel HIV-1 attachment inhibitor with improved pharmacokinetic properties.

PubMed

Yang, Zheng; Zadjura, Lisa M; Marino, Anthony M; D'Arienzo, Celia J; Malinowski, Jacek; Gesenberg, Christoph; Lin, Pin-Fang; Colonno, Richard J; Wang, Tao; Kadow, John F; Meanwell, Nicholas A; Hansel, Steven B

2010-04-01

Optimizing pharmacokinetic properties to improve oral exposure is a common theme in modern drug discovery. In the present work, in vitro Caco-2 permeability and microsomal half-life screens were utilized in an effort to guide the structure-activity relationship in order to improve the pharmacokinetic properties of novel HIV-1 attachment inhibitors. The relevance of the in vitro screens to in vivo pharmacokinetic properties was first demonstrated with a number of program compounds at the early stage of lead optimization. The Caco-2 permeability, tested at 200 microM, was quantitatively predictive of in vivo oral absorption, with complete absorption occurring at a Caco-2 permeability of 100 nm/s or higher. The liver microsomal half-life screen, conducted at 1 microM substrate concentration, can readily differentiate low-, intermediate-, and high-clearance compounds in rats, with a nearly 1:1 correlation in 12 out of 13 program compounds tested. Among the >100 compounds evaluated, BMS-488043 emerged as a lead, exhibiting a Caco-2 permeability of 178 nm/s and a microsomal half-life predictive of a low clearance (4 mL/min/kg) in humans. These in vitro characteristics translated well to the in vivo setting. The oral bioavailability of BMS-488043 in rats, dogs, and monkeys was 90%, 57%, and 60%, respectively. The clearance was low in all three species tested, with a terminal half-life ranging from 2.4 to 4.7 h. Furthermore, the oral exposure of BMS-488043 was significantly improved (6- to 12-fold in rats and monkeys) compared to the prototype compound BMS-378806 that had a suboptimal Caco-2 permeability (51 nm/s) and microsomal half-life. More importantly, the improvements in preclinical pharmacokinetics translated well to humans, leading to a >15-fold increase in the human oral exposure of BMS-488043 than BMS-378806 and enabling a clinical proof-of-concept for this novel class of anti-HIV agents. The current studies demonstrated the valuable role of in vitro ADME screens in improving oral pharmacokinetics at the lead optimization stage. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Repositioning FDA Drugs as Potential Cruzain Inhibitors from Trypanosoma cruzi: Virtual Screening, In Vitro and In Vivo Studies.

PubMed

Palos, Isidro; Lara-Ramirez, Edgar E; Lopez-Cedillo, Julio Cesar; Garcia-Perez, Carlos; Kashif, Muhammad; Bocanegra-Garcia, Virgilio; Nogueda-Torres, Benjamin; Rivera, Gildardo

2017-06-18

Chagas disease (CD) is a neglected disease caused by the parasite Trypanosoma cruzi , which affects underdeveloped countries. The current drugs of choice are nifurtimox and benznidazole, but both have severe adverse effects and less effectivity in chronic infections; therefore, the need to discover new drugs is essential. A computer-guided drug repositioning method was applied to identify potential FDA drugs (approved and withdrawn) as cruzain (Cz) inhibitors and trypanocidal effects were confirmed by in vitro and in vivo studies. 3180 FDA drugs were virtually screened using a structure-based approach. From a first molecular docking analysis, a set of 33 compounds with the best binding energies were selected. Subsequent consensus affinity binding, ligand amino acid contact clustering analysis, and ranked position were used to choose four known pharmacological compounds to be tested in vitro. Mouse blood samples infected with trypomastigotes from INC-5 and NINOA strains were used to test the trypanocidal effect of four selected compounds. Among these drugs, one fibrate antilipemic (etofyllin clofibrate) and three β-lactam antibiotics (piperacillin, cefoperazone, and flucloxacillin) showed better trypanocidal effects (LC 50 range 15.8-26.1 μg/mL) in comparison with benznidazole and nifurtimox (LC 50 range 33.1-46.7 μg/mL). A short-term in vivo evaluation of these compounds showed a reduction of parasitemia in infected mice (range 90-60%) at 6 h, but this was low compared to benznidazole (50%). This work suggests that four known FDA drugs could be used to design and obtain new trypanocidal agents.

Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

PubMed Central

Rezania, Alireza; Riedel, Michael J.; Wideman, Rhonda D.; Karanu, Francis; Ao, Ziliang; Warnock, Garth L.; Kieffer, Timothy J.

2011-01-01

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells. PMID:20971966

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine

2006-04-01

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Onlymore » MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.« less

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor

PubMed Central

Judson, Richard S.; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M.; Filer, Dayne L.; Houck, Keith A.; Martin, Matthew T.; Sipes, Nisha; Richard, Ann M.; Mansouri, Kamel; Setzer, R. Woodrow; Knudsen, Thomas B.; Crofton, Kevin M.; Thomas, Russell S.

2015-01-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. PMID:26272952

PRMT5 as a druggable target for glioblastoma therapy.

PubMed

Banasavadi-Siddegowda, Yeshavanth Kumar; Welker, Alessandra M; An, Min; Yang, Xiaozhi; Zhou, Wei; Shi, Guqin; Imitola, Jaime; Li, Chenglong; Hsu, Sigmund; Wang, Jiang; Phelps, Mitch; Zhang, Jianying; Beattie, Christine E; Baiocchi, Robert; Kaur, Balveen

2018-05-18

In spite of standard multimodal therapy consisting of surgical resection followed by radiation and concurrent chemotherapy, prognosis for glioblastoma (GBM) patients remains poor. The identification of both differentiated and undifferentiated "stem cell like" populations in the tumor highlights the significance of finding novel targets that affect the heterogeneous tumor cell population. Protein arginine methyltransferase 5 (PRMT5) is one such candidate gene whose nuclear expression correlates with poor survival and has been reported to be required for survival of differentiated GBM cells and self-renewal of undifferentiated GBM cells. In the current study we screened the specificity and efficacy of 4 novel PRMT5 inhibitors in the treatment of GBM. Efficacies of these inhibitors were screened using an in vitro GBM neurosphere model and an in vivo intracranial zebrafish model of glioma. Standard molecular biology methods were employed to investigate changes in cell cycle, growth, and senescence. In vitro and in vivo studies revealed that among the 4 PRMT5 inhibitors, treatment of GBM cells with compound 5 (CMP5) mirrored the effects of PRMT5 knockdown wherein it led to apoptosis of differentiated GBM cells and drove undifferentiated primary patient derived GBM cells into a nonreplicative senescent state. In vivo antitumor efficacy combined with the specificity of CMP5 underscores the importance of developing it for translation.

High-Throughput Toxicity Testing: New Strategies for ...

EPA Pesticide Factsheets

In recent years, the food industry has made progress in improving safety testing methods focused on microbial contaminants in order to promote food safety. However, food industry toxicologists must also assess the safety of food-relevant chemicals including pesticides, direct additives, and food contact substances. With the rapidly growing use of new food additives, as well as innovation in food contact substance development, an interest in exploring the use of high-throughput chemical safety testing approaches has emerged. Currently, the field of toxicology is undergoing a paradigm shift in how chemical hazards can be evaluated. Since there are tens of thousands of chemicals in use, many of which have little to no hazard information and there are limited resources (namely time and money) for testing these chemicals, it is necessary to prioritize which chemicals require further safety testing to better protect human health. Advances in biochemistry and computational toxicology have paved the way for animal-free (in vitro) high-throughput screening which can characterize chemical interactions with highly specific biological processes. Screening approaches are not novel; in fact, quantitative high-throughput screening (qHTS) methods that incorporate dose-response evaluation have been widely used in the pharmaceutical industry. For toxicological evaluation and prioritization, it is the throughput as well as the cost- and time-efficient nature of qHTS that makes it

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by In Silico Modeling and Virtual Screening Strategies to Combat Early Blight

NASA Astrophysics Data System (ADS)

Iftikhar, Sehrish; Shahid, Ahmad A.; Halim, Sobia A.; Wolters, Pieter J.; Vleeshouwers, Vivianne G. A. A.; Khan, Ajmal; Al-Harrasi, Ahmed; Ahmad, Shahbaz

2017-11-01

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening protocol. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify new and novel fungicides.

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by in Silico Modeling and Virtual Screening Strategies to Combat Early Blight

PubMed Central

Iftikhar, Sehrish; Shahid, Ahmad A.; Halim, Sobia A.; Wolters, Pieter J.; Vleeshouwers, Vivianne G. A. A.; Khan, Ajmal; Al-Harrasi, Ahmed; Ahmad, Shahbaz

2017-01-01

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify novel fungicides. PMID:29204422

Synthesis and characterization of a novel inhibitor of C-reactive protein-mediated proinflammatory effects.

PubMed

Kumaresan, Pappanaicken R; Devaraj, Sridevi; Huang, Wenzhe; Lau, Edmond Y; Liu, Ruiwu; Lam, Kit S; Jialal, Ishwarlal

2013-06-01

Numerous studies have shown that high C-reactive protein (CRP) levels predict cardiovascular disease and augur a poor prognosis in patients with acute coronary syndromes. Much in vitro and in vivo data support of a role for CRP in atherogenesis. There is an urgent need to develop inhibitors that specifically block the biological effects of CRP in vivo. The one-bead-one-compound (OBOC) combinatorial library method has been used to discover ligands against several biological targets. In this study, we use a novel fluorescence-based screening method to screen an OBOC combinatorial library for the discovery of peptides against human CRP. Human CRP was labeled with fluorescein isothiocyanate (FITC) and human serum albumin (HuSA) was labeled with phycoerythrin (PE) and used for screening. The OBOC library LWH-01 was synthesized on TentaGel resin beads using a standard solid-phase "split/mix" approach. By subtraction screening, eight peptides that bind specifically to CRP and not to HuSA were identified. In human aortic endothelial cells (HAECs) incubated with CRP, inhibitors CRPi-2, CRPi-3, and CRPi-6 significantly inhibited CRP-induced superoxide, cytokine release, and nuclear factor-κB (NFκB) activity. Molecular docking studies demonstrate that CRPi-2 interacts with the two Ca(2+) ions in the single subunit of CRP. The binding of CRPi-2 is reminiscent of choline binding. Future studies will examine the utility of this inhibitor in animal models and clinical trials.

Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration–HPLC-MS

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Gymnema sylvestre R. Br. (Asclepiadaceae) has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control) at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS) was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre. In this way, 9 compounds with higher enrichment factors (EFs) were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS) platform in the early anti-diabetic drug discovery stage. PMID:28496409

Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration-HPLC-MS.

PubMed

Chen, Guilin; Guo, Mingquan

2017-01-01

Gymnema sylvestre R. Br. (Asclepiadaceae) has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC 50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control) at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS) was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre . In this way, 9 compounds with higher enrichment factors (EFs) were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS) platform in the early anti-diabetic drug discovery stage.

Linking ToxCast Signatures with Functional Consequences: Proof-of-Concept Study using Known Inhibitors of Vascular Development

EPA Science Inventory

The USEPA’s ToxCast program is developing a novel approach to chemical toxicity testing using high-throughput screening (HTS) assays to rapidly test thousands of chemicals against hundreds of in vitro molecular targets. This approach is based on the premise that in vitro HTS bioa...

Identifying Functionally Linked Gene Modules Within Biological Pathways Assessed by ToxCast In Vitro Assays

EPA Science Inventory

The US EPA ToxCast program is using in vitro high-throughput screening assays to profile the bioactivity of environmental chemicals, with the ultimate goal of predicting in vivo toxicity. We hypothesize that in modeling toxicity it will be more constructive to understand the pert...

Human Pluripotent Stem Cell-Based Assay Predicts Developmental Toxicity Potential of ToxCast Chemicals (ACT meeting)

EPA Science Inventory

Worldwide initiatives to screen for toxicity potential among the thousands of chemicals currently in use require inexpensive and high-throughput in vitro models to meet their goals. The devTOX quickPredict platform is an in vitro human pluripotent stem cell-based assay used to as...

Virtual Liver: Evaluating the Impact of Hepatic Microdosimetry for ToxCast Chemicals

EPA Science Inventory

The U.S. EPA’s ToxCastTM program uses hundreds of high-throughput, in vitro assays to screen chemicals for potential toxicity. The assays are used to probe in vitro concentrations at which target cellular pathways and processes are perturbed by these chemicals. The U.S. EPA’s Vir...

Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production.

PubMed

Engelmann, Nancy J; Campbell, Jessica K; Rogers, Randy B; Rupassara, S Indumathie; Garlick, Peter J; Lila, Mary Ann; Erdman, John W

2010-09-22

Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors' knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

PubMed Central

2011-01-01

Background Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. Results Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. Conclusion In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints. PMID:21345205

Chemometrics Optimized Extraction Procedures, Phytosynergistic Blending and in vitro Screening of Natural Enzyme Inhibitors Amongst Leaves of Tulsi, Banyan and Jamun

PubMed Central

De, Baishakhi; Bhandari, Koushik; Singla, Rajeev K.; Katakam, Prakash; Samanta, Tanmoy; Kushwaha, Dilip Kumar; Gundamaraju, Rohit; Mitra, Analava

2015-01-01

Background: Tulsi, Banyan, and Jamun are popular Indian medicinal plants with notable hypoglycemic potentials. Now the work reports chemo-profiling of the three species with in-vitro screening approach for natural enzyme inhibitors (NEIs) against enzymes pathogenic for type 2 diabetes. Further along with the chemometrics optimized extraction process technology, phyto-synergistic studies of the composite polyherbal blends have also been reported. Objective: Chemometrically optimized extraction procedures, ratios of polyherbal composites to achieve phyto-synergistic actions, and in-vitro screening of NEIs amongst leaves of Tulsi, Banyan, and Jamun. Materials and Methods: The extraction process parameters of the leaves of three plant species (Ficus benghalensis, Syzigium cumini and Ocimum sanctum) were optimized by rotatable central composite design of chemometrics so as to get maximal yield of bio-actives. Phyto-blends of three species were prepared so as to achieve synergistic antidiabetic and antioxidant potentials and the ratios were optimized by chemometrics. Next, for in vitro screening of natural enzyme inhibitors the individual leaf extracts as well as composite blends were subjected to assay procedures to see their inhibitory potentials against the enzymes pathogenic in type 2 diabetes. The antioxidant potentials were also estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay. Results: Considering response surface methodology studies and from the solutions obtained using desirability function, it was found that hydro-ethanolic or methanolic solvent ratio of 52.46 ± 1.6 and at a temperature of 20.17 ± 0.6 gave an optimum yield of polyphenols with minimal chlorophyll leaching. The species also showed the presence of glycosides, alkaloids, and saponins. Composites in the ratios of 1:1:1 and 1:1:2 gave synergistic effects in terms of polyphenol yield and anti-oxidant potentials. All composites (1:1:1, 1:2:1, 2:1:1, 1:1:2) showed synergistic anti-oxidant actions. Inhibitory activities against the targeted enzymes expressed in terms of IC50 values have shown that hydro-ethanolic extracts in all cases whether individual species or composites in varying ratios gave higher IC50 values thus showing greater effectivity. Conclusion: Current research provides the state-of-the-art of search of NEIs amongst three species by in-vitro assays which can be further utilized for bioactivity-guided isolations of such enzyme inhibitors. Further, it reports the optimized phyto-blend ratios so as to achieve synergistic anti-oxidative actions. SUMMARY The current research work focuses on the optimization of the extraction process parameters and the ratios of phyto-synergistic blends of the leaves of three common medicinal plants viz. banyan, jamun and tulsi by chemometrics. Qualitative and quantitative chemo profiling of the extracts were done by different phytochemical tests and UV spectrophotometric methods. Enzymes like alpha amylase, alpha glucosidase, aldose reductase, dipeptidyl peptidase 4, angiotensin converting enzymes are found to be pathogenic in type 2 diabetes. In vitro screening of natural enzyme inhibitors amongst individual extracts and composite blends were carried out by different assay procedures and the potency expressed in terms of IC50 values. Antioxidant potentials were estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay. Hydroalcoholic solvent (50:50) gave maximal yield of bio-actives with minimal chlorophyll leaching. Hydroethanolic extract of tulsi showed maximal antioxidant effect. Though all composites showed synergism, maximal effects were shown by the composite (1:1:2) in terms of polyphenol yield, antioxidant effect and inhibitory actions against the targeted enzymes. Abbreviations used: DPP4- dipeptidyl peptidase 4; AR- aldose reductase; ACE- angiotensin converting enzyme; PPAR-γ- peroxisome proliferator activated receptor-γ; NEIs- natural enzyme inhibitors; BE- binding energy; GLP-1- Glucagon like peptide -1; ROS- Reactive oxygen species; CAT- catalase; GSH-Px- glutathione per-oxidase; SOD- superoxide dismutase; pNPG- para-nitro phenyl-α-D-gluco-pyranoside solution; DPPH- 1,1-diphenyl-2-picrylhydrazyl; RSM- Response surface methodology; CCD- central composite design; DMSO- dimethyl sulfoxide; HHL- hippuryl-L-histidyl-L-leucine; GPN-Tos- Gly-Pro p-nitroanilide toluenesulfonate salt; ESC- experimental scavenging capacity; TSC- theoretical scavenging capacity; FRAP- Ferric Reducing Assay Procedure; ABTS- 2, 2’- azinobis (3-ethylbenzothiazoline-6 – sulfonic acid. PMID:27013789

Cancer CRISPR Screens In Vivo.

PubMed

Chow, Ryan D; Chen, Sidi

2018-05-01

Clustered regularly interspaced short palindromic repeats (CRISPR) screening is a powerful toolset for investigating diverse biological processes. Most CRISPR screens to date have been performed with in vitro cultures or cellular transplant models. To interrogate cancer in animal models that more closely recapitulate the human disease, autochthonous direct in vivo CRISPR screens have recently been developed that can identify causative drivers in the native tissue microenvironment. By empowering multiplexed mutagenesis in fully immunocompetent animals, direct in vivo CRISPR screens enable the rapid generation of patient-specific avatars that can guide precision medicine. This Opinion article discusses the current status of in vivo CRISPR screens in cancer and offers perspectives on future applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Generation of a Close-to-Native In Vitro System to Study Lung Cells-Extracellular Matrix Crosstalk.

PubMed

Garlíková, Zuzana; Silva, Ana Catarina; Rabata, Anas; Potěšil, David; Ihnatová, Ivana; Dumková, Jana; Koledová, Zuzana; Zdráhal, Zbyněk; Vinarský, Vladimír; Hampl, Aleš; Pinto-do-Ó, Perpétua; Nascimento, Diana Santos

2018-01-01

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

PubMed

Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

In silico, in vitro and in vivo analyses of dipeptidyl peptidase IV inhibitory activity and the antidiabetic effect of sodium caseinate hydrolysate.

PubMed

Hsieh, Cheng-Hong; Wang, Tzu-Yuan; Hung, Chuan-Chuan; Jao, Chia-Ling; Hsieh, You-Liang; Wu, Si-Xian; Hsu, Kuo-Chiang

2016-02-01

The frequency (A), a novel in silico parameter, was developed by calculating the ratio of the number of truncated peptides with Xaa-proline and Xaa-alanine to all peptide fragments from a protein hydrolyzed with a specific protease. The highest in vitro DPP-IV inhibitory activity (72.7%) was observed in the hydrolysate of sodium caseinate by bromelain (Cas/BRO), and the constituent proteins of bovine casein also had relatively high A values (0.10-0.17) with BRO hydrolysis. 1CBR (the <1 kDa fraction of Cas/BRO) showed the greatest in vitro DPP-IV inhibitory activity of 77.5% and was used for in vivo test by high-fat diet-fed and low-dose streptozotocin-induced diabetic rats. The daily administration of 1CBR for 6 weeks was effective to improve glycaemic control in diabetic rats. The results indicate that the novel in silico method has the potential as a screening tool to predict dietary proteins to generate DPP-IV inhibitory and antidiabetic peptides.

US EPA - ToxCast and the Tox21 program: perspectives

EPA Science Inventory

ToxCast is a large-scale project being conducted by the U.S. EPA to screen ~2000 chemicals against a large battery of in vitro high-throughput screening (HTS) assays. ToxCast is complemented by the Tox21 project being jointly carried out by the U.S. NIH Chemical Genomics Center (...

RAN Translation as a Therapeutic in ALS

DTIC Science & Technology

2017-05-01

allow for HTS via CRISPR or drug screens to complement the in vitro screens using high-throughput microscopy or FACS. Figure 5: Mammalian G4C2...poly-GP in yeast (Figure 6A). [filler about RPS25 here?] This effect was further investigated in mammalian Hap1 cell lines with a CRISPR -mediated

SPECIES DIFFERENCES IN ANDROGEN AND ESTROGEN RECEPTOR STRUCTURE AND FUNCTION AMONG VERTEBRATES AND INVERTEBRATES FOR INTERSPECIES EXTRAPOLATION OF ENDOCRINE DISRUPTING CHEMICALS

EPA Science Inventory

In vitro screening assays designed to identify hormone minics or antagonists, including the EDSTAC Tier 1 Screening (TIS) Battery, typically use only mammalian estrogen (ER) and androgen receptors (AR). However, there is uncertainty concerning species differences in binding affin...

Using In Vitro High-Throughput Screening Data for Predicting Benzo[k]Fluoranthene Human Health Hazards

EPA Science Inventory

Today there are more than 80,000 chemicals in commerce and the environment. The potential human health risks are unknown for the vast majority of these chemicals as they lack human health risk assessments, toxicity reference values and risk screening values. We aim to use computa...

Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

EPA Science Inventory

An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

Unique Nanoparticle Optical Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Screening and Ranking

EPA Science Inventory

Nanoparticles (NPs) are novel materials having at least one dimension less than 100 nm and display unique physicochemical properties due to their nanoscale size. An emphasis has been placed on developing high throughput screening (HTS) assays to characterize and rank the toxiciti...

Extrapolation of mammalian-based ToxCast assay results to non-mammalian species to evaluate endocrine disruption

EPA Science Inventory

In vitro high-throughput screening (HTS) and in silico technologies have emerged as 21st century tools for chemical hazard identification. In 2007 the U.S. Environmental Protection Agency (EPA) launched the ToxCast Program, which has screened thousands of chemicals in hundreds of...

Cross-species extrapolation of mammalian-based ToxCast Data using Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS)

EPA Science Inventory

In vitro high-throughput screening (HTS) and in silico technologies have emerged as 21st century tools for chemical hazard identification. In 2007 the U.S. Environmental Protection Agency (EPA) launched the ToxCast Program, which has screened thousands of chemicals in hundreds of...

New Chemical/Biological Profiling and Informatics Approaches for Exploring Mutagenicity & Carcinogenicity: Updates of EPA ToxCast and Tox21 Programs

EPA Science Inventory

EPA’s National Center for Computational Toxicology is building capabilities to support a new paradigm for toxicity screening and prediction through harnessing of legacy toxicity data, creation of data linkages, and generation of new in vitro screening data. In association with EP...

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation

PubMed Central

Wells, Dagan; Kaur, Kulvinder; Grifo, Jamie; Glassner, Michael; Taylor, Jenny C; Fragouli, Elpida; Munne, Santiago

2014-01-01

Background The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Methods Embryo biopsy, whole genome amplification and semiconductor sequencing. Results A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. Conclusions This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability. PMID:25031024

Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes.

PubMed

Stadelmann, Britta; Scholl, Sabrina; Müller, Joachim; Hemphill, Andrew

2010-03-01

The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans▿

PubMed Central

Fu, Yue; Luo, Guanpingsheng; Spellberg, Brad J.; Edwards, John E.; Ibrahim, Ashraf S.

2008-01-01

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors. PMID:18178776

Evaluation of Pharmacokinetic Assumptions Using a 443 ...

EPA Pesticide Factsheets

With the increasing availability of high-throughput and in vitro data for untested chemicals, there is a need for pharmacokinetic (PK) models for in vitro to in vivo extrapolation (IVIVE). Though some PBPK models have been created for individual compounds using in vivo data, we are now able to rapidly parameterize generic PBPK models using in vitro data to allow IVIVE for chemicals tested for bioactivity via high-throughput screening. However, these new models are expected to have limited accuracy due to their simplicity and generalization of assumptions. We evaluated the assumptions and performance of a generic PBPK model (R package “httk”) parameterized by a library of in vitro PK data for 443 chemicals. We evaluate and calibrate Schmitt’s method by comparing the predicted volume of distribution (Vd) and tissue partition coefficients to in vivo measurements. The partition coefficients are initially over predicted, likely due to overestimation of partitioning into phospholipids in tissues and the lack of lipid partitioning in the in vitro measurements of the fraction unbound in plasma. Correcting for phospholipids and plasma binding improved the predictive ability (R2 to 0.52 for partition coefficients and 0.32 for Vd). We lacked enough data to evaluate the accuracy of changing the model structure to include tissue blood volumes and/or separate compartments for richly/poorly perfused tissues, therefore we evaluated the impact of these changes on model

In vitro screening for population variability in toxicity of pesticide-containing mixtures

PubMed Central

Abdo, Nour; Wetmore, Barbara A.; Chappell, Grace A.; Shea, Damian; Wright, Fred A.; Rusyna, Ivan

2016-01-01

Population-based human in vitro models offer exceptional opportunities for evaluating the potential hazard and mode of action of chemicals, as well as variability in responses to toxic insults among individuals. This study was designed to test the hypothesis that comparative population genomics with efficient in vitro experimental design can be used for evaluation of the potential for hazard, mode of action, and the extent of population variability in responses to chemical mixtures. We selected 146 lymphoblast cell lines from 4 ancestrally and geographically diverse human populations based on the availability of genome sequence and basal RNA-seq data. Cells were exposed to two pesticide mixtures – an environmental surface water sample comprised primarily of organochlorine pesticides and a laboratory-prepared mixture of 36 currently used pesticides – in concentration response and evaluated for cytotoxicity. On average, the two mixtures exhibited a similar range of in vitro cytotoxicity and showed considerable inter-individual variability across screened cell lines. However, when in vitroto-in vivo extrapolation (IVIVE) coupled with reverse dosimetry was employed to convert the in vitro cytotoxic concentrations to oral equivalent doses and compared to the upper bound of predicted human exposure, we found that a nominally more cytotoxic chlorinated pesticide mixture is expected to have greater margin of safety (more than 5 orders of magnitude) as compared to the current use pesticide mixture (less than 2 orders of magnitude) due primarily to differences in exposure predictions. Multivariate genome-wide association mapping revealed an association between the toxicity of current use pesticide mixture and a polymorphism in rs1947825 in C17orf54. We conclude that a combination of in vitro human population-based cytotoxicity screening followed by dosimetric adjustment and comparative population genomics analyses enables quantitative evaluation of human health hazard from complex environmental mixtures. Additionally, such an approach yields testable hypotheses regarding potential toxicity mechanisms. PMID:26386728

Interference of alpha-hemolytic streptococci isolated from tonsillar surface on beta-hemolytic streptococci (Streptococcus pyogenes)--a methodological study.

PubMed

Grahn, E; Holm, S E; Ekedahl, C; Roos, K

1983-07-01

The interference between alpha-streptococcal strains obtained from patients with repeated tonsillitis and a collection of group A streptococcal strains were studied. For this purpose three in vitro methods were designed and compared. The results obtained by a simple plating technique suitable for screening purposes were found to correlate well with those using more laborious techniques. In a limited scale some of the alpha- and beta-streptococcal combinations were tested under in vivo conditions using a tissue cage model allowing repeated sampling. In most instances agreement between the results of the in vitro and in vivo methods was registered. Several alpha-strains having inhibitory capacity to the majority of a collection of group A streptococci belonging to different serotypes were found, but also alpha-strains with an inhibitory activity restricted to few group A isolates within a certain serotype. Also beta-streptococcal strains with growth inhibiting activity towards some alpha-strains were found. As the methods were chosen to eliminate many of the unspecific inhibitory factors and the beta-hemolytic test strains showed a pattern of inhibition that varied for each of the reference alpha-strains the activity is most likely attributed to bacteriocin-like substances.

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

PubMed

Kang, Homan; Jeong, Sinyoung; Jo, Ahla; Chang, Hyejin; Yang, Jin-Kyoung; Jeong, Cheolhwan; Kyeong, San; Lee, Youn Woo; Samanta, Animesh; Maiti, Kaustabh Kumar; Cha, Myeong Geun; Kim, Taek-Keun; Lee, Sukmook; Jun, Bong-Hyun; Chang, Young-Tae; Chung, Junho; Lee, Ho-Young; Jeong, Dae Hong; Lee, Yoon-Sik

2018-02-01

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery

PubMed Central

Jiménez-Díaz, María Belén; Viera, Sara; Ibáñez, Javier; Mulet, Teresa; Magán-Marchal, Noemí; Garuti, Helen; Gómez, Vanessa; Cortés-Gil, Lorena; Martínez, Antonio; Ferrer, Santiago; Fraile, María Teresa; Calderón, Félix; Fernández, Esther; Shultz, Leonard D.; Leroy, Didier; Wilson, David M.; García-Bustos, José Francisco; Gamo, Francisco Javier; Angulo-Barturen, Iñigo

2013-01-01

The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task. PMID:23825598

Screening of Potential Free Radicals Scavenger and Antibacterial Activities of Purwoceng (Pimpinella alpina Molk).

PubMed

Wahyuningrum, Retno; Utami, Pri Iswati; Dhiani, Binar Asrining; Kumalasari, Malikhah; Kusumawardani, Rizka Sari

2016-11-01

Purwoceng ( Pimpinella alpina Molk) is a traditional medicinal plant used for its aphrodisiac values. This plant was originated Dieng Plateu, Central Java, Indonesia. Purwoceng has been reported to contain steroid, flavonoids, glycoside, saponins, tannins, and phenolic. Based on secondary metabolite compounds of Purwoceng herbs, a research need to be done to determine the other potential free radicals scavenger and antibacterial activities of Purwoceng. The objectives of this research are to screen the potential free radicals scavenger activity of in vitro using DPPH (1,1 diphenyl-2-picryl-hydrazil) radicals and NO• (nitric oxide) radicals, and antibacterial activity of Purwoceng. The extraction is done by a maceration method with petroleum ether, ethyl acetate, and ethanol solvent, respectively. Free radicals scavenger test was performed using DPPH radicals and NO• radicals, while antibacterial activity screening was performed using agar diffusion test. The results showed that ethyl acetate extract of Purwoceng has free radical scavenger activity with IC50 53.07 ppm lower than butylated hydroxytoluene. Ethyl acetate extract and ethanol extract of Purwoceng have antibacterial activity against Staphyloccus aureus , Escherichia coli , and MG42 bacterial isolate.

Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications.

PubMed

Kobayashi, Hiroki; Harada, Hiroko; Nakamura, Masaomi; Futamura, Yushi; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-Ichiro; Shin-Ya, Kazuo; Doi, Takayuki; Takahashi, Takashi; Natsume, Tohru; Imoto, Masaya; Sakakibara, Yasubumi

2012-04-05

Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

Using genetic methods to define the targets of compounds with antimalarial activity

PubMed Central

Flannery, Erika L.; Fidock, David A.; Winzeler, Elizabeth A.

2013-01-01

Although phenotypic cellular screening has been used to drive antimalarial drug discovery in recent years, in some cases target-based drug discovery remains more attractive. This is especially true when appropriate high-throughput cellular assays are lacking, as is the case for drug discovery efforts that aim to provide a replacement for primaquine (4-N-(6-methoxyquinolin-8-yl)pentane-1,4-diamine), the only drug that can block Plasmodium transmission to Anopheles mosquitoes and eliminate liver-stage hypnozoites. At present, however, there are no known chemically validated parasite protein targets that are important in all Plasmodium parasite developmental stages and that can be used in traditional biochemical compound screens. We propose that a plethora of novel, chemically validated, cross-stage antimalarial targets still remain to be discovered from the ~5,500 proteins encoded by the Plasmodium genomes. Here we discuss how in vitro evolution of drug-resistant strains of Plasmodium falciparum and subsequent whole-genome analysis can be used to find the targets of some of the many compounds discovered in whole-cell phenotypic screens. PMID:23927658

A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

PubMed

Wang, Yuru; Havel, Jocelyn; Beal, Peter A

2015-11-20

Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

PubMed

Röhm, Martina; Carle, Stefan; Maigler, Frank; Flamm, Johannes; Kramer, Viktoria; Mavoungou, Chrystelle; Schmid, Otmar; Schindowski, Katharina

2017-10-30

Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Preliminary phytochemical screening and In vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC

PubMed Central

2010-01-01

Background Many oxidative stress related diseases are as a result of accumulation of free radicals in the body. A lot of researches are going on worldwide directed towards finding natural antioxidants of plants origins. The aims of this study were to evaluate in vitro antioxidant activities and to screen for phytochemical constituents of Helichrysum longifolium DC. [Family Asteraceae] aqueous crude extract. Methods We assessed the antioxidant potential and phytochemical constituents of crude aqueous extract of Helichrysum longifolium using tests involving inhibition of superoxide anions, DPPH, H2O2, NO and ABTS. The flavonoid, proanthocyanidin and phenolic contents of the extract were also determined using standard phytochemical reaction methods. Results Phytochemical analyses revealed the presence of tannins, flavonoids, steroids and saponins. The total phenolic content of the aqueous leaf extract was 0.499 mg gallic acid equivalent/g of extract powder. The total flavonoid and proanthocyanidin contents of the plant were 0.705 and 0.005 mg gallic acid equivalent/g of extract powder respectively. The percentage inhibition of lipid peroxide at the initial stage of oxidation showed antioxidant activity of 87% compared to those of BHT (84.6%) and gallic acid (96%). Also, the percentage inhibition of malondialdehyde by the extract showed percentage inhibition of 78% comparable to those of BHT (72.24%) and Gallic (94.82%). Conclusions Our findings provide evidence that the crude aqueous extract of H. longifolium is a potential source of natural antioxidants, and this justified its uses in folkloric medicines. PMID:20470421

Characterization of Estrogen and Androgen Activity of Food Contact Materials by Different In Vitro Bioassays (YES, YAS, ERα and AR CALUX) and Chromatographic Analysis (GC-MS, HPLC-MS)

PubMed Central

Osorio, Veronica; Grininger, Angelika; Richter, Alexander; Bergmair, Johannes; Pyerin, Michael; Washüttl, Michael; Tacker, Manfred

2014-01-01

Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results. PMID:25000404

In vitro screening for potential chemical inhibitors of ...

EPA Pesticide Factsheets

Control of thyroid hormone (TH) signaling in vertebrates is dependent upon multiple key events including iodide uptake, hormone synthesis, metabolism and elimination, to maintain proper homeostasis of the hormones. Deiodinase enzymes interconvert THs between less active and more active forms via release of iodide from the substrate hormones. The activity of deiodinases has been identified as an important endpoint to include in the context of screening chemicals for thyroid hormone disruption. To address the lack of data regarding the potential for chemicals to inhibit these enzymes a research effort was initially focused on human deiodinase type 1 (D1). We utilized an adenovirus expression system for production of D1 enzyme, established robust assay parameters for non-radioactive determination of iodide release by the Sandell-Kolthoff method, and employed a 96-well plate format for screening chemical libraries. An initial set of 19 chemicals was used to establish the assay. Included in this set was the known D1 inhibitor 6-propylthiouracil (used as a positive control). Over 1800 unique chemicals primarily from the EPA’s ToxCast phase 1_v2, phase 2, and e1K chemical libraries were tested in the screening assay. Chemicals were initially screened at a single high concentration of 200 µM to identify potential D1 inhibitors. The majority of the chemicals did not inhibit D1 activity in this initial screen as defined as a response of less than 20% inhibition c

Novel 2-Phenoxyanilide Congeners Derived from a Hit Structure of the TCAMS: Synthesis and Evaluation of Their in Vitro Activity against Plasmodium falciparum.

PubMed

Weidner, Thomas; Nasereddin, Abed; Preu, Lutz; Grünefeld, Johann; Dzikowski, Ron; Kunick, Conrad

2016-02-17

The Tres Cantos Antimalarial Compound Set (TCAMS) is a publicly available compound library which contains 13533 hit structures with confirmed activity against Plasmodium falciparum, the infective agent responsible for malaria tropica. The TCAMS provides a variety of starting points for the investigation of new antiplasmodial drug leads. One of the promising compounds is TCMDC-137332, which seemed to be a good starting point due to its antiplasmodial potency and its predicted physicochemical properties. Several new analogues based on a 2-phenoxyanilide scaffold were synthesized by standard amide coupling reactions and were fully characterized regarding their identity and purity by spectroscopic and chromatographic methods. Furthermore, the results of the biological evaluation of all congeners against Plasmodium falciparum NF54 strains are presented. The findings of our in vitro screening could not confirm the presumed nanomolar antiplasmodial activity of TCMDC-137332 and its derivatives.

[Study on active constituents of traditional Chinese medicine reversing multidrug resistance of tumor cells in vitro].

PubMed

Zhang, H; Yang, L; Liu, S; Ren, L

2001-09-01

To screen drugs reversing multidrug resistance of tumor cells from active constituents of traditional Chinese medicine and to study the reversal action. The kill effects of the drugs on tumor cell lines in vitro were determined with MTT method. The Jin's formula was used to analyse the effect of drug combination. 5 micrograms/ml rhynchophylline, 2 micrograms/ml jatrorrhizine and 1.25 micrograms/ml indirulin could reverse multidrug resistance for vincristine on KBv200 cell line by 16.8, 5.1 and 4 fold respectively. 1.56-12.5 micrograms/ml curcumine combining with vincristine could sensitize antitumor effect both on KB and KBv200 cell lines. All rhynchophylline, jatrorrhizine and indirulin could reverse multidrug resistance for vincristine on KBv200 cell line. Curcumine combinating vincristine could sensitize antitumor effect both on kB and kBv200 cell lines.

Chemotaxonomic Characterization and in-Vitro Antimicrobial and Cytotoxic Activities of the Leaf Essential Oil of Curcuma longa Grown in Southern Nigeria

PubMed Central

Essien, Emmanuel E.; Newby, Jennifer Schmidt; Walker, Tameka M.; Setzer, William N.; Ekundayo, Olusegun

2015-01-01

Curcuma longa (turmeric) has been used in Chinese traditional medicine and Ayurvedic medicine for many years. Methods: The leaf essential oil of C. longa from southern Nigeria was obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS). The essential oil was screened for in vitro antibacterial, antifungal, and cytotoxic activities. The major components in C. longa leaf oil were ar-turmerone (63.4%), α-turmerone (13.7%), and β-turmerone (12.6%). A cluster analysis has revealed this to be a new essential oil chemotype of C. longa. The leaf oil showed notable antibacterial activity to Bacillus cereus and Staphylococcus aureus, antifungal activity to Aspergillus niger, and cytotoxic activity to Hs 578T (breast tumor) and PC-3 (prostate tumor) cells. The ar-turmerone-rich leaf essential oil of C. longa from Nigeria has shown potent biological activity and therapeutic promise. PMID:28930216

Multi-component identification and target cell-based screening of potential bioactive compounds in toad venom by UPLC coupled with high-resolution LTQ-Orbitrap MS and high-sensitivity Qtrap MS.

PubMed

Ren, Wei; Han, Lingyu; Luo, Mengyi; Bian, Baolin; Guan, Ming; Yang, Hui; Han, Chao; Li, Na; Li, Tuo; Li, Shilei; Zhang, Yangyang; Zhao, Zhenwen; Zhao, Haiyu

2018-04-28

Traditional Chinese medicines (TCMs) are undoubtedly treasured natural resources for discovering effective medicines in treating and preventing various diseases. However, it is still extremely difficult for screening the bioactive compounds due to the tremendous constituents in TCMs. In this work, the chemical composition of toad venom was comprehensively analyzed using ultra-high performance liquid chromatography (UPLC) coupled with high-resolution LTQ-Orbitrap mass spectrometry and 93 compounds were detected. Among them, 17 constituents were confirmed by standard substances and 8 constituents were detected in toad venom for the first time. Further, a compound database of toad venom containing the fullest compounds was further constructed using UPLC coupled with high-sensitivity Qtrap MS. Then a target cell-based approach for screening potential bioactive compounds from toad venom was developed by analyzing the target cell extracts. The reliability of this method was validated by negative controls and positive controls. In total, 17 components in toad venom were discovered to interact with the target cancer cells. Further, in vitro pharmacological trials were performed to confirm the anti-cancer activity of four of them. The results showed that the six bufogenins and seven bufotoxins detected in our research represented a promising resource to explore bufogenins/bufotoxins-based anticancer agents with low cardiotoxic effect. The target cell-based screening method coupled with the compound database of toad venom constructed by UPLC-Qtrap-MS with high sensitivity provide us a new strategy to rapidly screen and identify the potential bioactive constituents with low content in natural products, which was beneficial for drug discovery from other TCMs. ᅟ Graphical abstract.

Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

PubMed

Bheda, Poonam; Swatkoski, Stephen; Fiedler, Katherine L; Boeke, Jef D; Cotter, Robert J; Wolberger, Cynthia

2012-04-17

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

In vivo and in vitro hyperspectral imaging of cervical neoplasia

NASA Astrophysics Data System (ADS)

Wang, Chaojian; Zheng, Wenli; Bu, Yanggao; Chang, Shufang; Tong, Qingping; Zhang, Shiwu; Xu, Ronald X.

2014-02-01

Cervical cancer is a prevalent disease in many developing countries. Colposcopy is the most common approach for screening cervical intraepithelial neoplasia (CIN). However, its clinical efficacy heavily relies on the examiner's experience. Spectroscopy is a potentially effective method for noninvasive diagnosis of cervical neoplasia. In this paper, we introduce a hyperspectral imaging technique for noninvasive detection and quantitative analysis of cervical neoplasia. A hyperspectral camera is used to collect the reflectance images of the entire cervix under xenon lamp illumination, followed by standard colposcopy examination and cervical tissue biopsy at both normal and abnormal sites in different quadrants. The collected reflectance data are calibrated and the hyperspectral signals are extracted. Further spectral analysis and image processing works are carried out to classify tissue into different types based on the spectral characteristics at different stages of cervical intraepithelial neoplasia. The hyperspectral camera is also coupled with a lab microscope to acquire the hyperspectral transmittance images of the pathological slides. The in vivo and the in vitro imaging results are compared with clinical findings to assess the accuracy and efficacy of the method.

Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study.

PubMed

Yang, Zhihong; Lin, James; Zhang, John; Fong, Wai Ieng; Li, Pei; Zhao, Rong; Liu, Xiaohong; Podevin, William; Kuang, Yanping; Liu, Jiaen

2015-06-23

Recent advances in next-generation sequencing (NGS) have provided new methods for preimplantation genetic screening (PGS) of human embryos from in vitro fertilization (IVF) cycles. However, there is still limited information about clinical applications of NGS in IVF and PGS (IVF-PGS) treatments. The present study aimed to investigate the effects of NGS screening on clinical pregnancy and implantation outcomes for PGS patients in comparison to array comparative genomic hybridization (aCGH) screening. This study was performed in two phases. Phase I study evaluated the accuracy of NGS for aneuploidy screening in comparison to aCGH. Whole-genome amplification (WGA) products (n = 164) derived from previous IVF-PGS cycles (n = 38) were retrospectively analyzed with NGS. The NGS results were then compared with those of aCGH. Phase II study further compared clinical pregnancy and implantation outcomes between NGS and aCGH for IVF-PGS patients. A total of 172 patients at mean age 35.2 ± 3.5 years were randomized into two groups: 1) NGS (Group A): patients (n = 86) had embryos screened with NGS and 2) aCGH (Group B): patients (n = 86) had embryos screened with aCGH. For both groups, blastocysts were vitrified after trophectoderm biopsy. One to two euploid blastocysts were thawed and transferred to individual patients primarily based on the PGS results. Ongoing pregnancy and implantation rates were compared between the two study groups. NGS detected all types of aneuploidies of human blastocysts accurately and provided a 100 % 24-chromosome diagnosis consistency with the highly validated aCGH method. Moreover, NGS screening identified euploid blastocysts for transfer and resulted in similarly high ongoing pregnancy rates for PGS patients compared to aCGH screening (74.7 % vs. 69.2 %, respectively, p >0.05). The observed implantation rates were also comparable between the NGS and aCGH groups (70.5 % vs. 66.2 %, respectively, p >0.05). While NGS screening has been recently introduced to assist IVF patients, this is the first randomized clinical study on the efficiency of NGS for preimplantation genetic screening in comparison to aCGH. With the observed high accuracy of 24-chromosome diagnosis and the resulting high ongoing pregnancy and implantation rates, NGS has demonstrated an efficient, robust high-throughput technology for PGS.

A review of designer anabolic steroids in equine sports.

PubMed

Waller, Christopher C; McLeod, Malcolm D

2017-09-01

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in 'dietary' or 'nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

High-speed homogenization coupled with microwave-assisted extraction followed by liquid chromatography-tandem mass spectrometry for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria L. hairy root cultures.

PubMed

Jiao, Jiao; Gai, Qing-Yan; Zhang, Lin; Wang, Wei; Luo, Meng; Zu, Yuan-Gang; Fu, Yu-Jie

2015-06-01

A new, simple and efficient analysis method for fresh plant in vitro cultures-namely, high-speed homogenization coupled with microwave-assisted extraction (HSH-MAE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-was developed for simultaneous determination of six alkaloids and eight flavonoids in Isatis tinctoria hairy root cultures (ITHRCs). Compared with traditional methods, the proposed HSH-MAE offers the advantages of easy manipulation, higher efficiency, energy saving, and reduced waste. Cytohistological studies were conducted to clarify the mechanism of HSH-MAE at cellular/tissue levels. Moreover, the established LC-MS/MS method showed excellent linearity, precision, repeatability, and reproducibility. The HSH-MAE-LC-MS/MS method was also successfully applied for screening high-productivity ITHRCs. Overall, this study opened up a new avenue for the direct determination of secondary metabolic profiles from fresh plant in vitro cultures, which is valuable for improving quality control of plant cell/organ cultures and sheds light on the metabolomic analysis of biological samples. Graphical Abstract HSH-MAE-LC-MS/MS opened up a new avenue for the direct determination of alkaloids and flavonoids in fresh Isatis tinctoria hairy root cultures.

Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera

PubMed Central

Alhakmani, Fatma; Kumar, Sokindra; Khan, Shah Alam

2013-01-01

Objective To evaluate and compare the antioxidant potential and anti-inflammatory activity of ethanolic extract of flowers of Moringa oleifera (M. oleifera) grown in Oman. Methods Flowers of M. oleifera were collected in the month of December 2012 and identified by a botanist. Alcoholic extract of the dry pulverized flowers of M. oleifera were obtained by cold maceration method. The ethanolic flower extract was subjected to preliminary phytochemical screening as the reported methods. Folin-Ciocalteu reagent was used to estimate total phenolic content. DPPH was used to determine in-vitro antioxidant activity and anti-inflammatory activity of flowers was investigated by protein denaturation method. Results Phytochemical analysis of extract showed presence of major classes of phytochemicals such as tannins, alkaloids, flavonoids, cardiac glycosides etc. M. oleifera flowers were found to contain 19.31 mg/g of gallic acid equivalent of total phenolics in dry extract but exhibited moderate antioxidant activity. The anti-inflammatory activity of plant extract was significant and comparable with the standard drug diclofenac sodium. Conclusions The results of our study suggest that flowers of M. oleifera possess potent anti-inflammatory activity and are also a good source of natural antioxidants. Further study is needed to identify the chemical compounds responsible for their anti-inflammatory activity. PMID:23905019

Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation

USDA-ARS?s Scientific Manuscript database

In this study, plants (14) and essential oils (EO; 88) from plants that are naturalized to, or can be successfully grown in North America were evaluated in a batch culture in vitro screening experiments with ruminal fluid as potential anti-methanogenic additives for ruminant diets. Essential oils we...

CLONING AND IN VITRO EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR α FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the ma...

Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development

EPA Science Inventory

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens fo...

Comparison of in vitro estrogenic activity and estrogen concentrations in source and treated waters from 25 U.S. drinking water treatment plants

EPA Science Inventory

In vitro bioassays have been successfully used to screen for estrogenic activity in wastewater and surface water, however, few have been applied to treated drinking water. Here, extracts of source and treated drinking water samples were assayed for estrogenic activity using T47D...

In Vitro Assessment of Estrogenic Activity in Source and Treated Drinking Water Extracts from 25 U.S. Drinking Water Plants

EPA Science Inventory

The presence of estrogenic compounds in environmental water samples and their potential impact on fish, wildlife and human reproductive health has been of concern for some time. In vitro assays have been successfully used to screen for estrogenic activity in many types of water s...

Development of screen-printed tryptophan-kynurenine immunosensor for in vitro assay of kynurenine-mediated immunosuppression effect of cancer cells on activated T-cells.

PubMed

Karami, Pari; Majidi, Mir Reza; Johari-Ahar, Mohammad; Barar, Jaleh; Omidi, Yadollah

2017-06-15

The development of analytical methods that respond to the emerging need to perform rapid 'in situ' analyses of human metabolic pathways (HMPs) demonstrates disposable screen-printed electrodes (SPEs) as an alternative to the traditional tools. In the kynurenine pathway, one of the important HMPs, increased production of kynurenine (Kyn) as a main catabolite of tryptophan (Trp) degradation is involved in the immuno-editing process supporting cancer cells in escaping from the human immune system. In the current study, we demonstrate the development of a screen-printed potentiometric immunosensor for in vitro evaluation of Trp consumption and Kyn production controlled by cancer cells in response to the activated T-lymphocytes. To engineer this immunosensor, uniform layer of carboxylated multiwall carbon nanotubes (MWCNT) was deposited on gold screen-printed electrode (AuSPE), and afterwards monoclonal antibody (mAb) specific to l-kynurenine was covalently conjugated with the MWCNT modified AuSPE. The engineered immunosensor was examined in monitoring Trp consumption and Kyn production in metastatic (Calu-6, NCI-H1299, and HT29) and nonmetastatic (HepG2 and 1321NI) cancer cell lines. Without applying preparation and separation steps, this Trp-Kyn immunosensor offers an improved limit of detection (0.5nM and 120nM for Kyn and Trp detection, respectively) and a broad linear range of detection (LRD: 0.001-1µM and 1-100µM for Kyn, and 0.1-300µM for Trp detection). However, this immunosensor was successfully used for in situ analysis of Kyn that are produced during immuno-editing process in cell culture media, and could reveal that Trp consumption and Kyn production by highly metastatic cancer cells (HT29) were significantly higher than nonmetastatic HepG2 cancer cells. Owing to the screen-printed nature, such kind of biosensors have capability of being integrated into lab-on-a-chip (LOC), microfluidics, and micro total analysis systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Footprinting of Inhibitor Interactions of In Silico Identified Inhibitors of Trypanothione Reductase of Leishmania Parasite

PubMed Central

Venkatesan, Santhosh K.; Dubey, Vikash Kumar

2012-01-01

Structure-based virtual screening of NCI Diversity set II compounds was performed to indentify novel inhibitor scaffolds of trypanothione reductase (TR) from Leishmania infantum. The top 50 ranked hits were clustered using the AuPoSOM tool. Majority of the top-ranked compounds were Tricyclic. Clustering of hits yielded four major clusters each comprising varying number of subclusters differing in their mode of binding and orientation in the active site. Moreover, for the first time, we report selected alkaloids and dibenzothiazepines as inhibitors of Leishmania infantum TR. The mode of binding observed among the clusters also potentiates the probable in vitro inhibition kinetics and aids in defining key interaction which might contribute to the inhibition of enzymatic reduction of T[S] 2. The method provides scope for automation and integration into the virtual screening process employing docking softwares, for clustering the small molecule inhibitors based upon protein-ligand interactions. PMID:22550471

A method for direct assessment of tissue-nonspecific alkaline phosphatase (TNAP) inhibitors in blood samples.

PubMed

Sergienko, Eduard A; Sun, Qing; Ma, Chen-Ting

2013-01-01

Tissue nonspecific alkaline phosphatase (TNAP) is one of four human alkaline phosphatases (AP), a family of exocytic enzymes that catalyze hydrolysis of phospho-monoesters in bone, liver, kidney, and various other tissues. Overexpression of TNAP gives rise to excessive bone and soft tissue mineralization, including blood vessel calcification. Our prior screening campaigns have found several leads against this attractive therapeutic target using in vitro assay with a recombinant enzyme; these compounds were further optimized using medicinal chemistry approaches. To prioritize compounds for their use in animal models, we have designed and developed a biomarker assay for in situ detection of TNAP activity within human and mouse blood samples at physiological pH. This assay is suitable for screening compounds in 1,536-well plates using blood plasma from different mammalian species. The user may choose from two different substrates based on the need for greater assay simplicity or sensitivity.

Combinatorial microfluidic droplet engineering for biomimetic material synthesis

PubMed Central

Bawazer, Lukmaan A.; McNally, Ciara S.; Empson, Christopher J.; Marchant, William J.; Comyn, Tim P.; Niu, Xize; Cho, Soongwon; McPherson, Michael J.; Binks, Bernard P.; deMello, Andrew; Meldrum, Fiona C.

2016-01-01

Although droplet-based systems are used in a wide range of technologies, opportunities for systematically customizing their interface chemistries remain relatively unexplored. This article describes a new microfluidic strategy for rapidly tailoring emulsion droplet compositions and properties. The approach uses a simple platform for screening arrays of droplet-based microfluidic devices and couples this with combinatorial selection of the droplet compositions. Through the application of genetic algorithms over multiple screening rounds, droplets with target properties can be rapidly generated. The potential of this method is demonstrated by creating droplets with enhanced stability, where this is achieved by selecting carrier fluid chemistries that promote titanium dioxide formation at the droplet interfaces. The interface is a mixture of amorphous and crystalline phases, and the resulting composite droplets are biocompatible, supporting in vitro protein expression in their interiors. This general strategy will find widespread application in advancing emulsion properties for use in chemistry, biology, materials, and medicine. PMID:27730209

Synthesis, lipophilicity and antimicrobial activity evaluation of some new thiazolyl-oxadiazolines

PubMed Central

STOICA, CRISTINA IOANA; IONUȚ, IOANA; PÎRNĂU, ADRIAN; POP, CARMEN; ROTAR, ANCUȚA; VLASE, LAURIAN; ONIGA, SMARANDA; ONIGA, OVIDIU

2015-01-01

Background and aims Synthesis of new potential antimicrobial agents and evaluation of their lipophilicity. Methods Ten new thiazolyl-oxadiazoline derivatives were synthesized and their structures were validated by 1H-NMR and mass spectrometry. The lipophilicity of the compounds was evaluated using the principal component analysis (PCA) method. The necessary data for applying this method were obtained by reverse-phase thin-layer chromatography (RP-TLC). The antimicrobial activities were tested in vitro against four bacterial strains and one fungal strain. Results The lipophilicity varied with the structure but could not be correlated with the antimicrobial activity, since this was modest. Conclusions We have synthesized ten new heterocyclic compounds. After their physical and chemical characterization, we determined their lipophilicity and screened their antimicrobial activity. PMID:26733751

Nanotechnology-Based Surface Plasmon Resonance Affinity Biosensors for In Vitro Diagnostics

PubMed Central

Antiochia, Riccarda; Bollella, Paolo; Favero, Gabriele

2016-01-01

In the last decades, in vitro diagnostic devices (IVDDs) became a very important tool in medicine for an early and correct diagnosis, a proper screening of targeted population, and also assessing the efficiency of a specific therapy. In this review, the most recent developments regarding different configurations of surface plasmon resonance affinity biosensors modified by using several nanostructured materials for in vitro diagnostics are critically discussed. Both assembly and performances of the IVDDs tested in biological samples are reported and compared. PMID:27594884

Microengineered cell and tissue systems for drug screening and toxicology applications: Evolution of in-vitro liver technologies

PubMed Central

Usta, O. B.; McCarty, W. J.; Bale, S.; Hegde, M.; Jindal, R.; Bhushan, A.; Golberg, I.; Yarmush, M. L.

2015-01-01

The liver performs many key functions, the most prominent of which is serving as the metabolic hub of the body. For this reason, the liver is the focal point of many investigations aimed at understanding an organism’s toxicological response to endogenous and exogenous challenges. Because so many drug failures have involved direct liver toxicity or other organ toxicity from liver generated metabolites, the pharmaceutical industry has constantly sought superior, predictive in-vitro models that can more quickly and efficiently identify problematic drug candidates before they incur major development costs, and certainly before they are released to the public. In this broad review, we present a survey and critical comparison of in-vitro liver technologies along a broad spectrum, but focus on the current renewed push to develop “organs-on-a-chip”. One prominent set of conclusions from this review is that while a large body of recent work has steered the field towards an ever more comprehensive understanding of what is needed, the field remains in great need of several key advances, including establishment of standard characterization methods, enhanced technologies that mimic the in-vivo cellular environment, and better computational approaches to bridge the gap between the in-vitro and in-vivo results. PMID:26167518

Life-Stage Physiologically-Based Pharmacokinetic (PBPK) ...

EPA Pesticide Factsheets

This presentation discusses methods used to extrapolate from in vitro high-throughput screening (HTS) toxicity data for an endocrine pathway to in vivo for early life stages in humans, and the use of a life stage PBPK model to address rapidly changing physiological parameters. Adverse outcome pathways (AOPs), in this case endocrine disruption during development, provide a biologically-based framework for linking molecular initiating events triggered by chemical exposures to key events leading to adverse outcomes. The application of AOPs to human health risk assessment requires extrapolation of in vitro HTS toxicity data to in vivo exposures (IVIVE) in humans, which can be achieved through the use of a PBPK/PD model. Exposure scenarios for chemicals in the PBPK/PD model will consider both placental and lactational transfer of chemicals, with a focus on age dependent dosimetry during fetal development and after birth for a nursing infant. This talk proposes a universal life-stage computational model that incorporates changing physiological parameters to link environmental exposures to in vitro levels of HTS assays related to a developmental toxicological AOP for vascular disruption. In vitro toxicity endpoints discussed are based on two mechanisms: 1) Fetal vascular disruption, and 2) Neurodevelopmental toxicity induced by altering thyroid hormone levels in neonates via inhibition of thyroperoxidase in the thyroid gland. Application of our Life-stage computati

ToxCast Phase I

EPA Pesticide Factsheets

Background: Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use and the thousands of environmental chemicals lacking toxicity data. EPA's ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives: This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. Methods: We tested 309 mostly pesticide active chemicals in 467 assays across 9 technologies, including high-throughput cell-free assays and cell-based assays in multiple human primary cells and cell lines, plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Results: Chemicals display a broad spectrum of activity at the molecular and pathway levels. Many expected interactions are seen, including endocrine and xenobiotic metabolism enzyme activity. Chemicals range in promiscuity across pathways, from no activity to affecting dozens of pathways. We find a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also find associations between a small set in vitro ass

Signaling pathway cloud regulation for in silico screening and ranking of the potential geroprotective drugs

PubMed Central

Zhavoronkov, Alex; Buzdin, Anton A.; Garazha, Andrey V.; Borisov, Nikolay M.; Moskalev, Alexey A.

2014-01-01

The major challenges of aging research include absence of the comprehensive set of aging biomarkers, the time it takes to evaluate the effects of various interventions on longevity in humans and the difficulty extrapolating the results from model organisms to humans. To address these challenges we propose the in silico method for screening and ranking the possible geroprotectors followed by the high-throughput in vivo and in vitro validation. The proposed method evaluates the changes in the collection of activated or suppressed signaling pathways involved in aging and longevity, termed signaling pathway cloud, constructed using the gene expression data and epigenetic profiles of young and old patients' tissues. The possible interventions are selected and rated according to their ability to regulate age-related changes and minimize differences in the signaling pathway cloud. While many algorithmic solutions to simulating the induction of the old into young metabolic profiles in silico are possible, this flexible and scalable approach may potentially be used to predict the efficacy of the many drugs that may extend human longevity before conducting pre-clinical work and expensive clinical trials. PMID:24624136

Staged anticonvulsant screening for chronic epilepsy.

PubMed

Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung-Il; Roach, Bonnie; Pouliot, Wendy; Lu, Kimberly; Swiercz, Waldemar; Dudek, F Edward; Staley, Kevin J

2016-12-01

Current anticonvulsant screening programs are based on seizures evoked in normal animals. One-third of epileptic patients do not respond to the anticonvulsants discovered with these models. We evaluated a tiered program based on chronic epilepsy and spontaneous seizures, with compounds advancing from high-throughput in vitro models to low-throughput in vivo models. Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and lactate dehydrogenase release into culture media as rapid assays for seizure-like activity and cell death, respectively. Compounds that reduced these biochemical measures were retested with in vitro electrophysiological confirmation (i.e., second stage). The third stage involved crossover testing in the kainate model of chronic epilepsy, with blinded analysis of spontaneous seizures after continuous electrographic recordings. We screened 407 compound-concentration combinations. The cyclooxygenase inhibitor, celecoxib, had no effect on seizures evoked in normal brain tissue but demonstrated robust antiseizure activity in all tested models of chronic epilepsy. The use of organotypic hippocampal cultures, where epileptogenesis occurs on a compressed time scale, and where seizure-like activity and seizure-induced cell death can be easily quantified with biomarker assays, allowed us to circumvent the throughput limitations of in vivo chronic epilepsy models. Ability to rapidly screen compounds in a chronic model of epilepsy allowed us to find an anticonvulsant that would be missed by screening in acute models.

In Vitro Screen for Cyanide Antidotes

DTIC Science & Technology

1993-05-13

each others actions in the in yiro screen. Known cyanide antidotes (e.g., pyruvate, mercaptopyruvate, alpha - ketoglutarate , naloxone and flunarizine...generation, cytosolic-free calcium ) and inhibition of certain enzymes (catalase, superoxide dismutase and cytochrome oxidase) was evaluated for 39...cyanide, and for this reason other biochemical actions of cyanide [elevated cytosolic calcium (3), peroxide generation (4) and inhibition of

CLONING, EXPRESSION AND CHARACTERIZATION OF THE ANDROGEN RECEPTOR AND ISOLATION OF ESTROGEN RECEPTOR ALPHA FROM THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

EPA Science Inventory

In vitro screening assays designed to identify hormone mimics or antagonists, including those recommended for use in the EPA's Tier 1 screening battery, typically use mammalian estrogen (ER) and androgen receptors (AR) such as rat or human. Although we know that the amino acid s...

Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

PubMed Central

Halder, Vivek; Kombrink, Erich

2015-01-01

The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as “chemical genetics,” has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line. PMID:25688251

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation.

PubMed

Wells, Dagan; Kaur, Kulvinder; Grifo, Jamie; Glassner, Michael; Taylor, Jenny C; Fragouli, Elpida; Munne, Santiago

2014-08-01

The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Embryo biopsy, whole genome amplification and semiconductor sequencing. A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

In-vitro Antimicrobial Activities of Some Iranian Conifers

PubMed Central

Afsharzadeh, Maryam; Naderinasab, Mahboobe; Tayarani Najaran, Zahra; Barzin, Mohammad; Emami, Seyed Ahmad

2013-01-01

Male and female leaves and fruits of eleven different taxons of Iranian conifers (Cupressus sempervirens var. horizontalis, C. sempervirens var. sempervirens, C. sempervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, J. excelsa subsp. excelsa, J. excelsa subsp. polycarpos, J. foetidissima, J. oblonga, J. sabina, Platycladus orientalis and Taxus baccata) were collected from different localities of Iran, dried and extracted with methanol. The extracts were tested for their antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans. The extracts were screened qualitatively using four different methods, the disc diffusion, hole plate, cylinder agar diffusion and agar dilution methods, whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method. The best result was obtained by means of hole plate method in qualitative determination of antimicrobial activities of extracts and the greatest activity was found against S. aureus in all tested methods. PMID:24250573

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Occurrence and In Vitro Bioactivity of Estrogen, Androgen, and Glucocorticoid Compounds in a Nationwide Screen of United States Stream Waters.

PubMed

Conley, Justin M; Evans, Nicola; Cardon, Mary C; Rosenblum, Laura; Iwanowicz, Luke R; Hartig, Phillip C; Schenck, Kathleen M; Bradley, Paul M; Wilson, Vickie S

2017-05-02

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054-116 ng E2Eq L -1 ). There was a strong linear relationship (r 2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6-4.8 ng DHTEq L -1 ) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0-43 ng DexEq L -1 ); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

Occurrence and in vitro bioactivity of estrogen, androgen, and glucocorticoid compounds in a nationwide screen of United States stream waters

USGS Publications Warehouse

Conley, Justin M.; Evans, Nicola; Cardon, Mary C.; Rosenblum, Laura; Iwanowicz, Luke R.; Hartig, Phillip C.; Schenck, Kathleen M.; Bradley, Paul M.; Wilson, Vickie S.

2017-01-01

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054–116 ng E2Eq L–1). There was a strong linear relationship (r2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6–4.8 ng DHTEq L–1) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0–43 ng DexEq L–1); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

PubMed Central

Kustiawan, Paula M.; Puthong, Songchan; Arung, Enos T.; Chanchao, Chanpen

2014-01-01

Objective To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). Methods All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Results Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Conclusions Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s). PMID:25183275

Preimplantation genetic screening for all 24 chromosomes by microarray comparative genomic hybridization significantly increases implantation rates and clinical pregnancy rates in patients undergoing in vitro fertilization with poor prognosis

PubMed Central

Majumdar, Gaurav; Majumdar, Abha; Lall, Meena; Verma, Ishwar C.; Upadhyaya, Kailash C.

2016-01-01

CONTEXT: A majority of human embryos produced in vitro are aneuploid, especially in couples undergoing in vitro fertilization (IVF) with poor prognosis. Preimplantation genetic screening (PGS) for all 24 chromosomes has the potential to select the most euploid embryos for transfer in such cases. AIM: To study the efficacy of PGS for all 24 chromosomes by microarray comparative genomic hybridization (array CGH) in Indian couples undergoing IVF cycles with poor prognosis. SETTINGS AND DESIGN: A retrospective, case–control study was undertaken in an institution-based tertiary care IVF center to compare the clinical outcomes of twenty patients, who underwent 21 PGS cycles with poor prognosis, with 128 non-PGS patients in the control group, with the same inclusion criterion as for the PGS group. MATERIALS AND METHODS: Single cells were obtained by laser-assisted embryo biopsy from day 3 embryos and subsequently analyzed by array CGH for all 24 chromosomes. Once the array CGH results were available on the morning of day 5, only chromosomally normal embryos that had progressed to blastocyst stage were transferred. RESULTS: The implantation rate and clinical pregnancy rate (PR) per transfer were found to be significantly higher in the PGS group than in the control group (63.2% vs. 26.2%, P = 0.001 and 73.3% vs. 36.7%, P = 0.006, respectively), while the multiple PRs sharply declined from 31.9% to 9.1% in the PGS group. CONCLUSIONS: In this pilot study, we have shown that PGS by array CGH can improve the clinical outcome in patients undergoing IVF with poor prognosis. PMID:27382234

Preclinical discovery of candidate genes to guide pharmacogenetics during phase I development: the example of the novel anticancer agent ABT-751

PubMed Central

Innocenti, Federico; Ramírez, Jacqueline; Obel, Jennifer; Xiong, Julia; Mirkov, Snezana; Chiu, Yi-Lin; Katz, David A.; Carr, Robert A.; Zhang, Wei; Das, Soma; Adjei, Araba; Moyer, Ann M.; Chen, Pei Xian; Krivoshik, Andrew; Medina, Diane; Gordon, Gary B.; Ratain, Mark J.; Sahelijo, Leonardo; Weinshilboum, Richard M.; Fleming, Gini F.; Bhathena, Anahita

2013-01-01

Objective ABT-751, a novel orally available antitubulin agent, is mainly eliminated as inactive glucuronide (ABT-751G) and sulfate (ABT-751S) conjugates. We performed a pharmacogenetic investigation of ABT-751 pharmacokinetics using in-vitro data to guide the selection of genes for genotyping in a phase I trial of ABT-751. Methods UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes were screened for ABT-751 metabolite formation in vitro. Forty-seven cancer patients treated with ABT-751 were genotyped for 21 variants in these genes. Results UGT1A1, UGT1A4, UGT1A8, UGT2B7, and SULT1A1 were found to be involved in the formation of inactive ABT-751 glucuronide (ABT-751G) and sulfate (ABT-751S). SULT1A1 copy number (> 2) was associated with an average 34% increase in ABT-751 clearance (P= 0.044), an 18% reduction in ABT-751 AUC (P = 0.045), and a 50% increase in sulfation metabolic ratios (P=0.025). UGT1A8 rs6431558 was associated with a 28% increase in glucuronidation metabolic ratios (P =0.022), and UGT1A4*2 was associated with a 65% decrease in ABT-751 Ctrough (P = 0.009). Conclusion These results might represent the first example of a clinical pharmacokinetic effect of the SULT1A1 copy number variant on the clearance of a SULT1A1 substrate. A-priori selection of candidate genes guided by in-vitro metabolic screening enhanced our ability to identify genetic determinants of interpatient pharmacokinetic variability. PMID:23670235

In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens: Receptor-Based Bioassay Screens

PubMed Central

Ahn, Ki Chang; Zhao, Bin; Chen, Jiangang; Cherednichenko, Gennady; Sanmarti, Enio; Denison, Michael S.; Lasley, Bill; Pessah, Isaac N.; Kültz, Dietmar; Chang, Daniel P.Y.; Gee, Shirley J.; Hammock, Bruce D.

2008-01-01

Background Concerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects. Objectives In this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor–responsive and calcium signaling bioassays. Materials and methods We determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor–responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1). Results Some carbanilide compounds, including TCC (1–10 μM), enhanced estradiol (E2)-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1–10 μM) significantly enhanced the binding of [3H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca2+] in primary skeletal myotubes, but carbanilides had no effect. Conclusions Carbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca2+ mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS. PMID:18795164

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor.

PubMed

Judson, Richard S; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M; Filer, Dayne L; Houck, Keith A; Martin, Matthew T; Sipes, Nisha; Richard, Ann M; Mansouri, Kamel; Setzer, R Woodrow; Knudsen, Thomas B; Crofton, Kevin M; Thomas, Russell S

2015-11-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

Discrimination of skin sensitizers from non-sensitizers by interleukin-1α and interleukin-6 production on cultured human keratinocytes.

PubMed

Jung, Daun; Che, Jeong-Hwan; Lim, Kyung-Min; Chun, Young-Jin; Heo, Yong; Seok, Seung Hyeok

2016-09-01

In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Non-opioid analgesic drug flupirtine: Spectral analysis, DFT computations, in vitro bioactivity and molecular docking study

NASA Astrophysics Data System (ADS)

Leenaraj, D. R.; Hubert Joe, I.

2017-06-01

Spectral features of non-opioid analgesic drug flupirtine have been explored by the Fourier transform infrared, Raman and Nuclear magnetic resonance spectroscopic techniques combined with density functional theory computations. The bioactive conformer of flupirtine is stabilized by an intramolecular Csbnd H⋯N hydrogen bonding resulting by the steric strain of hydrogen atoms. Natural bond orbital and natural population analysis support this result. The charge redistribution also has been analyzed. Antimicrobial activities of flupirtine have been screened by agar well disc diffusion and molecular docking methods, which exposes the importance of triaminopyridine in flupirtine.

Cell-based flavivirus infection (CFI) assay for the evaluation of dengue antiviral candidates using high-content imaging.

PubMed

Tan, Kah Hin; Ki, Kitti Chan Wing; Watanabe, Satoru; Vasudevan, Subhash G; Krishnan, Manoj

2014-01-01

Large-scale screening of antiviral compounds that target dengue virus life cycle requires a robust cell-based assay that is rapid, easy to conduct, and sensitive enough to be able to assess viral infectivity and cell viability so that antiviral efficacy can be measured. In this chapter we describe a method that uses high-content imaging to evaluate the in vitro antiviral efficacy in a modification to the cell-based flavivirus immunodetection (CFI) assay that was described previously in Wang et al. (Antimicrob Agents Chemother 53(5):1823-1831, 2009).

Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.

2010-08-04

Methods to detect botulinum toxin, the most poisonous substance known, are reviewed. Current assays are being developed with two main objectives in mind: 1) to obtain sufficiently low detection limits to replace the mouse bioassay with an in vitro assay, and 2) to develop rapid assays for screening purposes that are as sensitive as possible while requiring an hour or less to process the sample an obtain the result. This review emphasizes the diverse analytical approaches and devices that have been developed over the last decade, while also briefly reviewing representative older immunoassays to provide background and context.

Recent Advances in Screening of Anti-Campylobacter Activity in Probiotics for Use in Poultry

PubMed Central

Saint-Cyr, Manuel J.; Guyard-Nicodème, Muriel; Messaoudi, Soumaya; Chemaly, Marianne; Cappelier, Jean-Michel; Dousset, Xavier; Haddad, Nabila

2016-01-01

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Campylobacter species involved in this infection usually include the thermotolerant species Campylobacter jejuni. The major reservoir for C. jejuni leading to human infections is commercial broiler chickens. Poultry flocks are frequently colonized by C. jejuni without any apparent symptoms. Risk assessment analyses have identified the handling and consumption of poultry meat as one of the most important sources of human campylobacteriosis, so elimination of Campylobacter in the poultry reservoir is a crucial step in the control of this foodborne infection. To date, the use of probiotics has demonstrated promising results to reduce Campylobacter colonization. This review provides recent insights into methods used for probiotic screening to reduce the prevalence and colonization of Campylobacter at the farm level. Different eukaryotic epithelial cell lines are employed to screen probiotics with an anti-Campylobacter activity and yield useful information about the inhibition mechanism involved. These in vitro virulence models involve only human intestinal or cervical cell lines whereas the use of avian cell lines could be a preliminary step to investigate mechanisms of C. jejuni colonization in poultry in the presence of probiotics. In addition, in vivo trials to evaluate the effect of probiotics on Campylobacter colonization are conducted, taking into account the complexity introduced by the host, the feed, and the microbiota. However, the heterogeneity of the protocols used and the short time duration of the experiments lead to results that are difficult to compare and draw conclusions at the slaughter-age of broilers. Nevertheless, the combined approach using complementary in vitro and in vivo tools (cell cultures and animal experiments) leads to a better characterization of probiotic strains and could be employed to assess reduced Campylobacter spp. colonization in chickens if some parameters are optimized. PMID:27303366