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

International Nuclear Information System (INIS)

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

2010-01-01

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 4 h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4 h 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

A rapid in vitro screening system for the identification and evaluation of anticancer drugs

International Nuclear Information System (INIS)

Kao, J.W.; Collins, J.L.

1989-01-01

We report the development of an in vitro screening system that can be used to identify new anticancer drugs that are specifically cytotoxic for dividing cells. The screening system takes advantage of the potential of many cell lines, including tumor cells, to stop dividing when they are plated at high cell density. The cytotoxic effects of anticancer drugs on dividing (i.e., cells plated at low cell density) and nondividing cells (i.e., cells plated at high cell density) is measured by the incorporation of 51Cr. This in vitro system was evaluated by measuring the cytotoxic effects of the anticancer drugs cisplatin, thiotepa, doxorubicin, methotrexate, and vinblastine on the cell lines B/C-N, ME-180, and MCF-7. In this in vitro system the concentrations of the anticancer drugs that produced significant cytotoxicity on only dividing cells are similar to the concentrations that are used clinically. The fact that this in vitro system is rapid, simple, applicable to many cell types, and able to predict effective concentrations of anticancer drugs should make it useful for the screening of new anticancer drugs and for the design of preclinical studies

In vitro screening of environmental chemicals for targeted testing prioritization: the ToxCast project.

Science.gov (United States)

Judson, Richard S; Houck, Keith A; Kavlock, Robert J; Knudsen, Thomas B; Martin, Matthew T; Mortensen, Holly M; Reif, David M; Rotroff, Daniel M; Shah, Imran; Richard, Ann M; Dix, David J

2010-04-01

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. The U.S. Environmental Protection Agency'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. 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. We tested 309 mostly pesticide active chemicals in 467 assays across nine 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. Chemicals displayed a broad spectrum of activity at the molecular and pathway levels. We saw many expected interactions, including endocrine and xenobiotic metabolism enzyme activity. Chemicals ranged in promiscuity across pathways, from no activity to affecting dozens of pathways. We found 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 found associations between a small set of in vitro assays and rodent liver lesion formation. This approach promises to provide meaningful data on the thousands of untested environmental chemicals and to guide targeted testing of environmental contaminants.

In Vitro screening of tomato genotypes for drought resistance using ...

African Journals Online (AJOL)

Drought is a major abiotic factor that limits plant growth and productivity. Tomato is an important vegetable crop and area under production is limited by irrigation water scarcity. Effort was made to screen tomato germplasm under in vitro condition using polyethylene glycol (PEG) at four concentrations (0, 20, 40 and 60 g/l) ...

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

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Xing Wang

2016-03-01

Full Text Available 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.

Hierarchical virtual screening approaches in small molecule drug discovery.

Science.gov (United States)

Kumar, Ashutosh; Zhang, Kam Y J

2015-01-01

Virtual screening has played a significant role in the discovery of small molecule inhibitors of therapeutic targets in last two decades. Various ligand and structure-based virtual screening approaches are employed to identify small molecule ligands for proteins of interest. These approaches are often combined in either hierarchical or parallel manner to take advantage of the strength and avoid the limitations associated with individual methods. Hierarchical combination of ligand and structure-based virtual screening approaches has received noteworthy success in numerous drug discovery campaigns. In hierarchical virtual screening, several filters using ligand and structure-based approaches are sequentially applied to reduce a large screening library to a number small enough for experimental testing. In this review, we focus on different hierarchical virtual screening strategies and their application in the discovery of small molecule modulators of important drug targets. Several virtual screening studies are discussed to demonstrate the successful application of hierarchical virtual screening in small molecule drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

Method for in vitro screening of aquatic fungicides

Science.gov (United States)

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 Silico Screening and In Vitro Activity Measurement of Javamide Analogues as Potential p38 MAPK Inhibitors

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Jae B. Park

2017-12-01

be a most potent p38 MAPK inhibitor among the tested compounds and the combining in silico and in vitro assay approach may be a useful and efficient solution as a functional screening approach in searching new lead compounds for targeted molecules.

Interpreting in vitro developmental toxicity test battery results: The consideration of toxicokinetics

NARCIS (Netherlands)

Bosgra, S.; Westerhout, J.

2015-01-01

In the EU collaborative project ChemScreen an alternative, in vitro assay-based test strategy was developed to screen compounds for reproductive toxicity. A toxicokinetic modeling approach was used to allow quantitative comparison between effective concentrations in the in vitro test battery and

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

Science.gov (United States)

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.

Complementing in vitro screening assays with in silico ...

Science.gov (United States)

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 under assay conditions but that can generate active metabolites under in vivo conditions. To address this potential issue, a case study will be presented to demonstrate the use of in silico tools to identify inactive parents with the ability to generate active metabolites. This case study used the results from an orthogonal assay designed to improve confidence in the identification of active chemicals tested across eighteen estrogen receptor (ER)-related in vitro assays by accounting for technological limitations inherent within each individual assay. From the 1,812 chemicals tested within the orthogonal assay, 1,398 were considered inactive. These inactive chemicals were analyzed using Chemaxon Metabolizer software to predict the first and second generation metabolites. From the nearly 1,400 inactive chemicals, over 2,200 first-generation (i.e., primary) metabolites and over 5,500 second-generation (i.e., secondary) metabolites were predicted. Nearly 70% of primary metabolites were immediately detoxified or converted to other metabolites, while over 70% of secondary metabolites remained stable. Among these predicted metabolites, those that are most likely to be produced and remain

An improved single cell ultrahigh throughput screening method based on in vitro compartmentalization.

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Fuqiang Ma

Full Text Available High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >10⁸ clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k(cat/K(M values approaching the diffusion-limited efficiency of ∼10⁸ M⁻¹s⁻¹.

In vitro and in vivo screening of azole fungicides for antiandrogenic effects

DEFF Research Database (Denmark)

Taxvig, Camilla; Vinggaard, Anne; Hass, Ulla

signs of feminization of the male offspring were investigated. Tebuconazole caused an increase in testicular 17alfa-hydroxyprogesterone and progesterone levels, and a decrease in testosterone levels in male fetuses. Epoxiconazole had no effect on any of the mesured hormonelevels. Furthermore...... and antiandrogenic effects both in vitro and in vivo. Two other azole fungicides, tebuconazole and epoxiconazole, have now been investigated for antiandrogenic effects in vitro and in vivo as well. The fungicides were screened in two well-established cell assays, including testing for agonistic and antagonistic...... effects on AR in transfected CHO cells, using an AR reporter gene assay. The compounds were also analyzed for effects on steroidogenesis in H295R cells, a human adrenocorticocarcinoma cell line, used to detect effects on steroid production. In vitro tebuconazole and epoxiconazole proved to be antagonists...

A Targeted Approach for Congenital Cytomegalovirus Screening Within Newborn Hearing Screening.

Science.gov (United States)

Fowler, Karen B; McCollister, Faye P; Sabo, Diane L; Shoup, Angela G; Owen, Kris E; Woodruff, Julie L; Cox, Edith; Mohamed, Lisa S; Choo, Daniel I; Boppana, Suresh B

2017-02-01

Congenital cytomegalovirus (cCMV) infection remains a leading cause of childhood hearing loss. Currently universal CMV screening at birth does not exist in the United States. An alternative approach could be testing infants who do not pass their newborn hearing screening (NHS) for cCMV. This study was undertaken to evaluate whether a targeted approach will identify infants with CMV-related sensorineural hearing loss (SNHL). Infants born at 7 US medical centers received NHS and were also screened for cCMV while in the newborn nursery. Infants who tested positive for CMV received further diagnostic audiologic evaluations to identify or confirm hearing loss. Between 2007 and 2012, 99 945 newborns were screened for both hearing impairment and cCMV. Overall, 7.0% of CMV-positive infants did not pass NHS compared with 0.9% of CMV-negative infants (P CMV-infected infants who passed their NHS had SNHL confirmed by further evaluation during early infancy. NHS in this cohort identified 57% of all CMV-related SNHL that occurred in the neonatal period. A targeted CMV approach that tests newborns who fail their NHS identified the majority of infants with CMV-related SNHL at birth. However, 43% of the infants with CMV-related SNHL in the neonatal period and cCMV infants who are at risk for late onset SNHL were not identified by NHS. Copyright © 2017 by the American Academy of Pediatrics.

Oral cancer screening: serum Raman spectroscopic approach

Science.gov (United States)

Sahu, Aditi K.; Dhoot, Suyash; Singh, Amandeep; Sawant, Sharada S.; Nandakumar, Nikhila; Talathi-Desai, Sneha; Garud, Mandavi; Pagare, Sandeep; Srivastava, Sanjeeva; Nair, Sudhir; Chaturvedi, Pankaj; Murali Krishna, C.

2015-11-01

Serum Raman spectroscopy (RS) has previously shown potential in oral cancer diagnosis and recurrence prediction. To evaluate the potential of serum RS in oral cancer screening, premalignant and cancer-specific detection was explored in the present study using 328 subjects belonging to healthy controls, premalignant, disease controls, and oral cancer groups. Spectra were acquired using a Raman microprobe. Spectral findings suggest changes in amino acids, lipids, protein, DNA, and β-carotene across the groups. A patient-wise approach was employed for data analysis using principal component linear discriminant analysis. In the first step, the classification among premalignant, disease control (nonoral cancer), oral cancer, and normal samples was evaluated in binary classification models. Thereafter, two screening-friendly classification approaches were explored to further evaluate the clinical utility of serum RS: a single four-group model and normal versus abnormal followed by determining the type of abnormality model. Results demonstrate the feasibility of premalignant and specific cancer detection. The normal versus abnormal model yields better sensitivity and specificity rates of 64 and 80% these rates are comparable to standard screening approaches. Prospectively, as the current screening procedure of visual inspection is useful mainly for high-risk populations, serum RS may serve as a useful adjunct for early and specific detection of oral precancers and cancer.

Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

Science.gov (United States)

Pulido, Sergio A; Muñoz, Diana L; Restrepo, Adriana M; Mesa, Carol V; Alzate, Juan F; Vélez, Iván D; Robledo, Sara M

2012-04-01

Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity

A computational design approach for virtual screening of peptide interactions across K+ channel families

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Craig A. Doupnik

2015-01-01

Full Text Available Ion channels represent a large family of membrane proteins with many being well established targets in pharmacotherapy. The ‘druggability’ of heteromeric channels comprised of different subunits remains obscure, due largely to a lack of channel-specific probes necessary to delineate their therapeutic potential in vivo. Our initial studies reported here, investigated the family of inwardly rectifying potassium (Kir channels given the availability of high resolution crystal structures for the eukaryotic constitutively active Kir2.2 channel. We describe a ‘limited’ homology modeling approach that can yield chimeric Kir channels having an outer vestibule structure representing nearly any known vertebrate or invertebrate channel. These computationally-derived channel structures were tested in silico for ‘docking’ to NMR structures of tertiapin (TPN, a 21 amino acid peptide found in bee venom. TPN is a highly selective and potent blocker for the epithelial rat Kir1.1 channel, but does not block human or zebrafish Kir1.1 channel isoforms. Our Kir1.1 channel-TPN docking experiments recapitulated published in vitro findings for TPN-sensitive and TPN-insensitive channels. Additionally, in silico site-directed mutagenesis identified ‘hot spots’ within the channel outer vestibule that mediate energetically favorable docking scores and correlate with sites previously identified with in vitro thermodynamic mutant-cycle analysis. These ‘proof-of-principle’ results establish a framework for virtual screening of re-engineered peptide toxins for interactions with computationally derived Kir channels that currently lack channel-specific blockers. When coupled with electrophysiological validation, this virtual screening approach may accelerate the drug discovery process, and can be readily applied to other ion channels families where high resolution structures are available.

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

Science.gov (United States)

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

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

Energy Technology Data Exchange (ETDEWEB)

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Cromwell, Evan F., E-mail: evan.cromwell@moldev.com [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Crittenden, Carole [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Wignall, Jessica A. [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); Wright, Fred A. [Department of Biostatistics, University of North Carolina, Chapel Hill, NC 27599 (United States); Rusyn, Ivan [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States)

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. A 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

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

International Nuclear Information System (INIS)

Sirenko, Oksana; Cromwell, Evan F.; Crittenden, Carole; Wignall, Jessica A.; Wright, Fred A.; Rusyn, Ivan

2013-01-01

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 2+ flux readouts synchronous with beating, and cell viability. A 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

A new in vitro screening system for anticancer drugs for the treatment of non-small cell lung cancer

International Nuclear Information System (INIS)

Hanauske, U.; Hanauske, A.R.; Clark, G.M.; Tsen, D.; Buchok, J.; Hoff, D.D. von

1989-01-01

We have evaluated a semiautomated radiometric assay (BACTEC 460 system) for screening of activity of anticancer drugs against human non-small cell lung cancer cell lines. Cells from seven cell lines were exposed to standard antineoplastic agents at four different concentrations using a 1-h incubation. Alpha 2-interferon was tested using a continuous incubation. In vitro drug activity was analyzed as a function of the clinically achievable serum concentration. Our results indicate that two cell lines (CALU-3, SK-MES-1) exhibit in vitro drug sensitivity patterns closest to those observed in clinical studies. These two cell lines might therefore be most useful for screening new anticancer compounds for activity against non-small cell lung cancer. The radiometric assay is a semiautomated system which has advantages over other, more time-consuming screening systems

An Efficient Approach to Screening Epigenome-Wide Data

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Meredith A. Ray

2016-01-01

Full Text Available Screening cytosine-phosphate-guanine dinucleotide (CpG DNA methylation sites in association with some covariate(s is desired due to high dimensionality. We incorporate surrogate variable analyses (SVAs into (ordinary or robust linear regressions and utilize training and testing samples for nested validation to screen CpG sites. SVA is to account for variations in the methylation not explained by the specified covariate(s and adjust for confounding effects. To make it easier to users, this screening method is built into a user-friendly R package, ttScreening, with efficient algorithms implemented. Various simulations were implemented to examine the robustness and sensitivity of the method compared to the classical approaches controlling for multiple testing: the false discovery rates-based (FDR-based and the Bonferroni-based methods. The proposed approach in general performs better and has the potential to control both types I and II errors. We applied ttScreening to 383,998 CpG sites in association with maternal smoking, one of the leading factors for cancer risk.

IN VITRO SCREENING OF LOCALLY ISOLATED LACTOBACILLUS SPECIES FOR PROBIOTIC PROPERTIES

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M. ASHRAF, M. ARSHAD, M. SIDDIQUE AND G. MUHAMMAD1

2009-10-01

Full Text Available The present study was conducted to determine the probiotic properties of locally isolated lactobacilli in-vitro conditions. For this purpose, intestinal contents (n=20 were collected from crop, gizzard, ileum and caecum of adult healthy chicks and conventional yogurt samples (n=20 were procured from the local market for the isolation of lactobacilli. These samples were mixed homogeneously in sterilized phosphate buffer saline (PBS separately. Samples from both sources were inoculated on deMan Rogosa and Sharpe (MRS agar. L. acidophilus 3, L. rhamnosus and L. salivarius were isolated from intestinal contents, while L. delbrucekii ssp bulgaricus and L. paracasei ssp paracasei 1 were isolated from yogurt samples. These lactobacilli were identified through standard API-50 CHL system and then screened for resistance against bile salt, acidic pH, gastric transit and ability to inhibit pathogens as well as survival under different storage temperatures. Tolerance level was found variable (P<0.05 among all the tested species of lactobacillus. All the tested species, except L. delbrucekii and L. paracasei, showed good survival (P<0.05. All lactobacilli inhibited the growth of E. coli and Staphylococcus aureus, except L. delbrucekii that showed significantly (P<0.05 low antimicrobial effect. The results showed that L. acidophilus 3, L. rhamnosus and L. salivarius fulfilled the criteria of in-vitro screening for probiotic properties.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

Science.gov (United States)

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

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

Science.gov (United States)

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...

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

Science.gov (United States)

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...

In vitro screening of the endocrine disrupting potency of brominated flame retardants and their metabolites

Energy Technology Data Exchange (ETDEWEB)

Hamers, T.; Kamstra, J.H. [Inst. for Environmental Studies (IVM), Amsterdam (Netherlands); Sonneveld, E. [BioDetection Systems (BDS), Amsterdam (Netherlands); Murk, A.J. [Wageningen Univ., Toxicology Group, Wageningen (Netherlands); Zegers, B.N.; Boon, J.P. [Royal Netherlands Inst. for Sea Research (NIOZ), Den Burg (Netherlands); Brouwer, A. [Umea Univ., Umea (Sweden)

2004-09-15

Substantial evidence is recently becoming available that brominated flame retardants (BFRs) are potential endocrine disruptors. The toxicological profile of BFRs, however, is too incomplete and insufficient to perform human and ecological risk assessment. To fill these gaps, the EU funded research program FIRE was started in December 2002. This program aims at the identification and toxicological characterization of the most potent and environmentally relevant BFRs and their possible risk for human and wildlife health. As part of a hazard identification approach, twenty seven BFRs have been selected within the framework of FIRE for pre-screening their endocrinedisrupting potencies. Selection of test compounds was based on a maximal variation in physicochemical characteristics of BFRs within the test set, allowing the establishment of quantitative structure-activity relationships (QSARs). In addition, environmental relevance (e.g. high production volumes and persistence) and availability for testing were used as selection criteria. BFRs were tested in seven different in vitro bioassays for their potency to interfere via estrogenic, thyroidal, androgenic, progestagenic, and Ah-receptor mediated pathways. Metabolisation rates of BFRs were determined using phenobarbital-induced rat liver microsomes. Finally, the endocrine disrupting potency of the metabolites was determined in the same in vitro bio-assays and compared to the potency of the parent compounds.

Comfortably engaging: which approach to alcohol screening should we use?

Science.gov (United States)

Vinson, Daniel C; Galliher, James M; Reidinger, Carol; Kappus, Jennifer A

2004-01-01

We wanted to compare 2 screening instruments for problem drinking, the CAGE and a single question, assessing frequency of use, patient and clinician comfort, and patient engagement in change. The study was a crossover, cluster-randomized clinical trial with 31 clinicians in Missouri and 13 in the American Academy of Family Physicians (AAFP) National Network for Family Practice and Primary Care Research; 2,800 patients provided data. The clinician was the unit of randomization. Clinicians decided whether to screen each patient; if they chose to screen, they used the screening approach assigned for that block of patients. The clinician and patient separately completed questionnaires immediately after the office visit to assess each one's comfort with screening (and any ensuing discussion) and the patient's engagement in change. Missouri clinicians screened more patients when assigned the single question (81%) than the CAGE (69%, P = .001 in weighted analysis). There was no difference among AAFP network clinicians (96% of patients screened with the CAGE, 97% with the single question). Eighty percent to 90% of clinicians and 70% of patients reported being comfortable with screening and the ensuing discussion, with no difference between approaches in either network. About one third of patients who were identified as problem drinkers reported thinking about or planning to change their drinking behavior, with no difference in engagement between screening approaches. Clinicians and patients reported similar comfort with the CAGE questions and the single-question screening tools for problem drinking, and the 2 instruments were equal in their ability to engage the patient. In Missouri, the single question was more likely to be used.

In vitro screening of snake venom against multidrug-resistant tuberculosis

Directory of Open Access Journals (Sweden)

Sujay Kumar Bhunia

2015-12-01

Full Text Available The re-emergence of multidrug-resistant tuberculosis (MDR-TB has brought to light the importance of screening effective novel drugs. In the present study, in vitro activities of different snake (Naja naja, Bungarus fasciatus, Daboia russelli russelli, Naja kaouthia venoms have been investigated against clinical isolate of MDR-TB strains. The treatment with all the venoms inhibited the mycobacterial growth for at least a week in common and two of them (Naja naja and Naja kaouthia showed significantly longer inhibition up to two weeks against the MDR-TB strain with single dose and a repetition of those two venoms exhibited inhibition up to more than four weeks.

Stem Cuttings as a Quick In Vitro Screening Method of Sodium Chloride Tolerance in Potato (Solanum) Genotypes

International Nuclear Information System (INIS)

Elhag, A. Z.; Mix-Wagnar, G.; Elbassam, N.; Horst, W.

2008-01-01

This study was conducted to find how far in vitro explants stem cuttings technique could be suitable for quick screening of NaCl tolerance solanum genotypes and to identify some aspects of their NaCl tolerance. Fifteen solanum genotypes were tested on four NaCl concentrations both in vitro and in vivo, two-node stem cuttings of in vitro produced explants were grown on Murashige and Skoog (MS) salts supplemented with four NaCl concentrations (0,40,80 and 120 mM) for six weeks in vitro. The other part of the in vitro grown explants were transplanted in Kick- Brauck- Manns pots containing sandy loam soil supplemented also with four NaCl concentration (0, 0.1, 0.2 and 0.3 NaCl, w/w) and grown further either for eight weeks or till harvest in a green house. Both experiments were in a completely randomized design with four replicates. The main stem length, shoot dry matter and tuber yield as well as mineral element (Na''+, K + , Ca''2''+ and Cl''-) were measured. The growth of all genotypes was affected by increasing of NaCl. There was a close correlation between growth response (length of explant main stem) in vitro and shoot dry matter and tuber yield in vivo (r=0.81** for dry matter and 0.72** for tuber yield. Na''+ and Cl''- concentrations in shoots wee inversely correlated with the vegetative growth (r=-0.73** for both in vitro and r=-0.89** and r=-0.88** in vivo, respectively). The genotypes showed varied ability to reduce the transport of Na''+ and Cl''- to the shoots, where by NaCl tolerant genotypes showed lower content of both elements than the sensitive ones. K''+ and Ca''2''+ concentrations were decreased with increasing NaCl concentration. The responses for mineral element (Na''+ and Cl - ) accumulation or restriction of explants in vitro and intact plants in vivo were also closely correlated (r=0.79** and 0.71**, respectively) especially at the medium NaCl concentrations (80 mM and 0.2% NaCl). The similar response of the explant and the intact plant

Quartz-Containing Ceramic Dusts: In vitro screening of the cytotoxic, genotoxic and pro-inflammatory potential of 5 factory samples

Energy Technology Data Exchange (ETDEWEB)

Ziemann, C; Creutzenberg, O [Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover (Germany); Jackson, P [CERAM Research Ltd., Stoke-on-Trent (United Kingdom); Brown, R [TOXSERVICES, Stretton (United Kingdom); Attik, G; Rihn, B H, E-mail: christina.ziemann@item.fraunhofer.d [Nancy-University, Faculte de Pharmacie, Nancy (France)

2009-02-01

Inhalation of some respirable crystalline silica (MMAD < approx. 4 mum) leads to inflammatory and malignant diseases. Comprehensive physicochemical/biological data and suitable in vitro/in vivo methods may distinguish between more or less harmful quartz-varieties. Within the European Collective Research Project SILICERAM an in vitro screening battery was established to evaluate cytotoxicity (LDH-release, MTT-assay), genotoxicity (Comet-assay) and pro-inflammatory potential (PGE{sub 2}-liberation, TNF-a mRNA expression) of 5 respirable quartz-containing dusts from ceramic plants: brickwork (BR: 7.8% quartz), tableware granulate/cast (TG/TC: 5.8%/3.1%), tiles (TI: 8.1%), refractory (RF: 3.7%). DQ12 (87% a-quartz) and Al{sub 2}O{sub 3} were used as particulate positive and negative controls, respectively. Primary rat alveolar macrophages and the macrophage cell line NR8383 served as model systems. Aluminium lactate was used as inhibitor of biologically active silica, enabling differentiation of silica- and non-specific toxicity. At 200mug/cm{sup 2} (2h) the dusts did not alter significantly LDH-release (except TC), whereas the MTT-assay demonstrated the mainly quartz-independent rank order: DQ12>RF>TG>Ti>BR>TC>Al{sub 2}O{sub 3}. DNA-damage was maximal for BR and TI followed by DQ12>TG>TC>RF>Al{sub 2}O{sub 3}. All dusts induced PGE{sub 2}-liberation (DQ12>BR>TC>TG>Ti>RF>Al{sub 2}O{sub 3}) at 50mug/cm{sup 2} (4h), but TNF-a mRNA (10mug/cm{sup 2}, 24h) was only increased by DQ12, TG (quartz-dependently), and TC. In conclusion, these in vitro tests were an adequate approach to screen the toxic potential of quartz-containing ceramic dusts, but the quartz-content was too low to differentiate the various quartz-varieties.

Screening and Selection of High Carotenoid Producing in Vitro Tomato Cell Culture Lines for [13C]-Carotenoid Production

OpenAIRE

Engelmann, Nancy J.; Campbell, Jessica K.; Rogers, Randy B.; Rupassara, S. Indumathie; Garlick, Peter J.; Lila, Mary Ann; Erdman, John W.

2010-01-01

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-lab...

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

International Nuclear Information System (INIS)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan; Durmus, Naside Gozde

2011-01-01

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

Energy Technology Data Exchange (ETDEWEB)

Xu Feng; Wu Jinhui; Wang Shuqi; Gurkan, Umut Atakan; Demirci, Utkan [Department of Medicine, Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Durmus, Naside Gozde, E-mail: udemirci@rics.bwh.harvard.edu [School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI (United States)

2011-09-15

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.

An in vitro model for screening estrogen activity of environmental samples after metabolism

Energy Technology Data Exchange (ETDEWEB)

Chahbane, N.; Schramm, K.W. [GSF - Forschungszentrum fuer Umwelt und Gesundheit Neuherberg GmbH, Oberschleissheim (Germany). Inst. fuer Oekologische Chemie; Kettrup, A. [Technische Univ. Muenchen, Freising (Germany). Lehrstuhl fuer Oekologische Chemie

2004-09-15

For a few years, yeast estrogen assay (YES) was accepted as a reliable and economic model for screening of environmental estrogens. Though the chemicals directly act with estrogen receptor (ER) can be filtered out by this model, there are still chemicals act with ER only after metabolism and some chemicals eliminate their estrogen activities after metabolism. That is to say, their metabolites exert or have stronger estrogen activities than themselves, which can be called bio-activation. In this case, for the lack of the metabolism enzyme system as human and other animals, only the assay with recombinant yeast cells is insufficient. So, it is necessary to combine the YES with metabolism procedure to evaluate the estrogen activities of these chemicals. The most common method used currently for in vitro metabolic activation in mutagenicity testing and also be applied to the estrogen screening field is S-9 mixture. Also, there is an attempt to develop a chemical model for cytochrome P450 as a bio-mimetic metabolic activation system. All these methods can be used as in vitro models for metabolism. Compare with these models, using whole H4II E cells for metabolism is an alternative and with superiorities. It has the excellence of short experiment period as all other in vitro models, but is much more close to the real surroundings as in vivo. Furthermore, the activity of 7-ethoxyresorufin-O-deethylase (EROD) can be easily measured during the whole incubation period for us to discuss the metabolic activities in a quantitative foundation, not only in qualitative. Methoxychlor is one of the chemicals with bio-activation ability. When directly used in the YES, it shows weak estrogen activity. But a main metabolite of methoxychlor, 2,2-bis (p-hydroxyphenyl) - 1,1,1-trichloroethane (HPTE) is a known estrogen mimic. For the long time using methoxychlor as a pesticide and its clear background, it is an ideal chemical to establish this in vitro system.

In vitro application of integrated selection index for screening drought tolerant genotypes in common wheat

Directory of Open Access Journals (Sweden)

Ezatollah FARSHADFAR

2016-10-01

Full Text Available This experiment was conducted on 20 wheat genotypes during 2010-2011 growing season at the Razi University, Kermanshah, Iran. A completely randomized design with six replications was used for callus induction and a 20 × 2 factorial experiment with three replications was used for response of genotypes to in vitro drought stress. ANOVA exhibited highly significant differences among the genotypes for callus growth rate, relative fresh mass growth, relative growth rate, callus water content, percent of callus chlorosis and proline content under stress condition (15 % PEG. PCA showed that the integrated selection index was correlated with callus growth index, relative fresh mass growth, relative growth rate and proline content indicating that these screening techniques can be useful for selecting drought tolerant genotypes. Screening drought tolerant genotypes and in vitro indicators of drought tolerance using mean rank, standard deviation of ranks and biplot analysis, discriminated genotypes 2, 18 and 10 as the most drought tolerant. Therefore they are recommended to be used as parents for genetic analysis, gene mapping and improvement of drought tolerance.

Development in Assay Methods for in Vitro Antimalarial Drug Efficacy Testing: A Systematic Review

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Shweta Sinha

2017-10-01

Full Text Available The emergence and spread of drug resistance are the major challenges in malaria eradication mission. Besides various strategies laid down by World Health Organization, such as vector management, source reduction, early case detection, prompt treatment, and development of new diagnostics and vaccines, nevertheless the need for new and efficacious drugs against malaria has become a critical priority on the global malaria research agenda. At several screening stages, millions of compounds are screened (1,000–2,000,000 compounds per screening campaign, before pre-clinical trials to select optimum lead. Carrying out in vitro screening of antimalarials is very difficult as different assay methods are subject to numerous sources of variability across different laboratories around the globe. Despite this, in vitro screening is an essential part of antimalarial drug development as it enables to resource various confounding factors such as host immune response and drug–drug interaction. Therefore, in this article, we try to illustrate the basic necessity behind in vitro study and how new methods are developed and subsequently adopted for high-throughput antimalarial drug screening and its application in achieving the next level of in vitro screening based on the current approaches (such as stem cells.

The screening approach for review of accident management programmes

International Nuclear Information System (INIS)

Misak, J.

1999-01-01

In this lecture the screening approach for review of accident management programmes are presented. It contains objective trees for accident management: logic structure of the approach; objectives and safety functions for accident management; safety principles

Comparison of a rational vs. high throughput approach for rapid salt screening and selection.

Science.gov (United States)

Collman, Benjamin M; Miller, Jonathan M; Seadeek, Christopher; Stambek, Julie A; Blackburn, Anthony C

2013-01-01

In recent years, high throughput (HT) screening has become the most widely used approach for early phase salt screening and selection in a drug discovery/development setting. The purpose of this study was to compare a rational approach for salt screening and selection to those results previously generated using a HT approach. The rational approach involved a much smaller number of initial trials (one salt synthesis attempt per counterion) that were selected based on a few strategic solubility determinations of the free form combined with a theoretical analysis of the ideal solvent solubility conditions for salt formation. Salt screening results for sertraline, tamoxifen, and trazodone using the rational approach were compared to those previously generated by HT screening. The rational approach produced similar results to HT screening, including identification of the commercially chosen salt forms, but with a fraction of the crystallization attempts. Moreover, the rational approach provided enough solid from the very initial crystallization of a salt for more thorough and reliable solid-state characterization and thus rapid decision-making. The crystallization techniques used in the rational approach mimic larger-scale process crystallization, allowing smoother technical transfer of the selected salt to the process chemist.

Newborn Congenital Cytomegalovirus Screening Based on Clinical Manifestations and Evaluation of DNA-based Assays for In Vitro Diagnostics.

Science.gov (United States)

Fujii, Tomoyuki; Oka, Akira; Morioka, Ichiro; Moriuchi, Hiroyuki; Koyano, Shin; Yamada, Hideto; Saito, Shigeru; Sameshima, Hiroshi; Nagamatsu, Takeshi; Tsuchida, Shinya; Inoue, Naoki

2017-10-01

To establish a strategy for congenital cytomegalovirus (cCMV) screening and to establish confirmatory assays approved as in vitro diagnostics by the regulatory authorities, we evaluated the clinical risks and performance of diagnostic assays developed by commercial companies, since cCMV infection has significant clinical consequences. Newborns with clinical manifestations considered to be consequences of cCMV infection (n = 575) were screened for the presence of cytomegalovirus (CMV) DNA in urine specimens collected onto filter paper placed in their diapers using the polymerase chain reaction-based assay reported previously. Liquid urine specimens were obtained from all of 20 CMV-positive newborns and 107 of the CMV-negative newborns identified in the screening. We used these 127 specimens, as well as 12 from cCMV cases identified in a previous study and 41 from healthy newborns, to compare the performance of 2 commercial assays and 1 in-house assay. The risk-based screening allowed the identification of cCMV cases at least 10-fold more efficiently than our previous universal screening, although there appears to be a limit to the identification of asymptomatically infected newborns. Although CMV-specific IgM during pregnancy was found frequently in mothers of cCMV newborns, CMV-IgM alone is not an effective diagnostic marker. The urine-filter-based assay and the 3 diagnostic assays yielded identical results. Although risk-based and universal newborn screening strategies for cCMV infection each have their respective advantages and disadvantages, urine-filter-based assay followed by confirmatory in vitro diagnostics assays is able to identify cCMV cases efficiently.

First-trimester screening for chromosomal abnormalities: advantages of an instant results approach.

Science.gov (United States)

Norton, Mary E

2010-09-01

Protocols that include first trimester screening for fetal chromosome abnormalities have become standard of care throughout the United States. Earlier screening allows for first trimester diagnostic testing in cases found to be at increased risk. However, first trimester screening requires coordination of the nuchal translucency ultrasound screening (NT) and biochemical screening, during early, specific, narrow, but slightly different gestational age ranges. Instant results can often be provided at the time of the NT ultrasound if preceded by the programs that perform the biochemical analyses; this optimizes the benefits of the first trimester approach while improving efficiency and communication with the patient. This article discusses the benefits and logistics of such an approach. Copyright 2010 Elsevier Inc. All rights reserved.

Preimplantation genetic screening.

Science.gov (United States)

Harper, Joyce C

2018-03-01

Preimplantation genetic diagnosis was first successfully performed in 1989 as an alternative to prenatal diagnosis for couples at risk of transmitting a genetic or chromosomal abnormality, such as cystic fibrosis, to their child. From embryos generated in vitro, biopsied cells are genetically tested. From the mid-1990s, this technology has been employed as an embryo selection tool for patients undergoing in vitro fertilisation, screening as many chromosomes as possible, in the hope that selecting chromosomally normal embryos will lead to higher implantation and decreased miscarriage rates. This procedure, preimplantation genetic screening, was initially performed using fluorescent in situ hybridisation, but 11 randomised controlled trials of screening using this technique showed no improvement in in vitro fertilisation delivery rates. Progress in genetic testing has led to the introduction of array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing for preimplantation genetic screening, and three small randomised controlled trials of preimplantation genetic screening using these new techniques indicate a modest benefit. Other trials are still in progress but, regardless of their results, preimplantation genetic screening is now being offered globally. In the near future, it is likely that sequencing will be used to screen the full genetic code of the embryo.

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

Data.gov (United States)

U.S. Environmental Protection Agency — In vitro bioactivity concentrations and chemical concentrations of estrogens, androgens, and glucocorticoids from a nationwide screen of United States stream water...

Approaches to virtual screening and screening library selection.

Science.gov (United States)

Wildman, Scott A

2013-01-01

The ease of access to virtual screening (VS) software in recent years has resulted in a large increase in literature reports. Over 300 publications in the last year report the use of virtual screening techniques to identify new chemical matter or present the development of new virtual screening techniques. The increased use is accompanied by a corresponding increase in misuse and misinterpretation of virtual screening results. This review aims to identify many of the common difficulties associated with virtual screening and allow researchers to better assess the reliability of their virtual screening effort.

Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro

International Nuclear Information System (INIS)

Emter, Roger; Ellis, Graham; Natsch, Andreas

2010-01-01

In vitro tests are needed to replace animal tests to screen for the skin sensitization potential of chemicals. Skin sensitizers are electrophilic molecules and the Nrf2-electrophile-sensing pathway comprising the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE) is emerging as a toxicity pathway induced by skin sensitizers. Previously, we screened a large set of chemicals in the reporter cell line AREc32, which contains an eight-fold repeat of the rat GSTA2 ARE-sequence upstream of a luciferase reporter gene in the human breast cancer cell line MCF7. This approach was now further developed to bring it closer to the conditions in the human skin and to propose a fully standardized assay. To this end, a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene was stably inserted into HaCaT keratinocytes. A standard operating procedure was developed whereby chemicals are routinely tested at 12 concentrations in triplicate for significant induction of gene activity. We report results from this novel assay on (i) a list of reference chemicals published by ECVAM, (ii) the ICCVAM list of chemicals for validation of alternative endpoints in the LLNA and (iii) on a more general list of 67 chemicals derived from the ICCVAM database. For comparison, peptide reactivity data are presented for the same chemicals. The results indicate a good predictive value of this approach for hazard identification. Its technical simplicity, the high-throughput format and the good predictivity may make this assay a candidate for rapid validation to meet the tight deadline to replace animal tests for skin sensitization by 2013 set by the European authorities.

A novel in vitro toxicological approach to identify chemicals with a prostate-mediated effect on male reproduction

Directory of Open Access Journals (Sweden)

S. Lorenzetti

2011-01-01

Full Text Available Prostate, an overlooked target in in vitro alternative methods, is critical for male fertility. Within the EU project ReProTect, the LNCaP cell line was used as a model system to screen chemicals affecting prostate by a tiered approach integrating two toxicological endpoints: cell viability and PSA secretion. A ReProTect training set of (anti androgenic chemicals affecting reproductive tissues were used. Androgens, and unexpectedly glufosinate ammonium, markedly increased PSA, whereas anti-androgens also increased PSA, but at a much lower magnitude than androgens. Our tiered approach properly discriminated androgenic compounds as well as yielded no false positives, as based on available toxicological evidences. The PSA secretion assay is directly linked to the prostate physiological function and it may integrate the information provided by mechanistic-based assays (i.e. AR binding and gene expression.

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

Science.gov (United States)

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�

[Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

Science.gov (United States)

Niimi, Hideki; Ogawa, Tomomi; Note, Rhougou; Hayashi, Shirou; Ueno, Tomohiro; Harada, Kenu; Uji, Yoshinori; Kitajima, Isao

2010-12-01

In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

Directory of Open Access Journals (Sweden)

Frederick Daidone

Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming

2015-01-01

that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell...... and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology....

Generation of peanut drought tolerant plants by pingyangmycin-mediated in vitro mutagenesis and hydroxyproline-resistance screening.

Directory of Open Access Journals (Sweden)

Jiongming Sui

Full Text Available In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP. After being extracted from mature seeds (cv. Huayu 20, the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation. The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS and nine individuals with higher protein content (>30% and 21 individuals with higher oil content (>58% were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

Determining potential locations for biomass valorization using a macro screening approach

Energy Technology Data Exchange (ETDEWEB)

Van Dael, M.; Van Passel, S.; Schreurs, E. [Research Group of Environmental Economics, Centre for Environmental Sciences, Hasselt University, Agoralaan Gebouw D, 3590 Diepenbeek (Belgium); Pelkmans, L.; Guisson, R. [VITO, Boeretang 200, 2400 Mol (Belgium); Swinnen, G. [Research Group of Marketing, Hasselt University, Agoralaan Gebouw D, 3590 Diepenbeek (Belgium)

2012-10-15

European policy states that by 2020 at least 20% of final energy consumption should come from renewable energy sources. Biomass as a renewable energy source cannot be disregarded in order to attain this target. In this study a macro screening approach is developed to determine potential locations for biomass valorization in a specified region. The approach consists of five steps: (1) criteria determination, (2) data gathering, (3) weight assignment, (4) final score, (5) spatial representation. The resulting outcome provides a first well balanced scan of the possibilities for energy production using regional biomass. This way policy makers and investors can be supported and motivated to study the possibilities of building energy production plants at specific locations in more detail, which can be described as a 'micro-screening'. In our case study the approach is applied to determine the potentially interesting locations to establish a biomass project. The region has been limited to the forty-four communities in the province of Limburg (Belgium). The macro screening approach has shown to be very effective since the amount of interesting locations has been reduced drastically.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

NARCIS (Netherlands)

Breier, J.M.; Gassmann, K.; Kayser, R.; Stegeman, H.; Groot, D.de; Fritsche, E.; Shafer, T.J.

2010-01-01

In vitro, high-throughput methods 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 ramifications for the accuracy,

Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening

International Nuclear Information System (INIS)

Gorrec, Fabrice; Palmer, Colin M.; Lebon, Guillaume; Warne, Tony

2011-01-01

Pi sampling, derived from the incomplete factorial approach, is an effort to maximize the diversity of macromolecular crystallization conditions and to facilitate the preparation of 96-condition initial screens. The Pi sampling method is derived from the incomplete factorial approach to macromolecular crystallization screen design. The resulting ‘Pi screens’ have a modular distribution of a given set of up to 36 stock solutions. Maximally diverse conditions can be produced by taking into account the properties of the chemicals used in the formulation and the concentrations of the corresponding solutions. The Pi sampling method has been implemented in a web-based application that generates screen formulations and recipes. It is particularly adapted to screens consisting of 96 different conditions. The flexibility and efficiency of Pi sampling is demonstrated by the crystallization of soluble proteins and of an integral membrane-protein sample

Hazard assessment of exhaust emissions - The next generation of fast and reliable tools for in vitro screening

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Rothen-Rutishauser, B.

2017-12-01

Hazard assessment of exhaust emissions - The next generation of fast and reliable tools for in vitro screening Barbara Rothen-Rutishauser Adolphe Merkle Institute, University of Fribourg, Switzerland; barbara.rothen@unifr.ch Pollution by vehicles is a major problem for the environment due to the various components in the exhaust gasses that are emitted into the atmosphere. A large number of epidemiological studies demonstrate the profound impact of vehicle emissions upon human health [1-3]. Such studies however, are unable to attribute a given subset of emissions to a certain adverse effect, which renders decision making difficult. Standardized protocols for exhaust toxicity assessment are lacking and it relies in many aspects on epidemiological and in vivo studies (animals), which are very time and cost-intensive and suffer from considerable ethical issues. An overview about the current state of research and clinical aspects in the field, as well as about the development of sophisticated in vitro approaches mimicking the inhalation of airborne particles / exhaust for the toxicological testing of engine emissions will be provided. Data will be presented that show that the combination of an air-liquid exposure system and 3D lung-cell culture model offers an adequate tool for fast and reliable investigations of complete exhaust toxicity as well as the effects of particulate fraction [4,5]. This approach yields important results for novel and improved emission technologies in the early stages of product development. [1] Donaldson et al. Part Fibre Toxicol 2005, 2: 10. [2] Ghio et al. J Toxicol Environ Health B Crit Rev 2012, 15: 1-21. [3] Peters et al. Res Rep Health Eff Inst 2009, 5-77. [4] Bisig et al. Emiss Control Sci Technol 2015, 1: 237-246. [5] Steiner et al. Atmos Environ 2013, 81: 380-388.

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

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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.

Towards the development of lipid multilayer microarrays for dose dependent in vitro delivery and screening

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Kusi-Appiah, Aubrey Emmanuel

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed especially for lipophilic materials. Small-molecule arrays present a promising approach. However, it has been a challenge to use them to obtain quantitative surface based dose-response curves in vitro, especially for lipophilic compounds. This thesis first introduces a simple novel method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The capability of controlling the dosage of the lipophilic molecules delivered to cells using the lipid multilayer microarray assay is further demonstrated using the nanointaglio printing method. This control enabled the variation of the volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. The surface supported lipid volume information was used to obtain EC-50 values for the response of HeLa cells to treatment with three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. Features with sub-cellular lateral dimensions were found to be necessary to obtain normal cell adhesion with HeLa cells. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which may be explained in terms of cell-adhesion playing a more important role in the surface-based assay. Finally, solution encapsulation was done for a library of hydrophilic silicon nanocrystals in order to set a solution standard for comparison with future surface supported delivery of the library. The

A practical approach to screen for authorised and unauthorised genetically modified plants.

Science.gov (United States)

Waiblinger, Hans-Ulrich; Grohmann, Lutz; Mankertz, Joachim; Engelbert, Dirk; Pietsch, Klaus

2010-03-01

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 x 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.

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

Science.gov (United States)

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.

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

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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.

Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches.

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Papadimitriou, Konstantinos; Zoumpopoulou, Georgia; Foligné, Benoit; Alexandraki, Voula; Kazou, Maria; Pot, Bruno; Tsakalidou, Effie

2015-01-01

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer's health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut-brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms.

Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches

Directory of Open Access Journals (Sweden)

Konstantinos ePapadimitriou

2015-02-01

Full Text Available Over the past decades the food industry has been revolutionized towards the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer’s health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut-brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms.

Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches

Science.gov (United States)

Papadimitriou, Konstantinos; Zoumpopoulou, Georgia; Foligné, Benoit; Alexandraki, Voula; Kazou, Maria; Pot, Bruno; Tsakalidou, Effie

2015-01-01

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer’s health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut–brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms. PMID:25741323

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity

International Nuclear Information System (INIS)

Cosgrove, Benjamin D.; King, Bracken M.; Hasan, Maya A.; Alexopoulos, Leonidas G.; Farazi, Paraskevi A.; Hendriks, Bart S.; Griffith, Linda G.; Sorger, Peter K.; Tidor, Bruce; Xu, Jinghai J.

2009-01-01

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFNγ, IL-1α, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1α, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.

Library fingerprints: a novel approach to the screening of virtual libraries.

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Klon, Anthony E; Diller, David J

2007-01-01

We propose a novel method to prioritize libraries for combinatorial synthesis and high-throughput screening that assesses the viability of a particular library on the basis of the aggregate physical-chemical properties of the compounds using a naïve Bayesian classifier. This approach prioritizes collections of related compounds according to the aggregate values of their physical-chemical parameters in contrast to single-compound screening. The method is also shown to be useful in screening existing noncombinatorial libraries when the compounds in these libraries have been previously clustered according to their molecular graphs. We show that the method used here is comparable or superior to the single-compound virtual screening of combinatorial libraries and noncombinatorial libraries and is superior to the pairwise Tanimoto similarity searching of a collection of combinatorial libraries.

Analysis and hit filtering of a very large library of compounds screened against Mycobacterium tuberculosis.

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Ekins, Sean; Kaneko, Takushi; Lipinski, Christopher A; Bradford, Justin; Dole, Krishna; Spektor, Anna; Gregory, Kellan; Blondeau, David; Ernst, Sylvia; Yang, Jeremy; Goncharoff, Nicko; Hohman, Moses M; Bunin, Barry A

2010-11-01

There is an urgent need for new drugs against tuberculosis which annually claims 1.7-1.8 million lives. One approach to identify potential leads is to screen in vitro small molecules against Mycobacterium tuberculosis (Mtb). Until recently there was no central repository to collect information on compounds screened. Consequently, it has been difficult to analyze molecular properties of compounds that inhibit the growth of Mtb in vitro. We have collected data from publically available sources on over 300 000 small molecules deposited in the Collaborative Drug Discovery TB Database. A cheminformatics analysis on these compounds indicates that inhibitors of the growth of Mtb have statistically higher mean logP, rule of 5 alerts, while also having lower HBD count, atom count and lower PSA (ChemAxon descriptors), compared to compounds that are classed as inactive. Additionally, Bayesian models for selecting Mtb active compounds were evaluated with over 100 000 compounds and, they demonstrated 10 fold enrichment over random for the top ranked 600 compounds. This represents a promising approach for finding compounds active against Mtb in whole cells screened under the same in vitro conditions. Various sets of Mtb hit molecules were also examined by various filtering rules used widely in the pharmaceutical industry to identify compounds with potentially reactive moieties. We found differences between the number of compounds flagged by these rules in Mtb datasets, malaria hits, FDA approved drugs and antibiotics. Combining these approaches may enable selection of compounds with increased probability of inhibition of whole cell Mtb activity.

[Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro].

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Brusentsev, E Iu; Igonina, T N; Amstislavskiĭ, S Ia

2014-01-01

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been consideredas laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

Screening for Modulatory Effects on Steroidogenesis Using the Human H295R Adrenocortical Cell Line: A Metabolomics Approach

NARCIS (Netherlands)

Rijk, J.C.W.; Peijnenburg, A.A.C.M.; Blokland, M.H.; Lommen, A.; Hoogenboom, L.A.P.; Bovee, T.F.H.

2012-01-01

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Science.gov (United States)

Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

2016-09-01

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

In vitro screening of durum wheat against water-stress mediated through polyethylene glycol

Directory of Open Access Journals (Sweden)

Nadia Sandra Kacem

2017-06-01

Full Text Available Three durum wheat (Triticum durum Desf. genotypes with three levels of drought tolerance were screened in order to evaluate their response to water stress at callus induction and plant regeneration levels. Significant differences were observed among the genotypes, and polyethylene glycol (PEG levels used, and their interactions were however, significant for all the studied characters. Increase in PEG concentration increased the time required for callus initiation and reduced the number of calli frequency of embryogenic structures and number of plants regenerated, showing the adverse effect of PEG on the somatic embryogenesis developmental., under in vitro conditions tested, and Djenah Khetifa was the most tolerant genotype, followed by Oued Zenati and Waha. This pattern was per their drought tolerance behavior under field conditions. Principal component analysis (PCA showed that 95.56% of the total variation was explained by the first two principal components. Biplot analysis allowed the stress-tolerant genotype to be distinguished from the two less tolerant genotypes. Time required for callus initiation was strongly negatively correlated with all other studied traits. These traits can be recommended as suitable selection criteria for screening drought-tolerant genotypes. The selected cells and plants will provide a tool for determining the mechanisms involved in tolerance to water stress.

Repositioning FDA Drugs as Potential Cruzain Inhibitors from Trypanosoma cruzi: Virtual Screening, In Vitro and In Vivo Studies

Directory of Open Access Journals (Sweden)

Isidro Palos

2017-06-01

Full Text Available 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 (LC50 range 15.8–26.1 μg/mL in comparison with benznidazole and nifurtimox (LC50 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.

Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production.

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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.

A semi-supervised approach using label propagation to support citation screening.

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Kontonatsios, Georgios; Brockmeier, Austin J; Przybyła, Piotr; McNaught, John; Mu, Tingting; Goulermas, John Y; Ananiadou, Sophia

2017-08-01

Citation screening, an integral process within systematic reviews that identifies citations relevant to the underlying research question, is a time-consuming and resource-intensive task. During the screening task, analysts manually assign a label to each citation, to designate whether a citation is eligible for inclusion in the review. Recently, several studies have explored the use of active learning in text classification to reduce the human workload involved in the screening task. However, existing approaches require a significant amount of manually labelled citations for the text classification to achieve a robust performance. In this paper, we propose a semi-supervised method that identifies relevant citations as early as possible in the screening process by exploiting the pairwise similarities between labelled and unlabelled citations to improve the classification performance without additional manual labelling effort. Our approach is based on the hypothesis that similar citations share the same label (e.g., if one citation should be included, then other similar citations should be included also). To calculate the similarity between labelled and unlabelled citations we investigate two different feature spaces, namely a bag-of-words and a spectral embedding based on the bag-of-words. The semi-supervised method propagates the classification codes of manually labelled citations to neighbouring unlabelled citations in the feature space. The automatically labelled citations are combined with the manually labelled citations to form an augmented training set. For evaluation purposes, we apply our method to reviews from clinical and public health. The results show that our semi-supervised method with label propagation achieves statistically significant improvements over two state-of-the-art active learning approaches across both clinical and public health reviews. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Is screen-and-treat approach suited for screening and management of precancerous cervical lesions in Sub-Saharan Africa?

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Fokom-Domgue, Joël; Vassilakos, Pierre; Petignat, Patrick

2014-08-01

The World Health Organization guidelines for screening and management of cervical precancerous lesions updated in 2013 made an emphasis on the use of the 'screen-and-treat' approach for cervical cancer prevention. In order to facilitate scaling-up in low income settings, most of these screen-and-treat strategies do not involve confirmatory biopsy. This yields a certain rate of overtreatment. In other words, a majority of people undergoing screen-and-treat intervention who are treated does not necessarily benefit from the treatment. Therefore, the issue of potential short term and long term complications of the recommended treatment procedures (cryotherapy and Loop Electrosurgical Excision Procedure) arises. This question has seldom been studied in resource poor countries, particularly in Sub-Saharan Africa where Human Immunodeficiency Virus infection is rampant in an epidemic fashion and where the procreative capacities are socially rewarding for women. We draw the attention of the scientific community and policy makers to the fact that the lack of evidence supporting the safety of these treatment procedures in African populations may have an impact on the acceptability of these strategies and therefore on the effectiveness of screening programs. Copyright © 2014 Elsevier Inc. All rights reserved.

Tele-cytology: An innovative approach for cervical cancer screening in resource-poor settings.

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Singh, Sandeep; Badaya, Sorabh

2016-01-01

Carcinoma cervix remains a leading cause of cancer mortality among women in countries lacking any screening program. The existing screening policy and approach via conventional cytology centered mainly in Tertiary Care Center, is totally unaffordable to Indian women, especially in the remote areas. This suggests the need of depolarizing the resources via generating the near real time modalities which could be used at the door step of the needy ones. For any screening modality to be effective it should be adequately sensitive, specific, reproducible, cheap, simple, affordable, and the most important is should be real time to ensure wide coverage and curtail loss to follow-up. Incorporating telecytology as a screening tool could make the dream come true. Telecytology is the interpretation of cytology material at a distance using digital images. Use of mobile telecytology unit housed in a van carrying satellite equipment and the automated image capturing systems is the central theme behind this idea. The imaging equipment would be carrying out the imaging of Papanicolaou smears prepared at the screening site and sending the images to the central laboratories situated at some tertiary care level. This concept could overcome the hindrance of trained cytology infrastructure in the resource poor settings and could provide an efficient and economical way of screening patients. There is possibility that the designed approach may not detect the entire women positive for the disease but if the desired objective was to diagnose as many cases as possible in resource poor setting, then this process offers an advantage over no screening at all.

In vitro screening of potato genotypes for osmotic stress tolerance

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Gelmesa Dandena

2017-02-01

genotypes. Most of the genotypes collected from Ethiopia were found to be susceptible to osmotic stress, except one farmers’ cultivar (Dadafa and two improved varieties (Zemen and Belete. Field evaluation of the tested materials under drought conditions would confirm the capacity of osmotic stress tolerant genotypes to perform well under drought-prone conditions and the potential interest of in vitro evaluation as a pre-screening component in potato breeding programs.

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

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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.

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

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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...

Screening and identification of potential PTP1B allosteric inhibitors using in silico and in vitro approaches.

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Shinde, Ranajit Nivrutti; Kumar, G Siva; Eqbal, Shahbaz; Sobhia, M Elizabeth

2018-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for Type 2 diabetes due to its specific role as a negative regulator of insulin signaling pathways. Discovery of active site directed PTP1B inhibitors is very challenging due to highly conserved nature of the active site and multiple charge requirements of the ligands, which makes them non-selective and non-permeable. Identification of the PTP1B allosteric site has opened up new avenues for discovering potent and selective ligands for therapeutic intervention. Interactions made by potent allosteric inhibitor in the presence of PTP1B were studied using Molecular Dynamics (MD). Computationally optimized models were used to build separate pharmacophore models of PTP1B and TCPTP, respectively. Based on the nature of interactions the target residues offered, a receptor based pharmacophore was developed. The pharmacophore considering conformational flexibility of the residues was used for the development of pharmacophore hypothesis to identify potentially active inhibitors by screening large compound databases. Two pharmacophore were successively used in the virtual screening protocol to identify potential selective and permeable inhibitors of PTP1B. Allosteric inhibition mechanism of these molecules was established using molecular docking and MD methods. The geometrical criteria values confirmed their ability to stabilize PTP1B in an open conformation. 23 molecules that were identified as potential inhibitors were screened for PTP1B inhibitory activity. After screening, 10 molecules which have good permeability values were identified as potential inhibitors of PTP1B. This study confirms that selective and permeable inhibitors can be identified by targeting allosteric site of PTP1B.

An in vitro approach for comparative interspecies metabolism of agrochemicals.

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Whalley, Paul M; Bartels, Michael; Bentley, Karin S; Corvaro, Marco; Funk, Dorothee; Himmelstein, Matthew W; Neumann, Birgit; Strupp, Christian; Zhang, Fagen; Mehta, Jyotigna

2017-08-01

The metabolism and elimination of a xenobiotic has a direct bearing on its potential to cause toxicity in an organism. The confidence with which data from safety studies can be extrapolated to humans depends, among other factors, upon knowing whether humans are systemically exposed to the same chemical entities (i.e. a parent compound and its metabolites) as the laboratory animals used to study toxicity. Ideally, to understand a metabolite in terms of safety, both the chemical structure and the systemic exposure would need to be determined. However, as systemic exposure data (i.e. blood concentration/time data of test material or metabolites) in humans will not be available for agrochemicals, an in vitro approach must be taken. This paper outlines an in vitro experimental approach for evaluating interspecies metabolic comparisons between humans and animal species used in safety studies. The aim is to ensure, where possible, that all potential human metabolites are also present in the species used in the safety studies. If a metabolite is only observed in human in vitro samples and is not present in a metabolic pathway defined in the toxicological species already, the toxicological relevance of this metabolite must be evaluated. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

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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

In vitro microfluidic models of tumor microenvironment to screen transport of drugs and nanoparticles.

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Ozcelikkale, Altug; Moon, Hye-Ran; Linnes, Michael; Han, Bumsoo

2017-09-01

Advances in nanotechnology have enabled numerous types of nanoparticles (NPs) to improve drug delivery to tumors. While many NP systems have been proposed, their clinical translation has been less than anticipated primarily due to failure of current preclinical evaluation techniques to adequately model the complex interactions between the NP and physiological barriers of tumor microenvironment. This review focuses on microfluidic tumor models for characterization of delivery efficacy and toxicity of cancer nanomedicine. Microfluidics offer significant advantages over traditional macroscale cell cultures by enabling recapitulation of tumor microenvironment through precise control of physiological cues such as hydrostatic pressure, shear stress, oxygen, and nutrient gradients. Microfluidic systems have recently started to be adapted for screening of drugs and NPs under physiologically relevant settings. So far the two primary application areas of microfluidics in this area have been high-throughput screening using traditional culture settings such as single cells or multicellular tumor spheroids, and mimicry of tumor microenvironment for study of cancer-related cell-cell and cell-matrix interactions. These microfluidic technologies are also useful in modeling specific steps in NP delivery to tumor and characterize NP transport properties and outcomes by systematic variation of physiological conditions. Ultimately, it will be possible to design drug-screening platforms uniquely tailored for individual patient physiology using microfluidics. These in vitro models can contribute to development of precision medicine by enabling rapid and patient-specific evaluation of cancer nanomedicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1460. doi: 10.1002/wnan.1460 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Photochemical binding of photoallergens to human serum albumin: a simple in vitro method for screening potential photoallergens

International Nuclear Information System (INIS)

Barratt, M.D.; Brown, K.R.

1985-01-01

A simple procedure employing UV spectroscopy is described for testing the ability of chemicals to form covalent conjugates with proteins after irradiation with the appropriate wavelength of light. A range of known photoallergens of widely differing structure has been tested using this procedure; results of these experiments, together with evidence from the scientific literature, provide a correlation between compounds known to be photoallergens and their ability to form covalent conjugates with proteins on irradiation with the appropriate wavelength of light. The method is proposed as an in vitro screening procedure for potential photoallergens. (Auth.)

In vitro antioxidant activity and phytochemical screening of Garhwal Himalaya medicinal plants

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Subhash Chandra

2016-08-01

Full Text Available Bistorta macrophylla, B.vaccinifolia and Persicaria polystachya are used for the treatment and prevention of many ailments including tuberculosis, inflammation, pyretic, fever, flue, lungs disorders, diarrhea, vomiting, arthritis, gout, kidney stones or hyperacidity and hypertension. This study was aimed to evaluate the possible in vitro antioxidant activity and phytochemical screening of B. macrophylla, B.vaccinifolia and P. polystachya. The results of antioxidant activity study of B. macrophylla showed maximum activity in the methanolic extracts at different concentration of 20, 40, 60, 80 and 100 µg/ml. The percent inhibition of writhing response by the extract was 36.18%, 44.72%, 59.21%. 67.08% and 83.39% respectively. In the present work a potent anti-oxidant activity of methanolic extract of the whole plants of B. macrophylla were demonstrated, validating the ethno pharmacological claims. These experimental findings would further establish the scientific basis of the traditional uses of the plant in the management of different conditions as well as control of different disease.

Novel approach to integrated DNA adductomics for the assessment of in vitro and in vivo environmental exposures.

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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.

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

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Conley, Justin M.; Evans, Nicola; Cardon, Mary C.; Rosenblum, Laura; Iwanowicz, Luke; 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.

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

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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.

Analysis of Chemical Bioactivity through In Vitro Profiling ...

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Safety assessment of drugs and environmental chemicals relies extensively on animal testing. However, the quantity of chemicals needing assessment and challenges of species extrapolation drive the development of alternative approaches. The EPA’s ToxCast and the multiagency Tox21 programs address this through use of an extensive in vitro screening program to generate data on a large library of important environmental chemicals. These in vitro assays encompass both cell-free, biochemical assays targeting proteins that may be potential molecular initiating events and cellular assays that provide coverage of critical signaling pathways and toxicity phenotypes. Effects on model organisms such as the developing zebrafish, are also part of the testing strategy. A variety of computational approaches are used to analyze the resulting complex data sets to gain insight in to inherent biological activity of chemicals and possible mechanisms of toxicity. Several case studies including identification of modulators of estrogen receptor and aromatic hydrocarbon receptor pathways with effects in primary human cell systems will be described. In addition, existing in vivo data from a subset of the chemicals was used to anchor predictive models using in vitro data for a number of adverse endpoints including reproductive and developmental toxicities. The strengths and weaknesses of this approach will be described. This work does not necessarily reflect official Agency policy. Pres

Design, synthesis, and validation of an in vitro platform peptide-whole cell screening assay using MTT reagent

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Sahar Ahmed

2017-05-01

Full Text Available An in vitro platform to perform peptide screening against different cancer cell lines was designed. The strategy for this screening relied on the design and detection of high-affinity cancer-targeting peptides based on the sequences of NGR and P160. Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager. For proof of concept, a peptide library (132 spots, and 66 different peptides was designed, synthesized, and screened against different cancer cell lines. The current strategy assists in the identification of positive and negative peptides as well as the relative binding between positive ones. Better binding peptide sequences of the NGR motif were demonstrated to show up to a 2.6-fold increase in CD13+ cell lines with insignificant binding to CD13− ones. Comparable results were observed for P160 peptide sequences, to which different peptides had increased binding, with an up to 3-fold increase relative to the native P160 peptide. Based on our results, new peptide sequences for cancer targeting were identified, and the developed strategy was applied to two different peptide libraries.

Chemometrics Optimized Extraction Procedures, Phytosynergistic Blending and in vitro Screening of Natural Enzyme Inhibitors Amongst Leaves of Tulsi, Banyan and Jamun.

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De, Baishakhi; Bhandari, Koushik; Singla, Rajeev K; Katakam, Prakash; Samanta, Tanmoy; Kushwaha, Dilip Kumar; Gundamaraju, Rohit; Mitra, Analava

2015-10-01

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. 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. 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. 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

Screening for EIA in India: enhancing effectiveness through ecological carrying capacity approach.

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Rajaram, T; Das, Ashutosh

2011-01-01

Developing countries across the world have embraced the policy of high economic growth as a means to reduce poverty. This economic growth largely based on industrial output is fast degrading the ecosystems, jeopardizing their long term sustainability. Environmental Impact Assessment (EIA) has long been recognized as a tool which can help in protecting the ecosystems and aid sustainable development. The Screening guidelines for EIA reflect the level of commitment the nation displays towards tightening its environmental protection system. The paper analyses the screening process for EIA in India and dissects the rationale behind the exclusions and thresholds set in the screening process. The screening process in India is compared with that of the European Union with the aim of understanding the extent of deviations from a screening approach in the context of better economic development. It is found that the Indian system excludes many activities from the purview of screening itself when compared to the EU. The constraints responsible for these exclusions are discussed and the shortcomings of the current command and control system of environmental management in India are also explained. It is suggested that an ecosystem carrying capacity based management system can provide significant inputs to enhance the effectiveness of EIA process from screening to monitoring. Copyright © 2010 Elsevier Ltd. All rights reserved.

Exploiting PubChem for Virtual Screening.

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Xie, Xiang-Qun

2010-12-01

IMPORTANCE OF THE FIELD: PubChem is a public molecular information repository, a scientific showcase of the NIH Roadmap Initiative. The PubChem database holds over 27 million records of unique chemical structures of compounds (CID) derived from nearly 70 million substance depositions (SID), and contains more than 449,000 bioassay records with over thousands of in vitro biochemical and cell-based screening bioassays established, with targeting more than 7000 proteins and genes linking to over 1.8 million of substances. AREAS COVERED IN THIS REVIEW: This review builds on recent PubChem-related computational chemistry research reported by other authors while providing readers with an overview of the PubChem database, focusing on its increasing role in cheminformatics, virtual screening and toxicity prediction modeling. WHAT THE READER WILL GAIN: These publicly available datasets in PubChem provide great opportunities for scientists to perform cheminformatics and virtual screening research for computer-aided drug design. However, the high volume and complexity of the datasets, in particular the bioassay-associated false positives/negatives and highly imbalanced datasets in PubChem, also creates major challenges. Several approaches regarding the modeling of PubChem datasets and development of virtual screening models for bioactivity and toxicity predictions are also reviewed. TAKE HOME MESSAGE: Novel data-mining cheminformatics tools and virtual screening algorithms are being developed and used to retrieve, annotate and analyze the large-scale and highly complex PubChem biological screening data for drug design.

Characterization of microbial metabolism of Syrah grape products in an in vitro colon model using targeted and non-targeted analytical approaches

OpenAIRE

Aura, Anna-Marja; Mattila, Ismo; Hyötyläinen, Tuulia; Gopalacharyulu, Peddinti; Cheynier, Veronique; Souquet, Jean-Marc; Bes, Magali; Le Bourvellec, Carine; Guyot, Sylvain; Orešič, Matej

2012-01-01

Purpose Syrah red grapes are used in the production of tannin-rich red wines. Tannins are high molecular weight molecules, proanthocyanidins (PAs), and poorly absorbed in the upper intestine. In this study, gut microbial metabolism of Syrah grape phenolic compounds was investigated. Methods Syrah grape pericarp was subjected to an enzymatic in vitro digestion model, and red wine and grape skin PA fraction were prepared. Microbial conversion was screened using an in vitro colon model with faec...

Reaching the Unreachable: Novel Approaches to Telemedicine Screening of Underserved Populations for Vitreoretinal Disease.

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Murchison, Ann P; Haller, Julia A; Mayro, Eileen; Hark, Lisa; Gower, Emily; Huisingh, Carrie; Rhodes, Lindsay; Friedman, David S; Lee, David J; Lam, Byron L

2017-07-01

Telemedicine involves electronic communication between a physician in one location and a patient in another location to provide remote medical care. Ophthalmologists are increasingly employing telemedicine, particularly in retinal disease screening and monitoring. Telemedicine has been utilized to decrease barriers to care and yield greater patient satisfaction and lower costs, while maintaining high sensitivity and specificity. This review discusses common patient barriers to eye care, innovative approaches to retinal disease screening and monitoring using telemedicine, and eye care policy initiatives needed to enact large-scale telemedicine eye disease screening programs.

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

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Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

2017-09-22

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

NASA Conjunction Assessment Organizational Approach and the Associated Determination of Screening Volume Sizes

Science.gov (United States)

Newman, Lauri K.; Hejduk, Matthew D.

2015-01-01

NASA is committed to safety of flight for all of its operational assets Performed by CARA at NASA GSFC for robotic satellites Focus of this briefing Performed by TOPO at NASA JSC for human spaceflight he Conjunction Assessment Risk Analysis (CARA) was stood up to offer this service to all NASA robotic satellites Currently provides service to 70 operational satellites NASA unmanned operational assets Other USG assets (USGS, USAF, NOAA) International partner assets Conjunction Assessment (CA) is the process of identifying close approaches between two orbiting objects; sometimes called conjunction screening The Joint Space Operations Center (JSpOC) a USAF unit at Vandenberg AFB, maintains the high accuracy catalog of space objects, screens CARA-supported assets against the catalog, performs OD tasking, and generates close approach data.

Comparing antioxidant capacity of purine alkaloids: a new, efficient trio for screening and discovering potential antioxidants in vitro and in vivo.

Science.gov (United States)

Tsoi, Bun; Yi, Ruo-Nan; Cao, Ling-Fang; Li, Shan-Bing; Tan, Rui-Rong; Chen, Min; Li, Xiao-Xiao; Wang, Chen; Li, Yi-Fang; Kurihara, Hiroshi; He, Rong-Rong

2015-06-01

The most commonly applied strategies for the evaluation of antioxidant capacity are the chemical- or cell-based approaches. However, the results obtained from these methods might not reflect the antioxidant ability of test samples within organisms. In this study, we propose a combination of experiments, including oxygen radical absorbance capacity (ORAC), cellular antioxidant activity assay (CAA), and the chick embryo model, as an efficient trio to evaluate antioxidant capacity of food components. Taking purine alkaloids as example, results demonstrate that chemical and cellular method might misinterpret their true ability on antioxidation. In chick embryo model, caffeine and theacrine can significantly improve vessel density on chorioallantoic membrane and myocardial apoptosis. The mechanism can be involving multiple targets within the organism. We believe that the trio proposed can be widely utilized in screening massive number of antioxidant in a cost-effective way. It will also help discovering new antioxidants that are easily being omitted due to their relatively poor in vitro activities. Copyright © 2014 Elsevier Ltd. All rights reserved.

Probiotic attributes of indigenous Lactobacillus spp. isolated from traditional fermented foods and beverages of north-western Himalayas using in vitro screening and principal component analysis.

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Kumari, Anila; Angmo, Kunzes; Monika; Bhalla, Tek Chand

2016-05-01

The present research was designed to explore indigenous probiotic Lactic acid bacteria from traditional fermented foods and beverages of North-western Himalayas for their probiotic potential. It was achieved through a step-by step approach focused on the technological characterization, evaluation of the probiotic traits and adherence ability. Fifty one LAB isolates from traditional fermented foods and beverages were initially screened for their technological properties and among them twenty isolates were selected. These isolates were further characterized and identified using 16S rRNA gene sequencing as Lactobacillus brevis (7 isolates), Lactobacillus casei (5), Lactobacillus paracasei (2), Lactobacillus buchneri (1), Lactobacillus plantarum (1) and Lactobacillus sp. (3). Identified isolates were evaluated by in vitro methods including survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, exopolysacharride production and haemolytic activity. The results of these experiments were used as input data for Principal Component Analysis; thus, to select the most promising probiotic isolates. Three isolates (L. brevis PLA2, L. paracasei PLA8 and L. brevis PLA16) were found to be most technological relevant and promising probiotic candidates in comparison to commercial probiotic strains. L. brevis PLA2 was selected as best isolate with probiotic potential by in vitro adherence to the human intestinal HT-29 cell line.

Can a Risk Factor Based Approach Safely Reduce Screening for Retinopathy of Prematurity?

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K. M. Friddle

2017-01-01

Full Text Available Objective. Current American retinopathy of prematurity (ROP screening guidelines is imprecise for infants ≥ 30 weeks with birth weights between 1500 and 2000 g. Our objective was to evaluate a risk factor based approach for screening premature infants at low risk for severe ROP. Study Design. We performed a 13-year review from Intermountain Health Care (IHC data. All neonates born at ≤32 weeks were reviewed to determine ROP screening and/or development of severe ROP. Severe ROP was defined by stage ≥ 3 or need for laser therapy. Regression analysis was used to identify significant risk factors for severe ROP. Results. We identified 4607 neonates ≤ 32 weeks gestation. Following exclusion for death, with no retinal exam or incomplete data, 2791 (61% were included in the study. Overall, severe ROP occurred in 260 (9.3%, but only 11/1601 ≥ 29 weeks (0.7%. All infants with severe ROP ≥ 29 weeks had at least 2 identified ROP risk factors. Implementation of this risk based screening strategy to the IHC population over the timeline of this study would have eliminated screening in 21% (343/1601 of the screened population. Conclusions. Limiting ROP screening for infants ≥ 29 and ≤ 32 weeks to only those with clinical risk factors could significantly reduce screening exams while identifying all infants with severe ROP.

Identification and Validation of a Potent Dual Inhibitor of the P. falciparum M1 and M17 Aminopeptidases Using Virtual Screening.

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Chiara Ruggeri

Full Text Available The Plasmodium falciparum PfA-M1 and PfA-M17 metalloaminopeptidases are validated drug targets for the discovery of antimalarial agents. In order to identify dual inhibitors of both proteins, we developed a hierarchical virtual screening approach, followed by in vitro evaluation of the highest scoring hits. Starting from the ZINC database of purchasable compounds, sequential 3D-pharmacophore and molecular docking steps were applied to filter the virtual 'hits'. At the end of virtual screening, 12 compounds were chosen and tested against the in vitro aminopeptidase activity of both PfA-M1 and PfA-M17. Two molecules showed significant inhibitory activity (low micromolar/nanomolar range against both proteins. Finally, the crystal structure of the most potent compound in complex with both PfA-M1 and PfA-M17 was solved, revealing the binding mode and validating our computational approach.

Evaluation and optimized selection of supersaturating drug delivery systems of posaconazole (BCS class 2b) in the gastrointestinal simulator (GIS): An in vitro-in silico-in vivo approach.

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Hens, Bart; Bermejo, Marival; Tsume, Yasuhiro; Gonzalez-Alvarez, Isabel; Ruan, Hao; Matsui, Kazuki; Amidon, Gregory E; Cavanagh, Katie L; Kuminek, Gislaine; Benninghoff, Gail; Fan, Jianghong; Rodríguez-Hornedo, Naír; Amidon, Gordon L

2018-03-30

Supersaturating drug delivery systems (SDDS) have been put forward in the recent decades in order to circumvent the issue of low aqueous solubility. Prior to the start of clinical trials, these enabling formulations should be adequately explored in in vitro/in silico studies in order to understand their in vivo performance and to select the most appropriate and effective formulation in terms of oral bioavailability and therapeutic outcome. The purpose of this work was to evaluate the in vivo performance of four different oral formulations of posaconazole (categorized as a biopharmaceutics classification system (BCS) class 2b compound) based on the in vitro concentrations in the gastrointestinal simulator (GIS), coupled with an in silico pharmacokinetic model to predict their systemic profiles. Recently published intraluminal and systemic concentrations of posaconazole for these formulations served as a reference to validate the in vitro and in silico results. Additionally, the morphology of the formed precipitate of posaconazole was visualized and characterized by optical microscopy studies and thermal analysis. This multidisciplinary work demonstrates an in vitro-in silico-in vivo approach that provides a scientific basis for screening SDDS by a user-friendly formulation predictive dissolution (fPD) device in order to rank these formulations towards their in vivo performance. Copyright © 2018. Published by Elsevier B.V.

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.

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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.

The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

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Bharani Manoharan

Full Text Available The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

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

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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.

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

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Phillips, Martin B.; Leonard, Jeremy A.; Grulke, Christopher M.; Chang, Daniel T.; Edwards, Stephen W.; Brooks, Raina; Goldsmith, Michael-Rock; El-Masri, Hisham; Tan, Yu-Mei

2015-01-01

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 knowledge on exposure, along with absorption, distribution, metabolism, and excretion (ADME) properties of chemicals. Objectives We developed a conceptual workflow to examine exposure and ADME properties in relation to an MIE. The utility of this workflow was evaluated using a previously established AOP, acetylcholinesterase (AChE) inhibition. Methods Thirty chemicals found to inhibit human AChE in the ToxCast™ assay were examined with respect to their exposure, absorption potential, and ability to cross the blood–brain barrier (BBB). Structures of active chemicals were compared against structures of 1,029 inactive chemicals to detect possible parent compounds that might have active metabolites. Results Application of the workflow screened 10 “low-priority” chemicals of 30 active chemicals. Fifty-two of the 1,029 inactive chemicals exhibited a similarity threshold of ≥ 75% with their nearest active neighbors. Of these 52 compounds, 30 were excluded due to poor absorption or distribution. The remaining 22 compounds may inhibit AChE in vivo either directly or as a result of metabolic activation. Conclusions The incorporation of exposure and ADME properties into the conceptual workflow eliminated 10 “low-priority” chemicals that may otherwise have undergone additional, resource-consuming analyses. Our workflow also increased confidence in interpretation of in vitro results by identifying possible “false negatives.” Citation Phillips MB, Leonard JA, Grulke CM, Chang DT, Edwards SW, Brooks R, Goldsmith MR, El-Masri H, Tan YM. 2016. A workflow to investigate exposure and pharmacokinetic influences on high-throughput in vitro chemical screening based on adverse outcome pathways. Environ

A novel approach for in vitro meat production.

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Pandurangan, Muthuraman; Kim, Doo Hwan

2015-07-01

The present review describes the possibility of in vitro meat production with the help of advanced co-culturing methods. In vitro meat production method could be a possible alternative for the conventional meat production. Originally, the research on in vitro meat production was initiated by the National Aeronautics and Space Administration (NASA) for space voyages. The required key qualities for accepting in vitro meat for consumption would be good efficiency ratio, increased protein synthesis rate in skeletal muscles, and mimicking the conventional meat qualities. In vitro culturing of meat is possible with the use of skeletal muscle tissue engineering, stem cell, cell co-culture, and tissue culture methods. Co-culture of myoblast and fibroblast is believed as one of the major techniques for in vitro meat production. In our lab, we have co-cultured myoblast and fibroblast. We believe that a billion pounds of in vitro meat could be produced from one animal for consumption. However, we require a great deal of research on in vitro meat production.

A multipumping flow system for in vitro screening of peroxynitrite scavengers.

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Ribeiro, Marta F T; Dias, Ana C B; Santos, João L M; Fernandes, Eduarda; Lima, José L F C; Zagatto, Elias A G

2007-09-01

Peroxynitrite anion is a reactive nitrogen species formed in vivo by the rapid, controlled diffusion reaction between nitric oxide and superoxide radicals. By reacting with several biological molecules, peroxynitrite may cause important cellular and tissue deleterious effects, which have been associated with many diseases. In this work, an automated flow-based procedure for the in vitro generation of peroxynitrite and subsequent screening of the scavenging activity of selected compounds is developed. This procedure involves a multipumping flow system (MPFS) and exploits the ability of compounds such as lipoic acid, dihydrolipoic acid, cysteine, reduced glutathione, oxidized glutathione, sulindac, and sulindac sulfone to inhibit the chemiluminescent reaction of luminol with peroxynitrite under physiological simulated conditions. Peroxynitrite was generated in the MPFS by the online reaction of acidified hydrogen peroxide with nitrite, followed by a subsequent stabilization by merging with a sodium hydroxide solution to rapidly quench the developing reaction. The pulsed flow and the timed synchronized insertion of sample and reagent solutions provided by the MPFS ensure the establishment of the reaction zone only inside the flow cell, thus allowing maximum chemiluminescence emission detection. The results obtained for the assayed compounds show that, with the exception of oxidized glutathione, all are highly potent scavengers of peroxynitrite at the studied concentrations.

Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives.

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Marugán, Carlos; Torres, Raquel; Lallena, María José

2015-01-01

Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

Morphologic abnormalities in 2-year-old children born after in vitro fertilization/intracytoplasmic sperm injection with preimplantation genetic screening: follow-up of a randomized controlled trial

NARCIS (Netherlands)

Beukers, Fenny; van der Heide, Maaike; Middelburg, Karin J.; Cobben, Jan Maarten; Mastenbroek, Sebastiaan; Breur, Rinske; van der Lee, Johanna H.; Hadders-Algra, Mijna; Bos, Arend F.; Kok, Joke H.; Houtzager, Bregje A.; Repping, Sjoerd; Twisk, Moniek; van der Veen, Fulco; Haadsma, Maaike; Heineman, Maas Jan; van Hoften, Jacorina; Jongbloed-Pereboom, Marjolein; Keating, Paul; Seggers, Jorien

2013-01-01

To evaluate the effect of preimplantation genetic screening (PGS) on morphologic outcome in children. Follow-up of a randomized controlled trial (RCT). University hospital. Two-year-old children born to mothers who participated in an RCT on the efficacy of PGS: 50 children born after in vitro

Non-animal photosafety screening for complex cosmetic ingredients with photochemical and photobiochemical assessment tools.

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Nishida, Hayato; Hirota, Morihiko; Seto, Yoshiki; Suzuki, Gen; Kato, Masashi; Kitagaki, Masato; Sugiyama, Mariko; Kouzuki, Hirokazu; Onoue, Satomi

2015-08-01

Previously, a non-animal screening approach was proposed for evaluating photosafety of cosmetic ingredients by means of in vitro photochemical and photobiochemical assays; however, complex cosmetic ingredients, such as plant extracts and polymers, could not be evaluated because their molecular weight is often poorly defined and so their molar concentration cannot be calculated. The aim of the present investigation was to establish a photosafety screen for complex cosmetic ingredients by using appropriately modified in vitro photosafety assays. Twenty plant extracts were selected as model materials on the basis of photosafety information, and their phototoxic potentials were assessed by means of ultraviolet (UV)/visible light (VIS) spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). The maximum UV/VIS absorption value was employed as a judgment factor for evaluating photoexcitability of samples, and the value of 1.0 was adopted as a tentative criterion for photosafety identification. The ROS/mROS assays were conducted at 50 μg/mL, and no false negative prediction was obtained. Furthermore, the ROS/mROS assays at 50 μg/mL had a similar predictive capacity to the ROS/mROS assays in the previous study. A systematic tiered approach for simple and rapid non-animal photosafety evaluation of complex cosmetic ingredients can be constructed using these modified in vitro photochemical assays. Copyright © 2015 Elsevier Inc. All rights reserved.

21 CFR 866.2420 - Oxidase screening test for gonorrhea.

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2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420... screening test for gonorrhea. (a) Identification. An oxidase screening test for gonorrhea is an in vitro... of gonorrhea. (b) Classification. Class III (premarket approval) (transitional device). (c) Date PMA...

Influence of approach velocity and mesh size on the entrainment and contact of a lowland river fish assemblage at a screened irrigation pump.

Directory of Open Access Journals (Sweden)

Craig A Boys

Full Text Available Fish screens can help prevent the entrainment or injury of fish at irrigation diversions, but only when designed appropriately. Design criteria cannot simply be transferred between sites or pump systems and need to be developed using an evidence-based approach with the needs of local species in mind. Laboratory testing is typically used to quantify fish responses at intake screens, but often limits the number of species that can studied and creates artificial conditions not directly applicable to screens in the wild. In this study a field-based approach was used to assess the appropriateness of different screen design attributes for the protection of a lowland river fish assemblage at an experimental irrigation pump. Direct netting of entrained fish was used along with sonar technology to quantify the probability of screen contact for a Murray-Darling Basin (Australia fish species. Two approach velocities (0.1 and 0.5 m.sec(-1 and different sizes of woven mesh (5, 10 and 20 mm were evaluated. Smaller fish (<150 mm in the assemblage were significantly more susceptible to entrainment and screen contact, especially at higher approach velocities. Mesh size appeared to have little impact on screen contact and entrainment, suggesting that approach velocity rather than mesh size is likely to be the primary consideration when developing screens. Until the effects of screen contacts on injury and survival of these species are better understood, it is recommended that approach velocities not exceed 0.1 m.sec(-1 when the desire is to protect the largest range of species and size classes for lowland river fish assemblages in the Murray-Darling Basin. The field method tested proved to be a useful approach that could compliment laboratory studies to refine fish screen design and facilitate field validation.

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

Energy Technology Data Exchange (ETDEWEB)

Riebeling, Christian [German Federal Institute for Risk Assessment (BfR), Department of Chemicals and Product Safety, Berlin (Germany); Wiemann, Martin [IBE R& D gGmbH, Institute for Lung Health, Münster (Germany); Schnekenburger, Jürgen [Biomedical Technology Center, Westfälische Wilhelms-University, Münster (Germany); Kuhlbusch, Thomas A.J. [Institute of Energy and Environmental Technology (IUTA) e.V., Air Quality & Sustainable Nanotechnology, Duisburg (Germany); Center for Nanointegration CENIDE, University of Duisburg-Essen, Duisburg, Germany, (Germany); Wohlleben, Wendel [BASF SE, Material Physics, Ludwigshafen (Germany); Luch, Andreas [German Federal Institute for Risk Assessment (BfR), Department of Chemicals and Product Safety, Berlin (Germany); Haase, Andrea, E-mail: andrea.haase@bfr.bund.de [German Federal Institute for Risk Assessment (BfR), Department of Chemicals and Product Safety, Berlin (Germany)

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 based 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. �

iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid β Combination for Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Takayuki Kondo

2017-11-01

Full Text Available In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer’s disease (AD. Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid β peptide (Aβ, a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aβ-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aβ effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aβ cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aβ effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development.

Cost-effectiveness of screening programs for Chlamydia trachomatis - A population-based dynamic approach

NARCIS (Netherlands)

Welte, R; Kretzschmar, M; Leidl, R; Van den Hoek, A; Jager, JC; Postma, MJ

2000-01-01

Background: Models commonly used for the economic assessment of chamydial screening programs do not consider population effects. Goal: To develop a novel dynamic approach for the economic evaluation of chlamydial prevention measures and to determine the cost-effectiveness of a general

In vitro screening of azole fungicides for antiandrogenic effects – comparison with in vivo effects

DEFF Research Database (Denmark)

testosterone and progesterone production. Epoxiconazole showed now effect. Furthermore tebuconazole increased the AGD in female pups and resulted in an increased number of nipples in male pups at PND 13, a tendency that was also seen for epoxiconazole, though it was not significant. In conclusion the results...... in H295R cells, a cell line, which produces a wide range of steroid hormones in measurable quantities, including testosterone, progesterone and estradiol, a property that makes it suitable as a screening assay to detect effects on steroidogenesis. In the in vitro tebuconazole and epoxiconazole showed...... antiandrogenic effects, and in the H295R cell assay, tebuconazole and epoxiconazole were like prochloraz able to inhibit testosterone and estradiol levels and increase progesterone levels. For the in vivo testing, a study was conducted testing the developmental effects on offspring after prenatal exposure...

New approaches to cervical cancer screening in Latin America and the Caribbean.

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Herrero, Rolando; Ferreccio, Catterina; Salmerón, Jorge; Almonte, Maribel; Sánchez, Gloria Ines; Lazcano-Ponce, Eduardo; Jerónimo, José

2008-08-19

Cervical cancer remains an important public health problem in the Latin America and Caribbean region (LAC), with an expected significant increase in disease burden in the next decades as a result of population ageing. Prophylactic human papillomavirus (HPV) vaccine is currently unaffordable in LAC countries. However, even if vaccination was implemented, an additional two decades will be required to observe its impact on HPV related disease and cancer. With some exceptions, cytology-based screening programs have been largely ineffective to control the problem in the region, and there is a need for new approaches to the organization of screening and for use of newly developed techniques. Several research groups in LAC have conducted research on new screening methods, some of which are summarized in this paper. A recommendation to reorganize screening programs is presented considering visual inspection for very low resource areas, improvement of cytology where it is operating successfully and HPV DNA testing followed by visual inspection with acetic acid (VIA) or cytology as soon as this method becomes technically and economically sustainable. This could be facilitated by the incorporation of new, low-cost HPV DNA testing methods and the use of self-collected vaginal specimens for selected groups of the population. An important requisite for screening based on HPV testing will be the quality assurance of the laboratory and the technique by validation and certification measures.

Screen-and-treat approaches for cervical cancer prevention in low-resource settings: a randomized controlled trial.

Science.gov (United States)

Denny, Lynette; Kuhn, Louise; De Souza, Michelle; Pollack, Amy E; Dupree, William; Wright, Thomas C

2005-11-02

Non-cytology-based screen-and-treat approaches for cervical cancer prevention have been developed for low-resource settings, but few have directly addressed efficacy. To determine the safety and efficacy of 2 screen-and-treat approaches for cervical cancer prevention that were designed to be more resource-appropriate than conventional cytology-based screening programs. Randomized clinical trial of 6555 nonpregnant women, aged 35 to 65 years, recruited through community outreach and conducted between June 2000 and December 2002 at ambulatory women's health clinics in Khayelitsha, South Africa. All patients were screened using human papillomavirus (HPV) DNA testing and visual inspection with acetic acid (VIA). Women were subsequently randomized to 1 of 3 groups: cryotherapy if she had a positive HPV DNA test result; cryotherapy if she had a positive VIA test result; or to delayed evaluation. Biopsy-confirmed high-grade cervical cancer precursor lesions and cancer at 6 and 12 months in the HPV DNA and VIA groups compared with the delayed evaluation (control) group; complications after cryotherapy. The prevalence of high-grade cervical intraepithelial neoplasia and cancer (CIN 2+) was significantly lower in the 2 screen-and-treat groups at 6 months after randomization than in the delayed evaluation group. At 6 months, CIN 2+ was diagnosed in 0.80% (95% confidence interval [CI], 0.40%-1.20%) of the women in the HPV DNA group and 2.23% (95% CI, 1.57%-2.89%) in the VIA group compared with 3.55% (95% CI, 2.71%-4.39%) in the delayed evaluation group (Pcryotherapy, major complications were rare. Both screen-and-treat approaches are safe and result in a lower prevalence of high-grade cervical cancer precursor lesions compared with delayed evaluation at both 6 and 12 months. Trial Registration http://clinicaltrials.gov Identifier: NCT00233727.

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Parallelogram approach using rat-human In vitro and rat in vivo toxicogenomics predicts acetaminophen-induced hepatotoxicity in humans

NARCIS (Netherlands)

Kienhuis, A.S.; Poll, M.C.G. van de; Wortelboer, H.; Herwijnen, M. van; Gottschalk, R.; Jong, C.H.C. de; Boorsma, A.; Paules, R.S.; Kleinjans, J.C.S.; Stierum, R.H.; Delft, J.H.M. van

2009-01-01

The frequent use of rodent hepatic in vitro systems in pharmacological and toxicological investigations challenges extrapolation of in vitro results to the situation in vivo and interspecies extrapolation from rodents to humans. The toxicogenomics approach may aid in evaluating relevance of these

A well-refined in vitro model derived from human embryonic stem cell for screening phytochemicals with midbrain dopaminergic differentiation-boosting potential for improving Parkinson's disease.

Science.gov (United States)

Hsieh, Wen-Ting; Chiang, Been-Huang

2014-07-09

Stimulation of endogenous neurogenesis is a potential approach to compensate for loss of dopaminergic neurons of substantia nigra compacta nigra (SNpc) in patients with Parkinson's disease (PD). This objective was to establish an in vitro model by differentiating pluripotent human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons for screening phytochemicals with mDA neurogenesis-boosting potentials. Consequently, a five-stage differentiation process was developed. The derived cells expressed many mDA markers including tyrosine hydroxylase (TH), β-III tubulin, and dopamine transporter (DAT). The voltage-gated ion channels and dopamine release were also examined for verifying neuron function, and the dopamine receptor agonists bromocriptine and 7-hydroxy-2-(dipropylamino)tetralin (7-OH-DPAT) were used to validate our model. Then, several potential phytochemicals including green tea catechins and ginsenosides were tested using the model. Finally, ginsenoside Rb1 was identified as the most potent phytochemical which is capable of upregulating neurotrophin expression and inducing mDA differentiation.

Evaluation of the antidiabetic property of aqueous leaves extract of Zanthoxylum armatum DC. using in vivo and in vitro approaches

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Carey Vana Rynjah

2018-01-01

Full Text Available The present study was designed to evaluate the antidiabetic potential of the aqueous leaves extract of Zanthoxylum armatum DC. leaves using in vivo and in vitro approaches. For in vivo studies, blood glucose level was monitored at different intervals after administration of varying doses of the extract for its hypoglycemic (100–6000 mg/kg b.w. and antihyperglycemic (250 mg/kg b.w. effect in normoglycemic and diabetic mice. In vitro enzymatic inhibition activity was tested against α-amylase, α- and β-glucosidase and lipase. Additionally hydroxyl radical, hydrogen peroxide scavenging assay and phytochemical screening were also performed. Element analysis of the plant was studied by Atomic Absorption Spectrometry (AAS and Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES. The plant extract showed significant hypoglycemic and antihyperglycemic effect in normoglycemic and diabetic mice. The IC50 values of extract for α-amylase, β-glucosidase, lipase, hydroxyl radical scavenging activity, hydrogen peroxide scavenging activity were 7.40 mg/ml, 0.30 mg/ml, 8.35 mg/ml, 3.25 mg/ml, 9.62 mg/ml respectively and the percentage of inhibition for α-glucosidase was 79.82% at 0.8 mg/ml. In vitro studies were compared with their respective standards. Elemental analysis revealed the presence of essential elements such as Mg, V, Fe, Cr, Zn, Cu, Mo, Mn, K, Ca, P and Sr which are all known to play a role in regulating blood glucose. The results demonstrate that Z. armatum aqueous leaves extract possess antidiabetic property in both in vivo and in vitro condition.

Spherical harmonics coefficients for ligand-based virtual screening of cyclooxygenase inhibitors.

Science.gov (United States)

Wang, Quan; Birod, Kerstin; Angioni, Carlo; Grösch, Sabine; Geppert, Tim; Schneider, Petra; Rupp, Matthias; Schneider, Gisbert

2011-01-01

Molecular descriptors are essential for many applications in computational chemistry, such as ligand-based similarity searching. Spherical harmonics have previously been suggested as comprehensive descriptors of molecular structure and properties. We investigate a spherical harmonics descriptor for shape-based virtual screening. We introduce and validate a partially rotation-invariant three-dimensional molecular shape descriptor based on the norm of spherical harmonics expansion coefficients. Using this molecular representation, we parameterize molecular surfaces, i.e., isosurfaces of spatial molecular property distributions. We validate the shape descriptor in a comprehensive retrospective virtual screening experiment. In a prospective study, we virtually screen a large compound library for cyclooxygenase inhibitors, using a self-organizing map as a pre-filter and the shape descriptor for candidate prioritization. 12 compounds were tested in vitro for direct enzyme inhibition and in a whole blood assay. Active compounds containing a triazole scaffold were identified as direct cyclooxygenase-1 inhibitors. This outcome corroborates the usefulness of spherical harmonics for representation of molecular shape in virtual screening of large compound collections. The combination of pharmacophore and shape-based filtering of screening candidates proved to be a straightforward approach to finding novel bioactive chemotypes with minimal experimental effort.

Cervical cancer screening and treatment of cervical intraepithelial neoplasia in female sex workers using “screen and treat” approach

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Joshi S

2015-05-01

Full Text Available Smita Joshi,1 Vinay Kulkarni,2 Trupti Darak,2 Uma Mahajan,1 Yogesh Srivastava,3 Sanjay Gupta,3 Sumitra Krishnan,1 Mahesh Mandolkar,2 Alok Chandra Bharti31Hirabai Cowasji Jehangir Medical Research Institute (HCJMRI, Jehangir Hospital Premises, Pune, Maharashtra, India; 2Prayas Health Group, Amrita Clinic, Pune, India; 3Institute for Cytology and Preventive Oncology, Indian Council of Medical Research, New Delhi, IndiaObjective: Female sex workers (FSWs are at an increased risk of human immunodeficiency virus (HIV as well as human papillomavirus (HPV infections and thus have an increased risk of cervical intraepithelial neoplasia (CIN and cervical cancer. We evaluated the feasibility of “screen and treat approach” for cervical cancer prevention and the performance of different screening tests among FSWs.Methods: Women were screened using cytology, VIA (visual inspection with acetic acid, and VILI (visual inspection with Lugol’s iodine and underwent colposcopy, biopsy, and immediate treatment using cold coagulation, if indicated, at the same visit.Results: We screened 300 FSWs of whom 200 (66.67% were HIV uninfected and 100 (33.34% were HIV infected. The overall prevalence of CIN 2–3 lesions was 4.7%. But all women with CIN 2–3 lesions were HIV infected, and thus the prevalence of CIN 2–3 lesions in HIV-infected FSWs was 14/100 (14%, 95% confidence interval: 7.2–20.8. All of them screened positive by all three screening tests. Cold coagulation was well tolerated, with no appreciable side effects.Conclusion: Cervical cancer prevention by “screen and treat” approach using VIA, followed by ablative treatment, in this high-risk group of women is feasible and can be implemented through various targeted intervention programs. Keywords: cytology, VIA, VILI, CIN, cold coagulation, cervical cancer, HPV, FSWs

Tiered High-Throughput Screening Approach to Identify ...

Science.gov (United States)

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Phenotypic screening approaches to develop Aurora kinase inhibitors: Drug Discovery perspectives

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Carlos eMarugán

2016-01-01

Full Text Available Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins such as Aurora A and B. Current drugs which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules, have several side effects (neutropenia, alopecia, emesis. Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype.We will briefly describe two multiplexing technologies (high-content imaging, microarrays and flow cytometry and two key processes for drug discovery research (assay development and validation following our own published industry quality standards. We will further focus on high-content imaging as a useful tool for phenotypic screening and will provide a concrete example of high-content imaging assay to detect Aurora A or B selective inhibitors discriminating the off-target effects related to inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors.

Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes

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Takehiro Nishikawa

2012-01-01

Full Text Available Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype and the protein translated from the information (phenotype, which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE system and a fluorescence-activated cell sorter (FACS used as a microcompartment, in vitro protein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized by in vitro protein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP and β-glucuronidase are described. We discuss the future directions for this method and its applications.

Systematic screening of imaging biomarkers for the Islets of Langerhans, among clinically available positron emission tomography tracers

International Nuclear Information System (INIS)

Karlsson, Filip; Antonodimitrakis, Pantelis Clewemar; Eriksson, Olof

2015-01-01

Introduction: Functional imaging could be utilized for visualizing pancreatic islets of Langerhans. Therefore, we present a stepwise algorithm for screening of clinically available positron emission tomography (PET) tracers for their use in imaging of the neuroendocrine pancreas in the context of diabetes. Methods: A stepwise procedure was developed for screening potential islet imaging agents. Suitable PET-tracer candidates were identified by their molecular mechanism of targeting. Clinical abdominal examinations were retrospectively analyzed for pancreatic uptake and retention. The target protein localization in the pancreas was assessed in silico by –omics approaches and the in vitro by binding assays to human pancreatic tissue. Results: Six putative candidates were identified and screened by using the stepwise procedure. Among the tested PET tracers, only [ 11 C]5-Hydroxy-tryptophan passed all steps. The remaining identified candidates were falsified as candidates and discarded following in silico and in vitro screening. Conclusions: Of the six clinically available PET tracers identified, [ 11 C]5-HTP was found to be a promising candidate for beta cell imaging, based on intensity of in vivo pancreatic uptake in humans, and islet specificity as assessed on human pancreatic cell preparations. The flow scheme described herein constitutes a methodology for evaluating putative islet imaging biomarkers among clinically available PET tracers

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

International Nuclear Information System (INIS)

Shin, Hyeong-Moo; Ernstoff, Alexi; Csiszar, Susan A.

2015-01-01

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 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

Identification of urinary biomarkers of exposure to di-(2-propylheptyl) phthalate using high-resolution mass spectrometry and two data-screening approaches.

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Shih, Chia-Lung; Liao, Pao-Mei; Hsu, Jen-Yi; Chung, Yi-Ning; Zgoda, Victor G; Liao, Pao-Chi

2018-02-01

Di-(2-propylheptyl) phthalate (DPHP) is a plasticizer used in polyvinyl chloride and vinyl chloride copolymer that has been suggested to be a toxicant in rats and may affect human health. Because the use of DPHP is increasing, the general German population is being exposed to DPHP. Toxicant metabolism is important for human toxicant exposure assessments. To date, the knowledge regarding DPHP metabolism has been limited, and only four metabolites have been identified in human urine. Ultra-performance liquid chromatography was coupled with Orbitrap high-resolution mass spectrometry (MS) and two data-screening approaches-the signal mining algorithm with isotope tracing (SMAIT) and the mass defect filter (MDF)-for DPHP metabolite candidate discovery. In total, 13 and 104 metabolite candidates were identified by the two approaches, respectively, in in vitro DPHP incubation samples. Of these candidates, 17 were validated as tentative exposure biomarkers using a rat model, 13 of which have not been reported in the literature. The two approaches generated rather different tentative DPHP exposure biomarkers, indicating that these approaches are complementary for discovering exposure biomarkers. Compared with the four previously reported DPHP metabolites, the three tentative novel biomarkers had higher peak intensity ratios, and two were confirmed as DPHP hydroxyl metabolites based on their MS/MS product ion profiles. These three tentative novel biomarkers should be further investigated for potential application in human exposure assessment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Photoreactivity of biologically active compounds. VII. Interaction of antimalarial drugs with melanin in vitro as part of phototoxicity screening.

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Kristensen, S; Orsteen, A L; Sande, S A; Tønnesen, H H

1994-10-01

The drugs commonly used in the treatment of malaria are photochemically unstable. Several of these compounds accumulate in melanin-rich tissues and cause toxic reactions which may be light induced. As part of the screening of the photochemical properties and phototoxic capabilities of antimalarials, the in vitro interaction of eight antimalarials with melanin was studied. The dissociation constant for the drug-melanin complex and the relative number of binding sites on melanin were estimated for six of the drugs using a curve-fitting program. The reaction rate for the formation of the melanin-drug complex was determined, and the complexes were further characterized by zeta potential measurements.

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

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Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

2017-08-24

Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

In Vitro Screening of Cytotoxic, Antimicrobial and Antioxidant ...

African Journals Online (AJOL)

Purpose: To evaluate the in vitro cytotoxic, antioxidant and antimicrobial activities of Clinacanthus nutans extracts and semi-fractions. Method: The plant was subjected to cold solvent extraction to produce petroleum ether, ethyl acetate and methanol crude extracts, followed by isolation using bioassay-guided fractionation.

Just-in-Time Compound Pooling Increases Primary Screening Capacity without Compromising Screening Quality.

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Elkin, L L; Harden, D G; Saldanha, S; Ferguson, H; Cheney, D L; Pieniazek, S N; Maloney, D P; Zewinski, J; O'Connell, J; Banks, M

2015-06-01

Compound pooling, or multiplexing more than one compound per well during primary high-throughput screening (HTS), is a controversial approach with a long history of limited success. Many issues with this approach likely arise from long-term storage of library plates containing complex mixtures of compounds at high concentrations. Due to the historical difficulties with using multiplexed library plates, primary HTS often uses a one-compound-one-well approach. However, as compound collections grow, innovative strategies are required to increase the capacity of primary screening campaigns. Toward this goal, we have developed a novel compound pooling method that increases screening capacity without compromising data quality. This method circumvents issues related to the long-term storage of complex compound mixtures by using acoustic dispensing to enable "just-in-time" compound pooling directly in the assay well immediately prior to assay. Using this method, we can pool two compounds per well, effectively doubling the capacity of a primary screen. Here, we present data from pilot studies using just-in-time pooling, as well as data from a large >2-million-compound screen using this approach. These data suggest that, for many targets, this method can be used to vastly increase screening capacity without significant reduction in the ability to detect screening hits. © 2015 Society for Laboratory Automation and Screening.

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

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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...

In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

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A. G. Nerkar

2009-01-01

Full Text Available Dihydrofolate reductase (DHFR is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino quinazolinones (Quinazolinone Shiff's bases QSB1-5 and pyridine-4-carbohydrazide Shiff's bases (ISB1-5 derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3 (4-(N, N-dimethyl-amino-phenyl Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute guidelines.

Screening sensitivity theory

International Nuclear Information System (INIS)

Oblow, E.M.; Perey, F.G.

1984-01-01

A comprehensive rigorous theory is developed for screening sensitivity coefficients in largescale modeling applications. The theory uses Bayesian inference and group theory to establish a probabilistic framework for solving an underdetermined system of linear equations. The underdetermined problem is directly related to statistical screening sensitivity theory as developed in recent years. Several examples of the new approach to screening are worked out in detail and comparisons are made with statistical approaches to the problem. The drawbacks of these latter methods are discussed at some length

(ENVIRONMENTAL SCIENCE and TECHNOLOGY) An Intuitive Approach for Predicting Human Risk with the Tox21 10k Library

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In vitro to in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human relevance have been limited. This is the first time IVIVE approaches and exposure comparisons have explored the entire Tox21 federal collaboration’s 10,000 chemi...

Prevalence, specificity and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments.

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Ylva Ivarsson

Full Text Available BACKGROUND: PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs, important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited. METHODOLOGY/PRINCIPAL FINDINGS: We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of in vivo cell-localization studies and in vitro dot blot and Surface Plasmon Resonance (SPR experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. In vitro specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands. CONCLUSIONS/SIGNIFICANCE: Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands.

Hierarchical screening for multiple mental disorders.

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Batterham, Philip J; Calear, Alison L; Sunderland, Matthew; Carragher, Natacha; Christensen, Helen; Mackinnon, Andrew J

2013-10-01

There is a need for brief, accurate screening when assessing multiple mental disorders. Two-stage hierarchical screening, consisting of brief pre-screening followed by a battery of disorder-specific scales for those who meet diagnostic criteria, may increase the efficiency of screening without sacrificing precision. This study tested whether more efficient screening could be gained using two-stage hierarchical screening than by administering multiple separate tests. Two Australian adult samples (N=1990) with high rates of psychopathology were recruited using Facebook advertising to examine four methods of hierarchical screening for four mental disorders: major depressive disorder, generalised anxiety disorder, panic disorder and social phobia. Using K6 scores to determine whether full screening was required did not increase screening efficiency. However, pre-screening based on two decision tree approaches or item gating led to considerable reductions in the mean number of items presented per disorder screened, with estimated item reductions of up to 54%. The sensitivity of these hierarchical methods approached 100% relative to the full screening battery. Further testing of the hierarchical screening approach based on clinical criteria and in other samples is warranted. The results demonstrate that a two-phase hierarchical approach to screening multiple mental disorders leads to considerable increases efficiency gains without reducing accuracy. Screening programs should take advantage of prescreeners based on gating items or decision trees to reduce the burden on respondents. © 2013 Elsevier B.V. All rights reserved.

Innovative approaches to cervical cancer screening for sex trade workers: an international scoping review.

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Thulien, Naomi S

2014-03-01

Female sex trade workers are among those at highest risk for developing and dying of cervical cancer, and yet many-particularly the most marginalized-are less likely than other women to be screened. This review summarizes global findings on innovative approaches to cervical cancer screening for female sex trade workers, highlights current gaps in the delivery of cervical cancer screening for female sex trade workers globally, and suggests areas for future research and policy development. A scoping review of peer-reviewed publications and grey literature was conducted. Medline (OVID), PubMed, EMBASE, and SCOPUS were searched for relevant studies written in English. There were no limitations placed on dates. Grey literature was identified by hand searching and through discussion with health care providers and community outreach workers currently working with sex trade workers. Twenty-five articles were deemed suitable for review. Articles detailing innovative ways for female sex trade workers to access cervical cancer screening were included. Articles about screening for sexually transmitted infections were also included if the findings could be generalized to screening for cervical cancer. Articles limited to exploring risk factors, knowledge, awareness, education, prevalence, and incidence of cervical cancer among sex trade workers were excluded from the review. Successful screening initiatives identified in the studies reviewed had unconventional hours of operation, understood the difference between street-based and venue-based sex trade workers, and/or used peers for outreach. Two significant gaps in health care service delivery were highlighted in this review: the limited use of unorthodox hours and the nearly exclusive practice of providing sexually transmitted infection screening for female sex trade workers without cervical cancer screening. In addition, although street-based (as opposed to venue-based) sex trade workers are likely at higher risk for

Virtual screening approach to identifying influenza virus neuraminidase inhibitors using molecular docking combined with machine-learning-based scoring function.

Science.gov (United States)

Zhang, Li; Ai, Hai-Xin; Li, Shi-Meng; Qi, Meng-Yuan; Zhao, Jian; Zhao, Qi; Liu, Hong-Sheng

2017-10-10

In recent years, an epidemic of the highly pathogenic avian influenza H7N9 virus has persisted in China, with a high mortality rate. To develop novel anti-influenza therapies, we have constructed a machine-learning-based scoring function (RF-NA-Score) for the effective virtual screening of lead compounds targeting the viral neuraminidase (NA) protein. RF-NA-Score is more accurate than RF-Score, with a root-mean-square error of 1.46, Pearson's correlation coefficient of 0.707, and Spearman's rank correlation coefficient of 0.707 in a 5-fold cross-validation study. The performance of RF-NA-Score in a docking-based virtual screening of NA inhibitors was evaluated with a dataset containing 281 NA inhibitors and 322 noninhibitors. Compared with other docking-rescoring virtual screening strategies, rescoring with RF-NA-Score significantly improved the efficiency of virtual screening, and a strategy that averaged the scores given by RF-NA-Score, based on the binding conformations predicted with AutoDock, AutoDock Vina, and LeDock, was shown to be the best strategy. This strategy was then applied to the virtual screening of NA inhibitors in the SPECS database. The 100 selected compounds were tested in an in vitro H7N9 NA inhibition assay, and two compounds with novel scaffolds showed moderate inhibitory activities. These results indicate that RF-NA-Score improves the efficiency of virtual screening for NA inhibitors, and can be used successfully to identify new NA inhibitor scaffolds. Scoring functions specific for other drug targets could also be established with the same method.

High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

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Heather L Martin

Full Text Available Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs.

Science.gov (United States)

Guo, Jinchao; Chen, Lili; Liu, Xin; Gao, Ying; Zhang, Dabing; Yang, Litao

2012-06-01

Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyceshygroscopicus (bar) and Streptomyces viridochromogenes (pat), and Cry1Ab, Cry1Ac, Cry1A(b/c), mCry3A, and Cry3Bb1 derived from Bacillus thuringiensis. The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80targetcopies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program. Copyright © 2011 Elsevier Ltd. All rights reserved.

Non-animal photosafety assessment approaches for cosmetics based on the photochemical and photobiochemical properties.

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Onoue, Satomi; Suzuki, Gen; Kato, Masashi; Hirota, Morihiko; Nishida, Hayato; Kitagaki, Masato; Kouzuki, Hirokazu; Yamada, Shizuo

2013-12-01

The main purpose of the present study was to establish a non-animal photosafety assessment approach for cosmetics using in vitro photochemical and photobiochemical screening systems. Fifty-one cosmetics, pharmaceutics and other chemicals were selected as model chemicals on the basis of animal and/or clinical photosafety information. The model chemicals were assessed in terms of photochemical properties by UV/VIS spectral analysis, reactive oxygen species (ROS) assay and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Most phototoxins exhibited potent UV/VIS absorption with molar extinction coefficients of over 1000M(-1)cm(-1), although false-negative prediction occurred for 2 cosmetic phototoxins owing to weak UV/VIS absorption. Among all the cosmetic ingredients, ca. 42% of tested chemicals were non-testable in the ROS assay because of low water solubility; thereby, micellar ROS (mROS) assay using a solubilizing surfactant was employed for follow-up screening. Upon combination use of ROS and mROS assays, the individual specificity was 88.2%, and the positive and negative predictivities were estimated to be 94.4% and 100%, respectively. In the 3T3 NRU PT, 3 cosmetics and 4 drugs were incorrectly predicted not to be phototoxic, although some of them were typical photoallergens. Thus, these in vitro screening systems individually provide false predictions; however, a systematic tiered approach using these assays could provide reliable photosafety assessment without any false-negatives. The combined use of in vitro assays might enable simple and fast non-animal photosafety evaluation of cosmetic ingredients. Copyright © 2013 Elsevier Ltd. All rights reserved.

Screening agars for MRSA: evaluation of a stepwise diagnostic approach with two different selective agars for the screening for methicillin-resistant Staphylococcus aureus (MRSA).

Science.gov (United States)

Micheel, Volker; Hogan, Benedikt; Köller, Thomas; Warnke, Philipp; Crusius, Sabine; Hinz, Rebecca; Hagen, Ralf Matthias; Schwarz, Norbert Georg; Frickmann, Hagen

2015-01-01

Colonization with methicillin-resistant Staphylococcus aureus (MRSA) poses a hygiene risk that does not spare field hospitals or military medical field camps during military deployments. Diagnostic options for unambiguously identifying MRSA isolates are usually scarce in military environments. In this study, we assessed the stepwise application of two different selective agars for the specific identification of MRSA in screening analyses. Nasal swabs from 1541 volunteers were subjected to thioglycollate broth enrichment and subsequently screened on CHROMagar MRSA selective agar for the identification of MRSA. The MRSA identity of suspicious-looking colonies was confirmed afterwards or excluded by another selective agar, chromID MRSA. All isolates from the selective agars with MRSA-specific colony morphology were identified by biochemical methods and mass spectrometry. The initial CHROMagar MRSA screening identified suspicious colonies in 36 out of 1541 samples. A total of 25 of these 36 isolates showed MRSA-like growth on chromID agar. Out of these 25 isolates, 24 were confirmed as MRSA, while one isolate was identified as Staphylococcus kloosii. From the 11 strains that did not show suspicious growth on chromID agar, 3 were methicillin-sensitive Staphylococcus aureus (MSSA, with one instance of co-colonization with Corynebacterium spp.), 2 were confirmed as MRSA (with 1 instance of co-colonization with MSSA), 2 were lost during passaging and could not be re-cultured, one could not be identified by the applied approaches, and the remaining 3 strains were identified as Staphylococcus saprophyticus, Staphylococcus hominis (co-colonized with Macrococcus caseolyticus) and Staphylococcus cohnii, respectively. The application of the selective agar CHROMagar MRSA alone proved to be too non-specific to allow for a reliable diagnosis of the presence of MRSA. The combined use of two selective agars in a stepwise approach reduced this non-specificity with an acceptably low

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

Science.gov (United States)

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

2012-09-01

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. The identification of phytoconstituents and assay of antioxidant, thrombolytic, cytotoxic, antimicrobial activities were conducted using specific standard in vitro procedures. 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. The present findings suggest that, the plant widely available in Bangladesh, could be a prominent source of medicinally important natural compounds.

In Vitro and in Vivo Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives

Directory of Open Access Journals (Sweden)

Barbara Guantario

2018-03-01

Full Text Available Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 Enterococcus gallinarum, 3 E. casseliflavus, 14 Leuconostoc mesenteroides, 19 Lactobacillus pentosus, 7 L. coryniformis, and 2 L. oligofermentans. The L. pentosus and L. coryniformis strains were subjected to further screening to evaluate their probiotic potential, using a combination of in vitro and in vivo approaches. The majority of them showed high survival rates under in vitro simulated gastro-intestinal conditions, and positive antimicrobial activity against Salmonella enterica serovar Typhimurium, Listeria monocytogenes and enterotoxigenic Escherichia coli (ETEC pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three L. coryniformis strains were selected as very good performers in the initial in vitro testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth in vitro. Parallel screening employing the simplified model organism Caenorhabditis elegans, fed the Lactobacillus strains as a food source, revealed that one L. pentosus and one L. coryniformis strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial

Staged anticonvulsant screening for chronic epilepsy.

Science.gov (United States)

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.

Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.

Directory of Open Access Journals (Sweden)

Anthony Arnoldo

2008-02-01

Full Text Available Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS, a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

Phytochemical screening and antimalarial activity of some plants traditionally used in Indonesia

Directory of Open Access Journals (Sweden)

Syamsudin Abdillah

2015-06-01

Full Text Available Objective: To evaluate ethanolic extracts of phytochemical screening, in vitro and in vivo antiplasmodial activities of 15 plants used as antimalarial in Sei Kepayang, North Sumatra. Methods: Extraction was done through maceration with 70% ethanol and screened against chemical content, in vitro test anti-plasmodium against Plasmodium falciparum 3D7 strain and in vivo test in mice infected Plasmodium berghei. Results: The results showed that the plant extract contained a group of saponins, flavonoids, alkaloids, quinone, sterols, triterpene, tannins and cumarine. However, extract of Momordica charantia, Carica papaya, Garcinia atroviridis, Alstonia scholaris, Smallanthus sonchifolia and Cassia siamea had strong anti-plasmodium activity both in vitro and in vivo. Conclusions: In vitro and in vivo antiplasmodial activities of 15 plants are used as antimalarial in Sei Kepayang, North Sumatra. All the plants have in vitro and in vivo anti-plasmodium activity except Orthosiphon stamineus and Luffa cylindrica (ED50 > 1 000 mg/kg body weight and IC50 > 100 μg/mL, respectively.

One step versus two step approach for gestational diabetes screening: systematic review and meta-analysis of the randomized trials.

Science.gov (United States)

Saccone, Gabriele; Caissutti, Claudia; Khalifeh, Adeeb; Meltzer, Sara; Scifres, Christina; Simhan, Hyagriv N; Kelekci, Sefa; Sevket, Osman; Berghella, Vincenzo

2017-12-03

To compare both the prevalence of gestational diabetes mellitus (GDM) as well as maternal and neonatal outcomes by either the one-step or the two-step approaches. Electronic databases were searched from their inception until June 2017. We included all randomized controlled trials (RCTs) comparing the one-step with the two-step approaches for the screening and diagnosis of GDM. The primary outcome was the incidence of GDM. Three RCTs (n = 2333 participants) were included in the meta-analysis. 910 were randomized to the one step approach (75 g, 2 hrs), and 1423 to the two step approach. No significant difference in the incidence of GDM was found comparing the one step versus the two step approaches (8.4 versus 4.3%; relative risk (RR) 1.64, 95%CI 0.77-3.48). Women screened with the one step approach had a significantly lower risk of preterm birth (PTB) (3.7 versus 7.6%; RR 0.49, 95%CI 0.27-0.88), cesarean delivery (16.3 versus 22.0%; RR 0.74, 95%CI 0.56-0.99), macrosomia (2.9 versus 6.9%; RR 0.43, 95%CI 0.22-0.82), neonatal hypoglycemia (1.7 versus 4.5%; RR 0.38, 95%CI 0.16-0.90), and admission to neonatal intensive care unit (NICU) (4.4 versus 9.0%; RR 0.49, 95%CI 0.29-0.84), compared to those randomized to screening with the two step approach. The one and the two step approaches were not associated with a significant difference in the incidence of GDM. However, the one step approach was associated with better maternal and perinatal outcomes.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Science.gov (United States)

Hop, Cornelis E C A; Cole, Mark J; Davidson, Ralph E; Duignan, David B; Federico, James; Janiszewski, John S; Jenkins, Kelly; Krueger, Suzanne; Lebowitz, Rebecca; Liston, Theodore E; Mitchell, Walter; Snyder, Mark; Steyn, Stefan J; Soglia, John R; Taylor, Christine; Troutman, Matt D; Umland, John; West, Michael; Whalen, Kevin M; Zelesky, Veronica; Zhao, Sabrina X

2008-11-01

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Approaching the Hard-to-Reach in Organized Colorectal Cancer Screening: an Overview of Individual, Provider and System Level Coping Strategies

Directory of Open Access Journals (Sweden)

Jason Liwen Huang

2017-06-01

Full Text Available Background: Despite the proven effectiveness of colorectal cancer (CRC screening on reduction of CRC mortality, the uptake of CRC screening remains low. Participation rate is one of determinants for the success of organized population-based screening program. This review aims to identify those who are hard-to-reach, and summarize the strategies to increase their screening rate from individual, provider and system levels. Methods: A systematic search of electronic English databases was conducted on the factors and strategies of uptake in CRC screening for the hard-to-reach population up to May 2017. Discussion: The coverage rate and participation rate are two indexes to identify the hard-to-reach population in organized CRC screening program. However, the homeless, new immigrants, people with severe mental illness, the jail intimates, and people with characteristics including lower education levels and/or low socioeconomic status, living in rural/remote areas, without insurance, and racial minorities are usually recognized as hard-to-reach populations. For them, organized screening programs offer a better coverage, while novel invitation approaches for eligible individuals and multiple strategies from primary care physicians are still needed to enhance screening rates among subjects who are hard-to-reach. Suggestions implied the effectiveness of interventions at the system level, including linkages to general practice; use of decision making tools; enlisting supports from coalition; and the continuum from screening to diagnosis and treatment. Conclusion: Organized CRC screening offers a system access to approach the hard-to-reach populations. To increase their uptake, multiple and novel strategies from individual, provider and system levels should be applied. For policymakers, public healthcare providers and community stakeholders, it is a test to tailor their potential needs and increase their participation rates through continuous efforts to

Human in vitro 3D co-culture model to engineer vascularized bone-mimicking tissues combining computational tools and statistical experimental approach.

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Bersini, Simone; Gilardi, Mara; Arrigoni, Chiara; Talò, Giuseppe; Zamai, Moreno; Zagra, Luigi; Caiolfa, Valeria; Moretti, Matteo

2016-01-01

The generation of functional, vascularized tissues is a key challenge for both tissue engineering applications and the development of advanced in vitro models analyzing interactions among circulating cells, endothelium and organ-specific microenvironments. Since vascularization is a complex process guided by multiple synergic factors, it is critical to analyze the specific role that different experimental parameters play in the generation of physiological tissues. Our goals were to design a novel meso-scale model bridging the gap between microfluidic and macro-scale studies, and high-throughput screen the effects of multiple variables on the vascularization of bone-mimicking tissues. We investigated the influence of endothelial cell (EC) density (3-5 Mcells/ml), cell ratio among ECs, mesenchymal stem cells (MSCs) and osteo-differentiated MSCs (1:1:0, 10:1:0, 10:1:1), culture medium (endothelial, endothelial + angiopoietin-1, 1:1 endothelial/osteo), hydrogel type (100%fibrin, 60%fibrin+40%collagen), tissue geometry (2 × 2 × 2, 2 × 2 × 5 mm(3)). We optimized the geometry and oxygen gradient inside hydrogels through computational simulations and we analyzed microvascular network features including total network length/area and vascular branch number/length. Particularly, we employed the "Design of Experiment" statistical approach to identify key differences among experimental conditions. We combined the generation of 3D functional tissue units with the fine control over the local microenvironment (e.g. oxygen gradients), and developed an effective strategy to enable the high-throughput screening of multiple experimental parameters. Our approach allowed to identify synergic correlations among critical parameters driving microvascular network development within a bone-mimicking environment and could be translated to any vascularized tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioactive screening and in vitro antioxidant assessment of Nauclea latifolia leaf decoction

Science.gov (United States)

Iheagwam, Franklyn Nonso; Nsedu, Emmanuel Israel; Kayode, Kazeem Oyindamola; Emiloju, Opeyemi Christianah; Ogunlana, Olubanke Olujoke; Chinedu, Shalom Nwodo

2018-04-01

The phytochemical constituents and antioxidant properties of Nauclea latifolia leaf decoction were investigated. Dried leaves were extracted in ethanol. Qualitative and quantitative phytochemical analysis was determined spectrometrically. The antioxidant activities were examined in vitro using 2,2-diphenyl-1-picrylhydrazyl radical, total antioxidant capacity and ferric reducing antioxidant power assays. Phytochemical screening confirmed the presence of flavonoids, alkaloids, anthocyanins, betacyanins, phenols, saponins, terpenoids, cardiac glycosides and quinones. The total lycopene, β-carotene, phenolics, flavonoid and alkaloid content were found to be 0.038 ± 0.01 mg CAE/g, 0.120 ± 0.04 mg CAE/g, 58.08 ± 0.58 mg GAE/g, 10.75 ± 0.17 mg RE/g and 0.32 ± 0.08% respectively. N. latifolia ethanol leaf extract demonstrated effective antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl with an IC50 of 2.58 ± 0.08 mg/mL compared to 0.86 ± 0.02 mg/mL and < 0.01 ± 0.01 mg/mL for butylated hydroxytoluene and ascorbic acid respectively. Total antioxidant capacity and ferric reducing antioxidant power of the extract were 73.81 ± 2.27 and 1314.45 ± 197.64 mg AAE/g respectively. Excellent positive correlations between the phenolic content and antioxidant activities of the extract were observed. The leaf of N. latifolia is of therapeutic value and may be exploited for its rich antioxidant components.

Advancing early detection of autism spectrum disorder by applying an integrated two-stage screening approach

NARCIS (Netherlands)

Oosterling, Iris J.; Wensing, Michel; Swinkels, Sophie H.; van der Gaag, Rutger Jan; Visser, Janne C.; Woudenberg, Tim; Minderaa, Ruud; Steenhuis, Mark-Peter; Buitelaar, Jan K.

Background: Few field trials exist on the impact of implementing guidelines for the early detection of autism spectrum disorders (ASD). The aims of the present study were to develop and evaluate a clinically relevant integrated early detection programme based on the two-stage screening approach of

Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

Energy Technology Data Exchange (ETDEWEB)

Vanparys, Caroline, E-mail: caroline.vanparys@ua.ac.be [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)

2010-09-15

In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

International Nuclear Information System (INIS)

Vanparys, Caroline; Depiereux, Sophie; Nadzialek, Stephanie; Robbens, Johan; Blust, Ronny; Kestemont, Patrick; De Coen, Wim

2010-01-01

In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC 50 value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R 2 = 0.98), the estrogen receptor (ER) binding (R 2 = 0.84) and the ER transcription activation assay (R 2 = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of

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

Science.gov (United States)

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.

A high-throughput screening approach to discovering good forms of biologically inspired visual representation.

Science.gov (United States)

Pinto, Nicolas; Doukhan, David; DiCarlo, James J; Cox, David D

2009-11-01

While many models of biological object recognition share a common set of "broad-stroke" properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model--e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit) is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct "parts" have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor). In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision.

A high-throughput screening approach to discovering good forms of biologically inspired visual representation.

Directory of Open Access Journals (Sweden)

Nicolas Pinto

2009-11-01

Full Text Available While many models of biological object recognition share a common set of "broad-stroke" properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model--e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct "parts" have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor. In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision.

In vitro systems in pneumocystis research

DEFF Research Database (Denmark)

Dei-Cas, E; Cailliez, J C; Lundgren, Bettina

1996-01-01

as well as elimination of host cell debris from parasite extracts. In other investigations, such as transmission, infectivity, life cycle, biochemical, in vitro culture or drug-screening studies, viable and infectious Pneumocystis organisms are urgently required. However, there is no generally accepted...

Identification of LasR Ligands through a Virtual Screening Approach

DEFF Research Database (Denmark)

Skovstrup, Søren; Le Quement, Sebastian Thordal; Hansen, Thomas

2013-01-01

as an attractive therapeutic target for the next generation of antimicrobial agents. In the present study, a virtual screening workflow combining pharmacophore‐ and structure‐based approaches was used to identify new LasR ligands. Five novel inducers and three inhibitors of LasR activity were validated...... experimentally by use of a cell‐based assay. Interestingly, these compounds are molecularly distinct from the native signal molecule, N‐3‐oxododecanoyl‐L‐homoserine lactone (OHN), and may serve as lead structures for the design of new drugs. The binding modes of these compounds to the OHN binding site in Las......R were predicted and used to identify the key interactions that contribute to the induction and inhibition of LasR activity....

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

Science.gov (United States)

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 evaluation of medicinal, antioxidant activities and phytochemical screening of iphiona grantioides and pluchea arguta subsp

International Nuclear Information System (INIS)

Naveed, S.; Ibrar, M.

2016-01-01

The crude ethanolic extracts (EE) derived from different parts of Iphiona grantioides (flower, leaf, stem and root) and Pluchea arguta subsp glabra Qaiser (leaf, stem and root) were screened In vitro for possible antioxidant, antibacterial, antifungal activities and phytochemical profile. Results for phytochemical screening revealed the presence of various important primary and secondary metabolites including carbohydrates, protein, essential oils, tannins, flavonoids, saponins, glycosides, phytosterol, tri-terphenoids and volatile oil in the ethanolic extracts (EE) of different parts of I. grantioides and P. arguta. Significant antibacterial activity was observed for flower and leaf (EE) of I. grantioides against Klebsiela pneumonia and Escherichia coli, while moderate antibacterial activity was showed by extracts from other parts of this plant. Similarly P. arguta leaf (EE) showed significant inhibitory activity against Escherichia coli, Pseudomonas aeroginosa and Micrococcus luteus than its stem and roots extract. The crude ethanolic extract of all parts of I. grantioides and P. arguta proved a rich source of fungicidal potential. Both the plants proved to have antioxidant potential by exhibiting DPPH radical scavenging activity. So it may be concluded from the present investigations that I. grantioides and P. arguta, both have a wide range of antibacterial, antifungal and antioxidant properties and can be utilized for the development of antimicrobial drugs for human beings, animals and as safe pesticides in agricultural practices as well as could be an important source of natural radical scavengers. (author)

New approaches to screening infrastructure investments in multi-reservoir systems- Evaluating proposed dams in Ethiopia and Kenya

Science.gov (United States)

Harou, J. J.; Geressu, R. T.; Hurford, A. P.

2014-12-01

Two approaches have been used traditionally to screen infrastructure investments in multi-reservoir systems: scenario analysis of a few simulated designs and deterministic optimization, sometimes using hydro-economic models or screening optimization models. Simulation models realistically represent proposed water systems and can easily include multiple performance metrics; however each prospective system operating rules need to be formulated and simulated for each proposed design (time consuming. Optimization models have been used to overcome this burden. Screening optimization models use integer or non-linear programming and can be challenging to apply to large and/or multi-objective systems. Hydro-economic models that use deterministic (implicit stochastic) optimization must be modified to examine each different plan and they cannot always reproduce realistic or politically acceptable system operations. In this presentation we demonstrate the application of a new screening approach to multi-reservoir systems where operating rules and new assets (dams) are simultaneously optimized in a multi-criteria context. Results are not least cost investment plans that satisfy reliability or other engineering constraints, but rather Pareto-optimal sets of asset portfolios that work well under historical and/or future scenarios. This is achieved by using stakeholder-built simulation models linked to multi-criteria search algorithms (e.g. many objective evolutionary algorithms, MOEA). Typical output is demonstrated through two case-studies on the Tana and Blue Nile rivers where operating rules and reservoir assets are efficiently screened together considering stakeholder-defined metrics. The focus on the Tana system is how reservoir operating rules and new irrigation schemes should be co-managed to limit ecological damages. On the Nile system, we identify Blue Nile river reservoir capacities that least negatively impact downstream Nile nations. Limitations and new directions of

Screening for chronic comorbid diseases in people with HIV: the need for a strategic approach.

Science.gov (United States)

Peters, B; Post, F; Wierzbicki, A S; Phillips, A; Power, L; Das, S; Johnson, M; Moyle, G; Hughes, L; Wilkins, E; McCloskey, E; Compston, J; Di Angelantonio, E

2013-01-01

Among people living with HIV, the proportion of deaths attributed to chronic noninfectious comorbid diseases has increased over the past 15 years. This is partly a result of increased longevity in the era of highly active antiretroviral therapy (HAART), and also because HIV infection is related, causally or otherwise, to several chronic conditions. These comorbidities include conditions that are strongly associated with modifiable risk factors, such as cardiovascular disease (CVD), diabetes, and renal and bone diseases, and increasingly management guidelines for HIV recommend risk evaluation for these conditions. The uptake of these screening approaches is often limited by the resources required for their application, and hence the management of risk reduction in most HIV-infected populations falls below a reasonable standard. The situation is compounded by the fact that few risk calculators have been adjusted for specific use in HIV infection. There is substantial overlap of risk factors for the four common comorbid diseases listed above that are especially relevant in HIV infection, and this offers an opportunity to develop a simple screening approach that encompasses the key risk factors for lifestyle-related chronic disease in people with HIV infection. This would identify those patients who require more in-depth investigation, and facilitate a stepwise approach to targeted management. Such a tool could improve communication between patient and clinician. A significant proportion of people with HIV are sufficiently engaged with their care to participate in health promotion and take the lead in using patient-centric screening measures. Health-based social networking offers a mechanism for dissemination of such a tool and is able to embed educational messages and support within the process. © 2012 British HIV Association.

Quality management in European screening laboratories in blood establishments: A view of current approaches and trends.

Science.gov (United States)

Pereira, Paulo; Westgard, James O; Encarnação, Pedro; Seghatchian, Jerard; de Sousa, Gracinda

2015-04-01

The screening laboratory has a critical role in the post-transfusion safety. The success of its targets and efficiency depends on the management system used. Even though the European Union directive 2002/98/EC requires a quality management system in blood establishments, its requirements for screening laboratories are generic. Complementary approaches are needed to implement a quality management system focused on screening laboratories. This article briefly discusses the current good manufacturing practices and good laboratory practices, as well as the trends in quality management system standards. ISO 9001 is widely accepted in some European Union blood establishments as the quality management standard, however this is not synonymous of its successful application. The ISO "risk-based thinking" is interrelated with the quality risk-management process of the EuBIS "Standards and criteria for the inspection of blood establishments". ISO 15189 should be the next step on the quality assurance of a screening laboratory, since it is focused on medical laboratory. To standardize the quality management systems in blood establishments' screening laboratories, new national and European claims focused on technical requirements following ISO 15189 is needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity

Energy Technology Data Exchange (ETDEWEB)

Boisvert, Annie; Jones, Steven; Issop, Leeyah [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Erythropel, Hanno C. [Department of Chemical Engineering, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Papadopoulos, Vassilios [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Culty, Martine, E-mail: martine.culty@mcgill.ca [The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Medicine, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada); Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, Canada H4A 3J1 (Canada)

2016-10-15

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. - Highlights: • Phthalate plasticizers exert toxic effects on male reproduction

In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity

International Nuclear Information System (INIS)

Boisvert, Annie; Jones, Steven; Issop, Leeyah; Erythropel, Hanno C.; Papadopoulos, Vassilios; Culty, Martine

2016-01-01

Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. - Highlights: • Phthalate plasticizers exert toxic effects on male reproduction

In silico study of in vitro GPCR assays by QSAR modeling

Science.gov (United States)

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...

Analysis of ToxCast data for food-relevant compounds by comparison with in vivo data using the RISK21 approach

Science.gov (United States)

The ToxCast program has generated a wealth of in vitro high throughput screening data, and best approaches for the interpretation and use of these data remain undetermined. We present case studies comparing the ToxCast and in vivo toxicity data for two food contact substances us...

Does brain slices from pentylenetetrazole-kindled mice provide a more predictive screening model for antiepileptic drugs?

DEFF Research Database (Denmark)

Hansen, Suzanne L.; Sterjev, Zoran; Werngreen, Marie

2012-01-01

The cortical wedge is a commonly applied model for in vitro screening of new antiepileptic drugs (AEDs) and has been extensively used in characterization of well-known AEDs. However, the predictive validity of this model as a screening model has been questioned as, e.g., carbamazepine has been...... screening model for AEDs. To this end, we compared the in vitro and in vivo pharmacological profile of several selected AEDs (phenobarbital, phenytoin, tiagabine, fosphenytoin, valproate, and carbamazepine) along with citalopram using the PTZ-kindled model and brain slices from naïve, saline...

Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.

Science.gov (United States)

Martins da Silva, Sarah J; Brown, Sean G; Sutton, Keith; King, Louise V; Ruso, Halil; Gray, David W; Wyatt, Paul G; Kelly, Mark C; Barratt, Christopher L R; Hope, Anthony G

2017-05-01

Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

Science.gov (United States)

Turk, Samo; Kovac, Andreja; Boniface, Audrey; Bostock, Julieanne M; Chopra, Ian; Blanot, Didier; Gobec, Stanislav

2009-03-01

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

A "genome-to-lead" approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1-like dopamine receptors.

Directory of Open Access Journals (Sweden)

Jason M Meyer

2012-01-01

Full Text Available BACKGROUND: Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides. METHODOLOGY/PRINCIPAL FINDINGS: We describe a "genome-to-lead" approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2 from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D(1-like (Gα(s-coupled receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC(50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM. Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC(50 = 5.8±1.5 nM and norepinephrine (EC(50 = 760±180 nM, while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as "hits," and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D(1 dopamine receptor (hD(1 revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2

Assessment of pyrrolizidine alkaloid-induced toxicity in an in vitro screening model.

Science.gov (United States)

Li, Yan Hong; Kan, Winnie Lai Ting; Li, Na; Lin, Ge

2013-11-25

Pyrrolizidine alkaloids (PAs) are a group of heterocyclic phytotoxins present in a wide range of plants. The consumption of PA-containing medicinal herbs or PA-contaminated foodstuffs has long been reported to cause human hepatotoxicity. However, the degrees of hepatotoxicity of different PAs are unknown, which makes it difficult to determine a universal threshold of toxic dose of individual PAs for safe regulation of PA-containing natural products. The aim of the present study is to develop a simple and convenient in vitro model to assess the hepatotoxicity of different PAs. Six common cytotoxicity assays were used to evaluate the hepatotoxicity of different PAs in human hepatocellular carcinoma HepG2 cells. The combination of MTT and bromodeoxyuridine incorporation (BrdU) assays demonstrated to be a suitable method to evaluate the toxic potencies of various PAs in HepG2 cells, and the results indicated that otonecine-type PA (clivorine: IC₂₀=0.013 ± 0.004 mM (MTT), 0.066 ± 0.031 mM (BrdU)) exhibited significantly higher cytotoxic and anti-proliferative effects than retronecine-type PA (retrorsine: IC₂₀=0.27 ± 0.07 mM (MTT), 0.19 ± 0.03 mM (BrdU)). While as expected, the known less toxic platyphylline-type PA (platyphylline: IC₂₀=0.85 ± 0.11 mM (MTT), 1.01 ± 0.40 mM (BrdU)) exhibited significantly less toxicity. The different cytotoxic and anti-proliferative potencies of various PAs in the same retronecine-type could also be discriminated by using the combined MTT and BrdU assays. In addition, the developed assays were further utilized to test alkaloid extract of Gynura segetum, a senecionine and seneciphylline-containing herb, the overall cytotoxicity of two PAs in the extract was comparable to that of these two PAs tested individually. Using the developed in vitro model, the cytotoxicity of different PAs and the extract of a PA-containing herb were investigated in parallel in one system, and their different hepatotoxic potencies were determined

In vitro mutagenesis and production of agronomically useful potato variants

International Nuclear Information System (INIS)

Das, A.; Gosal, S.S.; Sidhu, J.S.; Dhaliwal, H.S.

2001-01-01

In vitro grown shoot cultures of two Indian potato varieties 'Kufri jyoti' and 'Kufri Chandramukhi' were subjected to gamma irradiation at 20 and 40 Gy. The irradiated shoot cultures were subcultured to yield a generation of plantlets. After 4-6 weeks of incubation, these shoots were transferred onto MS medium supplemented with benzylaminopurine, BAP (10mg/1) and sucrose (8% w/v) and incubated at 20 deg. C. The M 1 V 3 plants were screened in vitro for late blight resistance by detached leaf method. The resistant plants were screened in M 1 V 4 generation by artificial inoculation of sporangial inoculum on the pot sown plants. Chlorophyll persistence is a simple screening method for heat tolerance. Chlorophyll persistence of different plantlets showed that the percentage of injury was less in the case of plants, which had been obtained from irradiated material. In the case of control plants, there was one hundred-percent damage to the plants. The mutation frequency was calculated for characters like late blight resistance and heat tolerance (in vitro microtuberisation and chlorophyll persistence). The gamma ray dose of 40 Gy was observed to produce a higher mutation frequency

3D Printing of Tissue Engineered Constructs for in vitro Modeling of Disease Progression and Drug Screening

Science.gov (United States)

Vanderburgh, Joseph; Sterling, Julie A.

2016-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D versus 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design (CAD) file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs (TECs) that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs. PMID:27169894

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet

Science.gov (United States)

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet (Presented by Dr. Marilyn J. Aardema, Chief Scientific Advisor, Toxicology, Dr. Leon Stankowski, et. al. (6/28/2012)

Assessment of xenoestrogenic exposure by a biomarker approach: application of the E-Screen bioassay to determine estrogenic response of serum extracts

Directory of Open Access Journals (Sweden)

Weihe Pal

2003-10-01

Full Text Available Abstract Background Epidemiological documentation of endocrine disruption is complicated by imprecise exposure assessment, especially when exposures are mixed. Even if the estrogenic activity of all compounds were known, the combined effect of possible additive and/or inhibiting interaction of xenoestrogens in a biological sample may be difficult to predict from chemical analysis of single compounds alone. Thus, analysis of mixtures allows evaluation of combined effects of chemicals each present at low concentrations. Methods We have developed an optimized in vitro E-Screen test to assess the combined functional estrogenic response of human serum. The xenoestrogens in serum were separated from endogenous steroids and pharmaceuticals by solid-phase extraction followed by fractionation by high-performance liquid chromatography. After dissolution of the isolated fraction in ethanol-DMSO, the reconstituted extract was added with estrogen-depleted fetal calf serum to MCF-7 cells, the growth of which is stimulated by estrogen. After a 6-day incubation on a microwell plate, cell proliferation was assessed and compared with the effect of a 17-beta-estradiol standard. Results and conclusions To determine the applicability of this approach, we assessed the estrogenicity of serum samples from 30 pregnant and 60 non-pregnant Danish women thought to be exposed only to low levels of endocrine disruptors. We also studied 211 serum samples from pregnant Faroese women, whose marine diet included whale blubber that contain a high concentration of persistent halogenated pollutants. The estrogenicity of the serum from Danish controls exceeded the background in 22.7 % of the cases, while the same was true for 68.1 % of the Faroese samples. The increased estrogenicity response did not correlate with the lipid-based concentrations of individual suspected endocrine disruptors in the Faroese samples. When added along with the estradiol standard, an indication of an

Rapid Screening of In-Vitro Regenerated Plantlets of Four Nigerian ...

African Journals Online (AJOL)

Prof. Ogunji

Tissue culture technique provides a rapid means of studying plant-pathogen interaction in a controlled ... Keywords: cowpea, in-vitro regeneration, hormones, Fusarium wilt, protocols. .... solanacearum and evaluation of tomato in Nepal.

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

Science.gov (United States)

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.

Cost-effectiveness analysis of preimplantation genetic screening and in vitro fertilization versus expectant management in patients with unexplained recurrent pregnancy loss.

Science.gov (United States)

Murugappan, Gayathree; Ohno, Mika S; Lathi, Ruth B

2015-05-01

To determine whether in vitro fertilization with preimplantation genetic screening (IVF/PGS) is cost effective compared with expectant management in achieving live birth for patients with unexplained recurrent pregnancy loss (RPL). Decision analytic model comparing costs and clinical outcomes. Academic recurrent pregnancy loss programs. Women with unexplained RPL. IVF/PGS with 24-chromosome screening and expectant management. Cost per live birth. The IVF/PGS strategy had a live-birth rate of 53% and a clinical miscarriage rate of 7%. Expectant management had a live-birth rate of 67% and clinical miscarriage rate of 24%. The IVF/PGS strategy was 100-fold more expensive, costing $45,300 per live birth compared with $418 per live birth with expectant management. In this model, IVF/PGS was not a cost-effective strategy for increasing live birth. Furthermore, the live-birth rate with IVF/PGS needs to be 91% to be cost effective compared with expectant management. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A Learning Collaborative Approach to Improve Primary Care STI Screening.

Science.gov (United States)

McKee, M Diane; Alderman, Elizabeth; York, Deborah V; Blank, Arthur E; Briggs, Rahil D; Hoidal, Kelsey E S; Kus, Christopher; Lechuga, Claudia; Mann, Marie; Meissner, Paul; Patel, Nisha; Racine, Andrew D

2017-10-01

The Bronx Ongoing Pediatric Screening (BOPS) project sought to improve screening for sexual activity and sexually transmitted infections (gonorrhea and chlamydia [GCC] and HIV) in a primary care network, employing a modified learning collaborative, real-time clinical data feedback to practices, improvement coaching, and a pay-for-quality monetary incentive. Outcomes are compared for 11 BOPS-participating sites and 10 non-participating sites. The quarterly median rate for documenting sexual activity status increased from 55% to 88% (BOPS sites) and from 13% to 74% (non-BOPS sites). GCC screening of sexually active youth increased at BOPS and non-BOPS sites. Screening at non-health care maintenance visits improved more at BOPS than non-BOPS sites. Data from nonparticipating sites suggests that introduction of an adolescent EMR template or other factors improved screening rates regardless of BOPS participation; BOPS activities appear to promote additional improvement of screening during non-health maintenance visits.

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Directory of Open Access Journals (Sweden)

Mutsuki Amano

Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities.

Science.gov (United States)

Schmidt, Béla Z; Lehmann, Martin; Gutbier, Simon; Nembo, Erastus; Noel, Sabrina; Smirnova, Lena; Forsby, Anna; Hescheler, Jürgen; Avci, Hasan X; Hartung, Thomas; Leist, Marcel; Kobolák, Julianna; Dinnyés, András

2017-01-01

Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Breast Cancer Screening in Denmark

DEFF Research Database (Denmark)

Jørgensen, Karsten Juhl; Gøtzsche, Peter C; Kalager, Mette

2017-01-01

Background: Effective breast cancer screening should detect early-stage cancer and prevent advanced disease. Objective: To assess the association between screening and the size of detected tumors and to estimate overdiagnosis (detection of tumors that would not become clinically relevant). Design......) and nonadvanced (≤20 mm) breast cancer tumors in screened and nonscreened women were measured. Two approaches were used to estimate the amount of overdiagnosis: comparing the incidence of advanced and nonadvanced tumors among women aged 50 to 84 years in screening and nonscreening areas; and comparing...... rate ratio, 1.49 [95% CI, 1.43 to 1.54]). The first estimation approach found that 271 invasive breast cancer tumors and 179 ductal carcinoma in situ (DCIS) lesions were overdiagnosed in 2010 (overdiagnosis rate of 24.4% [including DCIS] and 14.7% [excluding DCIS]). The second approach, which accounted...

Screening and syndromic approaches to identify gonorrhea and chlamydial infection among women.

Science.gov (United States)

Sloan, N L; Winikoff, B; Haberland, N; Coggins, C; Elias, C

2000-03-01

The standard diagnostic tools to identify sexually transmitted infections are often expensive and have laboratory and infrastructure requirements that make them unavailable to family planning and primary health-care clinics in developing countries. Therefore, inexpensive, accessible tools that rely on symptoms, signs, and/or risk factors have been developed to identify and treat reproductive tract infections without the need for laboratory diagnostics. Studies were reviewed that used standard diagnostic tests to identify gonorrhea and cervical chlamydial infection among women and that provided adequate information about the usefulness of the tools for screening. Aggregation of the studies' results suggest that risk factors, algorithms, and risk scoring for syndromic management are poor indicators of gonorrhea and chlamydial infection in samples of both low and high prevalence and, consequently, are not effective mechanisms with which to identify or manage these conditions. The development and evaluation of other approaches to identify gonorrhea and chlamydial infections, including inexpensive and simple laboratory screening tools, periodic universal treatment, and other alternatives must be given priority.

Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology.

Science.gov (United States)

Meyer, Golo M; Maurer, Hans H; Meyer, Markus R

2016-01-01

This paper reviews MS approaches applied to metabolism studies, structure elucidation and qualitative or quantitative screening of drugs (of abuse) and/or their metabolites. Applications in clinical and forensic toxicology were included using blood plasma or serum, urine, in vitro samples, liquids, solids or plant material. Techniques covered are liquid chromatography coupled to low-resolution and high-resolution multiple stage mass analyzers. Only PubMed listed studies published in English between January 2008 and January 2015 were considered. Approaches are discussed focusing on sample preparation and mass spectral settings. Comments on advantages and limitations of these techniques complete the review.

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

International Nuclear Information System (INIS)

Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine; Tissier, Marie-Helene; Bonnet, Pierre-Antoine; Vian, Laurence; Fabre, Isabelle; Ourlin, Jean-Claude

2006-01-01

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. Only 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

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

Science.gov (United States)

Blaazer, Antoni R; Orrling, Kristina M; Shanmugham, Anitha; Jansen, Chimed; Maes, Louis; Edink, Ewald; Sterk, Geert Jan; Siderius, Marco; England, Paul; Bailey, David; de Esch, Iwan J P; Leurs, Rob

2015-01-01

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization. © 2014 Society for Laboratory Automation and Screening.

In-Vitro Antibacterial Activities And Preliminary Phytochemical ...

African Journals Online (AJOL)

Studies on the in-vitro antibacterial activities and phytochemical screening of the aqueous and ethanolic extracts of Zingiber officinale (ginger) against some clinical bacterial isolates (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) obtained from ear and urine samples were carried out using ...

A Tiered Approach to Retinopathy of Prematurity Screening (TARP) Using a Weight Gain Predictive Model and a Telemedicine System.

Science.gov (United States)

Gurwin, Jaclyn; Tomlinson, Lauren A; Quinn, Graham E; Ying, Gui-Shuang; Baumritter, Agnieshka; Binenbaum, Gil

2017-01-05

The Telemedicine Approaches to Evaluating Acute-Phase Retinopathy of Prematurity (e-ROP) Study telemedicine system of remote fundus image grading and The Children's Hospital of Philadelphia Retinopathy of Prematurity (CHOP-ROP) postnatal weight gain predictive model are 2 approaches for improving ROP screening efficiency. Current screening has low specificity for severe ROP. To describe a tiered approach to ROP screening (TARP) for identifying children who develop severe ROP using telemedicine and a predictive model synergistically. This investigation was a post hoc analysis of a cohort in the e-ROP Study (a multicenter prospective telemedicine study) and the Postnatal Growth and Retinopathy of Prematurity (G-ROP) Study (a multicenter retrospective cohort study). The setting was neonatal intensive care units at The Children's Hospital of Philadelphia and the Hospital of the University of Pennsylvania. Participants in the e-ROP Study were premature infants with a birth weight less than 1251 g and a known ROP outcome enrolled between May 25, 2011, and October 31, 2013. The G-ROP Study enrolled all infants undergoing ROP examinations with a known ROP outcome who were born between January 1, 2006, and December 31, 2011. The mean outcomes were the sensitivity for type 1 ROP, reductions in infants requiring imaging or examinations, numbers of imaging sessions and examinations, and total clinical encounters (imaging sessions and examinations combined). The following 4 screening approaches were evaluated: ROUTINE (only diagnostic examinations by an ophthalmologist), CHOP-ROP (birth weight and gestational age, with weekly weight gain initiating examinations when the risk cut point is surpassed), e-ROP IMAGING (trained reader grading of type 1 or 2 ROP initiates diagnostic examinations), and TARP (CHOP-ROP alarm initiates imaging, and imaging finding of severe ROP initiates diagnostic examinations). A total of 242 infants were included in the study, with a median birth

The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification.

Science.gov (United States)

Swyka, Robert A; Berkowitz, David B

2017-12-14

The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The in situ enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol interfaces an organic reaction layer with an adjacent aqueous layer containing reporting enzymes that act upon the organic reaction product, giving rise to a spectroscopic signal. ISES allows the experimentalist to rapidly glean information on the relative rates of a set of parallel organic/organometallic reactions under investigation, without the need to quench the reactions or draw aliquots. In certain cases, the real-time enzymatic readout also provides information on sense and magnitude of enantioselectivity and substrate specificity. This article contains protocols for single-well (relative rate) and double-well (relative rate/enantiomeric excess) ISES, in addition to a colorimetric ISES protocol and a miniaturized double-well procedure. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Directory of Open Access Journals (Sweden)

Erick Vaz Guimarães

2009-03-01

Full Text Available Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

Science.gov (United States)

Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

1988-02-01

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

Evaluation of two-year Jewish genetic disease screening program in Atlanta: insight into community genetic screening approaches.

Science.gov (United States)

Shao, Yunru; Liu, Shuling; Grinzaid, Karen

2015-04-01

Improvements in genetic testing technologies have led to the development of expanded carrier screening panels for the Ashkenazi Jewish population; however, there are major inconsistencies in current screening practices. A 2-year pilot program was launched in Atlanta in 2010 to promote and facilitate screening for 19 Jewish genetic diseases. We analyzed data from this program, including participant demographics and outreach efforts. This retrospective analysis is based on a de-identified dataset of 724 screenees. Data were obtained through medical chart review and questionnaires and included demographic information, screening results, response to outreach efforts, and follow-up behavior and preferences. We applied descriptive analysis, chi-square tests, and logistic regression to analyze the data and compare findings with published literature. The majority of participants indicated that they were not pregnant or did not have a partner who was pregnant were affiliated with Jewish organizations and reported 100 % AJ ancestry. Overall, carrier frequency was 1 in 3.9. Friends, rabbis, and family members were the most common influencers of the decision to receive screening. People who were older, had a history of pregnancy, and had been previously screened were more likely to educate others (all p influencers who then encouraged screening in the target population. Educating influencers and increasing overall awareness were the most effective outreach strategies.

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

International Nuclear Information System (INIS)

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

2011-01-01

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.

Computational approaches to screen candidate ligands with anti- Parkinson's activity using R programming.

Science.gov (United States)

Jayadeepa, R M; Niveditha, M S

2012-01-01

It is estimated that by 2050 over 100 million people will be affected by the Parkinson's disease (PD). We propose various computational approaches to screen suitable candidate ligand with anti-Parkinson's activity from phytochemicals. Five different types of dopamine receptors have been identified in the brain, D1-D5. Dopamine receptor D3 was selected as the target receptor. The D3 receptor exists in areas of the brain outside the basal ganglia, such as the limbic system, and thus may play a role in the cognitive and emotional changes noted in Parkinson's disease. A ligand library of 100 molecules with anti-Parkinson's activity was collected from literature survey. Nature is the best combinatorial chemist and possibly has answers to all diseases of mankind. Failure of some synthetic drugs and its side effects have prompted many researches to go back to ancient healing methods which use herbal medicines to give relief. Hence, the candidate ligands with anti-Parkinson's were selected from herbal sources through literature survey. Lipinski rules were applied to screen the suitable molecules for the study, the resulting 88 molecules were energy minimized, and subjected to docking using Autodock Vina. The top eleven molecules were screened according to the docking score generated by Autodock Vina Commercial drug Ropinirole was computed similarly and was compared with the 11 phytochemicals score, the screened molecules were subjected to toxicity analysis and to verify toxic property of phytochemicals. R Programming was applied to remove the bias from the top eleven molecules. Using cluster analysis and Confusion Matrix two phytochemicals were computationally selected namely Rosmarinic acid and Gingkolide A for further studies on the disease Parkinson's.

A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

Science.gov (United States)

Qin, Chong-Zhen; Ren, Xian; Tan, Zhi-Rong; Chen, Yao; Yin, Ji-Ye; Yu, Jing; Qu, Jian; Zhou, Hong-Hao; Liu, Zhao-Qian

2014-02-01

A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.

An integrated in silico/in vitro approach to assess the xenoestrogenic potential of Alternaria mycotoxins and metabolites.

Science.gov (United States)

Dellafiora, Luca; Warth, Benedikt; Schmidt, Verena; Del Favero, Giorgia; Mikula, Hannes; Fröhlich, Johannes; Marko, Doris

2018-05-15

Xenoestrogenic mycotoxins may contaminate food and feed posing a public health issue. Besides the zearalenone group, the Alternaria toxin alternariol (AOH) has been described as a potential mycoestrogen. However, the estrogenicity of Alternaria toxins is still largely overlooked and further data are needed to better describe the group toxicity. In the frame of risk assessment, mixed in silico/in vitro approaches already proved to be effective first-line analytical tools. An integrated in silico/in vitro approach was used to investigate the effects of metabolic and chemical modifications on the estrogenicity of AOH. Among the considered modifications, methylation was found critical for enhancing estrogenicity (as seen for alternariol monomethyl ether (AME)) while hydroxylation and glucuronidation had the opposite effect (as seen for 4-hydroxy AOH and 4-hydroxy AME). The structure-activity relationship analysis provided the structural rationale. Our results provide insights to design more efficient risk assessment studies expanding knowledge over the group toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Maximizing gain in high-throughput screening using conformal prediction.

Science.gov (United States)

Svensson, Fredrik; Afzal, Avid M; Norinder, Ulf; Bender, Andreas

2018-02-21

Iterative screening has emerged as a promising approach to increase the efficiency of screening campaigns compared to traditional high throughput approaches. By learning from a subset of the compound library, inferences on what compounds to screen next can be made by predictive models, resulting in more efficient screening. One way to evaluate screening is to consider the cost of screening compared to the gain associated with finding an active compound. In this work, we introduce a conformal predictor coupled with a gain-cost function with the aim to maximise gain in iterative screening. Using this setup we were able to show that by evaluating the predictions on the training data, very accurate predictions on what settings will produce the highest gain on the test data can be made. We evaluate the approach on 12 bioactivity datasets from PubChem training the models using 20% of the data. Depending on the settings of the gain-cost function, the settings generating the maximum gain were accurately identified in 8-10 out of the 12 datasets. Broadly, our approach can predict what strategy generates the highest gain based on the results of the cost-gain evaluation: to screen the compounds predicted to be active, to screen all the remaining data, or not to screen any additional compounds. When the algorithm indicates that the predicted active compounds should be screened, our approach also indicates what confidence level to apply in order to maximize gain. Hence, our approach facilitates decision-making and allocation of the resources where they deliver the most value by indicating in advance the likely outcome of a screening campaign.

Malignant transformation in vitro: criteria, biological markers, and application in environmental screening of carcinogens

International Nuclear Information System (INIS)

Borek, C.

1979-01-01

Biological markers which distinguish malignantly transformed fibroblasts from their normal counterpart include pleomorphic morphology, lowered requirement for nutritional factors, loss of density inhibition of growth, complex topography as discernible by scanning electron microscopy, loss in surface proteins, incomplete glycosylation of membrane glycolylipids and glycoproteins, increased production of specific proteases, decreased organization of the cytoskeleton, and acquisition of neoantigens. Several of these markers are not consistently found in transformed epithelial cells and therefore cannot serve to distinguish unequivocally neoplastic epithelial cells from the normal counterparts. The only criteria associated with the transformed nature of both fibroblasts and epithelial cells are the ability of the cells to proliferate in semisolid medium and to induce tumors in appropriate hosts. In vitro systems represent a powerful tool for screening the mutagenic/oncogenic potential of physical, chemical, and environmental agents. Fibroblasts rather than epithelial cells are preferred for this purpose at the present time because of the clear-cut phenotypic differences between the normal and the transformed cells. These systems have been useful in establishing that malignant transformation can be induced by doses as low as 1 rad of X rays or 0.1 rad of neutrons, and that fractionation at low dose levelsleads to enhanced transformation. They have been useful in identifying a large number of hazardous chemicals and in evaluating the relationship between the mutagenic and carcinogenic potential of radiation and chemicals

In vitro methods for hazard assessment of industrial chemicals – opportunities and challenges

Directory of Open Access Journals (Sweden)

Chin Lin eWong

2015-05-01

Full Text Available Allergic contact dermatitis (ACD is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases (OSDs, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay (LLNA is the ‘method of choice’ for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals.

Prenatal screening for chromosomal abnormalities in IVF patients that opted for preimplantation genetic screening/diagnosis (PGS/D): a need for revised algorithms in the era of personalized medicine.

Science.gov (United States)

Takyi, Afua; Santolaya-Forgas, Joaquin

2017-06-01

Obstetricians offer prenatal screening for most common chromosomal abnormalities to all pregnant women including those that had in vitro fertilization (IVF) and preimplantation genetic screening/diagnosis (PGS/D). We propose that free fetal DNA in maternal circulation together with the second trimester maternal serum alfa feto protein (MSAFP) and ultrasound imaging is the best prenatal screening test for chromosomal abnormalities and congenital anomalies in IVF-PGD/S patients because risk estimations from all other prenatal screening algorithms for chromosomal abnormalities depend heavily on maternal age which is irrelevant in PGS/D patients.

Data mining a small molecule drug screening representative subset from NIH PubChem.

Science.gov (United States)

Xie, Xiang-Qun; Chen, Jian-Zhong

2008-03-01

PubChem is a scientific showcase of the NIH Roadmap Initiatives. It is a compound repository created to facilitate information exchange and data sharing among the NIH Roadmap-funded Molecular Library Screening Center Network (MLSCN) and the scientific community. However, PubChem has more than 10 million records of compound information. It will be challenging to conduct a drug screening of the whole database of millions of compounds. Thus, the purpose of the present study was to develop a data mining cheminformatics approach in order to construct a representative and structure-diverse sublibrary from the large PubChem database. In this study, a new chemical diverse representative subset, rePubChem, was selected by whole-molecule chemistry-space matrix calculation using the cell-based partition algorithm. The representative subset was generated and was then subjected to evaluations by compound property analyses based on 1D and 2D molecular descriptors. The new subset was also examined and assessed for self-similarity analysis based on 2D molecular fingerprints in comparing with the source compound library. The new subset has a much smaller library size (540K compounds) with minimum similarity and redundancy without loss of the structural diversity and basic molecular properties of its parent library (5.3 million compounds). The new representative subset library generated could be a valuable structure-diverse compound resource for in silico virtual screening and in vitro HTS drug screening. In addition, the established subset generation method of using the combined cell-based chemistry-space partition metrics with pairwised 2D fingerprint-based similarity search approaches will also be important to a broad scientific community interested in acquiring structurally diverse compounds for efficient drug screening, building representative virtual combinatorial chemistry libraries for syntheses, and data mining large compound databases like the PubChem library in general.

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches

Energy Technology Data Exchange (ETDEWEB)

Muczynski, V. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); Cravedi, J.P. [INRA, INP, Université de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Lehraiki, A.; Levacher, C.; Moison, D.; Lecureuil, C.; Messiaen, S. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); Perdu, E. [INRA, INP, Université de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Frydman, R. [Service de Gynécologie-Obstétrique, Hôpital A. Béclère, Université Paris Sud F-92141 Clamart (France); Habert, R. [Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unité 967, F-92265, Fontenay aux Roses (France); and others

2012-05-15

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10{sup −5} M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7–12 weeks) of gestation and cultured in the presence or not of 10{sup −5} M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with {sup 14}C-MEHP. A 10{sup −5} M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10{sup −5} M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells. -- Highlights: ► 10{sup −5} M of MEHP impairs germ cell development in the human fetal testis. ► Organotypic culture is a suitable approach to investigate phthalate effects in human. ► MEHP is not metabolized in the human fetal testis. ► In mice, MEHP triggers similar effects both in vivo and in vitro.

A multi-infrastructure gateway for virtual drug screening

NARCIS (Netherlands)

Jaghoori, Mohammad Mahdi; van Altena, Allard J.; Bleijlevens, Boris; Ramezani, Sara; Font, Juan Luis; Olabarriaga, Silvia D.

2015-01-01

In computer-aided drug design, software tools are used to narrow down possible drug candidates, thereby reducing the amount of expensive in vitro research, by a process called virtual screening. This process includes large computations that require advanced computing infrastructure; however, using

A high-resolution peak fractionation approach for streamlined screening of nuclear-factor-E2-related factor-2 activators in Salvia miltiorrhiza.

Science.gov (United States)

Zhang, Hui; Luo, Li-Ping; Song, Hui-Peng; Hao, Hai-Ping; Zhou, Ping; Qi, Lian-Wen; Li, Ping; Chen, Jun

2014-01-24

Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components. Copyright © 2013 Elsevier B.V. All rights reserved.

Combinational approach using solid dispersion and semi-solid matrix technology to enhance in vitro dissolution of telmisartan

Directory of Open Access Journals (Sweden)

Syed Faisal Ali

2016-02-01

Full Text Available The present investigation was focused to formulate semi-solid capsules (SSCs of hydrophobic drug telmisartan (TLMS by encapsulating semi-solid matrix of its solid dispersion (SD in HPMC capsules. The combinational approach was used to reduce the lag time in drug release and improvise its dissolution. SDs of TLMS was prepared using hot fusion method by varying the combinations of Pluronic-F68, Gelucire 50/13 and Plasdone S630. A total of nine batches (SD1-SD9 were characterized for micromeritic properties, in vitro dissolution behavior and surface characterization. SD4 with 52.43% cumulative drug release (CDR in phosphate buffer, pH 7.4, in 120 min, t50% 44.2 min and DE30min 96.76% was selected for the development of semi-solid capsules. Differential scanning calorimetry of SD4 revealed molecular dispersion of TLMS in Pluronic-F68. SD4 was formulated into SSCs using Gelucire 44/14 and PEG 400 as semi-solid components and PEG 6000 as a suspending agent to achieve reduction in lag time for effective drug dissolution. SSC6 showed maximum in vitro drug dissolution 97.49 % in phosphate buffer, pH 7.4 with in 20 min that was almost a three folds reduction in the time required to achieve similar dissolution by SD. Thus, SSCs present an excellent approach to enhance in vitro dissolution as well as to reduce the lag time of dissolution for poorly water soluble drugs especially to those therapeutic classes that are intended for faster onset of action. Developed approach based on HPMC capsules provided a better alternative to target delivery of telmisartan to the vegetarian population.

Free-Form Deformation Approach for Registration of Visible and Infrared Facial Images in Fever Screening

Directory of Open Access Journals (Sweden)

Yedukondala Narendra Dwith Chenna

2018-01-01

Full Text Available Fever screening based on infrared (IR thermographs (IRTs is an approach that has been implemented during infectious disease pandemics, such as Ebola and Severe Acute Respiratory Syndrome. A recently published international standard indicates that regions medially adjacent to the inner canthi provide accurate estimates of core body temperature and are preferred sites for fever screening. Therefore, rapid, automated identification of the canthi regions within facial IR images may greatly facilitate rapid fever screening of asymptomatic travelers. However, it is more difficult to accurately identify the canthi regions from IR images than from visible images that are rich with exploitable features. In this study, we developed and evaluated techniques for multi-modality image registration (MMIR of simultaneously captured visible and IR facial images for fever screening. We used free form deformation (FFD models based on edge maps to improve registration accuracy after an affine transformation. Two widely used FFD models in medical image registration based on the Demons and cubic B-spline algorithms were qualitatively compared. The results showed that the Demons algorithm outperformed the cubic B-spline algorithm, likely due to overfitting of outliers by the latter method. The quantitative measure of registration accuracy, obtained through selected control point correspondence, was within 2.8 ± 1.2 mm, which enables accurate and automatic localization of canthi regions in the IR images for temperature measurement.

Yield and Efficiency of Mental Health Screening: A Comparison of Screening Protocols at Intake to Prison.

Science.gov (United States)

Martin, Michael S; Potter, Beth K; Crocker, Anne G; Wells, George A; Colman, Ian

2016-01-01

The value of screening for mental illness has increasingly been questioned in low prevalence settings due to high false positive rates. However, since false positive rates are related to prevalence, screening may be more effective in higher prevalence settings, including correctional institutions. We compared the yield (i.e. newly detected cases) and efficiency (i.e. false positives) of five screening protocols to detect mental illness in prisons against the use of mental health history taking (the prior approach to detecting mental illness). We estimated the accuracy of the six approaches to detect an Axis I disorder among a sample of 467 newly admitted male inmates (83.1% participation rate). Mental health history taking identified only 41.0% (95% CI 32.1, 50.6) of all inmates with mental illness. Screening protocols identified between 61.9 and 85.7% of all cases, but referred between 2 and 3 additional individuals who did not have a mental illness for every additional case detected compared to the mental health history taking approach. In low prevalence settings (i.e. 10% or less) the screening protocols would have had between 4.6 and 16.2 false positives per true positive. While screening may not be practical in low prevalence settings, it may be beneficial in jails and prisons where the prevalence of mental illness is higher. Further consideration of the context in which screening is being implemented, and of the impacts of policies and clinical practices on the benefits and harms of screening is needed to determine the effectiveness of screening in these settings.

Yield and Efficiency of Mental Health Screening: A Comparison of Screening Protocols at Intake to Prison.

Directory of Open Access Journals (Sweden)

Michael S Martin

Full Text Available The value of screening for mental illness has increasingly been questioned in low prevalence settings due to high false positive rates. However, since false positive rates are related to prevalence, screening may be more effective in higher prevalence settings, including correctional institutions. We compared the yield (i.e. newly detected cases and efficiency (i.e. false positives of five screening protocols to detect mental illness in prisons against the use of mental health history taking (the prior approach to detecting mental illness.We estimated the accuracy of the six approaches to detect an Axis I disorder among a sample of 467 newly admitted male inmates (83.1% participation rate. Mental health history taking identified only 41.0% (95% CI 32.1, 50.6 of all inmates with mental illness. Screening protocols identified between 61.9 and 85.7% of all cases, but referred between 2 and 3 additional individuals who did not have a mental illness for every additional case detected compared to the mental health history taking approach. In low prevalence settings (i.e. 10% or less the screening protocols would have had between 4.6 and 16.2 false positives per true positive.While screening may not be practical in low prevalence settings, it may be beneficial in jails and prisons where the prevalence of mental illness is higher. Further consideration of the context in which screening is being implemented, and of the impacts of policies and clinical practices on the benefits and harms of screening is needed to determine the effectiveness of screening in these settings.

Repositioning the substrate activity screening (SAS) approach as a fragment-based method for identification of weak binders.

Science.gov (United States)

Gladysz, Rafaela; Cleenewerck, Matthias; Joossens, Jurgen; Lambeir, Anne-Marie; Augustyns, Koen; Van der Veken, Pieter

2014-10-13

Fragment-based drug discovery (FBDD) has evolved into an established approach for "hit" identification. Typically, most applications of FBDD depend on specialised cost- and time-intensive biophysical techniques. The substrate activity screening (SAS) approach has been proposed as a relatively cheap and straightforward alternative for identification of fragments for enzyme inhibitors. We have investigated SAS for the discovery of inhibitors of oncology target urokinase (uPA). Although our results support the key hypotheses of SAS, we also encountered a number of unreported limitations. In response, we propose an efficient modified methodology: "MSAS" (modified substrate activity screening). MSAS circumvents the limitations of SAS and broadens its scope by providing additional fragments and more coherent SAR data. As well as presenting and validating MSAS, this study expands existing SAR knowledge for the S1 pocket of uPA and reports new reversible and irreversible uPA inhibitor scaffolds. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals

DEFF Research Database (Denmark)

van der Burg, Bart; Wedebye, Eva Bay; Dietrich, Daniel R.

2015-01-01

to validate the test panel using mechanistic approaches. We are actively engaged in promoting regulatory acceptance of the tools developed as an essential step towards practical application, including case studies for read-across purposes. With this approach, a significant saving in animal use and associated......There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use. The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner preferably using validated existing tools and place them in an innovative alternative testing...

Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens

Energy Technology Data Exchange (ETDEWEB)

Chaikuad, Apirat, E-mail: apirat.chaikuad@sgc.ox.ac.uk [University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ (United Kingdom); Knapp, Stefan [University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ (United Kingdom); Johann Wolfgang Goethe-University, Building N240 Room 3.03, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main (Germany); Delft, Frank von, E-mail: apirat.chaikuad@sgc.ox.ac.uk [University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ (United Kingdom)

2015-07-28

An alternative strategy for PEG sampling is suggested through the use of four newly defined PEG smears to enhance chemical space in reduced screens with a benefit towards protein crystallization. The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

Technical Update: Preimplantation Genetic Diagnosis and Screening.

Science.gov (United States)

Dahdouh, Elias M; Balayla, Jacques; Audibert, François; Wilson, R Douglas; Audibert, François; Brock, Jo-Ann; Campagnolo, Carla; Carroll, June; Chong, Karen; Gagnon, Alain; Johnson, Jo-Ann; MacDonald, William; Okun, Nanette; Pastuck, Melanie; Vallée-Pouliot, Karine

2015-05-01

To update and review the techniques and indications of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Discussion about the genetic and technical aspects of preimplantation reproductive techniques, particularly those using new cytogenetic technologies and embryo-stage biopsy. Clinical outcomes of reproductive techniques following the use of PGD and PGS are included. This update does not discuss in detail the adverse outcomes that have been recorded in association with assisted reproductive technologies. Published literature was retrieved through searches of The Cochrane Library and Medline in April 2014 using appropriate controlled vocabulary (aneuploidy, blastocyst/physiology, genetic diseases, preimplantation diagnosis/methods, fertilization in vitro) and key words (e.g., preimplantation genetic diagnosis, preimplantation genetic screening, comprehensive chromosome screening, aCGH, SNP microarray, qPCR, and embryo selection). Results were restricted to systematic reviews, randomized controlled trials/controlled clinical trials, and observational studies published from 1990 to April 2014. There were no language restrictions. Searches were updated on a regular basis and incorporated in the update to January 2015. Additional publications were identified from the bibliographies of retrieved articles. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. The quality of evidence in this document was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care. (Table 1) BENEFITS, HARMS, AND COSTS: This update will educate readers about new preimplantation genetic concepts, directions, and technologies. The major harms and costs identified are those of assisted reproductive

Determination of genetic toxicity and potential carcinogenicity in vitro--challenges post the Seventh Amendment to the European Cosmetics Directive.

Science.gov (United States)

Tweats, D J; Scott, A D; Westmoreland, C; Carmichael, P L

2007-01-01

Genetic toxicology and its role in the detection of carcinogens is currently undergoing a period of reappraisal. There is an increasing interest in developing alternatives to animal testing and the three R's of reduction, refinement and replacement are the basis for EU and national animal protection laws the Seventh Amendment to the EU Cosmetics Directive will ban the marketing of cosmetic/personal care products that contain ingredients that have been tested in animal models. Thus in vivo tests such as the bone marrow micronucleus test, which has a key role in current testing strategies for genotoxicity, will not be available for this class of products. The attrition rate for new, valuable and safe chemicals tested in an in vitro-only testing battery, using the in vitro tests currently established for genotoxicity screening, will greatly increase once this legislation is in place. In addition there has been an explosion of knowledge concerning the cellular and molecular events leading to carcinogenesis. This knowledge has not yet been fully factored into screening chemicals for properties that are not directly linked to mutation induction. Thus there is a pressing need for new, more accurate approaches to determine genotoxicity and carcinogenicity. However, a considerable challenge is presented for these new approaches to be universally accepted and new tests sufficiently validated by March 2009 when the animal testing and marketing bans associated with the Seventh Amendment are due to come into force. This commentary brings together ideas and approaches from several international workshops and meetings to consider these issues.

Phytochemical characteristics and in vitro antibacterial activity of ...

African Journals Online (AJOL)

The aqueous and ethanol stem bark-extracts of Caesalpinia pulcherrima (Pride of Barbados) were screened for phyto-constituents and in vitro antimicrobial activity on clinical isolates of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Salmonella typhi and Klebsiella pneumoniae using ...

TfR Binding Peptide Screened by Phage Display Technology ...

African Journals Online (AJOL)

Purpose: To screen an hTfR affinity peptide and investigate its activity in vitro. Methods: hTfR ... Keywords: Peptide, hTfR, Transferrin receptor, Phage display technology, Enhanced green ..... mediated uptake of peptides that bind the human.

Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

International Nuclear Information System (INIS)

Chen, Li; Schmidt, Emmett V; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark; Benes, Cyril; Lacy-Hulbert, Adam

2013-01-01

The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens

In vitro models to evaluate the permeability of poorly soluble drug entities: Challenges and perspectives

DEFF Research Database (Denmark)

Buckley, S. T.; Fischer, S. M.; Fricker, G.

2012-01-01

The application of in vitro models in drug permeability studies represents a useful screening tool for assessing the biopharmaceutical appropriateness of new chemical entities (NCEs). Of note, there remains an ever-increasing number of NCEs which exhibit poor aqueous solubility. However, in their......The application of in vitro models in drug permeability studies represents a useful screening tool for assessing the biopharmaceutical appropriateness of new chemical entities (NCEs). Of note, there remains an ever-increasing number of NCEs which exhibit poor aqueous solubility. However...

Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

Science.gov (United States)

Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

2012-01-01

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

A Tox21 Approach to Altered Epigenetic Landscapes: Assessing Epigenetic Toxicity Pathways Leading to Altered Gene Expression and Oncogenic Transformation In Vitro

Directory of Open Access Journals (Sweden)

Craig L. Parfett

2017-06-01

A, UHRF1, CTCF, HOTAIR and ANRIL were found to have experimental evidence showing that functional perturbations played “driver” roles in human cellular transformation. Measurement of epigenotoxicants presents challenges for short-term carcinogenicity testing, especially in the high-throughput modes emphasized in the Tox21 chemicals testing approach. There is need to develop and validate in vitro tests to detect both, locus-specific, and genome-wide, epigenetic alterations with causal links to oncogenic cellular phenotypes. Some recent examples of cell-based high throughput chemical screening assays are presented that have been applied or have shown potential for application to epigenetic endpoints.

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library.

Science.gov (United States)

Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G; Zhang, Ying

2016-01-01

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

Contact-based ligand-clustering approach for the identification of active compounds in virtual screening

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Mantsyzov AB

2012-09-01

Full Text Available Alexey B Mantsyzov,1 Guillaume Bouvier,2 Nathalie Evrard-Todeschi,1 Gildas Bertho11Université Paris Descartes, Sorbonne, Paris, France; 2Institut Pasteur, Paris, FranceAbstract: Evaluation of docking results is one of the most important problems for virtual screening and in silico drug design. Modern approaches for the identification of active compounds in a large data set of docked molecules use energy scoring functions. One of the general and most significant limitations of these methods relates to inaccurate binding energy estimation, which results in false scoring of docked compounds. Automatic analysis of poses using self-organizing maps (AuPosSOM represents an alternative approach for the evaluation of docking results based on the clustering of compounds by the similarity of their contacts with the receptor. A scoring function was developed for the identification of the active compounds in the AuPosSOM clustered dataset. In addition, the AuPosSOM efficiency for the clustering of compounds and the identification of key contacts considered as important for its activity, were also improved. Benchmark tests for several targets revealed that together with the developed scoring function, AuPosSOM represents a good alternative to the energy-based scoring functions for the evaluation of docking results.Keywords: scoring, docking, virtual screening, CAR, AuPosSOM

A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery

Science.gov (United States)

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

A new in vivo screening paradigm to accelerate antimalarial drug discovery.

Directory of Open Access Journals (Sweden)

María Belén Jiménez-Díaz

Full Text Available 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

In vitro differentiation of mouse embryonic stem cells into functional ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

In vitro cytotoxic screening of selected Saudi medicinal plants.

Science.gov (United States)

Almehdar, Hussein; Abdallah, Hossam M; Osman, Abdel-Moneim M; Abdel-Sattar, Essam A

2012-04-01

Many natural products from plants have been identified to exert anticancer activity. It might be expected to be a challenge to look at the Saudi plants in order to discover new sources for new molecules which may have anticancer activity. The methanolic extracts of forty species of plants traditionally used in Saudi Arabia for the treatment of a variety of diseases were tested in vitro for their potential anticancer activity on different human cancer cell lines. The cytotoxic activity of the methanolic extracts of the tested plants were determined using three human cancer cell lines, namely, breast cancer (MCF7), hepatocellular carcinoma (HEPG2), and cervix cancer (HELA) cells. In addition, human normal melanocyte (HFB4) was used as normal nonmalignant cells. Sulforhodamine B colorimetric assay was used to evaluate the in vitro cytotoxic activity of the different extracts. The growth inhibition of 50% (IC(50)) for each extract was calculated from the optical density of treated and untreated cells. Doxorubicin, a broad-spectrum anticancer drug, was used as the positive control. Nine plant extracts were chosen for further fractionation based on their activity and availability. Interesting cytotoxic activity was observed for Hypoestes forskaolii, Withania somnifera, Solanum glabratum, Adenium obesum, Pistacia vera oleoresin, Caralluma quadrangula, Eulophia petersii, Phragmanthera austroarabica, and Asparagus officinalis. Other extracts showed poor activity.

Sniffer dogs as part of a bimodal bionic research approach to develop a lung cancer screening.

Science.gov (United States)

Boedeker, Enole; Friedel, Godehard; Walles, Thorsten

2012-05-01

Lung cancer (LC) continues to represent a heavy burden for health care systems worldwide. Epidemiological studies predict that its role will increase in the near future. While patient prognosis is strongly associated with tumour stage and early detection of disease, no screening test exists so far. It has been suggested that electronic sensor devices, commonly referred to as 'electronic noses', may be applicable to identify cancer-specific volatile organic compounds in the breath of patients and therefore may represent promising screening technologies. However, three decades of research did not bring forward a clinically applicable device. Here, we propose a new research approach by involving specially trained sniffer dogs into research strategies by making use of their ability to identify LC in the breath sample of patients.

A desirability-based multi objective approach for the virtual screening discovery of broad-spectrum anti-gastric cancer agents.

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Yunierkis Perez-Castillo

Full Text Available Gastric cancer is the third leading cause of cancer-related mortality worldwide and despite advances in prevention, diagnosis and therapy, it is still regarded as a global health concern. The efficacy of the therapies for gastric cancer is limited by a poor response to currently available therapeutic regimens. One of the reasons that may explain these poor clinical outcomes is the highly heterogeneous nature of this disease. In this sense, it is essential to discover new molecular agents capable of targeting various gastric cancer subtypes simultaneously. Here, we present a multi-objective approach for the ligand-based virtual screening discovery of chemical compounds simultaneously active against the gastric cancer cell lines AGS, NCI-N87 and SNU-1. The proposed approach relays in a novel methodology based on the development of ensemble models for the bioactivity prediction against each individual gastric cancer cell line. The methodology includes the aggregation of one ensemble per cell line using a desirability-based algorithm into virtual screening protocols. Our research leads to the proposal of a multi-targeted virtual screening protocol able to achieve high enrichment of known chemicals with anti-gastric cancer activity. Specifically, our results indicate that, using the proposed protocol, it is possible to retrieve almost 20 more times multi-targeted compounds in the first 1% of the ranked list than what is expected from a uniform distribution of the active ones in the virtual screening database. More importantly, the proposed protocol attains an outstanding initial enrichment of known multi-targeted anti-gastric cancer agents.

Raman spectroscopic approach to monitor the in vitro cyclization of creatine → creatinine

Science.gov (United States)

Gangopadhyay, Debraj; Sharma, Poornima; Singh, Sachin Kumar; Singh, Pushkar; Tarcea, Nicolae; Deckert, Volker; Popp, Jürgen; Singh, Ranjan K.

2015-01-01

The creatine → creatinine cyclization, an important metabolic phenomenon has been initiated in vitro at acidic pH and studied through Raman spectroscopic and DFT approach. The equilibrium composition of neutral, zwitterionic and protonated microspecies of creatine has been monitored with time as the reaction proceeds. Time series Raman spectra show clear signature of creatinine formation at pH 3 after ∼240 min at room temperature and reaction is faster at higher temperature. The spectra at pH 1 and pH 5 do not show such signature up to 270 min implying faster reaction rate at pH 3.

The Effect of Screen Size on Mobile Phone User Comprehension of Health Information and Application Structure: An Experimental Approach.

Science.gov (United States)

Al Ghamdi, Ebtisam; Yunus, Faisal; Da'ar, Omar; El-Metwally, Ashraf; Khalifa, Mohamed; Aldossari, Bakheet; Househ, Mowafa

2016-01-01

This research analyzes the impact of mobile phone screen size on user comprehension of health information and application structure. Applying experimental approach, we asked randomly selected users to read content and conduct tasks on a commonly used diabetes mobile application using three different mobile phone screen sizes. We timed and tracked a number of parameters, including correctness, effectiveness of completing tasks, content ease of reading, clarity of information organization, and comprehension. The impact of screen size on user comprehension/retention, clarity of information organization, and reading time were mixed. It is assumed on first glance that mobile screen size would affect all qualities of information reading and comprehension, including clarity of displayed information organization, reading time and user comprehension/retention of displayed information, but actually the screen size, in this experimental research, did not have significant impact on user comprehension/retention of the content or on understanding the application structure. However, it did have significant impact on clarity of information organization and reading time. Participants with larger screen size took shorter time reading the content with a significant difference in the ease of reading. While there was no significant difference in the comprehension of information or the application structures, there were a higher task completion rate and a lower number of errors with the bigger screen size. Screen size does not directly affect user comprehension of health information. However, it does affect clarity of information organization, reading time and user's ability to recall information.

Screening of metal-organic frameworks for carbon dioxide capture from flue gas using a combined experimental and modeling approach.

Science.gov (United States)

Yazaydin, A Ozgür; Snurr, Randall Q; Park, Tae-Hong; Koh, Kyoungmoo; Liu, Jian; Levan, M Douglas; Benin, Annabelle I; Jakubczak, Paulina; Lanuza, Mary; Galloway, Douglas B; Low, John J; Willis, Richard R

2009-12-30

A diverse collection of 14 metal-organic frameworks (MOFs) was screened for CO(2) capture from flue gas using a combined experimental and modeling approach. Adsorption measurements are reported for the screened MOFs at room temperature up to 1 bar. These data are used to validate a generalized strategy for molecular modeling of CO(2) and other small molecules in MOFs. MOFs possessing a high density of open metal sites are found to adsorb significant amounts of CO(2) even at low pressure. An excellent correlation is found between the heat of adsorption and the amount of CO(2) adsorbed below 1 bar. Molecular modeling can aid in selection of adsorbents for CO(2) capture from flue gas by screening a large number of MOFs.

Phytochemical and in vitro screening of some Ficus and Morus spp. for hypolipidaemic and antioxidant activities and in vivo assessment of Ficus mysorensis (Roth).

Science.gov (United States)

Awad, Nagwa E; Seida, Ahmed A; Hamed, Manal A; Mahmoud, Ahlam H; Elbatanony, Marwa M

2012-01-01

Phytochemical screening of air-dried leaves and fruit juice of certain Ficus and Morus spp. have been studied. In an in vitro study, the ethanol and hexane extracts of the investigated plants were evaluated against hyperlipidaemia by estimating the rate limiting enzyme of cholesterol biothenysis; β-hydroxy-β-methylglutaryl coenzyme A reductase (HMG-CoA reductase). The antioxidant activity was evaluated by reduction of DPPH(-) free radical. Extra phytochemical screening of Ficus extracts was undertaken, which recorded potent hypolipidaemic and antioxidant activities. The more pronounced extract, Ficus mysorensis (hexane extract), was evaluated in vivo by estimation of the lipid profile and certain antioxidant parameters in hypercholesterolemic rats. The hexane fraction was chromatographed and six isolated compounds were identified. Furthermore, its saponifiable fraction was identified by a MS/MS technique. In conclusion, F. mysorensis recorded hypolipidaemic and antioxidant effects. Detailed studies of the isolated compounds must be undertaken for an evaluation against hypercholesterolemia and free radical elevation.

Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

Science.gov (United States)

Handral, Harish K; Tong, Huei Jinn; Islam, Intekhab; Sriram, Gopu; Rosa, Vinicus; Cao, Tong

2016-10-01

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

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Itsuki Anzai

2016-08-01

Full Text Available Dominant mutations in Cu/Zn-superoxide dismutase (SOD1 gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS. A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

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

Energy Technology Data Exchange (ETDEWEB)

Doherty, Kimberly R., E-mail: kimberly.doherty@quintiles.com; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Shell, Scott A.; Bacus, Sarah

2015-05-15

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. 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

In vitro screening of soil bacteria for inhibiting phytopathogenic fungi ...

African Journals Online (AJOL)

At present, the greatest interest resides with the development and application of specific biocontrol agent for the control of diseases on plant and this form the focus of this work. Several soil bacteria were evaluated in vitro for their effectiveness on the basis of their ability to suppress fungi in plate inhibition assays. 51 strains ...

Screening of Lactobacillus plantarum isolated from fermented idli ...

African Journals Online (AJOL)

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to screen eight potential probiotic Lactobacillus plantarum strains from fermented idli batter using in vitro assays such as bile tolerance, acid tolerance, transit ...

What next for preimplantation genetic screening? A polar body approach!

NARCIS (Netherlands)

Geraedts, Joep; Collins, John; Gianaroli, Luca; Goossens, Veerle; Handyside, Alan; Harper, Joyce; Montag, Markus; Repping, Sjoerd; Schmutzler, Andreas

2010-01-01

Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live

"To Screen or not to Screen": Comparing the Health and Economic Benefits of Early Peanut Introduction Strategies in Five Countries.

Science.gov (United States)

Shaker, Marcus; Stukus, David; Chan, Edmond S; Fleischer, David M; Spergel, Jonathan M; Greenhawt, Matthew

2018-03-30

Early peanut introduction (EPI) in the first year of life is associated with reduced risk of developing peanut allergy in children with either severe eczema and/or egg allergy. However, EPI recommendations differ among countries with formal guidelines. Using simulation and Markov modeling over a 20-year horizon to attempt to explore optimal EPI strategies applied to the US population, we compared high-risk infant specific IgE peanut screening (US/Canadian) with the Australiasian Society for Clinical Immunology and Allergy (Australia/New Zealand) (ASCIA) and the United Kingdom Department of Health (UKDOH) published EPI approaches. Screening peanut skin testing of all children with early onset eczema and/or egg allergy before in-office peanut introduction was dominated by a no-screen approach, in terms of number of cases of peanut allergy prevented, QALY's, and healthcare costs, though screening resulted in a slightly lower rate of allergic reactions to peanut per-patient in high-risk children. Considering costs of peanut allergy in high-risk children, the per-patient cost of early introduction without screening over the model horizon was $6,556.69 (95%CI, $6,512.76-$6,600.62), compared with a cost of $7,576.32 (95%CI, $7,531.38-$7,621.26) for skin test screening prior to introduction. From a US societal perspective, screening prior to introduction cost $654,115,322 and resulted in 3,208 additional peanut allergy diagnoses. Both screening and non-screening approaches dominated deliberately delayed peanut introduction. A no-screening approach for EPI has superior health and economic benefits in terms of number of peanut allergy cases prevented, QALY's, and total health care costs compared to screening and in-office peanut introduction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A new approach to data evaluation in the non-target screening of organic trace substances in water analysis.

Science.gov (United States)

Müller, Alexander; Schulz, Wolfgang; Ruck, Wolfgang K L; Weber, Walter H

2011-11-01

Non-target screening via high performance liquid chromatography-mass spectrometry (HPLC-MS) has gained increasingly in importance for monitoring organic trace substances in water resources targeted for the production of drinking water. In this article a new approach for evaluating the data from non-target HPLC-MS screening in water is introduced and its advantages are demonstrated using the supply of drinking water as an example. The crucial difference between this and other approaches is the comparison of samples based on compounds (features) determined by their full scan data. In so doing, we take advantage of the temporal, spatial, or process-based relationships among the samples by applying the set operators, UNION, INTERSECT, and COMPLEMENT to the features of each sample. This approach regards all compounds, detectable by the used analytical method. That is the fundamental meaning of non-target screening, which includes all analytical information from the applied technique for further data evaluation. In the given example, in just one step, all detected features (1729) of a landfill leachate sample could be examined for their relevant influences on water purification respectively drinking water. This study shows that 1721 out of 1729 features were not relevant for the water purification. Only eight features could be determined in the untreated water and three of them were found in the final drinking water after ozonation. In so doing, it was possible to identify 1-adamantylamine as contamination of the landfill in the drinking water at a concentration in the range of 20 ng L(-1). To support the identification of relevant compounds and their transformation products, the DAIOS database (Database-Assisted Identification of Organic Substances) was used. This database concept includes some functions such as product ion search to increase the efficiency of the database query after the screening. To identify related transformation products the database function

Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials.

Science.gov (United States)

Clippinger, Amy J; Ahluwalia, Arti; Allen, David; Bonner, James C; Casey, Warren; Castranova, Vincent; David, Raymond M; Halappanavar, Sabina; Hotchkiss, Jon A; Jarabek, Annie M; Maier, Monika; Polk, William; Rothen-Rutishauser, Barbara; Sayes, Christie M; Sayre, Phil; Sharma, Monita; Stone, Vicki

2016-07-01

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

Energy Technology Data Exchange (ETDEWEB)

Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States); Zhu, Hao [The Rutgers Center for Computational and Integrative Biology, Rutgers University, Camden, NJ (United States); Department of Chemistry, Rutgers University, Camden, NJ (United States); Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia [NovaMechanics Ltd., Nicosia (Cyprus); Rusyn, Ivan, E-mail: iir@unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Tropsha, Alexander, E-mail: alex_tropsha@unc.edu [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States)

2013-10-01

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function. �

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

International Nuclear Information System (INIS)

Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh; Zhu, Hao; Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia; Rusyn, Ivan; Tropsha, Alexander

2013-01-01

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R 2 = 0.71, STL R 2 = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R 2 = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function. • The results

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

Science.gov (United States)

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.

Application of ToxCast High-Throughput Screening and ...

Science.gov (United States)

Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

In vitro and in vivo approaches to study osteocyte biology.

Science.gov (United States)

Kalajzic, Ivo; Matthews, Brya G; Torreggiani, Elena; Harris, Marie A; Divieti Pajevic, Paola; Harris, Stephen E

2013-06-01

Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

Impact ionization in GaAs: A screened exchange density-functional approach

International Nuclear Information System (INIS)

Picozzi, S.; Asahi, R.; Geller, C.B.; Continenza, A.; Freeman, A.J.

2001-01-01

Results are presented of a fully ab initio calculation of impact ionization rates in GaAs within the density functional theory framework, using a screened-exchange formalism and the highly precise all-electron full-potential linearized augmented plane wave method. The calculated impact ionization rates show a marked orientation dependence in k space, indicating the strong restrictions imposed by the conservation of energy and momentum. This anisotropy diminishes as the impacting electron energy increases. A Keldysh type fit performed on the energy-dependent rate shows a rather soft edge and a threshold energy greater than the direct band gap. The consistency with available Monte Carlo and empirical pseudopotential calculations shows the reliability of our approach and paves the way to ab initio calculations of pair production rates in new and more complex materials

Screening based approach and dehydrogenation kinetics for MgH2: Guide to find suitable dopant using first-principles approach.

Science.gov (United States)

Kumar, E Mathan; Rajkamal, A; Thapa, Ranjit

2017-11-14

First-principles based calculations are performed to investigate the dehydrogenation kinetics considering doping at various layers of MgH 2 (110) surface. Doping at first and second layer of MgH 2 (110) has a significant role in lowering the H 2 desorption (from surface) barrier energy, whereas the doping at third layer has no impact on the barrier energy. Molecular dynamics calculations are also performed to check the bonding strength, clusterization, and system stability. We study in details about the influence of doping on dehydrogenation, considering the screening factors such as formation enthalpy, bulk modulus, and gravimetric density. Screening based approach assist in finding Al and Sc as the best possible dopant in lowering of desorption temperature, while preserving similar gravimetric density and Bulk modulus as of pure MgH 2 system. The electron localization function plot and population analysis illustrate that the bond between Dopant-Hydrogen is mainly covalent, which weaken the Mg-Hydrogen bonds. Overall we observed that Al as dopant is suitable and surface doping can help in lowering the desorption temperature. So layer dependent doping studies can help to find the best possible reversible hydride based hydrogen storage materials.

The health system and population health implications of large-scale diabetes screening in India: a microsimulation model of alternative approaches.

Directory of Open Access Journals (Sweden)

Sanjay Basu

2015-05-01

Full Text Available Like a growing number of rapidly developing countries, India has begun to develop a system for large-scale community-based screening for diabetes. We sought to identify the implications of using alternative screening instruments to detect people with undiagnosed type 2 diabetes among diverse populations across India.We developed and validated a microsimulation model that incorporated data from 58 studies from across the country into a nationally representative sample of Indians aged 25-65 y old. We estimated the diagnostic and health system implications of three major survey-based screening instruments and random glucometer-based screening. Of the 567 million Indians eligible for screening, depending on which of four screening approaches is utilized, between 158 and 306 million would be expected to screen as "high risk" for type 2 diabetes, and be referred for confirmatory testing. Between 26 million and 37 million of these people would be expected to meet international diagnostic criteria for diabetes, but between 126 million and 273 million would be "false positives." The ratio of false positives to true positives varied from 3.9 (when using random glucose screening to 8.2 (when using a survey-based screening instrument in our model. The cost per case found would be expected to be from US$5.28 (when using random glucose screening to US$17.06 (when using a survey-based screening instrument, presenting a total cost of between US$169 and US$567 million. The major limitation of our analysis is its dependence on published cohort studies that are unlikely fully to capture the poorest and most rural areas of the country. Because these areas are thought to have the lowest diabetes prevalence, this may result in overestimation of the efficacy and health benefits of screening.Large-scale community-based screening is anticipated to produce a large number of false-positive results, particularly if using currently available survey-based screening

Pesticide Cumulative Risk Assessment: Framework for Screening Analysis

Science.gov (United States)

This document provides guidance on how to screen groups of pesticides for cumulative evaluation using a two-step approach: begin with evaluation of available toxicological information and, if necessary, follow up with a risk-based screening approach.

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

Directory of Open Access Journals (Sweden)

Elad Tako

2016-11-01

Full Text Available 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.

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

Science.gov (United States)

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

ScreenBEAM: a novel meta-analysis algorithm for functional genomics screens via Bayesian hierarchical modeling.

Science.gov (United States)

Yu, Jiyang; Silva, Jose; Califano, Andrea

2016-01-15

Functional genomics (FG) screens, using RNAi or CRISPR technology, have become a standard tool for systematic, genome-wide loss-of-function studies for therapeutic target discovery. As in many large-scale assays, however, off-target effects, variable reagents' potency and experimental noise must be accounted for appropriately control for false positives. Indeed, rigorous statistical analysis of high-throughput FG screening data remains challenging, particularly when integrative analyses are used to combine multiple sh/sgRNAs targeting the same gene in the library. We use large RNAi and CRISPR repositories that are publicly available to evaluate a novel meta-analysis approach for FG screens via Bayesian hierarchical modeling, Screening Bayesian Evaluation and Analysis Method (ScreenBEAM). Results from our analysis show that the proposed strategy, which seamlessly combines all available data, robustly outperforms classical algorithms developed for microarray data sets as well as recent approaches designed for next generation sequencing technologies. Remarkably, the ScreenBEAM algorithm works well even when the quality of FG screens is relatively low, which accounts for about 80-95% of the public datasets. R package and source code are available at: https://github.com/jyyu/ScreenBEAM. ac2248@columbia.edu, jose.silva@mssm.edu, yujiyang@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

Science.gov (United States)

Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

2018-01-01

The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

Computer Screen Use Detection Using Smart Eyeglasses

Directory of Open Access Journals (Sweden)

Florian Wahl

2017-05-01

Full Text Available Screen use can influence the circadian phase and cause eye strain. Smart eyeglasses with an integrated color light sensor can detect screen use. We present a screen use detection approach based on a light sensor embedded into the bridge of smart eyeglasses. By calculating the light intensity at the user’s eyes for different screens and content types, we found only computer screens to have a significant impact on the circadian phase. Our screen use detection is based on ratios between color channels and used a linear support vector machine to detect screen use. We validated our detection approach in three studies. A test bench was built to detect screen use under different ambient light sources and intensities in a controlled environment. In a lab study, we evaluated recognition performance for different ambient light intensities. By using participant-independent models, we achieved an ROC AUC above 0.9 for ambient light intensities below 200 lx. In a study of typical ADLs, screen use was detected with an average ROC AUC of 0.83 assuming screen use for 30% of the time.

Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example.

Science.gov (United States)

Dimitrov, S; Detroyer, A; Piroird, C; Gomes, C; Eilstein, J; Pauloin, T; Kuseva, C; Ivanova, H; Popova, I; Karakolev, Y; Ringeissen, S; Mekenyan, O

2016-12-01

When searching for alternative methods to animal testing, confidently rescaling an in vitro result to the corresponding in vivo classification is still a challenging problem. Although one of the most important factors affecting good correlation is sample characteristics, they are very rarely integrated into correlation studies. Usually, in these studies, it is implicitly assumed that both compared values are error-free numbers, which they are not. In this work, we propose a general methodology to analyze and integrate data variability and thus confidence estimation when rescaling from one test to another. The methodology is demonstrated through the case study of rescaling the in vitro Direct Peptide Reactivity Assay (DPRA) reactivity to the in vivo Local Lymph Node Assay (LLNA) skin sensitization potency classifications. In a first step, a comprehensive statistical analysis evaluating the reliability and variability of LLNA and DPRA as such was done. These results allowed us to link the concept of gray zones and confidence probability, which in turn represents a new perspective for a more precise knowledge of the classification of chemicals within their in vivo OR in vitro test. Next, the novelty and practical value of our methodology introducing variability into the threshold optimization between the in vitro AND in vivo test resides in the fact that it attributes a confidence probability to the predicted classification. The methodology, classification and screening approach presented in this study are not restricted to skin sensitization only. They could be helpful also for fate, toxicity and health hazard assessment where plenty of in vitro and in chemico assays and/or QSARs models are available. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Phytochemicals Screening and In Vitro Antibacterial Activity of Elaeis guineensis Leaves Extracts Against Human Pathogenic Bacteria

International Nuclear Information System (INIS)

Noorshilawati Abdul Aziz; Umi Nadhirah Halim; Nur Suraya Abdullah

2015-01-01

Chloroform and methanol extracts of Elaeis guineensis leaves were investigated for in vitro antibacterial activity against the human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Four different concentrations of both extracts consists of 50, 100, 200 and 300 mg/ ml were prepared for antibacterial activity using disc diffusion method. The results revealed that chloroform and methonal extract showed high toxicity against all bacterial strain tested. However, both extracts is more effective and exhibit better inhibiting activity against gram positive bacteria, S. aureus compared to gram negative bacteria (E. coli and P. aeruginosa). Methanol extract of Elaeis guineensis leaves shows greater inhibition zone compared to chloroform extract as phyto chemical screening revealed that this extracts contain terpenoids, tannins and saponin. The highest antibacterial activity was exhibited by 300 mg/ ml methanolic extracts against S. aureus which inhibited 10.67 ± 0.33 mm of the diameter zone. Followed by 200 mg/ ml methanolic extracts and 300 mg/ ml chloroform extracts against S. aureus which inhibited 9.17 ± 0.17 mm and 8.33 ± 1.67 mm respectively. This result revealed the potentials of Elaeis guineensis as antibacterial agent in combating infections from human pathogenic bacteria. However, further studies, including identification and purification of the active compounds, will need to be pursued. (author)

Screening for collusion: a spatial statistics approach

NARCIS (Netherlands)

Heijnen, P.; Haan, M.A.; Soetevent, A.R.

2012-01-01

We develop a method to screen for local cartels. We first test whether there is statistical evidence of clustering of outlets that score high on some characteristic that is consistent with collusive behavior. If so, we determine in a second step the most suspicious regions where further antitrust

Screening for collusion: a spatial statistics approach

NARCIS (Netherlands)

Heijnen, P.; Haan, M.A.; Soetevent, A.R.

2015-01-01

We develop a method to screen for local cartels. We first test whether there is statistical evidence of clustering of outlets that score high on some characteristic that is consistent with collusive behavior. If so, we determine in a second step the most suspicious regions where further antitrust

Screening for collusion : A spatial statistics approach

NARCIS (Netherlands)

Heijnen, Pim; Haan, Marco A.; Soetevent, Adriaan R.

2015-01-01

We develop a method to screen for local cartels. We first test whether there is statistical evidence of clustering of outlets that score high on some characteristic that is consistent with collusive behavior. If so, we determine in a second step the most suspicious regions where further antitrust

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.

Science.gov (United States)

Faria, Jorge M S; Sena, Inês; Vieira da Silva, Inês; Ribeiro, Bruno; Barbosa, Pedro; Ascensão, Lia; Bennett, Richard N; Mota, Manuel; Figueiredo, A Cristina

2015-06-01

Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all β- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

WISDOM-II: Screening against multiple targets implicated in malaria using computational grid infrastructures

Directory of Open Access Journals (Sweden)

Kenyon Colin

2009-05-01

Full Text Available Abstract Background Despite continuous efforts of the international community to reduce the impact of malaria on developing countries, no significant progress has been made in the recent years and the discovery of new drugs is more than ever needed. Out of the many proteins involved in the metabolic activities of the Plasmodium parasite, some are promising targets to carry out rational drug discovery. Motivation Recent years have witnessed the emergence of grids, which are highly distributed computing infrastructures particularly well fitted for embarrassingly parallel computations like docking. In 2005, a first attempt at using grids for large-scale virtual screening focused on plasmepsins and ended up in the identification of previously unknown scaffolds, which were confirmed in vitro to be active plasmepsin inhibitors. Following this success, a second deployment took place in the fall of 2006 focussing on one well known target, dihydrofolate reductase (DHFR, and on a new promising one, glutathione-S-transferase. Methods In silico drug design, especially vHTS is a widely and well-accepted technology in lead identification and lead optimization. This approach, therefore builds, upon the progress made in computational chemistry to achieve more accurate in silico docking and in information technology to design and operate large scale grid infrastructures. Results On the computational side, a sustained infrastructure has been developed: docking at large scale, using different strategies in result analysis, storing of the results on the fly into MySQL databases and application of molecular dynamics refinement are MM-PBSA and MM-GBSA rescoring. The modeling results obtained are very promising. Based on the modeling results, In vitro results are underway for all the targets against which screening is performed. Conclusion The current paper describes the rational drug discovery activity at large scale, especially molecular docking using FlexX software

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

Science.gov (United States)

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.

Estimating radionuclide air concentrations near buildings: a screening approach

International Nuclear Information System (INIS)

Miller, C.W.; Yildiran, M.

1984-01-01

For some facilities that routinely release small amounts of radionuclides to the atmosphere, such as hospitals, research laboratories, contaminated clothing laundries, and others, it is necessary to estimate the dose to persons very near the buildings from which the releases occur. Such facilities need simple screening procedures which provide reasonable assurance that as long as the calculated dose is less than some fraction of a relevant dose limit no individual will receive a dose in excess of that limit. Screening procedures have been proposed for persons living within hundreds of meters to a few kilometers from a source of radioactive effluent. This paper examines a screening technique for estimating long-term average radionuclide air concentrations within approximately 100 m of a building from which the release occurs. The technique is based on a modified gaussion plume model (HB model) which considers the influence of the tallest building within 100 m and is independant of atmospheric stability and downwind distance. 4 references, 2 tables

Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.

Science.gov (United States)

T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen

2017-06-01

Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound

In vitro Inhibition of Mycobacterium smegmatis and Mycobacterium ...

African Journals Online (AJOL)

Some Nigerian plants used in traditional medicine to treat tuberculosis and/or some of its symptoms were screened for in vitro activity against Mycobacterium smegmatis and a clinical isolate of Mycobacterium tuberculosis. Only 3 of the 6 crude methanolic extracts of the 6 plant species exhibited inhibitory activities against ...

Staged anticonvulsant screening for chronic epilepsy

OpenAIRE

Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung‐Il; Roach, Bonnie; Pouliot, Wendy; Lu, Kimberly; Swiercz, Waldemar; Dudek, F. Edward; Staley, Kevin J.

2016-01-01

Abstract Objective: 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. Methods: Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and l...

Screening diagnostic program breast cancer

International Nuclear Information System (INIS)

Portnoj, L.M.; Zhakova, I.I.; Budnikova, N.V.; Rukhlyadko, E.D.

1995-01-01

The authors propose their screening program for detection of breast cancer. It includes the entire complex of present-day screening diagnostic methods, starting from an original system for the formation of groups at risk of breast cancer and completed by the direct diagnostic model of detection of the condition, oriented at a differentiated approach to the use of mammographic techniques. The proposed organizational and methodologic screening measures are both economic and diagnostically effective, thus meeting the principal requirements to screening programs. Screening of 8541 risk-groups patients helped detect 867 nodular formations, 244 of which were cancer and 623 benign formations. 8 refs., 3 figs.,

In vitro gastrointestinal digestion increases the translocation of polystyrene nanoparticles in an in vitro intestinal co-culture model.

Science.gov (United States)

Walczak, Agata P; Kramer, Evelien; Hendriksen, Peter J M; Helsdingen, Richard; van der Zande, Meike; Rietjens, Ivonne M C M; Bouwmeester, Hans

2015-01-01

The conditions of the gastrointestinal tract may change the physicochemical properties of nanoparticles (NPs) and therewith the bioavailability of orally taken NPs. Therefore, we assessed the impact of in vitro gastrointestinal digestion on the protein corona of polystyrene NPs (PS-NPs) and their subsequent translocation across an in vitro intestinal barrier. A co-culture of intestinal Caco-2 and HT29-MTX cells was exposed to 50 nm PS-NPs of different charges (positive and negative) in two forms: pristine and digested in an in vitro gastrointestinal digestion model. In vitro digestion significantly increased the translocation of all, except the "neutral", PS-NPs. Upon in vitro digestion, translocation was 4-fold higher for positively charged NPs and 80- and 1.7-fold higher for two types of negatively charged NPs. Digestion significantly reduced the amount of protein in the corona of three out of four types of NPs. This reduction of proteins was 4.8-fold for "neutral", 3.5-fold for positively charged and 1.8-fold for one type of negatively charged PS-NPs. In vitro digestion also affected the composition of the protein corona of PS-NPs by decreasing the presence of higher molecular weight proteins and shifting the protein content of the corona to low molecular weight proteins. These findings are the first to report that in vitro gastrointestinal digestion significantly affects the protein corona and significantly increases the in vitro translocation of differently charged PS-NPs. These findings stress the importance of including the in vitro digestion in future in vitro intestinal translocation screening studies for risk assessment of orally taken NPs.

Prediction of pharmacokinetic and toxicological parameters of a 4-phenylcoumarin isolated from geopropolis: In silico and in vitro approaches.

Science.gov (United States)

da Cunha, Marcos Guilherme; Franco, Gilson César Nobre; Franchin, Marcelo; Beutler, John A; de Alencar, Severino Matias; Ikegaki, Masaharu; Rosalen, Pedro Luiz

2016-11-30

In silico and in vitro methodologies have been used as important tools in the drug discovery process, including from natural sources. The aim of this study was to predict pharmacokinetic and toxicity (ADME/Tox) properties of a coumarin isolated from geopropolis using in silico and in vitro approaches. Cinnamoyloxy-mammeisin (CNM) isolated from Brazilian M. scutellaris geopropolis was evaluated for its pharmacokinetic parameters by in silico models (ACD/Percepta™ and MetaDrug™ software). Genotoxicity was assessed by in vitro DNA damage signaling PCR array. CNM did not pass all parameters of Lipinski's rule of five, with a predicted low oral bioavailability and high plasma protein binding, but with good predicted blood brain barrier penetration. CNM was predicted to show low affinity to cytochrome P450 family members. Furthermore, the predicted Ames test indicated potential mutagenicity of CNM. Also, the probability of toxicity for organs and tissues was classified as moderate and high for liver and kidney, and moderate and low for skin and eye irritation, respectively. The PCR array analysis showed that CNM significantly upregulated about 7% of all DNA damage-related genes. By exploring the biological function of these genes, it was found that the predicted CNM genotoxicity is likely to be mediated by apoptosis. The predicted ADME/Tox profile suggests that external use of CNM may be preferable to systemic exposure, while its genotoxicity was characterized by the upregulation of apoptosis-related genes after treatment. The combined use of in silico and in vitro approaches to evaluate these parameters generated useful hypotheses to guide further preclinical studies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of a Combined In Vitro Physiologically Based Kinetic (PBK) and Monte Carlo Modelling Approach to Predict Interindividual Human Variation in Phenol-Induced Developmental Toxicity.

Science.gov (United States)

Strikwold, Marije; Spenkelink, Bert; Woutersen, Ruud A; Rietjens, Ivonne M C M; Punt, Ans

2017-06-01

With our recently developed in vitro physiologically based kinetic (PBK) modelling approach, we could extrapolate in vitro toxicity data to human toxicity values applying PBK-based reverse dosimetry. Ideally information on kinetic differences among human individuals within a population should be considered. In the present study, we demonstrated a modelling approach that integrated in vitro toxicity data, PBK modelling and Monte Carlo simulations to obtain insight in interindividual human kinetic variation and derive chemical specific adjustment factors (CSAFs) for phenol-induced developmental toxicity. The present study revealed that UGT1A6 is the primary enzyme responsible for the glucuronidation of phenol in humans followed by UGT1A9. Monte Carlo simulations were performed taking into account interindividual variation in glucuronidation by these specific UGTs and in the oral absorption coefficient. Linking Monte Carlo simulations with PBK modelling, population variability in the maximum plasma concentration of phenol for the human population could be predicted. This approach provided a CSAF for interindividual variation of 2.0 which covers the 99th percentile of the population, which is lower than the default safety factor of 3.16 for interindividual human kinetic differences. Dividing the dose-response curve data obtained with in vitro PBK-based reverse dosimetry, with the CSAF provided a dose-response curve that reflects the consequences of the interindividual variability in phenol kinetics for the developmental toxicity of phenol. The strength of the presented approach is that it provides insight in the effect of interindividual variation in kinetics for phenol-induced developmental toxicity, based on only in vitro and in silico testing. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Screening physical health? Yes! But...: nurses' views on physical health screening in mental health care.

Science.gov (United States)

Happell, Brenda; Scott, David; Nankivell, Janette; Platania-Phung, Chris

2013-08-01

To explore nurses' views on the role of nurses in screening and monitoring for physical care of consumers with serious mental illness, at a regional mental health care service. People with serious mental illness experience heightened incidence of preventable and treatable physical illnesses such as cardiovascular disease and diabetes. Screening and monitoring are considered universal clinical safeguards. Nurses can potentially facilitate systematic screening, but their views on physical health care practices are rarely investigated. Qualitative exploratory study. Focus group interviews with 38 nurses of a regional mental health care service district of Australia. To facilitate discussion, participants were presented with a screening system, called the Health Improvement Profile (HIP), as an exemplar of screening of physical health risks by nurses. Inductive data analysis and theme development were guided by a thematic analysis framework. Nurses argued that treatable and preventable physical health problems were common. Four main themes were identified: screening - essential for good practice; the policy-practice gap; 'screening then what?' and, is HIP the answer? Screening and monitoring were considered crucial to proper diagnosis and treatment, however, were not performed systematically or consistently. Nurse readiness for an enhanced role in screening was shaped by: role and responsibility issues, legal liability concerns, funding and staff shortages. Participants were concerned that lack of follow up would limit effectiveness of these interventions. Screening was considered an important clinical step in effective diagnosis and treatment; however, identified barriers need to be addressed to ensure screening is part of a systemic approach to improve physical health of consumers with serious mental illness. Nurses have potential to influence improvement in physical health outcomes for consumers of mental health services. Such potential can only be realised if a

In vitro and biomechanical screening of polyethylene glycol and poly(trimethylene carbonate) block copolymers for annulus fibrosus repair.

Science.gov (United States)

Long, Rose G; Rotman, Stijn G; Hom, Warren W; Assael, Dylan J; Illien-Jünger, Svenja; Grijpma, Dirk W; Iatridis, James C

2018-02-01

Herniated intervertebral discs (IVDs) are a common cause of back and neck pain. There is an unmet clinical need to seal annulus fibrosus (AF) defects, as discectomy surgeries address acute pain but are complicated by reherniation and recurrent pain. Copolymers of polyethylene glycol with trimethylene carbonate (TMC) and hexamethylene diisocyanate (HDI) end-groups were formulated as AF sealants as the HDI form covalent bonds with native AF tissue. TMC adhesives were evaluated and optimized using the design criteria: stable size, strong adherence to AF tissue, high cytocompatibility, restoration of IVD biomechanics to intact levels following in situ repair, and low extrusion risk. TMC adhesives had high adhesion strength as assessed with a pushout test (150 kPa), and low degradation rates over 3 weeks in vitro. Both TMC adhesives had shear moduli (220 and 490 kPa) similar to, but somewhat higher than, AF tissue. The adhesive with three TMC moieties per branch (TMC3) was selected for additional in situ testing because it best matched AF shear properties. TMC3 restored torsional stiffness, torsional hysteresis area and axial range of motion to intact states. However, in a failure test of compressive deformation under fixed 5 ° flexion, some herniation risk was observed with failure strength of 5.9 MPa compared with 13.5 MPa for intact samples; TMC3 herniated under cyclic organ culture testing. These TMC adhesives performed well during in vitro and in situ testing, but additional optimization to enhance failure strength is required to further this material to advanced screening tests, such as long-term degradation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Current Status of Comprehensive Chromosome Screening for Elective Single-Embryo Transfer

Directory of Open Access Journals (Sweden)

Ming-Yih Wu

2014-01-01

Full Text Available Most in vitro fertilization (IVF experts and infertility patients agree that the most ideal assisted reproductive technology (ART outcome is to have a healthy, full-term singleton born. To this end, the most reliable policy is the single-embryo transfer (SET. However, unsatisfactory results in IVF may result from plenty of factors, in which aneuploidy associated with advanced maternal age is a major hurdle. Throughout the past few years, we have got a big leap in advancement of the genetic screening of embryos on aneuploidy, translocation, or mutations. This facilitates a higher success rate in IVF accompanied by the policy of elective SET (eSET. As the cost is lowering while the scale of genome characterization continues to be up over the recent years, the contemporary technologies on trophectoderm biopsy and freezing-thaw, comprehensive chromosome screening (CCS with eSET appear to be getting more and more popular for modern IVF centers. Furthermore, evidence has showen that, by these avant-garde techniques (trophectoderm biopsy, vitrification, and CCS, older infertile women with the help of eSET may have an opportunity to increase the success of their live birth rates approaching those reported in younger infertility patients.

Testing Precision Screening for Breast Cancer

Science.gov (United States)

An NCI research article about individualized approaches that could help identify those at risk of breast cancer who need to be screened and testing screening intervals that are appropriate for each person’s level of risk.

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

Energy Technology Data Exchange (ETDEWEB)

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com [Molecular Devices, LLC, Sunnyvale, CA (United States); Grimm, Fabian A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Ryan, Kristen R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Iwata, Yasuhiro; Chiu, Weihsueh A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Parham, Frederick [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Wignall, Jessica A. [ICF, Fairfax, VA (United States); Anson, Blake [Cellular Dynamics International, Madison, WI (United States); Cromwell, Evan F. [Protein Fluidics, Inc., Burlingame, CA (United States); Behl, Mamta [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Rusyn, Ivan [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Tice, Raymond R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States)

2017-05-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca{sup 2+} flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights: �

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

International Nuclear Information System (INIS)

Sirenko, Oksana; Grimm, Fabian A.; Ryan, Kristen R.; Iwata, Yasuhiro; Chiu, Weihsueh A.; Parham, Frederick; Wignall, Jessica A.; Anson, Blake; Cromwell, Evan F.; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R.

2017-01-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca 2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights: �

Research perspectives for pre-screening alternatives to animal experimentation

International Nuclear Information System (INIS)

Walum, Erik; Hedander, Jan; Garberg, Per

2005-01-01

The MEIC study revealed a high predictivity of in vitro cytotoxicity data for human acute systemic toxicity. The idea, put forward by several authors, that compounds that show high cytotoxicity should not need further testing for confirmation but could be assumed toxic also in vivo provides a convenient concept for the selection of the most relevant compounds for further studies in large sets of chemicals, as in the REACH program. The automated techniques applied in high throughput screening (HTS) by the pharmaceutical and biotech industries to select hits in extensive compound collections represent an opportunity to significantly increase the capacity of cytotoxicity testing. Furthermore, it has been suggested that a combination of cytotoxicity data and some basic biokinetic information would greatly improve the accuracy in the extrapolation from in vitro to in vivo and thus make it possible to identify additional toxic compounds that might have escaped in the initial screen. Such information, which can be obtained in a medium throughput screening mode (MTS), includes biotransformation, absorption and some aspects of distribution. The measurement of the net flux of a compound over a cellular barrier, as the one formed in culture by human Caco-2 cells, gives useful, but limited, information on both gut absorption and blood-brain barrier penetration. The test procedures discussed here, as well as other supplementary in vitro tests, cannot always easily be described in terms of animal-based test replacements. In those instances, the necessary test validation cannot be carried out using animal reference data, and prediction models may have to be adapted to new ideas. Consequently, concepts of prospective validation to supplement the now well-established retrospective validation have to be developed

Service innovation: a comparison of two approaches for physical screening of psychiatric inpatients.

Science.gov (United States)

Harrison, Mark Richard; McMillan, Catherine Frances; Dickinson, Timothy

2012-06-01

Psychiatric medications have clear links to obesity, diabetes, dyslipidaemia, hypertension, hyperprolactinaemia and movement disorders. These disorders are a common cause of morbidity and mortality in psychiatric patients but physical screening by health services is often haphazard. We report the findings of an audit of physical screening across two hospital wards. Each ward undertook a process of service improvement. One ward modified the admissions proforma and the other developed a discharge screening clinic. The effectiveness of each of these interventions was then compared through a reaudit of practice across both wards. At baseline, screening was performed inconsistently and infrequently. On average, the modified admissions proforma increased screening rates by 4.7% compared to 30.7% for discharge screening clinics. The discharge screening clinic demonstrated statistically significant improvements in screening rates and effectively delivered health promotion advice. Discharge screening clinics are significantly more likely than improved admissions procedures to detect clinically significant abnormalities. If these abnormalities are detected and treated then the long-term physical health of psychiatric patients may be improved.

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

Science.gov (United States)

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.

Clozapine-induced agranulocytosis: Evidence for an immune-mediated mechanism from a patient-specific in-vitro approach

Energy Technology Data Exchange (ETDEWEB)

Regen, Francesca; Herzog, Irmelin; Hahn, Eric; Ruehl, Claudia; Le Bret, Nathalie; Dettling, Michael; Heuser, Isabella [Dept. of Psychiatry, Campus Benjamin Franklin, Charité, University Medicine Berlin (Germany); Hellmann-Regen, Julian, E-mail: julian.hellmann@charite.de [Dept. of Psychiatry, Campus Benjamin Franklin, Charité, University Medicine Berlin (Germany); Berlin Institute of Health (BIH), Berlin (Germany)

2017-02-01

Use of the atypical antipsychotic clozapine (CZP) is compromised by the risk of potentially fatal agranulocytosis/granulocytopenia (CIAG). To address this, we have established a simple, personalized cell culture-based strategy to identify CIAG-susceptible patients, hypothesizing that an immunogenic and possibly haptene-based mechanism underlies CIAG pathophysiology. To detect a putative haptene-induced response to CZP in vitro exposure, a traditional lymphocyte stimulation assay was adapted and applied to patient-specific peripheral blood-derived mononuclear cells (PBMC). 6 patients with a history of CIAG, 6 patients under CZP treatment (without CIAG) and 12 matched healthy controls were studied. In vitro CZP exposure, even at strikingly low levels, resulted in significantly increased proliferation rates only in CIAG patients' PBMC. Other parameters including cell viability and mitogen-induced proliferation were also affected by in vitro CZP exposure, yet there was no significant difference between the groups. This personalized approach is a starting point for further investigations into a putative haptene-based mechanism underlying CIAG development, and may facilitate the future development of predictive testing. - Highlights: • Clozapine induces proliferation in PBMCs from patients with a history of CIAG. • Simple, PBMC-based assay results in robust effects of physiological clozapine levels. • Haptene-based mechanisms discussed to underlie clozapine-induced proliferation.

Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

Science.gov (United States)

Cassells, Alan C

2012-01-01

The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

Psychiatric screening and interventions for minor refugees in Europe: an overview of approaches and tools.

Science.gov (United States)

Horlings, Annerieke; Hein, Irma

2018-02-01

Currently hundreds of thousands of minor refugees entered Europe. This group has been exposed to traumatic events pre-, during, and post-migration and is at increased risk of developing psychiatric disorders. In this article, we describe the results of our literature search on screening and interventions for post-traumatic stress disorder (PTSD) in minor refugees, in order to make recommendations for clinical practice. Results show that studies on diagnostic accuracy of assessment instruments and efficacy of mental healthcare interventions in this population are lacking. Traumatic experiences pre-flight, during the flight and at resettlement, superimposed by parental PTSD, and other contextual factors, might lead to more than 25% of minor refugees developing PTSD. To enhance the number of minor refugees recognized with PTSD, we recommend the use of a brief screening instrument. A public health approach, focusing on environmental supportive factors is the first step in treatment for this group, followed by short-term psychological group interventions focusing on psycho-education and stress reduction. Minor refugees with no improvement in PTSD symptoms by these interventions need referral to specialized mental health care services. Mental health providers should be culturally competent. What is Known: • Post-traumatic stress disorder, anxiety, sleeping problems, and depression are the most common psychiatric disorders in minor refugees. • Evidence based methods on screening and interventions in minor refugees with psychiatric disorders are lacking. What is New: • In the absence of validated screening tools a best practice reliable, quick and child-friendly tool is presented. • A layered system for mental health care and psychosocial support in minor refugees is explained.

Interactions among biotic and abiotic factors affect the reliability of tungsten microneedles puncturing in vitro and in vivo peripheral nerves: A hybrid computational approach

Energy Technology Data Exchange (ETDEWEB)

Sergi, Pier Nicola, E-mail: p.sergi@sssup.it [Translational Neural Engineering Laboratory, The Biorobotics Institute, Scuola Superiore Sant' Anna, Viale Rinaldo Piaggio 34, Pontedera, 56025 (Italy); Jensen, Winnie [Department of Health Science and Technology, Fredrik Bajers Vej 7, 9220 Aalborg (Denmark); Yoshida, Ken [Department of Biomedical Engineering, Indiana University - Purdue University Indianapolis, 723 W. Michigan St., SL220, Indianapolis, IN 46202 (United States)

2016-02-01

Tungsten is an elective material to produce slender and stiff microneedles able to enter soft tissues and minimize puncture wounds. In particular, tungsten microneedles are used to puncture peripheral nerves and insert neural interfaces, bridging the gap between the nervous system and robotic devices (e.g., hand prostheses). Unfortunately, microneedles fail during the puncture process and this failure is not dependent on stiffness or fracture toughness of the constituent material. In addition, the microneedles' performances decrease during in vivo trials with respect to the in vitro ones. This further effect is independent on internal biotic effects, while it seems to be related to external biotic causes. Since the exact synergy of phenomena decreasing the in vivo reliability is still not known, this work explored the connection between in vitro and in vivo behavior of tungsten microneedles through the study of interactions between biotic and abiotic factors. A hybrid computational approach, simultaneously using theoretical relationships and in silico models of nerves, was implemented to model the change of reliability varying the microneedle diameter, and to predict in vivo performances by using in vitro reliability and local differences between in vivo and in vitro mechanical response of nerves. - Highlights: • We provide phenomenological Finite Element (FE) models of peripheral nerves to study the interactions with W microneedles • We provide a general interaction-based approach to model the reliability of slender microneedles • We evaluate the reliability of W microneedels to puncture in vivo nerves • We provide a novel synergistic hybrid approach (theory + simulations) involving interactions among biotic and abiotic factors • We validate the hybrid approach by using experimental data from literature.

Interactions among biotic and abiotic factors affect the reliability of tungsten microneedles puncturing in vitro and in vivo peripheral nerves: A hybrid computational approach

International Nuclear Information System (INIS)

Sergi, Pier Nicola; Jensen, Winnie; Yoshida, Ken

2016-01-01

Tungsten is an elective material to produce slender and stiff microneedles able to enter soft tissues and minimize puncture wounds. In particular, tungsten microneedles are used to puncture peripheral nerves and insert neural interfaces, bridging the gap between the nervous system and robotic devices (e.g., hand prostheses). Unfortunately, microneedles fail during the puncture process and this failure is not dependent on stiffness or fracture toughness of the constituent material. In addition, the microneedles' performances decrease during in vivo trials with respect to the in vitro ones. This further effect is independent on internal biotic effects, while it seems to be related to external biotic causes. Since the exact synergy of phenomena decreasing the in vivo reliability is still not known, this work explored the connection between in vitro and in vivo behavior of tungsten microneedles through the study of interactions between biotic and abiotic factors. A hybrid computational approach, simultaneously using theoretical relationships and in silico models of nerves, was implemented to model the change of reliability varying the microneedle diameter, and to predict in vivo performances by using in vitro reliability and local differences between in vivo and in vitro mechanical response of nerves. - Highlights: • We provide phenomenological Finite Element (FE) models of peripheral nerves to study the interactions with W microneedles • We provide a general interaction-based approach to model the reliability of slender microneedles • We evaluate the reliability of W microneedels to puncture in vivo nerves • We provide a novel synergistic hybrid approach (theory + simulations) involving interactions among biotic and abiotic factors • We validate the hybrid approach by using experimental data from literature

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

Science.gov (United States)

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.

Studies of Cream Seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan Efficacy Trial: In Vitro and In Vivo Screening Tools Reflect Human Studies and Predict Beneficial Results from Iron Biofortified Beans.

Science.gov (United States)

Tako, Elad; Reed, Spenser; Anandaraman, Amrutha; Beebe, Steve E; Hart, Jonathan J; Glahn, Raymond P

2015-01-01

of these in vitro and in vivo screening tools to guide studies aimed to develop and evaluate biofortified staple food crops. This approach has the potential to more effectively utilize research funds and provides a means to monitor the nutritional quality of the Fe-biofortified crops once released to farmers.

Industrial screening programs for workers

International Nuclear Information System (INIS)

Lavine, M.P.

1982-01-01

Industrial screening efforts to identify classes of workers who are more susceptible to workplace hazards, by virtue of their fertility, genetic, or lifestyle characteristics, represent a relatively new approach to reducing workplace risks. Screening has already raised some important economic, legal, social, medical, and moral questions. Employers, employees, administrative agencies, and the courts are offering different, often conflicting answers. Ultimately the acceptability of various screening schemes rests upon judgments about how a society justifies the distribution of risk. The questions that industrial screening programs raise are only partially answered by empirical evidence; the rest is a matter of values

Advances in Assays and Analytical Approaches for Botulinum Toxin Detection

Energy Technology Data Exchange (ETDEWEB)

Grate, Jay W.; Ozanich, Richard M.; Warner, Marvin G.; Bruckner-Lea, Cindy J.; Marks, James D.

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.

Availability and accessibility of subsidized mammogram screening program in peninsular Malaysia: A preliminary study using travel impedance approach.

Science.gov (United States)

Mahmud, Aidalina; Aljunid, Syed Mohamed

2018-01-01

Access to healthcare is essential in the pursuit of universal health coverage. Components of access are availability, accessibility (spatial and non-spatial), affordability and acceptability. Measuring spatial accessibility is common approach to evaluating access to health care. This study aimed to determine the availability and spatial accessibility of subsidised mammogram screening in Peninsular Malaysia. Availability was determined from the number and distribution of facilities. Spatial accessibility was determined using the travel impedance approach to represent the revealed access as opposed to potential access measured by other spatial measurement methods. The driving distance of return trips from the respondent's residence to the facilities was determined using a mapping application. The travel expenditure was estimated by multiplying the total travel distance by a standardised travel allowance rate, plus parking fees. Respondents in this study were 344 breast cancer patients who received treatment at 4 referral hospitals between 2015 and 2016. In terms of availability, there were at least 6 major entities which provided subsidised mammogram programs. Facilities with mammogram involved with these programs were located more densely in the central and west coast region of the Peninsula. The ratio of mammogram facility to the target population of women aged 40-74 years ranged between 1: 10,000 and 1:80,000. In terms of accessibility, of the 3.6% of the respondents had undergone mammogram screening, their mean travel distance was 53.4 km (SD = 34.5, range 8-112 km) and the mean travel expenditure was RM 38.97 (SD = 24.00, range RM7.60-78.40). Among those who did not go for mammogram screening, the estimated travel distance and expenditure had a skewed distribution with median travel distance of 22.0 km (IQR 12.0, 42.0, range 2.0-340.0) and the median travel cost of RM 17.40 (IQR 10.40, 30.00, range 3.40-240.00). Higher travel impedance was noted among those who

The development of high-content screening (HCS) technology and its importance to drug discovery.

Science.gov (United States)

Fraietta, Ivan; Gasparri, Fabio

2016-01-01

High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development. Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology. Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.

An in vitro metabolomics approach to identify hepatotoxicity biomarkers in human L02 liver cells treated with pekinenal, a natural compound.

Science.gov (United States)

Shi, Jiexia; Zhou, Jing; Ma, Hongyue; Guo, Hongbo; Ni, Zuyao; Duan, Jin'ao; Tao, Weiwei; Qian, Dawei

2016-02-01

An in vitro cell metabolomics study was performed on human L02 liver cells to investigate the toxic biomarkers of pekinenal from the herb Euphorbia pekinensis Rupr. Pekinenal significantly induced L02 cell damage, which was characterised by necrosis and apoptosis. Metabolomics combined with data pattern recognition showed that pekinenal significantly altered the profiles of more than 1299 endogenous metabolites with variable importance in the projection (VIP) > 1. Further, screening correlation coefficients between the intensities of all metabolites and the extent of L02 cell damage (MTT) identified 12 biomarker hits: ten were downregulated and two were upregulated. Among these hits, LysoPC(18:1(9Z)/(11Z)), PC(22:0/15:0) and PC(20:1(11Z)/14:1(9Z)) were disordered, implying the initiation of inflammation and cell damage. Several fatty acids (FAs) (3-hydroxytetradecanedioic acid, pivaloylcarnitine and eicosapentaenoyl ethanolamide) decreased due to fatty acid oxidation. Dihydroceramide and Cer(d18:0/14:0) were also altered and are associated with apoptosis. Additional examination of the levels of intracellular reactive oxygen species (ROS) and two eicosanoids (PGE2, PGF2α) in the cell supernatant confirmed the fatty acid oxidation and arachidonic acid metabolism pathways, respectively. In summary, cell metabolomics is a highly efficient approach for identifying toxic biomarkers and helping understand toxicity mechanisms and predict herb-induced liver injury.

Using In Vitro High-Throughput Screening Data for Predicting ...

Science.gov (United States)

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. By coupling qHTS data with adverse outcome pathways (AOPs) we can use ontologies to make predictions about potential hazards and to identify those assays which are sufficient to infer these same hazards. Once those assays are identified, we can use bootstrap natural spline-based metaregression to integrate the evidence across multiple replicates or assays (if a combination of assays are together necessary to be sufficient). In this pilot, we demonstrate how we were able to identify that benzo[k]fluoranthene (B[k]F) may induce DNA damage and steatosis using qHTS data and two separate AOPs. We also demonstrate how bootstrap natural spline-based metaregression can be used to integrate the data across multiple assay replicates to generate a concentration-response curve. We used this analysis to calculate an internal point of departure of 0.751µM and risk-specific concentrations of 0.378µM for both 1:1,000 and 1:10,000 additive risk for B[k]F induced DNA damage based on the p53 assay. Based on the available evidence, we

Introducing a open-quote modular close-quote approach to fish screen installation

International Nuclear Information System (INIS)

Taft, E.P.; Winchell, F.C.; Cook, T.C.; Sullivan, C.W.

1992-01-01

A new fish screen design-the modular inclined screen-promises to offer a versatile and cost-effective solution for fish protection in many situation. In an effort to provide the hydroelectric industry with more cost-effective alternatives to existing fish screen designs, the Electric Power Research Institute (EPRI) recently has undertaken several research projects. One focus of EPRI's research has been on the development and testing of high-velocity fish screens. This program has led to the development of a new screening concept, which shows promise for providing effective protection for a wide range of fish species at hydro plants, steam generating facilities, and irrigation diversions. The concept, known as the Modular Inclined Screen (MIS), currently is being evaluated in laboratory studies prior to field application. The screen is of open-quotes modularclose quotes design so as to provide the flexibility necessary for application at a broad range of water intakes. The module is suitable for installation in penstocks, canals, and head pond intakes. The MIS module consists of an entrance with a trashrack, dewatering stoplog slots, an inclined wedgewire screen set at a shallow angle of 10 to 20 degrees to the flow, and a bypass for diverting fish to a transport pipe. The screen is mounted on a pivot shaft so that it can be cleaned via rotation and backflushing. The module is completely enclosed, and is designed to operate at water velocities ranging from 2 to 10 feet per second depending on the fish species and life stages to be protected. Laboratory studies are under way to evaluate the design configuration that yields the best hydraulic conditions for safe fish passage, and the biological effectiveness of this design in diverting selected fish species to the bypass

Screening the banana biodiversity for drought tolerance: can an in vitro growth model and proteomics be used as a tool to discover tolerant varieties and understand homeostasis.

Science.gov (United States)

Vanhove, Anne-Catherine; Vermaelen, Wesley; Panis, Bart; Swennen, Rony; Carpentier, Sebastien C

2012-01-01

There is a great need for research aimed at understanding drought tolerance, screening for drought tolerant varieties and breeding crops with an improved water use efficiency. Bananas and plantains are a major staple food and export product with a worldwide production of over 135 million tonnes per year. Water however is the most limiting abiotic factor in banana production. A screening of the Musa biodiversity has not yet been performed. We at KU Leuven host the Musa International Germplasm collection with over 1200 accessions. To screen the Musa biodiversity for drought tolerant varieties, we developed a screening test for in vitro plants. Five varieties representing different genomic constitutions in banana (AAAh, AAA, AAB, AABp, and ABB) were selected and subjected to a mild osmotic stress. The ABB variety showed the smallest stress induced growth reduction. To get an insight into the acclimation and the accomplishment of homeostasis, the leaf proteome of this variety was characterized via 2D DIGE. After extraction of the leaf proteome of six control and six stressed plants, 2600 spots could be distinguished. A PCA analysis indicates that control and stressed plants can blindly be classified based on their proteome. One hundred and twelve proteins were significantly more abundant in the stressed plants and 18 proteins were significantly more abundant in control plants (FDR α 0.05). Twenty four differential proteins could be identified. The proteome analysis clearly shows that there is a new balance in the stressed plants and that the respiration, metabolism of ROS and several dehydrogenases involved in NAD/NADH homeostasis play an important role.

Phonic Attenuation due to Screen-Barriers

Directory of Open Access Journals (Sweden)

Vasile Bacria

2011-10-01

Full Text Available The technique of noise decreasing admits two basic approaches: an active approach and a passive one. In the frame of passive method one can count the employment of screen-barriers. In this paper we present some considerations on sound attenuation due to screen-barriers emphasizing the elements which influence it. The elucidation of these elements is made by measurements. The obtained results can be applied in every other practical situation concerning the protection against noise.

Synthesis of New 3-(2-Chloroquinolin-3-yl)-5-Phenylisoxazole Derivatives via Click-Chemistry Approach

International Nuclear Information System (INIS)

Fernández-Galleguillos, Carlos; Saavedra, Luis A.; Gutierrez, Margarita

2014-01-01

Herein, we report the synthesis of new substituted 3-(2-chloroquinolin-3-yl)-5-phenylisoxazole (3a-j) by click chemistry in good to moderate yields. This approach is based on the regioselective copper(I)-catalyzed cycloaddition between different nitrile oxides derived from 2-chloroquinoline- 3-carbaldehyde (2a-j) and phenylacetylene. Finally these derivatives were screened for their antibacterial evaluation in vitro against three Gram-negative clinical bacteria: Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii using standard methods. (author)

Synthesis of New 3-(2-Chloroquinolin-3-yl)-5-Phenylisoxazole Derivatives via Click-Chemistry Approach

Energy Technology Data Exchange (ETDEWEB)

Fernandez-Galleguillos, Carlos; Saavedra, Luis A.; Gutierrez, Margarita [Laboratorio de Sintesis Organica, Instituto de Quimica de Recursos Naturales, Universidad de Talca (Chile)

2014-02-15

Herein, we report the synthesis of new substituted 3-(2-chloroquinolin-3-yl)-5-phenylisoxazole (3a-j) by click chemistry in good to moderate yields. This approach is based on the regioselective copper(I)-catalyzed cycloaddition between different nitrile oxides derived from 2-chloroquinoline- 3-carbaldehyde (2a-j) and phenylacetylene. Finally these derivatives were screened for their antibacterial evaluation in vitro against three Gram-negative clinical bacteria: Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii using standard methods. (author)

A rational approach to heavy-atom derivative screening

International Nuclear Information System (INIS)

Joyce, M. Gordon; Radaev, Sergei; Sun, Peter D.

2010-01-01

In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom compound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the process of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing

A rational approach to heavy-atom derivative screening

Energy Technology Data Exchange (ETDEWEB)

Joyce, M. Gordon; Radaev, Sergei; Sun, Peter D., E-mail: psun@nih.gov [Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, Maryland 20852 (United States)

2010-04-01

In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom compound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the process of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing.

The TSCA interagency testing committee`s approaches to screening and scoring chemicals and chemical groups: 1977-1983

Energy Technology Data Exchange (ETDEWEB)

Walker, J.D. [Environmental Protection Agency, Washington, DC (United States)

1990-12-31

This paper describes the TSCA interagency testing committee`s (ITC) approaches to screening and scoring chemicals and chemical groups between 1977 and 1983. During this time the ITC conducted five scoring exercises to select chemicals and chemical groups for detailed review and to determine which of these chemicals and chemical groups should be added to the TSCA Section 4(e) Priority Testing List. 29 refs., 1 fig., 2 tabs.

Integration of in vitro biorelevant dissolution and in silico PBPK model of carvedilol to predict bioequivalence of oral drug products.

Science.gov (United States)

Ibarra, Manuel; Valiante, Cristian; Sopeña, Patricia; Schiavo, Alejandra; Lorier, Marianela; Vázquez, Marta; Fagiolino, Pietro

2018-06-15

Bioequivalence implementation in developing countries where a high proportion of similar drug products are being marketed has found several obstacles, impeding regulatory agencies to move forward with this policy. Biopharmaceutical quality of these products, several of which are massively prescribed, remains unknown. In this context, an in vitro-in silico-in vivo approach is proposed as a mean to screen product performance and target specific formulations for bioequivalence assessment. By coupling in vitro biorelevant dissolution testing in USP-4 Apparatus (flow-through cell) with physiologically-based pharmacokinetic (PBPK) modeling in PK-Sim® software (Bayer, Germany), the performance of seven similar products of carvedilol tablets containing 25 mg available in the Uruguayan market were compared with the brand-name drug Dilatrend®. In silico simulations for Dilatrend® were compared with published results of bioequivalence studies performed in fasting conditions allowing model development through a learning and confirming process. Single-dose pharmacokinetic profiles were then simulated for the brand-name drug and two similar drug products selected according to in vitro observations, in a virtual Caucasian population of 1000 subjects (50% male, aged between 18 and 50 years with standard body-weights). Population bioequivalence ratios were estimated revealing that in vitro differences in drug release would have a major impact in carvedilol maximum plasma concentration, leading to a non-bioequivalence outcome. Predictions support the need to perform in vivo bioequivalence for these products of extensive use. Application of the in vitro-in silico-in vivo approach stands as an interesting alternative to tackle and reduce drug product variability in biopharmaceutical quality. Copyright © 2018 Elsevier B.V. All rights reserved.

The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

Science.gov (United States)

Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

2010-04-01

We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

Novel inhibitors to Taenia solium Cu/Zn superoxide dismutase identified by virtual screening

Science.gov (United States)

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.

Beliefs Underlying Messages of Anti-Cancer-Screening

Science.gov (United States)

Okuhara, Tsuyoshi; Ishikawa, Hirono; Okada, Masahumi; Kato, Mio; Kiuchi, Takahiro

2018-02-26

Background: Cancer screening rates are lower in Japan than in Western countries. Meanwhile, anti-cancer-screening activists take to the internet to spread their messages that cancer screening has little or no efficacy, poses substantial health risks such as side effects from radiation exposure, and that people should forgo cancer screening. We applied a qualitative approach to explore the beliefs underlying the messages of anti-cancer-screening websites, by focusing on perceived value the beliefs provided to those who held them. Methods: We conducted online searches using Google Japan and Yahoo! Japan, targeting websites we classified as “pro,” “anti,” or “neutral” depending on their claims. We applied a dual analytic approach- inductive thematic analysis and deductive interpretative analysis- to the textual data of the anti websites. Results: Of the 88 websites analyzed, five themes that correspond to beliefs were identified: destruction of common knowledge, denial of standard cancer control, education about right cancer control, education about hidden truths, and sense of superiority that only I know the truth. Authors of anti websites ascribed two values (“safety of people” and “self-esteem”) to their beliefs. Conclusion: The beliefs of authors of anti-cancer-screening websites were supposed to be strong. It would be better to target in cancer screening promotion not outright screening refusers but screening hesitant people who are more amenable to changing their attitudes toward screening. The possible means to persuade them were discussed. Creative Commons Attribution License

RNAi Screening in Spodoptera frugiperda.

Science.gov (United States)

Ghosh, Subhanita; Singh, Gatikrushna; Sachdev, Bindiya; Kumar, Ajit; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

2016-01-01

RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.

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

Science.gov (United States)

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.

Drug discovery for schistosomiasis: hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening.

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Maha-Hamadien Abdulla

2009-07-01

Full Text Available Praziquantel (PZQ is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism screening with adult worms in vitro and/or animal models of disease-tools that limit automation and throughput with modern microtiter plate-formatted compound libraries.A partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to 'reposition' (re-profile drugs as anti-schistosomals with potential savings in development timelines and costs.Multiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of 'hit' drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource.To accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that

Chemical biology drug sensitivity screen identifies sunitinib as synergistic agent with disulfiram in prostate cancer cells.

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Kirsi Ketola

Full Text Available Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects.In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression.Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell growth. Disulfiram

A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

Science.gov (United States)

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.

High-Throughput Screening as a Supplemental Tool for the Development of Advanced Emission Control Catalysts: Methodological Approaches and Data Processing

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Andreas Sundermann

2016-01-01

Full Text Available A high-throughput (HT screening platform developed at hte with the application focus on automotive catalysis is described. hte HT units are configured for performing steady-state testing, as well as dynamic tests with fast feed switches, such as lean/rich excursions for the evaluation of NOx storage capacity and efficiency of lean NOx traps (LNT, ammonia storage capacity for selective catalytic reduction (SCR, evaluation of oxygen storage capacity (OSC, as well as lambda sweep tests for screening of three-way catalysts (TWC. Even though catalysts are screened on a rather small scale (~100 mg powder, experience showed that dosing rather complex gas mixtures in concentrations close to that found in real exhaust for the given application is mandatory to generate relevant data. The objective of this work is to give additional insight into HT technology. In the industrial research laboratory, HT screening has matured to become a reliable approach for rapid screening of both reaction parameter spaces, as well as material properties relevant for exhaust gas catalyst development. Due to the speed of optimized screening involving 48 parallel reactors, automated handling of primary data is an imported requirement. Software for data reduction, like estimation of light-off temperature, needs to be robust and handle results for diverse sample libraries in an unattended fashion. In combination with the statistical design of experiment and multivariate data analysis, HT testing has become a valuable enhancement to automotive catalyst development.

High-throughput fragment screening by affinity LC-MS.

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Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

2013-02-01

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

[Methodology of Screening New Antibiotics: Present Status and Prospects].

Science.gov (United States)

Trenin, A S

2015-01-01

Due to extensive distribution of pathogen resistance to available pharmaceuticals and serious problems in the treatment of various infections and tumor diseases, the necessity of new antibiotics is urgent. The basic methodological approaches to chemical synthesis of antibiotics and screening of new antibiotics among natural products, mainly among microbial secondary metabolites, are considered in the review. Since the natural compounds are very much diverse, screening of such substances gives a good opportunity to discover antibiotics of various chemical structure and mechanism of action. Such an approach followed by chemical or biological transformation, is capable of providing the health care with new effective pharmaceuticals. The review is mainly concentrated on screening of natural products and methodological problems, such as: isolation of microbial producers from the habitats, cultivation of microorganisms producing appropriate substances, isolation and chemical characterization of microbial metabolites, identification of the biological activity of the metabolites. The main attention is paid to the problems of microbial secondary metabolism and design of new models for screening biologically active compounds. The last achievements in the field of antibiotics and most perspective approaches to future investigations are discussed. The main methodological approach to isolation and cultivation of the producers remains actual and needs constant improvement. The increase of the screening efficiency can be achieved by more rapid chemical identification of antibiotics and design of new screening models based on the biological activity detection.

Screen printing as a scalable and low-cost approach for rigid and flexible thin-film transistors using separated carbon nanotubes.

Science.gov (United States)

Cao, Xuan; Chen, Haitian; Gu, Xiaofei; Liu, Bilu; Wang, Wenli; Cao, Yu; Wu, Fanqi; Zhou, Chongwu

2014-12-23

Semiconducting single-wall carbon nanotubes are very promising materials in printed electronics due to their excellent mechanical and electrical property, outstanding printability, and great potential for flexible electronics. Nonetheless, developing scalable and low-cost approaches for manufacturing fully printed high-performance single-wall carbon nanotube thin-film transistors remains a major challenge. Here we report that screen printing, which is a simple, scalable, and cost-effective technique, can be used to produce both rigid and flexible thin-film transistors using separated single-wall carbon nanotubes. Our fully printed top-gated nanotube thin-film transistors on rigid and flexible substrates exhibit decent performance, with mobility up to 7.67 cm2 V(-1) s(-1), on/off ratio of 10(4)∼10(5), minimal hysteresis, and low operation voltage (transistors (bent with radius of curvature down to 3 mm) and driving capability for organic light-emitting diode have been demonstrated. Given the high performance of the fully screen-printed single-wall carbon nanotube thin-film transistors, we believe screen printing stands as a low-cost, scalable, and reliable approach to manufacture high-performance nanotube thin-film transistors for application in display electronics. Moreover, this technique may be used to fabricate thin-film transistors based on other materials for large-area flexible macroelectronics, and low-cost display electronics.

Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC.

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Aiyegoro, Olayinka A; Okoh, Anthony I

2010-05-14

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. 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. 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%). 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.

Bacterial and fungal keratitis in Upper Egypt: In vitro screening of enzymes, toxins and antifungal activity

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Abdullah A Gharamah

2014-01-01

Full Text Available Purpose: This work was conducted to study the ability of bacterial and fungal isolates from keratitis cases in Upper Egypt to produce enzymes, toxins, and to test the isolated fungal species sensitivity to some therapeutic agents. Materials and Methods: One hundred and fifteen patients clinically diagnosed to have microbial keratitis were investigated. From these cases, 37 bacterial isolates and 25 fungal isolates were screened for their ability to produce extra-cellular enzymes in solid media. In addition, the ability of fungal isolates to produce mycotoxins and their sensitivity to 4 antifungal agents were tested. Results: Protease, lipase, hemolysins, urease, phosphatase, and catalase were detected respectively in 48.65%, 37.84%, 59.46%, 43.24%, 67.57%, and 100% out of 37 bacterial isolates tested. Out of 25 fungal isolates tested during the present study, 80% were positive for protease, 84% for lipase and urease, 28% for blood hemolysis, and 100% for phosphatase and catalase enzymes. Thirteen fungal isolates were able to produce detectable amounts of 7 mycotoxins in culture medium (aflatoxins (B1, B2, G1, and G2, sterigmatocystin, fumagillin, diacetoxyscirpenol, zearalenone, T-2 toxin, and trichodermin. Among the antifungal agents tested in this study, terbinafine showed the highest effect against most isolates in vitro. Conclusion: In conclusion, the ability of bacterial and fungal isolates to produce extracellular enzymes and toxins may be aid in the invasion and destruction of eye tissues, which, in turn, lead to vision loss.

Polyester monomers lack ability to bind and activate both androgenic and estrogenic receptors as determined by in vitro and in silico methods.

Science.gov (United States)

Osimitz, Thomas G; Welsh, William J; Ai, Ni; Toole, Colleen

2015-01-01

The paper presents results from the screening of seven monomers used by Eastman Chemical to make various polymers. Ethylene glycol, diethylene glycol, polytetramethylene glycol, isophthalic acid, monosodium-5-sulfoisophthalic acid, 1,4-cyclohexanedicarboxylic acid, and dimethylcyclohexanedicarboxylate were screened for potential androgenicity or estrogenicity. The following studies were conducted: QSAR for binding to the AR and ER, in vitro Androgen Receptor Binding Assay, in vitro Estrogen Receptor Binding Assays (alpha and beta isoforms), in vitro Androgen Receptor Transactivation Assay in human cells, and in vitro Estrogen Receptor Transactivation Assay in human cells. None of the QSAR models predicted that any of the monomers possessed appreciable binding affinity for either AR or ER. Binding assays showed no evidence of interaction with either the AR or the alpha or beta ER receptors. Similarly, the AR and ER transactivation assays were negative. Moreover, six of the seven monomers have been subjected to 13-week and developmental toxicity studies in rats with no androgen- or estrogen-related effects being noted. Given the negative results of the in vitro screening assays (except PMG which demonstrated cytotoxicity) as well as available repeated dose and developmental and reproductive studies, the data suggest that none of the monomers tested exhibit androgenic or estrogenic hazards. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

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Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

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Prerna Grover

Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

Studying inhibition of calcium oxalate stone formation: an in vitro approach for screening hydrogen sulfide and its metabolites

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S. Vaitheeswari

2015-06-01

Full Text Available ABSTRACTPurpose:Calcium oxalate urolithiasis is one of the most common urinary tract diseases and is of high prevalence. The present study proposes to evaluate the antilithiatic property of hydrogen sulfide and its metabolites like thiosulfate & sulfate in an in vitro model.Materials and Methods:The antilithiatic activity of sodium hydrogen sulfide (NaSH, sodium thiosulfate (Na2S2O3 and sodium sulfate (Na2SO4 on the kinetics of calcium oxalate crystal formation was investigated both in physiological buffer and in urine from normal and recurrent stone forming volunteers. The stones were characterized by optical and spectroscopic techniques.Results:The stones were characterized to be monoclinic, prismatic and bipyramidal habit which is of calcium monohydrate and dihydrate nature. The FTIR displayed fingerprint corresponding to calcium oxalate in the control while in NaSH treated, S=O vibrations were visible in the spectrum. The order of percentage inhibition was NaSH>Na2S2O3>Na2SO4.Conclusion:Our study indicates that sodium hydrogen sulfide and its metabolite thiosulfate are inhibitors of calcium oxalate stone agglomeration which makes them unstable both in physiological buffer and in urine. This effect is attributed to pH changes and complexing of calcium by S2O32-and SO42- moiety produced by the test compounds.

Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms

NARCIS (Netherlands)

Bosmans, Lien; De Bruijn, I.; de Mot, Rene; Readers, Hans; Lievens, Bart

2016-01-01

Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens.Weshowed thatwhen using the samemedium, but different agar compositions, the activity of a bacterial antagonist against Agrobacteriumwas strongly affected.

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

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Greta Zubaite

2017-02-01

Full Text Available Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.

In vitro screening of organotin compounds and sediment extracts for cytotoxicity to fish cells.

Science.gov (United States)

Giltrap, Michelle; Macken, Ailbhe; McHugh, Brendan; McGovern, Evin; Foley, Barry; Davoren, Maria

2011-01-01

The present study reports an in vitro screening method for contaminants in sediment samples utilizing an RTG-2 cell line. This technique integrates cytotoxicity testing with analytical chemistry with the aim of achieving a toxicity evaluation of the sediment sample. The toxic effect of individual organotin (OT) compounds and their presence in the sediment sample is the focus of the present study; however, other contaminants are also discussed. The following OT compounds: tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and a sediment solvent extract are exposed to the RTG-2 fish cell line. Both the alamar blue (AB) and neutral red (NR) assays are used to assess cytotoxicity after 24-h and 96-h exposure. Methodology for preparation of a sediment solvent extract suitable for biological testing and analytical determination is also described. With the RTG-2 cells, the AB and NR assays had comparable sensitivity for each individual OT compound exposure after 24 h, with TPT being the most toxic compound tested. The individual OT compound concentrations required to induce a 50% toxic effect on the cells (369 ng ml⁻¹ TBT, 1,905 ng ml⁻¹ DBT) did not equate to the concentrations of these contaminants present in the sediment extract that induced a 50% effect on the cells (294 ng ml⁻¹ TBT, 109 ng ml⁻¹ DBT). The solvent extract therefore exhibited a greater toxicity, and this suggests that the toxic effects observed were not due to OT compounds alone. The presence of other contaminants in the solvent extract is confirmed with chemical analysis, warranting further toxicity testing of contaminant mixtures and exposure to the cell line to further elucidate a complete toxicity evaluation. © 2010 SETAC.

Screening for Anti-lipase Properties of 37 Traditional Chinese Medicinal Herbs

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Cheng-Dong Zheng

2010-06-01

Conclusion: The results support the view that herbs represent a rich source of anti-lipase compounds. The screening of the methanolic extracts of 37 Chinese medicinal plants in vitro led to the identification of several extracts with potential activity against PPL, in particular, P. vulgaris and R. palmatum. We also found that several monomeric chemicals in these herbs exhibited good or moderate activity against PPL. To the best of our knowledge, these traditional Chinese herbal medicines or phytochemicals have not been previously screened for their lipase inhibitory activity.

Charge degeneracy removal in the screened hydrogen atom

International Nuclear Information System (INIS)

Penna, Andre L A; Diniz, Joao B; Oliveira, Fernando A

2009-01-01

We derive an analytical model for the states of the screened hydrogen atom by using a new charge degeneracy removal approach. Starting from the nonzero Thomas-Fermi parameter q, we show that screening effect is due to breaks of the charge degeneracy in each quantum level of the hydrogen atom. The charge degeneracy removal reparametrizes the atomic system through the effective nuclear charge α n,l and the appearance of a dual charge γ n,l for each quantum level. In this approach, we show that the screening of a quantum state depends hierarchically on the screening from all previous quantum states with the same angular quantum numbers. The excited state energies E n,l (q) are analytically found taking into account the contribution of this new charge degeneracy for each quantum level. Finally, we also have estimated accurate critical screening parameters q* n,l for the bound-unbound transition.

European position statement on lung cancer screening

DEFF Research Database (Denmark)

Oudkerk, Matthijs; Devaraj, Anand; Vliegenthart, Rozemarijn

2017-01-01

Lung cancer screening with low-dose CT can save lives. This European Union (EU) position statement presents the available evidence and the major issues that need to be addressed to ensure the successful implementation of low-dose CT lung cancer screening in Europe. This statement identified...... specific actions required by the European lung cancer screening community to adopt before the implementation of low-dose CT lung cancer screening. This position statement recommends the following actions: a risk stratification approach should be used for future lung cancer low-dose CT programmes...... need to set a timeline for implementing lung cancer screening....

In vivo and In vitro neurochemical-based assessments of wastewater effluents from the Maumee River area of concern

International Nuclear Information System (INIS)

Arini, Adeline; Cavallin, Jenna E.; Berninger, Jason P.; Marfil-Vega, Ruth; Mills, Marc; Villeneuve, Daniel L.; Basu, Niladri

2016-01-01

Wastewater treatment plant (WWTP) effluents contain potentially neuroactive chemicals though few methods are available to screen for the presence of such agents. Here, two parallel approaches (in vivo and in vitro) were used to assess WWTP exposure-related changes to neurochemistry. First, fathead minnows (FHM, Pimephales promelas) were caged for four days along a WWTP discharge zone into the Maumee River (Ohio, USA). Grab water samples were collected and extracts obtained for the detection of alkylphenols, bisphenol A (BPA) and steroid hormones. Second, the extracts were then used as a source of in vitro exposure to brain tissues from FHM and four additional species relevant to the Great Lakes ecosystem (rainbow trout (RT), river otter (RO), bald eagle (BE) and human (HU)). The ability of the wastewater (in vivo) or extracts (in vitro) to interact with enzymes (monoamine oxidase (MAO) and glutamine synthetase (GS)) and receptors (dopamine (D2) and N-methyl-D-aspartate receptor (NMDA)) involved in dopamine and glutamate-dependent neurotransmission were examined on brain homogenates. In vivo exposure of FHM led to significant decreases of NMDA receptor binding in females (24–42%), and increases of MAO activity in males (2.8- to 3.2-fold). In vitro, alkylphenol-targeted extracts significantly inhibited D2 (66% in FHM) and NMDA (24–54% in HU and RT) receptor binding, and induced MAO activity in RT, RO, and BE brains. Steroid hormone-targeted extracts inhibited GS activity in all species except FHM. BPA-targeted extracts caused a MAO inhibition in FHM, RT and BE brains. Using both in vivo and in vitro approaches, this study shows that WWTP effluents contain agents that can interact with neurochemicals important in reproduction and other neurological functions. Additional work is needed to better resolve in vitro to in vivo extrapolations (IVIVE) as well as cross-species differences. - Highlights: • We conducted in vivo and in vitro neurochemical

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

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Neil O Carragher

2011-04-01

Full Text Available 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.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

International Nuclear Information System (INIS)

Spaggiari, Dany; Daali, Youssef; Rudaz, Serge

2016-01-01

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC 50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC 50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC 50 values generated 10-fold faster

Magnetic Particle / Magnetic Resonance Imaging: In-Vitro MPI-Guided Real Time Catheter Tracking and 4D Angioplasty Using a Road Map and Blood Pool Tracer Approach.

Science.gov (United States)

Salamon, Johannes; Hofmann, Martin; Jung, Caroline; Kaul, Michael Gerhard; Werner, Franziska; Them, Kolja; Reimer, Rudolph; Nielsen, Peter; Vom Scheidt, Annika; Adam, Gerhard; Knopp, Tobias; Ittrich, Harald

2016-01-01

In-vitro evaluation of the feasibility of 4D real time tracking of endovascular devices and stenosis treatment with a magnetic particle imaging (MPI) / magnetic resonance imaging (MRI) road map approach and an MPI-guided approach using a blood pool tracer. A guide wire and angioplasty-catheter were labeled with a thin layer of magnetic lacquer. For real time MPI a custom made software framework was developed. A stenotic vessel phantom filled with saline or superparamagnetic iron oxide nanoparticles (MM4) was equipped with bimodal fiducial markers for co-registration in preclinical 7T MRI and MPI. In-vitro angioplasty was performed inflating the balloon with saline or MM4. MPI data were acquired using a field of view of 37.3×37.3×18.6 mm3 and a frame rate of 46 volumes/sec. Analysis of the magnetic lacquer-marks on the devices were performed with electron microscopy, atomic absorption spectrometry and micro-computed tomography. Magnetic marks allowed for MPI/MRI guidance of interventional devices. Bimodal fiducial markers enable MPI/MRI image fusion for MRI based roadmapping. MRI roadmapping and the blood pool tracer approach facilitate MPI real time monitoring of in-vitro angioplasty. Successful angioplasty was verified with MPI and MRI. Magnetic marks consist of micrometer sized ferromagnetic plates mainly composed of iron and iron oxide. 4D real time MP imaging, tracking and guiding of endovascular instruments and in-vitro angioplasty is feasible. In addition to an approach that requires a blood pool tracer, MRI based roadmapping might emerge as a promising tool for radiation free 4D MPI-guided interventions.

Magnetic Particle / Magnetic Resonance Imaging: In-Vitro MPI-Guided Real Time Catheter Tracking and 4D Angioplasty Using a Road Map and Blood Pool Tracer Approach.

Directory of Open Access Journals (Sweden)

Johannes Salamon

Full Text Available In-vitro evaluation of the feasibility of 4D real time tracking of endovascular devices and stenosis treatment with a magnetic particle imaging (MPI / magnetic resonance imaging (MRI road map approach and an MPI-guided approach using a blood pool tracer.A guide wire and angioplasty-catheter were labeled with a thin layer of magnetic lacquer. For real time MPI a custom made software framework was developed. A stenotic vessel phantom filled with saline or superparamagnetic iron oxide nanoparticles (MM4 was equipped with bimodal fiducial markers for co-registration in preclinical 7T MRI and MPI. In-vitro angioplasty was performed inflating the balloon with saline or MM4. MPI data were acquired using a field of view of 37.3×37.3×18.6 mm3 and a frame rate of 46 volumes/sec. Analysis of the magnetic lacquer-marks on the devices were performed with electron microscopy, atomic absorption spectrometry and micro-computed tomography.Magnetic marks allowed for MPI/MRI guidance of interventional devices. Bimodal fiducial markers enable MPI/MRI image fusion for MRI based roadmapping. MRI roadmapping and the blood pool tracer approach facilitate MPI real time monitoring of in-vitro angioplasty. Successful angioplasty was verified with MPI and MRI. Magnetic marks consist of micrometer sized ferromagnetic plates mainly composed of iron and iron oxide.4D real time MP imaging, tracking and guiding of endovascular instruments and in-vitro angioplasty is feasible. In addition to an approach that requires a blood pool tracer, MRI based roadmapping might emerge as a promising tool for radiation free 4D MPI-guided interventions.

Integrated in vitro approaches to assess the bioaccessibility and bioavailability of silicon-biofortified leafy vegetables and preliminary effects on bone.

Science.gov (United States)

D'Imperio, Massimiliano; Brunetti, Giacomina; Gigante, Isabella; Serio, Francesco; Santamaria, Pietro; Cardinali, Angela; Colucci, Silvia; Minervini, Fiorenza

2017-03-01

Food industries are increasingly oriented toward new foods to improve nutritional status and/or to combat nutritional deficiency diseases. In this context, silicon biofortification could be an innovative tool for obtaining new foods with possible positive effects on bone mineralization. In this paper, an alternative and quick in vitro approach was applied in order to evaluate the potential health-promoting effects of five silicon-biofortified leafy vegetables (tatsoi, mizuna, purslane, Swiss chard and chicory) on bone mineralization compared with a commercial silicon supplement. The silicon bioaccessibility and bioavailability of the five leafy vegetables (biofortified or not) and of the supplement were assessed by applying a protocol consisting of in vitro gastrointestinal digestion coupled with a Caco-2 cell model. Silicon bioaccessibility ranged from 0.89 to 8.18 mg/L and bioavailability ranged from 111 to 206 μg/L of Si for both vegetables and supplement. Furthermore, the bioavailable fractions were tested on a human osteoblast cell model following the expression of type 1 collagen and alkaline phosphatase. The results obtained highlighted that the bioavailable fraction of biofortified purslane and Swiss chard improved the expression of both osteoblast markers compared with the supplement and other vegetables. These results underline the potentially beneficial effect of biofortified leafy vegetables and also indicate the usefulness of in vitro approaches for selecting the best vegetable with positive bone effects for further in vivo research.

Identification of novel antitubulin agents by using a virtual screening approach based on a 7-point pharmacophore model of the tubulin colchi-site.

Science.gov (United States)

Massarotti, Alberto; Theeramunkong, Sewan; Mesenzani, Ornella; Caldarelli, Antonio; Genazzani, Armando A; Tron, Gian Cesare

2011-12-01

Tubulin inhibition represents an established target in the field of anticancer research, and over the last 20 years, an intensive search for new antimicrotubule agents has occurred. Indeed, in silico models have been presented that might aid the discovery of novel agents. Among these, a 7-point pharmacophore model has been recently proposed. As a formal proof of this model, we carried out a ligand-based virtual screening on the colchicine-binding site. In vitro testing demonstrated that two compounds displayed a cytotoxic profile on neuroblastoma cancer cells (SH-SY5H) and one had an antitubulinic profile. © 2011 John Wiley & Sons A/S.

Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

Directory of Open Access Journals (Sweden)

Pathan AAK

2016-05-01

Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the �

Biotransformation of the mycotoxin enniatin B1 in pigs: A comparative in vitro and in vivo approach.

Science.gov (United States)

Ivanova, Lada; Uhlig, Silvio; Devreese, Mathias; Croubels, Siska; Fæste, Christiane Kruse

2017-07-01

Enniatins (ENNs) are hexadepsipeptidic mycotoxins produced by Fusarium species. They occur in mg/kg levels in grain from Northern climate areas. Major ENNs such as ENN B and B1 have shown considerable cytotoxicity in different in vitro test systems. To adequately assess exposure and in vivo toxicity the toxicokinetic properties need to be investigated. The present study describes the metabolism of ENN B1 both in vitro and in vivo in pigs, comparing metabolites found in vitro in experiments with liver microsomes from different pig strains to those found in the plasma of pigs after single oral or intravenous application of ENN B1. Metabolites of hepatic biotransformation were tentatively identified and characterised by high performance liquid chromatography coupled to ion trap and high-resolution mass spectrometry, as well as chemical derivatisations. Kinetic parameters of metabolite formation and elimination were determined. Metabolite formation was higher when ENN B1 was absorbed from the gut compared to intravenous administration indicating pre-systemic metabolism of ENN B1 after oral uptake. The in vitro approach resulted in the detection of ten ENN B1 metabolites, while six were detected in in vivo samples. The putative ENN B1 metabolites were products of hydroxylation, carbonylation, carboxylation and oxidative demethylation reactions. Copyright © 2017. Published by Elsevier Ltd.

Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

OpenAIRE

Lewis, Russell E.; Liao, Guangling; Young, Katherine; Douglas, Cameron; Kontoyiannis, Dimitrios P.

2014-01-01

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus f...

Impact of preimplantation genetic screening on donor oocyte-recipient cycles in the United States.

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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 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.

CoMiniGut—a small volume in vitro colon model for the screening of gut microbial fermentation processes

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Khakimov, Bekzod; Nielsen, Sebastian; Sørensen, Helena; van den Berg, Frans; Nielsen, Dennis Sandris

2018-01-01

Driven by the growing recognition of the influence of the gut microbiota (GM) on human health and disease, there is a rapidly increasing interest in understanding how dietary components, pharmaceuticals and pre- and probiotics influence GM. In vitro colon models represent an attractive tool for this purpose. With the dual objective of facilitating the investigation of rare and expensive compounds, as well as an increased throughput, we have developed a prototype in vitro parallel gut microbial fermentation screening tool with a working volume of only 5 ml consisting of five parallel reactor units that can be expanded with multiples of five to increase throughput. This allows e.g., the investigation of interpersonal variations in gut microbial dynamics and the acquisition of larger data sets with enhanced statistical inference. The functionality of the in vitro colon model, Copenhagen MiniGut (CoMiniGut) was first demonstrated in experiments with two common prebiotics using the oligosaccharide inulin and the disaccharide lactulose at 1% (w/v). We then investigated fermentation of the scarce and expensive human milk oligosaccharides (HMOs) 3-Fucosyllactose, 3-Sialyllactose, 6-Sialyllactose and the more common Fructooligosaccharide in fermentations with infant gut microbial communities. Investigations of microbial community composition dynamics in the CoMiniGut reactors by MiSeq-based 16S rRNA gene amplicon high throughput sequencing showed excellent experimental reproducibility and allowed us to extract significant differences in gut microbial composition after 24 h of fermentation for all investigated substrates and fecal donors. Furthermore, short chain fatty acids (SCFAs) were quantified for all treatments and donors. Fermentations with inulin and lactulose showed that inulin leads to a microbiota dominated by obligate anaerobes, with high relative abundance of Bacteroidetes, while the more easily fermented lactulose leads to higher relative abundance of

CoMiniGut-a small volume in vitro colon model for the screening of gut microbial fermentation processes.

Science.gov (United States)

Wiese, Maria; Khakimov, Bekzod; Nielsen, Sebastian; Sørensen, Helena; van den Berg, Frans; Nielsen, Dennis Sandris

2018-01-01

Driven by the growing recognition of the influence of the gut microbiota (GM) on human health and disease, there is a rapidly increasing interest in understanding how dietary components, pharmaceuticals and pre- and probiotics influence GM. In vitro colon models represent an attractive tool for this purpose. With the dual objective of facilitating the investigation of rare and expensive compounds, as well as an increased throughput, we have developed a prototype in vitro parallel gut microbial fermentation screening tool with a working volume of only 5 ml consisting of five parallel reactor units that can be expanded with multiples of five to increase throughput. This allows e.g., the investigation of interpersonal variations in gut microbial dynamics and the acquisition of larger data sets with enhanced statistical inference. The functionality of the in vitro colon model, Copenhagen MiniGut (CoMiniGut) was first demonstrated in experiments with two common prebiotics using the oligosaccharide inulin and the disaccharide lactulose at 1% (w/v). We then investigated fermentation of the scarce and expensive human milk oligosaccharides (HMOs) 3-Fucosyllactose, 3-Sialyllactose, 6-Sialyllactose and the more common Fructooligosaccharide in fermentations with infant gut microbial communities. Investigations of microbial community composition dynamics in the CoMiniGut reactors by MiSeq-based 16S rRNA gene amplicon high throughput sequencing showed excellent experimental reproducibility and allowed us to extract significant differences in gut microbial composition after 24 h of fermentation for all investigated substrates and fecal donors. Furthermore, short chain fatty acids (SCFAs) were quantified for all treatments and donors. Fermentations with inulin and lactulose showed that inulin leads to a microbiota dominated by obligate anaerobes, with high relative abundance of Bacteroidetes, while the more easily fermented lactulose leads to higher relative abundance of

CoMiniGut—a small volume in vitro colon model for the screening of gut microbial fermentation processes

Directory of Open Access Journals (Sweden)

Maria Wiese

2018-01-01

Full Text Available Driven by the growing recognition of the influence of the gut microbiota (GM on human health and disease, there is a rapidly increasing interest in understanding how dietary components, pharmaceuticals and pre- and probiotics influence GM. In vitro colon models represent an attractive tool for this purpose. With the dual objective of facilitating the investigation of rare and expensive compounds, as well as an increased throughput, we have developed a prototype in vitro parallel gut microbial fermentation screening tool with a working volume of only 5 ml consisting of five parallel reactor units that can be expanded with multiples of five to increase throughput. This allows e.g., the investigation of interpersonal variations in gut microbial dynamics and the acquisition of larger data sets with enhanced statistical inference. The functionality of the in vitro colon model, Copenhagen MiniGut (CoMiniGut was first demonstrated in experiments with two common prebiotics using the oligosaccharide inulin and the disaccharide lactulose at 1% (w/v. We then investigated fermentation of the scarce and expensive human milk oligosaccharides (HMOs 3-Fucosyllactose, 3-Sialyllactose, 6-Sialyllactose and the more common Fructooligosaccharide in fermentations with infant gut microbial communities. Investigations of microbial community composition dynamics in the CoMiniGut reactors by MiSeq-based 16S rRNA gene amplicon high throughput sequencing showed excellent experimental reproducibility and allowed us to extract significant differences in gut microbial composition after 24 h of fermentation for all investigated substrates and fecal donors. Furthermore, short chain fatty acids (SCFAs were quantified for all treatments and donors. Fermentations with inulin and lactulose showed that inulin leads to a microbiota dominated by obligate anaerobes, with high relative abundance of Bacteroidetes, while the more easily fermented lactulose leads to higher relative

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

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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...

Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis

NARCIS (Netherlands)

Rijke, de E.; Essers, M.L.; Rijk, J.C.W.; Thevis, M.; Bovee, T.F.H.; Ginkel, van L.A.; Sterk, S.S.

2013-01-01

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC)

Pharmacokinetic–pharmacodynamic modelling of drug‐induced QTc interval prolongation in man: prediction from in vitro human ether‐à‐go‐go‐related gene binding and functional inhibition assays and conscious dog studies

Science.gov (United States)

Dubois, V F S; Casarotto, E; Danhof, M

2016-01-01

Background and Purpose Functional measures of human ether‐à‐go‐go‐related gene (hERG; Kv11.1) channel inhibition have been prioritized as an in vitro screening tool for candidate molecules. However, it is unclear how these results can be translated to humans. Here, we explore how data on drug binding and functional inhibition in vitro relate to QT prolongation in vivo. Using cisapride, sotalol and moxifloxacin as paradigm compounds, we assessed the relationship between drug concentrations, binding, functional measures and in vivo effects in preclinical species and humans. Experimental Approach Pharmacokinetic–pharmacodynamic modelling was used to characterize the drug effects in hERG functional patch clamp, hERG radio‐labelled dofetilide displacement experiments and QT interval in conscious dogs. Data were analysed in parallel to identify potential correlations between pharmacological activity in vitro and in vivo. Key Results An Emax model could not be used due to large variability in the functional patch clamp assay. Dofetilide displacement revealed that binding curves are unrelated to the in vivo potency estimates for QTc prolongation in dogs and humans. Mean in vitro potency estimates ranged from 99.9 nM for cisapride to 1030 μM for moxifloxacin. Conclusions and Implications The lack of standardized protocols for in vitro assays leads to significant differences in experimental conditions, making the assessment of in vitro–in vivo correlations unreliable. Identification of an accurate safety window during the screening of candidate molecules requires a quantitative framework that disentangles system‐ from drug‐specific properties under physiological conditions, enabling translation of the results to humans. Similar considerations will be relevant for the comprehensive in vitro pro‐arrhythmia assay initiative. PMID:27427789

In Vitro Approaches to Evaluation of Sun Protectin Factor

Czech Academy of Sciences Publication Activity Database

Bendová, H.; Akrman, J.; Krejčí, A.; Kubáč, L.; Jírová, D.; Kejlová, K.; Kolářová, H.; Brabec, Marek; Malý, M.

2007-01-01

Roč. 21, č. 7 (2007), s. 1268-1275 ISSN 0887-2333 Source of funding: V - iné verejné zdroje Keywords : sunscreens * in vitro * SPF * nonparametric model Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 2.193, year: 2007

Knowledge of Chlamydia trachomatis among men and women approached to participate in community-based screening, Scotland, UK

Directory of Open Access Journals (Sweden)

Hart Graham J

2010-12-01

Full Text Available Abstract Background Poor awareness and knowledge of Chlamydia trachomatis could be a barrier to uptake of screening. This study aimed to determine the level of awareness and knowledge of chlamydia among young people who were being approached in a variety of community settings and offered opportunistic screening. Methods Men and women aged 16-24 years were approached in education, health and fitness, and workplace settings and invited to complete a self-administered questionnaire then provide a urine sample for chlamydia testing. Follow-up semi-structured interviews with 24 respondents were carried out after test results were received. Results 363 questionnaires were completed (43.5% from men. Whilst awareness of chlamydia was high, knowledge decreased as questions became increasingly focussed so that around half of respondents were unaware of the asymptomatic nature of chlamydia infections. Men's knowledge of symptoms was consistently lower than women's, with most men failing to identify unusual discharge as a symptom in men (men 58.3%, female 45.8%, p = 0.019; fewer men knew unusual discharge was a symptom among women (men 65.3% female 21.4%, p Conclusions Despite scientific gains in understanding chlamydia infection, public understanding remains limited. Greater efforts are required to translate scientific evidence to the public. An improvement in knowledge may maximise gains from interventions to improve detection.

Knowledge of Chlamydia trachomatis among men and women approached to participate in community-based screening, Scotland, UK.

Science.gov (United States)

Lorimer, Karen; Hart, Graham J

2010-12-30

Poor awareness and knowledge of Chlamydia trachomatis could be a barrier to uptake of screening. This study aimed to determine the level of awareness and knowledge of chlamydia among young people who were being approached in a variety of community settings and offered opportunistic screening. Men and women aged 16-24 years were approached in education, health and fitness, and workplace settings and invited to complete a self-administered questionnaire then provide a urine sample for chlamydia testing. Follow-up semi-structured interviews with 24 respondents were carried out after test results were received. 363 questionnaires were completed (43.5% from men). Whilst awareness of chlamydia was high, knowledge decreased as questions became increasingly focussed so that around half of respondents were unaware of the asymptomatic nature of chlamydia infections. Men's knowledge of symptoms was consistently lower than women's, with most men failing to identify unusual discharge as a symptom in men (men 58.3%, female 45.8%, p = 0.019); fewer men knew unusual discharge was a symptom among women (men 65.3% female 21.4%, p participation in the study. Despite scientific gains in understanding chlamydia infection, public understanding remains limited. Greater efforts are required to translate scientific evidence to the public. An improvement in knowledge may maximise gains from interventions to improve detection.

Voluntary pulmonary function screening with GOLD standard: an effective and simple approach to detect lung obstruction.

Science.gov (United States)

Wang, Shengyu; Gong, Wei; Tian, Yao; Yang, Min

2015-11-01

The prevalence of lung obstruction is probably underestimated. Early detection and screening may alter the course and prognosis associated with lung disease. We investigated the effectiveness of voluntary lung function screening program and the agreement between the Global Initiative for Chronic Obstructive Lung Disease (GOLD) and the lower limit of normal (LLN) standard for public screening in Xi'an China. Pulmonary function testing was conducted on volunteers recruited from eight community centers in Xi'an, China between July and August 2012. Participants underwent three forced vital capacity (FVC) maneuvers. The maneuver with the best FEV1 was retained. Participants filled out a medical history survey before undergoing pulmonary function testing. Patients that self-reported lung disease on the health survey were excluded from the analysis. A total of 803 volunteers participated in this study. And 722 participants (93.8%) did not self-report chronic lung disease and were analyzed. Of these participants, 143 subjects (19.8%) were diagnosed by GOLD standard and 134 subjects (18.6%) had obstruction with LLN definition. GOLD definition can identify more asymptomatic subjects (19.1%) with respect to LLN. GOLD definition can detect more lung obstruction in elder subjects compared with young people, the difference is significant (P=0.0007). The overall agreement between the 2 methods was good: the kappa estimate was 0.822. The agreement in subjects aged 40-49, 50-59 and 60-69 years was good: the kappa estimate was 0.82, 0.936 and 0.907 respectively and the agreement in subjects aged 18-29 was inferior: the kappa estimate was only 0.555. Voluntary lung function screening program with GOLD standard may be a simple and effective approach to ensuring high yield detection of lung obstruction in subjects aged 40-69.

Effect of the trehalose levels on the screening of yeast as probiotic by in vivo and in vitro assays Efeito da trealose na seleção de leveduras para uso como probióticos utilizando testes in vitro e in vivo

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Flaviano S. Martins

2008-03-01

Full Text Available 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.Probióticos são definidos como microrganismos (bactérias e leveduras que exercem um efeito benéfico na saúde do hospedeiro quando ingeridos em quantidades adequadas. A seleção desses agentes bioterapêuticos normalmente é feita por testes in vitro simulando o ambiente gastrointestinal que determina a capacidade de sobrevivência no trato digestivo. Neste trabalho, a possibilidade de extrapolação dos dados obtidos nos testes in vitro para as condições in vivo foi estudada utilizando cinco linhagens de Saccharomyces cerevisiae isoladas da floresta Atlântica brasileira. O conteúdo de trealose e a sobrevivência após a exposição a diversos estresses fisiológicos geralmente encontrados no trato gastrointestinal de humanos foram determinados para as cinco linhagens e os resultados comparados com a Saccharomyces boulardii, um probiótico conhecido. Esses resultados

Digital screen visits in home care services

DEFF Research Database (Denmark)

Zarakit, Mohamad; Nors Hansen, Louise; Evron, Lotte Orr

2017-01-01

with participant observation of three selected screen visits with older patients with a minority ethnic background. Analysis: thematic analysis based on a hermeneutic approach. Primarily results indicate that older patients with a minority ethnic background are screened out during the recruitment phase for digital...

The influence of the negative-positive ratio and screening database size on the performance of machine learning-based virtual screening.

Science.gov (United States)

Kurczab, Rafał; Bojarski, Andrzej J

2017-01-01

The machine learning-based virtual screening of molecular databases is a commonly used approach to identify hits. However, many aspects associated with training predictive models can influence the final performance and, consequently, the number of hits found. Thus, we performed a systematic study of the simultaneous influence of the proportion of negatives to positives in the testing set, the size of screening databases and the type of molecular representations on the effectiveness of classification. The results obtained for eight protein targets, five machine learning algorithms (SMO, Naïve Bayes, Ibk, J48 and Random Forest), two types of molecular fingerprints (MACCS and CDK FP) and eight screening databases with different numbers of molecules confirmed our previous findings that increases in the ratio of negative to positive training instances greatly influenced most of the investigated parameters of the ML methods in simulated virtual screening experiments. However, the performance of screening was shown to also be highly dependent on the molecular library dimension. Generally, with the increasing size of the screened database, the optimal training ratio also increased, and this ratio can be rationalized using the proposed cost-effectiveness threshold approach. To increase the performance of machine learning-based virtual screening, the training set should be constructed in a way that considers the size of the screening database.

Influencing Cancer Screening Participation Rates—Providing a Combined Cancer Screening Program (a ‘One Stop’ Shop Could Be a Potential Answer

Directory of Open Access Journals (Sweden)

Amanda Bobridge

2017-12-01

Full Text Available IntroductionParticipation in established cancer screening programs remains variable. Therefore, a renewed focus on how to increase screening uptake, including addressing structural barriers such as time, travel, and cost is needed. One approach could be the provision of combined cancer screening, where multiple screening tests are provided at the same time and location (essentially a ‘One Stop’ screening shop. This cohort study explored both cancer screening behavior and the acceptability of a combined screening approach.MethodsParticipants of the North Western Adelaide Health Study (NWAHS, South Australia were invited to participate in a questionnaire about cancer screening behaviors and the acceptability of a proposed ‘One Stop’ cancer screening shop. Data were collected from 10th August 2015 to 18th January 2016, weighted for selection probability, age, and sex and analyzed using descriptive and multivariable logistic regression analysis.Results1,562 people, 52% female (mean age 54.1 years ± 15.2 participated. Reported screening participation was low, the highest being for Pap Smear (34.4%. Common reasons for screening participation were preventing sickness (56.1%, CI 53.2–59.0%, maintaining health (51%, CI 48–53.9%, and free program provision (30.9%, CI 28.2–33.6%. Females were less likely to state that screening is not beneficial [OR 0.37 (CI 0.21–0.66, p < 0.001] and to cite sickness prevention [OR 2.10 (CI 1.46–3.00, p < 0.001] and free program [OR 1.75 (CI 1.22–2.51, p < 0.003] as reasons for screening participation. Of those who did not participate, 34.6% (CI 30.3–39.1% stated that there was nothing that discouraged them from participation, with 55- to 64-year olds [OR 0.24 (CI 0.07–0.74, p < 0.04] being less likely to cite this reason. 21% (CI 17.2–24.8% thought they did not need screening, while a smaller proportion stated not having time (6.9%, CI 4.9–9.7% and the costs associated

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

Science.gov (United States)

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 ...

Polyphenol bioavailability in nuts and seeds by an in vitro dialyzability approach.

Science.gov (United States)

Herbello-Hermelo, Paloma; Lamas, Juan Pablo; Lores, Marta; Domínguez-González, Raquel; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2018-07-15

An in vitro dialyzability approach has been undertaken to elucidate the bioavailable fraction of the total polyphenols (TPs) of edible nuts and seeds. The TP contents in samples and in dialyzates were assessed by the Folin-Ciocalteu spectrophotometric method. Antioxidant activity was determined in selected samples, using a modified method against Trolox®. TPs and antioxidant activity in nuts/seeds were determined after applying a pressurized liquid extraction sample pre-treatment. High dialyzability ratios were assessed in most nuts/seeds (TP dialyzability percentages within the 25-91% range). The highest TP dialyzability ratios were found in raw Brazil nuts (81 ± 5%), toasted pistachios (88 ± 9%), and fried cashews (89 ± 9%), whereas TPs in pumpkin seeds were found to be very low (TPs were not detected in the dialyzable fraction). TP dialyzability was correlated with the copper content in nuts and seeds. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro performances of hypocotyl and cotyledon explants of tomato ...

African Journals Online (AJOL)

A plant tissue culture technique is a good method for the evaluation and screening of plant genotypes for salt tolerance. In vitro evaluations of sodium chloride (NaCl) effects on two tomato cultivars (Pearl and Beril) were investigated with four NaCl levels (0, 25, 50 and 75 mM) using hypocotyl and cotyledon explants.

High-throughput screening for various classes of doping agents using a new 'dilute-and-shoot' liquid chromatography-tandem mass spectrometry multi-target approach.

Science.gov (United States)

Guddat, S; Solymos, E; Orlovius, A; Thomas, A; Sigmund, G; Geyer, H; Thevis, M; Schänzer, W

2011-01-01

A new multi-target approach based on liquid chromatography--electrospray ionization tandem mass spectrometry (LC-(ESI)-MS/MS) is presented to screen for various classes of prohibited substances using direct injection of urine specimens. With a highly sensitive new generation hybrid mass spectrometer classic groups of drugs--for example, diuretics, beta2-agonists--stimulants and narcotics are detectable at concentration levels far below the required limits. Additionally, more challenging and various new target compounds could be implemented. Model compounds of stimulant conjugates were studied to investigate a possible screening without complex sample preparation. As a main achievement, the integration of the plasma volume expanders dextran and hydroxyethyl starch (HES), commonly analyzed in time-consuming, stand-alone procedures, is accomplished. To screen for relatively new prohibited compounds, a common metabolite of the selective androgen receptor modulator (SARMs) andarine, a metabolite of growth hormone releasing peptide (GHRP-2), and 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) are analyzed. Following a completely new approach, conjugates of di(2-ethylhexyl) phthalate (DEHP) metabolites are monitored to detect abnormally high levels of plasticizers indicating for illicit blood transfusion. The assay was fully validated for qualitative purposes considering the parameters specificity, intra- (3.2-16.6%) and inter-day precision (0.4-19.9%) at low, medium and high concentration, robustness, limit of detection (1-70 ng/ml, dextran: 30 µg/ml, HES: 10 µg/ml) and ion suppression/enhancement effects. The analyses of post-administration and routine doping control samples demonstrates the applicability of the method for sports drug testing. This straightforward and reliable approach accomplishes the combination of different screening procedures resulting in a high-throughput method that increases the efficiency of the labs daily work. Copyright © 2011 John

High-throughput screening of chemicals as functional ...

Science.gov (United States)

Identifying chemicals that provide a specific function within a product, yet have minimal impact on the human body or environment, is the goal of most formulation chemists and engineers practicing green chemistry. We present a methodology to identify potential chemical functional substitutes from large libraries of chemicals using machine learning based models. We collect and analyze publicly available information on the function of chemicals in consumer products or industrial processes to identify a suite of harmonized function categories suitable for modeling. We use structural and physicochemical descriptors for these chemicals to build 41 quantitative structure–use relationship (QSUR) models for harmonized function categories using random forest classification. We apply these models to screen a library of nearly 6400 chemicals with available structure information for potential functional substitutes. Using our Functional Use database (FUse), we could identify uses for 3121 chemicals; 4412 predicted functional uses had a probability of 80% or greater. We demonstrate the potential application of the models to high-throughput (HT) screening for “candidate alternatives” by merging the valid functional substitute classifications with hazard metrics developed from HT screening assays for bioactivity. A descriptor set could be obtained for 6356 Tox21 chemicals that have undergone a battery of HT in vitro bioactivity screening assays. By applying QSURs, we wer

A novel way to grow hemozoin-like crystals in vitro and its use to screen for hemozoin inhibiting antimalarial compounds.

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Vincent Thomas

Full Text Available BACKGROUND: Hemozoin crystals are normally formed in vivo by Plasmodium parasites to detoxify free heme released after hemoglobin digestion during its intraerythrocytic stage. Inhibition of hemozoin formation by various drugs results in free heme concentration toxic for the parasites. As a consequence, in vitro assays have been developed to screen and select candidate antimalarial drugs based on their capacity to inhibit hemozoin formation. In this report we describe new ways to form hemozoin-like crystals that were incidentally discovered during research in the field of prion inactivation. METHODS: We investigated the use of a new assay based on naturally occurring "self-replicating" particles and previously described as presenting resistance to decontamination comparable to prions. The nature of these particles was determined using electron microscopy, Maldi-Tof analysis and X-ray diffraction. They were compared to synthetic hemozoin and to hemozoin obtained from Plasmodium falciparum. We then used the assay to evaluate the capacity of various antimalarial and anti-prion compounds to inhibit "self-replication" (crystallisation of these particles. RESULTS: We identified these particles as being similar to ferriprotoporphyrin IX crystal and confirmed the ability of these particles to serve as nuclei for growth of new hemozoin-like crystals (HLC. HLC are morphologically similar to natural and synthetic hemozoin. Growth of HLC in a simple assay format confirmed inhibition by quinolines antimalarials at potencies described in the literature. Interestingly, artemisinins and tetracyclines also seemed to inhibit HLC growth. CONCLUSIONS: The described HLC assay is simple and easy to perform and may have the potential to be used as an additional tool to screen antimalarial drugs for their hemozoin inhibiting activity. As already described by others, drugs that inhibit hemozoin crystal formation have also the potential to inhibit misfolded proteins

A practical approach to radiological evaluation of CT lung cancer screening examinations

NARCIS (Netherlands)

Xie, Xueqian; Heuvelmans, Marjolein A.; van Ooijen, Peter M. A.; Oudkerk, Matthijs; Vliegenthart, Rozemarijn

2013-01-01

Lung cancer is the most common cause of cancer-related death in the world. The Dutch-Belgian Randomized Lung Cancer Screening Trial (Dutch acronym: NELSON) was launched to investigate whether screening for lung cancer by low-dose multidetector computed tomography (CT) in high-risk patients will lead

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

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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.

Effect of carbohydrate source on the in vitro germination of Elaeis ...

African Journals Online (AJOL)

uche okafor

2016-07-20

Jul 20, 2016 ... and starch were screened for the most suitable carbon source for the in vitro culture of oil palm (Elaeis guineensis Jacq. ..... environmentally friendly substitute for fossil fuel and a source of commercial vegetable oil. Conflict of Interests. The authors have not declared any conflict of interests. REFERENCES.

In vitro Antimicrobial Activity ofCitrus aurantifolia and its Phytochemical screening

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Rafi khan Pathan

2012-05-01

Full Text Available Objective: To evaluate the antimicrobial efficacy of Citrus aurantifolia Linn (CA against some microorganisms - bacteria and fungus were Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Pseudomonas spp, Aspergillus niger, Aspergillus fumigates, Mucor spp and Pencillium. Methods: 100 毺 l of 10 mg CA were assessed against eight test microorganisms by agar well Diffusion Method. Gentamicin and Ketoconazole 10 mg/ml were used as standards. A different solvent was used to obtain CA leaf extract by using maceration technique. Results: %yield obtained for dried leaf extract of CA with chloroform, ethanol, acetone, petroleum ether and aqueous ethanol was approximately 15%, 18%, 09%, 11% and 24% respectively. Due to its high yield value hydroalcoholic extract of CA was used for estimating the antimicrobial activity and its phytochemical screening. Phytochemical screening of CA plant reveals the presence of Alkaloids, carbohydrates, flavonoids, steroids and tannins. Conclusions: The study demonstrates that the hydroalcoholic extract of CA leaf exhibit antibacterial activity on Klebsiella pneumonia, Pseudomonas sp, Staphylococcus aureus and antifungal activity among Aspergillus niger, Aspergillus fumigates, Mucor species. These recognized a good support to the use of this plant in herbal medicine and as base for the development of new drugs and phytomedicine.

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

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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.

Preliminary phytochemical screening and In vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC

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Okoh Anthony I

2010-05-01

Full Text Available Abstract 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.

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae

International Nuclear Information System (INIS)

Formagio, A.S.N.; Vieira, M.C.; Volobuff, C.R.F.; Silva, M.S.; Matos, A.I.; Cardoso, C.A.L.; Foglio, M.A.; Carvalho, J.E.

2015-01-01

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI 50 ) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI 50 values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs

In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae

Energy Technology Data Exchange (ETDEWEB)

Formagio, A.S.N.; Vieira, M.C. [Faculdade de Ciências Agrárias, Universidade Federal da Grande Dourados, Dourados, MS (Brazil); Volobuff, C.R.F.; Silva, M.S. [Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourados, Dourados, MS (Brazil); Matos, A.I. [Faculdade de Ciências, Universidade de Lisboa, Lisboa (Portugal); Cardoso, C.A.L. [Curso de Química, Universidade Estadual do Mato Grosso do Sul, Dourados, MS (Brazil); Foglio, M.A.; Carvalho, J.E. [Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, Campinas, SP (Brazil)

2015-02-13

The aim of this research was to investigate the antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species in vitro. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for acetylcholinesterase (AchE) inhibitors using Ellman's reagent. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI{sub 50}) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 µg/mL, respectively. In addition, the Annona crassiflora and A. coriacea seed extracts were the most active among the tested extracts and the most effective against the tumor cell lines, with GI{sub 50} values below 8.90 µg/mL. The A. cacans extract displayed the lowest activity. Based on the microplate assay, the percent AchE inhibition of the extracts ranged from 12 to 52%, and the A. coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the A. crassiflora seed samples. The A. coriacea seeds contained ferulic and sinapic acid. Overall, the results indicated that A. crassiflora and A. coriacea extracts have antiproliferative and anticholinesterase properties, which opens up new possibilities for alternative pharmacotherapy drugs.

The in vitro screening of aromatic amides as potential inhibitors of poly (ADP-ribose) polymerase

International Nuclear Information System (INIS)

Brown, D.M.; Horsman, M.R.; Lee, W.W.; Brown, J.M.

1984-01-01

It is now well established that the chromosomal enzyme poly (ADP-ribose) polymerase (ADPRP) is involved in the repair of DNA damage caused by ionizing radiation and alkylating agents, although the mechanisms involved are still not clear. ADPRP inhibitors include thymidine, nicotinamides, benzamides and methyl xanthines. The authors have demonstrated that these compounds are effective inhibitors of X-ray-induced potentially lethal damage repair (PLDR). More recently, they have shown that the cytotoxicity of the bifunctional alkylating L-phenylalanine mustard (L-PAM) was enhanced in vitro and in vivo by 3-aminobenzamide, nicotinamide and caffeine, although in the latter case pharmacokinetic changes could have contributed to the enhanced killing. The authors have examined a series of substituted carbocyclic and heterocyclic aromatic amides as potential inhibitors of ADPRP. The effect of these compounds on ADPRP activity in vitro as well as their effect on the repair of X-ray and alkylation damage in vitro are presented

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis

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Moy Richard L

2008-07-01

Full Text Available Abstract Background Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis. Results We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis. Conclusion Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

How Phenotypic Screening Influenced Drug Discovery: Lessons from Five Years of Practice.

Science.gov (United States)

Haasen, Dorothea; Schopfer, Ulrich; Antczak, Christophe; Guy, Chantale; Fuchs, Florian; Selzer, Paul

Since 2011, phenotypic screening has been a trend in the pharmaceutical industry as well as in academia. This renaissance was triggered by analyses that suggested that phenotypic screening is a superior strategy to discover first-in-class drugs. Despite these promises and considerable investments, pharmaceutical research organizations have encountered considerable challenges with the approach. Few success stories have emerged in the past 5 years and companies are questioning their investment in this area. In this contribution, we outline what we have learned about success factors and challenges of phenotypic screening. We then describe how our efforts in phenotypic screening have influenced our approach to drug discovery in general. We predict that concepts from phenotypic screening will be incorporated into target-based approaches and will thus remain influential beyond the current trend.

How should we screen for depression following a natural disaster? An ROC approach to post-disaster screening in adolescents and adults.

Science.gov (United States)

Cohen, Joseph R; Adams, Zachary W; Menon, Suvarna V; Youngstrom, Eric A; Bunnell, Brian E; Acierno, Ron; Ruggiero, Kenneth J; Danielson, Carla Kmett

2016-09-15

The present study's aim was to provide the foundation for an efficient, empirically based protocol for depression screening following a natural disaster. Utilizing a Receiver Operating Characteristic (ROC) analytic approach, the study tested a) what specific disaster-related stressors (i.e., property damage, loss of basic services) and individual-related constructs (i.e., PTSD symptoms, trauma history, social support) conveyed the greatest risk for post-natural disaster depression, b) specific cutoff scores across these measures, and c) whether the significance or cutoff scores for each construct varied between adolescents and adults. Structured phone-based clinical interviews were conducted with 2000 adolescents who lived through a tornado and 1543 adults who survived a hurricane. Findings suggested that in both adolescents and adults, individual-related constructs forecasted greater risk for depressive symptoms following a natural disaster compared to disaster-related stressors. Furthermore, trauma history and PTSD symptoms were particularly strong indicators for adolescent depressive symptoms compared to adult depressive symptoms. Adolescents and adults who reported vulnerable scores for social support, trauma history, and lifetime PTSD symptoms were approximately twice as likely to present as depressed following the natural disaster. Findings from the present study were limited to post-disaster assessments and based on self-reported functioning 6-12 months following the natural disaster. The present study synthesizes the extensive body of research on post-disaster functioning by providing a clear framework for which questions may be most important to ask when screening for depression following a natural disaster. Copyright © 2016 Elsevier B.V. All rights reserved.

Recombinant Kinase Production and Fragment Screening by NMR Spectroscopy.

Science.gov (United States)

Han, Byeonggu; Ahn, Hee-Chul

2016-01-01

During the past decade fragment-based drug discovery (FBDD) has rapidly evolved and several drugs or drug candidates developed by FBDD approach are clinically in use or in clinical trials. For example, vemurafenib, a V600E mutated BRAF inhibitor, was developed by utilizing FBDD approach and approved by FDA in 2011. In FBDD, screening of fragments is the starting step for identification of hits and lead generation. Fragment screening usually relies on biophysical techniques by which the protein-bound small molecules can be detected. NMR spectroscopy has been extensively used to study the molecular interaction between the protein and the ligand, and has many advantages in fragment screening over other biophysical techniques. This chapter describes the practical aspects of fragment screening by saturation transfer difference NMR.

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

Science.gov (United States)

Picollo, Federico; Battiato, Alfio; Bernardi, Ettore; Plaitano, Marilena; Franchino, Claudio; Gosso, Sara; Pasquarelli, Alberto; Carbone, Emilio; Olivero, Paolo; Carabelli, Valentina

2016-02-01

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.

Comparison of first-tier cell-free DNA screening for common aneuploidies with conventional publically funded screening.

Science.gov (United States)

Langlois, Sylvie; Johnson, JoAnn; Audibert, François; Gekas, Jean; Forest, Jean-Claude; Caron, André; Harrington, Keli; Pastuck, Melanie; Meddour, Hasna; Tétu, Amélie; Little, Julian; Rousseau, François

2017-12-01

This study evaluates the impact of offering cell-free DNA (cfDNA) screening as a first-tier test for trisomies 21 and 18. This is a prospective study of pregnant women undergoing conventional prenatal screening who were offered cfDNA screening in the first trimester with clinical outcomes obtained on all pregnancies. A total of 1198 pregnant women were recruited. The detection rate of trisomy 21 with standard screening was 83% with a false positive rate (FPR) of 5.5% compared with 100% detection and 0% FPR for cfDNA screening. The FPR of cfDNA screening for trisomies 18 and 13 was 0.09% for each. Two percent of women underwent an invasive diagnostic procedure based on screening or ultrasound findings; without the cfDNA screening, it could have been as high as 6.8%. Amongst the 640 women with negative cfDNA results and a nuchal translucency (NT) ultrasound, only 3 had an NT greater or equal to 3.5 mm: one had a normal outcome and two lost their pregnancy before 20 weeks. cfDNA screening has the potential to be a highly effective first-tier screening approach leading to a significant reduction of invasive diagnostic procedures. For women with a negative cfDNA screening result, NT measurement has limited clinical utility. © 2017 John Wiley & Sons, Ltd.

Ligand efficiency based approach for efficient virtual screening of compound libraries.

Science.gov (United States)

Ke, Yi-Yu; Coumar, Mohane Selvaraj; Shiao, Hui-Yi; Wang, Wen-Chieh; Chen, Chieh-Wen; Song, Jen-Shin; Chen, Chun-Hwa; Lin, Wen-Hsing; Wu, Szu-Huei; Hsu, John T A; Chang, Chung-Ming; Hsieh, Hsing-Pang

2014-08-18

Here we report for the first time the use of fit quality (FQ), a ligand efficiency (LE) based measure for virtual screening (VS) of compound libraries. The LE based VS protocol was used to screen an in-house database of 125,000 compounds to identify aurora kinase A inhibitors. First, 20 known aurora kinase inhibitors were docked to aurora kinase A crystal structure (PDB ID: 2W1C); and the conformations of docked ligand were used to create a pharmacophore (PH) model. The PH model was used to screen the database compounds, and rank (PH rank) them based on the predicted IC50 values. Next, LE_Scale, a weight-dependant LE function, was derived from 294 known aurora kinase inhibitors. Using the fit quality (FQ = LE/LE_Scale) score derived from the LE_Scale function, the database compounds were reranked (PH_FQ rank) and the top 151 (0.12% of database) compounds were assessed for aurora kinase A inhibition biochemically. This VS protocol led to the identification of 7 novel hits, with compound 5 showing aurora kinase A IC50 = 1.29 μM. Furthermore, testing of 5 against a panel of 31 kinase reveals that it is selective toward aurora kinase A & B, with <50% inhibition for other kinases at 10 μM concentrations and is a suitable candidate for further development. Incorporation of FQ score in the VS protocol not only helped identify a novel aurora kinase inhibitor, 5, but also increased the hit rate of the VS protocol by improving the enrichment factor (EF) for FQ based screening (EF = 828), compared to PH based screening (EF = 237) alone. The LE based VS protocol disclosed here could be applied to other targets for hit identification in an efficient manner. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Screening of photosynthetic pigments for herbicidal activity with a new computational molecular approach.

Science.gov (United States)

Krishnaraj, R Navanietha; Chandran, Saravanan; Pal, Parimal; Berchmans, Sheela

2013-12-01

There is an immense interest among the researchers to identify new herbicides which are effective against the herbs without affecting the environment. In this work, photosynthetic pigments are used as the ligands to predict their herbicidal activity. The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a good target for the herbicides. Homology modeling of the target enzyme is done using Modeler 9.11 and the model is validated. Docking studies were performed with AutoDock Vina algorithm to predict the binding of the natural pigments such as β-carotene, chlorophyll a, chlorophyll b, phycoerythrin and phycocyanin to the target. β-carotene, phycoerythrin and phycocyanin have higher binding energies indicating the herbicidal activity of the pigments. This work reports a procedure to screen herbicides with computational molecular approach. These pigments will serve as potential bioherbicides in the future.

Virtual screening of compound libraries.

Science.gov (United States)

Cerqueira, Nuno M F S A; Sousa, Sérgio F; Fernandes, Pedro A; Ramos, Maria João

2009-01-01

During the last decade, Virtual Screening (VS) has definitively established itself as an important part of the drug discovery and development process. VS involves the selection of likely drug candidates from large libraries of chemical structures by using computational methodologies, but the generic definition of VS encompasses many different methodologies. This chapter provides an introduction to the field by reviewing a variety of important aspects, including the different types of virtual screening methods, and the several steps required for a successful virtual screening campaign within a state-of-the-art approach, from target selection to postfilter application. This analysis is further complemented with a small collection important VS success stories.

A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

Science.gov (United States)

Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

2016-05-09

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Biotechnological approaches to enhance salidroside, rosin and its derivatives production in selected Rhodiola spp. in vitro cultures.

Science.gov (United States)

Grech-Baran, Marta; Sykłowska-Baranek, Katarzyna; Pietrosiuk, Agnieszka

Rhodiola (Crassulaceae) an arctic-alpine plant, is extensively used in traditional folk medicine in Asian and European countries. A number of investigations have demonstrated that Rhodiola preparations exhibit adaptogenic, neuroprotective, anti-tumour, cardioprotective, and anti-depressant effects. The main compounds responsible for these activities are believed to be salidroside, rosin and its derivatives which became the target of biotechnological investigations. This review summarizes the results of the diverse biotechnological approaches undertaken to enhance the production of salidroside, rosin and its derivatives in callus, cell suspension and organ in vitro cultures of selected Rhodiola species.

American Indian Men's Perceptions of Breast Cancer Screening for American Indian Women.

Science.gov (United States)

Filippi, Melissa K; Pacheco, Joseph; James, Aimee S; Brown, Travis; Ndikum-Moffor, Florence; Choi, Won S; Greiner, K Allen; Daley, Christine M

2014-01-01

Screening, especially screening mammography, is vital for decreasing breast cancer incidence and mortality. Screening rates in American Indian women are low compared to other racial/ethnic groups. In addition, American Indian women are diagnosed at more advanced stages and have lower 5-year survival rate than others. To better address the screening rates of American Indian women, focus groups (N=8) were conducted with American Indian men (N=42) to explore their perceptions of breast cancer screening for American Indian women. Our intent was to understand men's support level toward screening. Using a community-based participatory approach, focus groups were audio-taped, transcribed verbatim, and analyzed using a text analysis approach developed by our team. Topics discussed included breast cancer and screening knowledge, barriers to screening, and suggestions to improve screening rates. These findings can guide strategies to improve knowledge and awareness, communication among families and health care providers, and screening rates in American Indian communities.

In vitro screening for aryl hydrocarbon receptor agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in vivo mouse liver cytochrome P450-1A induction by propanil, diuron and linuron.

Science.gov (United States)

Takeuchi, Shinji; Iida, Mitsuru; Yabushita, Hisatoshi; Matsuda, Tadashi; Kojima, Hiroyuki

2008-12-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation and differentiation. In this study, we have developed a highly sensitive AhR-mediated reporter cell line, DR-EcoScreen cells, which are mouse hepatoma Hepa1c1c7 cells stably transfected with a reporter plasmid containing seven copies of dioxin-responsive element. Using these DR-EcoScreen cells, we performed the reporter gene assay and characterized the AhR agonistic activities of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 6 ureas, and 45 others). Eleven of the 200 pesticides (acifluorfen-methyl, bifenox, chlorpyrifos, isoxathion, quinalphos, chlorpropham, diethofencarb, propanil, diuron, linuron, and prochloraz) showed AhR-mediated transcriptional activity. In particular, three herbicides (propanil, diuron, and linuron) have a common chemical structure and showed more potent agonistic activity than other pesticides. To investigate the in vivo effects, we examined the gene expression of AhR-inducible cytochrome P450 1As (CYP1As) in the liver of female C57BL/6 mice intraperitoneally injected with these three herbicides (300 mg kg(-1)) by quantitative RT-PCR, resulting in induction of significant high levels of CYP1A1 and CYP1A2 mRNAs. This indicates that propanil, diuron and linuron possess AhR-mediated transactivation effect in vivo as well as in vitro. Through the present study, we demonstrated that DR-EcoScreen cells are useful for sensitive, rapid and simple identification of AhR agonists among a large number of environmental chemicals.

A study to evaluate the potential of an in silico approach for predicting dipeptidyl peptidase-IV inhibitory activity in vitro of protein hydrolysates.

Science.gov (United States)

Wang, Tzu-Yuan; Hsieh, Cheng-Hong; Hung, Chuan-Chuan; Jao, Chia-Ling; Lin, Pei-Yi; Hsieh, You-Liang; Hsu, Kuo-Chiang

2017-11-01

A total of 294 edible protein sequences and 5 commercial proteases listed in the BIOPEP database were analyzed in silico. The frequency (A), a parameter in silico described previously, was examined further to calculating the ratio of truncated peptides with Xaa-proline and/or Xaa-alanine to all peptide fragments in a protein hydrolyzed with a protease, using the BIOPEP database. Then the in vitro DPP-IV inhibitory activity was determined using the same 15 protein and protease combinations to evaluate their relationship. The result shows that A values considering the number of Xaa-proline+Xaa-alanine exhibited a strong correlation with in vitro DPP-IV inhibition rates by Pearson's correlation analysis (r=0.6993; Psilico approach is effective to predict DPP-IV inhibitory activities in vitro of protein hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prospective Assessment of Virtual Screening Heuristics Derived Using a Novel Fusion Score.

Science.gov (United States)

Pertusi, Dante A; O'Donnell, Gregory; Homsher, Michelle F; Solly, Kelli; Patel, Amita; Stahler, Shannon L; Riley, Daniel; Finley, Michael F; Finger, Eleftheria N; Adam, Gregory C; Meng, Juncai; Bell, David J; Zuck, Paul D; Hudak, Edward M; Weber, Michael J; Nothstein, Jennifer E; Locco, Louis; Quinn, Carissa; Amoss, Adam; Squadroni, Brian; Hartnett, Michelle; Heo, Mee Ra; White, Tara; May, S Alex; Boots, Evelyn; Roberts, Kenneth; Cocchiarella, Patrick; Wolicki, Alex; Kreamer, Anthony; Kutchukian, Peter S; Wassermann, Anne Mai; Uebele, Victor N; Glick, Meir; Rusinko, Andrew; Culberson, J Christopher

2017-09-01

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

Energy Technology Data Exchange (ETDEWEB)

Spaggiari, Dany, E-mail: dany.spaggiari@unige.ch [School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland); Daali, Youssef, E-mail: youssef.daali@hcuge.ch [Clinical Pharmacology and Toxicology Service, Geneva University Hospitals, Rue Gabrielle Perret-Gentil, 1211 Genève 14 (Switzerland); Rudaz, Serge, E-mail: serge.rudaz@unige.ch [School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland); Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland)

2016-07-01

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC{sub 50} values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values generated 10

FROM THE CONVENTIONAL TO THE MILDER APPROACHES TO OVARIAN STIMULATION FOR IN VITRO FERTILIZATION

Directory of Open Access Journals (Sweden)

Eda Vrtačnik-Bokal

2018-02-01

Full Text Available Background: In-vitro fertilization (IVF is performed with oocytes collected in natural and stimulated cycles. Different approaches to ovarian stimulation have been employed worldwide. Fol- lowing the introduction of gonadotrophin realising hormone (GnRH antagonists and strategies to reduce multiple birth such as single embryo transfer, there is an interest in the revival of natural and mild approaches to ovarian stimulation in IVF. Particulary mild ovarian stimulation aims to achieve cost-effective, patient-friendly regimens which optimize the balance between outcomes and risks of treatment. Methods: Pubmed was searched up to 2009 for papers on natural, modified natural, mild and con- ventional IVF cycles. Results: Data discussed in this review do not allow any conclusions to be drawn regarding the most optimal mild ovarian stimulation protocol. However, it is absolutelly clear that introduction of GnRH antagonists into clinical practice has allowed for the introduction of milder stimulation approaches for IVF treatment because of preventing premature luteinising hor- mone (LH rise by competitive blockade of the GnRH receptors. Studies also show that mild exogenous gonadotrophins interference with the decrease in follicle-stimulating hormone ((FSH levels in the mid-folicular phase was sufficient to override the selection of a single dominant follicle and enhance the most mature follicles to growth due to their increased sensitivity for FSH and acquired responsiveness to LH. Conclusions: The implementation of mild stimulation and modified natural cycle into standard clinical practise appears to be justified, although more prospective studies are needed to find the most appropriate mild stimulation approaches.

Utilization of International Association of Diabetes and Pregnancy Study Groups criteria vs. a two-step approach to screening for gestational diabetes mellitus in Chinese women with twin pregnancies.

Science.gov (United States)

Liu, X; Chen, Y; Zhou, Q; Shi, H; Cheng, W W

2015-03-01

To evaluate prevalence and pregnancy outcomes using the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria and screening protocol vs. a standard two-step screening approach for gestational diabetes mellitus in Chinese twin pregnancies. A retrospective cohort study for pregnancies during 2007-2013 was performed in a tertiary hospital in Shanghai, China. Data were abstracted from the medical records of twin pregnancies delivered at the hospital. During the period 2007-2011, this hospital used a two-step approach with a 50 g screening with a cut-off value of ≥ 7.8 mmol/l followed by a 100 g diagnostic oral glucose tolerance test (OGTT) utilizing Carpenter-Coustan criteria. In 2012-2013, the hospital switched to the IADPSG protocol of universal 75 g OGTT. Among 1461 twin pregnancies, 643 were screened utilizing IADPSG criteria and 818 using the two-step protocol. Gestational diabetes mellitus was diagnosed more frequently in the IADPSG group than in the two-step group [20.4% and 7.0%, respectively; adjusted odds ratio (aOR) = 3.22; 95% confidence interval (CI) = 2.30-4.52]. During the IADPSG period, the incidence of pre-eclampsia was 38% lower in non-gestational diabetes mellitus affected pregnancies compared with the two-step period (aOR = 0.62; 95% CI = 0.44-0.87). We observed no significant differences in most perinatal outcomes between the two groups. Compared with a standard two-step approach to screening and diagnosis, the IADPSG screening method resulted in a three-fold increase in the incidence of gestational diabetes mellitus in twin pregnancies, with a 38% lower risk of pre-eclampsia but no significant difference in most perinatal outcomes in non-gestational diabetes mellitus affected pregnancies. © 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.

Machine Learning Approaches Toward Building Predictive Models for Small Molecule Modulators of miRNA and Its Utility in Virtual Screening of Molecular Databases.

Science.gov (United States)

Periwal, Vinita; Scaria, Vinod

2017-01-01

The ubiquitous role of microRNAs (miRNAs) in a number of pathological processes has suggested that they could act as potential drug targets. RNA-binding small molecules offer an attractive means for modulating miRNA function. The availability of bioassay data sets for a variety of biological assays and molecules in public domain provides a new opportunity toward utilizing them to create models and further utilize them for in silico virtual screening approaches to prioritize or assign potential functions for small molecules. Here, we describe a computational strategy based on machine learning for creation of predictive models from high-throughput biological screens for virtual screening of small molecules with the potential to inhibit microRNAs. Such models could be potentially used for computational prioritization of small molecules before performing high-throughput biological assay.

Validation approach for a fast and simple targeted screening method for 75 antibiotics in meat and aquaculture products using LC-MS/MS.

Science.gov (United States)

Dubreil, Estelle; Gautier, Sophie; Fourmond, Marie-Pierre; Bessiral, Mélaine; Gaugain, Murielle; Verdon, Eric; Pessel, Dominique

2017-04-01

An approach is described to validate a fast and simple targeted screening method for antibiotic analysis in meat and aquaculture products by LC-MS/MS. The strategy of validation was applied for a panel of 75 antibiotics belonging to different families, i.e., penicillins, cephalosporins, sulfonamides, macrolides, quinolones and phenicols. The samples were extracted once with acetonitrile, concentrated by evaporation and injected into the LC-MS/MS system. The approach chosen for the validation was based on the Community Reference Laboratory (CRL) guidelines for the validation of screening qualitative methods. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest, generally the maximum residue limit (MRL). A robustness study was also performed to test the influence of different factors. The validation showed that the method is valid to detect and identify 73 antibiotics of the 75 antibiotics studied in meat and aquaculture products at the validation levels.

Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect.

Science.gov (United States)

Presti, I; D'Orazio, G; Labra, M; La Ferla, B; Mezzasalma, V; Bizzaro, G; Giardina, S; Michelotti, A; Tursi, F; Vassallo, M; Di Gennaro, P

2015-07-01

Probiotic ingestion is recommended as a preventive approach to maintain the balance of the intestinal microbiota and to enhance the human well-being. During the whole life of each individual, the gut microbiota composition could be altered by lifestyle, diet, antibiotic therapies and other stress conditions, which may lead to acute and chronic disorders. Hence, probiotics can be administered for the prevention or treatment of some disorders, including lactose malabsorption, acute diarrhoea, irritable bowel syndrome, necrotizing enterocolitis and mild forms of inflammatory bowel disease. The probiotic-mediated effect is an important issue that needs to be addressed in relation to strain-specific probiotic properties. In this work, the probiotic properties of new Lactobacillus and Bifidobacterium strains were screened, and their effects in vitro were evaluated. They were screened for probiotic properties by determining their tolerance to low pH and to bile salts, antibiotic sensitivity, antimicrobial activity and vitamin B8, B9 and B12 production, and by considering their ability to increase the antioxidant potential and to modulate the inflammatory status of systemic-miming cell lines in vitro. Three out of the examined strains presenting the most performant probiotic properties, as Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070 and Bifidobacterium animalis subsp. lactis PBSO75, were evaluated for their effects also on human intestinal HT-29 cell line. The obtained results support the possibility to move to another level of study, that is, the oral administration of these probiotical strains to patients with acute and chronic gut disorders, by in vivo experiments.

Implementation and Characterization of Dynamic Genetic Networks in Vitro

OpenAIRE

Niederholtmeyer, Henrike Marie

2015-01-01

Transcription and translation (TX-TL) can be performed in vitro, outside of cells, allowing the assembly and analysis of genetic networks. This approach to engineering biological networks in a less complex and more controllable environment could one day allow rapid prototyping of network designs before implementing them in living cells. Furthermore, the in vitro approach provides insight into how natural biological systems are built and is instructive to define the rules for engineering biolo...

Screening uptake rates and the clinical and cost effectiveness of screening for gestational diabetes mellitus in primary versus secondary care: study protocol for a randomised controlled trial.

LENUS (Irish Health Repository)

O Dea, Angela

2014-01-17

The risks associated with gestational diabetes mellitus (GDM) are well recognized, and there is increasing evidence to support treatment of the condition. However, clear guidance on the ideal approach to screening for GDM is lacking. Professional groups continue to debate whether selective screening (based on risk factors) or universal screening is the most appropriate approach. Additionally, there is ongoing debate about what levels of glucose abnormalities during pregnancy respond best to treatment and which maternal and neonatal outcomes benefit most from treatment. Furthermore, the implications of possible screening options on health care costs are not well established. In response to this uncertainty there have been repeated calls for well-designed, randomised trials to determine the efficacy of screening, diagnosis, and management plans for GDM. We describe a randomised controlled trial to investigate screening uptake rates and the clinical and cost effectiveness of screening in primary versus secondary care settings. The objective of this study is to assess screening uptake rates, and the clinical and cost effectiveness of screening for GDM in primary versus secondary care.

Quantifying Overdiagnosis in Cancer Screening: A Systematic Review to Evaluate the Methodology.

Science.gov (United States)

Ripping, Theodora M; Ten Haaf, Kevin; Verbeek, André L M; van Ravesteyn, Nicolien T; Broeders, Mireille J M

2017-10-01

Overdiagnosis is the main harm of cancer screening programs but is difficult to quantify. This review aims to evaluate existing approaches to estimate the magnitude of overdiagnosis in cancer screening in order to gain insight into the strengths and limitations of these approaches and to provide researchers with guidance to obtain reliable estimates of overdiagnosis in cancer screening. A systematic review was done of primary research studies in PubMed that were published before January 1, 2016, and quantified overdiagnosis in breast cancer screening. The studies meeting inclusion criteria were then categorized by their methods to adjust for lead time and to obtain an unscreened reference population. For each approach, we provide an overview of the data required, assumptions made, limitations, and strengths. A total of 442 studies were identified in the initial search. Forty studies met the inclusion criteria for the qualitative review. We grouped the approaches to adjust for lead time in two main categories: the lead time approach and the excess incidence approach. The lead time approach was further subdivided into the mean lead time approach, lead time distribution approach, and natural history modeling. The excess incidence approach was subdivided into the cumulative incidence approach and early vs late-stage cancer approach. The approaches used to obtain an unscreened reference population were grouped into the following categories: control group of a randomized controlled trial, nonattenders, control region, extrapolation of a prescreening trend, uninvited groups, adjustment for the effect of screening, and natural history modeling. Each approach to adjust for lead time and obtain an unscreened reference population has its own strengths and limitations, which should be taken into consideration when estimating overdiagnosis. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Population newborn screening for inherited metabolic disease: current UK perspectives.

Science.gov (United States)

Green, A; Pollitt, R J

1999-06-01

Some of the generally accepted criteria for screening programmes are inappropriate for newborn metabolic screening as they ignore the family dimension and the importance of timely genetic information. Uncritical application of such criteria creates special difficulties for screening by tandem mass spectrometry, which can detect a range diseases with widely different natural histories and responsiveness to treatment. Further difficulties arise from increasing demands for direct proof of the effects of screening on long-term morbidity and mortality. The randomized controlled trial is held to be the gold standard, but for ethical and practical reasons it will be impossible to achieve for such relatively rare diseases. This approach also oversimplifies the complex matrix of costs and benefits of newborn metabolic screening. A more workable approach could involve Bayesian synthesis, combining quantitative performance data from carefully designed prospective pilot studies of screening with existing experience of the natural history, diagnosis, and management of the individual disorders concerned.

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1.

Science.gov (United States)

Seifert, Alexander; Antonovici, Mihaela; Hauer, Bernhard; Pleiss, Jürgen

2011-06-14

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti-tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self-sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild-type enzyme converts (4R)-limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio-, stereo- and chemoselectivity, compared to wild-type. From this library the variant with the highest perillyl alcohol ratio was fine-tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)-limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TeraSCREEN: multi-frequency multi-mode Terahertz screening for border checks

Science.gov (United States)

Alexander, Naomi E.; Alderman, Byron; Allona, Fernando; Frijlink, Peter; Gonzalo, Ramón; Hägelen, Manfred; Ibáñez, Asier; Krozer, Viktor; Langford, Marian L.; Limiti, Ernesto; Platt, Duncan; Schikora, Marek; Wang, Hui; Weber, Marc Andree

2014-06-01

The challenge for any security screening system is to identify potentially harmful objects such as weapons and explosives concealed under clothing. Classical border and security checkpoints are no longer capable of fulfilling the demands of today's ever growing security requirements, especially with respect to the high throughput generally required which entails a high detection rate of threat material and a low false alarm rate. TeraSCREEN proposes to develop an innovative concept of multi-frequency multi-mode Terahertz and millimeter-wave detection with new automatic detection and classification functionalities. The system developed will demonstrate, at a live control point, the safe automatic detection and classification of objects concealed under clothing, whilst respecting privacy and increasing current throughput rates. This innovative screening system will combine multi-frequency, multi-mode images taken by passive and active subsystems which will scan the subjects and obtain complementary spatial and spectral information, thus allowing for automatic threat recognition. The TeraSCREEN project, which will run from 2013 to 2016, has received funding from the European Union's Seventh Framework Programme under the Security Call. This paper will describe the project objectives and approach.

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models.

Directory of Open Access Journals (Sweden)

Freija Van den Driessche

Full Text Available Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

Abnormal ovarian cancer screening test result: women's informational, psychological and practical needs.

Science.gov (United States)

Ryan, Patricia Y; Graves, Kristi D; Pavlik, Edward J; Andrykowski, Michael A

2007-01-01

Considerable effort has been devoted to the identification of cost-effective approaches to screening for ovarian cancer (OC). Transvaginal ultrasound (TVS) is one such screening approach. Approximately 5-7% of routine TVS screening tests yield abnormal results. Some women experience significant distress after receipt of an abnormal TVS screening test. Four focus groups provided in-depth, qualitative data regarding the informational, psychological, and practical needs of women after the receipt of an abnormal TVS result. Through question and content analytic procedures, we identified four themes: anticipation, emotional response, role of the screening technician, and impact of prior cancer experiences. Results provide initial guidance toward development of interventions to promote adaptive responses after receipt of an abnormal cancer screening test result.

In Vitro Androgen Bioassays as a Detection Method for Designer Androgens

Directory of Open Access Journals (Sweden)

Alison K. Heather

2013-02-01

Full Text Available 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.

Early Detection and Screening for Breast Cancer.

Science.gov (United States)

Coleman, Cathy

2017-05-01

To review the history, current status, and future trends related to breast cancer screening. Peer-reviewed articles, web sites, and textbooks. Breast cancer remains a complex, heterogeneous disease. Serial screening with mammography is the most effective method to detect early stage disease and decrease mortality. Although politics and economics may inhibit organized mammography screening programs in many countries, the judicious use of proficient clinical and self-breast examination can also identify small tumors leading to reduced morbidity. Oncology nurses have exciting opportunities to lead, facilitate, and advocate for delivery of high-quality screening services targeting individuals and communities. A practical approach is needed to translate the complexities and controversies surrounding breast cancer screening into improved care outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

Vasa previa screening strategies: a decision and cost-effectiveness analysis.

Science.gov (United States)

Sinkey, R G; Odibo, A O

2018-05-22

The aim of this study is to perform a decision and cost-effectiveness analysis comparing four screening strategies for the antenatal diagnosis of vasa previa among singleton pregnancies. A decision-analytic model was constructed comparing vasa previa screening strategies. Published probabilities and costs were applied to four transvaginal screening scenarios which occurred at the time of mid-trimester ultrasound: no screening, ultrasound-indicated screening, screening pregnancies conceived by in vitro fertilization (IVF), and universal screening. Ultrasound-indicated screening was defined as performing a transvaginal ultrasound at the time of routine anatomy ultrasound in response to one of the following sonographic findings associated with an increased risk of vasa previa: low-lying placenta, marginal or velamentous cord insertion, or bilobed or succenturiate lobed placenta. The primary outcome was cost per quality adjusted life years (QALY) in U.S. dollars. The analysis was from a healthcare system perspective with a willingness to pay (WTP) threshold of $100,000 per QALY selected. One-way and multivariate sensitivity analyses (Monte-Carlo simulation) were performed. This decision-analytic model demonstrated that screening pregnancies conceived by IVF was the most cost-effective strategy with an incremental cost effectiveness ratio (ICER) of $29,186.50 / QALY. Ultrasound-indicated screening was the second most cost-effective with an ICER of $56,096.77 / QALY. These data were robust to all one-way and multivariate sensitivity analyses performed. Within our baseline assumptions, transvaginal ultrasound screening for vasa previa appears to be most cost-effective when performed among IVF pregnancies. However, both IVF and ultrasound-indicated screening strategies fall within contemporary willingness-to-pay thresholds, suggesting that both strategies may be appropriate to apply in clinical practice. This article is protected by copyright. All rights reserved. This

Fish screens at hydroelectric diversions

International Nuclear Information System (INIS)

Ott, R.F.

1994-01-01

Preventing downstream migrating fish from entering the turbines at hydroelectric projects is a standard mitigation goal of state and federal fishery management agencies. The object is to minimize the adverse impacts to the fish associated with the exclusion and passage through the bypass water conveyance facilities. In the western United States, most of the fishery management agencies have fish screen design criteria that focus on the approach and transportational velocities, maximum opening dimensions of the screen material, and the cleaning standards. Recently, more attention has been given to fish behavioral traits such as attraction and sustained and darting swimming speed, which has resulted in more attention to the position of the screens to the flow and the length of time the downstream migrants are exposed to the screens. Criteria for length of time of exposure, size and position of bypass, flow and velocities in the bypass entrances, discharge requirements back into the receiving water, and exposure to predation have created unique challenges to the fish screen designer. This paper discusses some of the more recent types of fixed fish screens that are being installed at hydroelectric plants that meet these challenges

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening.

Science.gov (United States)

An, Steven S; Askovich, Peter S; Zarembinski, Thomas I; Ahn, Kwangmi; Peltier, John M; von Rechenberg, Moritz; Sahasrabudhe, Sudhir; Fredberg, Jeffrey J

2011-01-13

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Directory of Open Access Journals (Sweden)

von Rechenberg Moritz

2011-01-01

Full Text Available Abstract Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20 and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM. Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Results Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. Conclusions This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

Science.gov (United States)

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Fluorescence-based high-throughput screening of dicer cleavage activity.

Science.gov (United States)

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

A tailored intervention to promote uptake of retinal screening among young adults with type 2 diabetes - an intervention mapping approach.

Science.gov (United States)

Lake, Amelia J; Browne, Jessica L; Abraham, Charles; Tumino, Dee; Hines, Carolyn; Rees, Gwyneth; Speight, Jane

2018-05-31

-based approach to the development of a simple health intervention designed to promote uptake of screening in accordance with national guidelines. The methods and findings illustrate how Intervention Mapping can be employed to develop tailored retinal screening promotion materials for specific priority populations. This paper has implications for future program planners and is intended to assist those wishing to use Intervention Mapping to create similar theoretically-driven, tailored resources.

Biological Evaluations of an Off-Stream Channel, Horizontal Flat-Plate Fish Screen-The Farmers Screen

Science.gov (United States)

Mesa, Matthew G.; Rose, Brien P.; Copeland, Elizabeth S.

2010-01-01

Screens are commonly installed at water diversion sites to reduce entrainment of fish. Recently, the Farmers Irrigation District in Hood River, Oregon, developed a new flat-plate screen design that offers passive operation and may result in reduced operation and installation costs to irrigators. To evaluate the performance (its biological effect on fish) of this type of screen, two size classes of juvenile coho salmon (Oncorhynchus kistuch) were released over a small version of this screen in the field-the Herman Creek screen. The performance of the screen was evaluated over a range of inflow [0.02 to 0.42 m3/s (cubic meters per second)] and diversion flows (0.02 to 0.34 m3/s) at different weir wall heights. The mean approach velocities for the screen ranged from 0 to 5 cm/s (centimeters per second) and mean sweeping velocities ranged from 36 to 178 cm/s. Water depths over the screen surface ranged from 1 to 25 centimeters and were directly related to weir wall height and inflow. Passage of juvenile coho salmon over the screen under a variety of hydraulic conditions did not severely injure them or cause delayed mortality. For all fish, the mean percentage of body surface area that was injured after passage over the screen ranged from about 0.4 to 3.0%. This occurred even though many fish contacted the screen surface during passage. No fish were observed becoming impinged on the screen surface (greater than 1 second contact with the screen). When operated within its design criteria (diversion flows of about 0.28 m3/s), the screen provided safe and effective downstream passage of juvenile salmonids under a variety of hydraulic conditions. However, we do not recommend operating the screen at inflows less than 0.14 m3/s (5 ft3/s) because water depth can get quite shallow and the screen can completely dewater, particularly at very low flows.

Acclimatization of in Vitro-derived Dendrobium

Directory of Open Access Journals (Sweden)

Jaime A. Teixeira da Silva

2017-05-01

Full Text Available The successful ex vitro establishment of Dendrobium plantlets raised in vitro determines the quality of the end product (cut flowers or potted plants in commercial production for economic gain. When in vitro Dendrobium plantlets are transplanted from the culture room to greenhouse conditions, they may desiccate or wilt rapidly and can die as a result of changes in the environment, unless substantial precautions are taken to adapt plantlets to a new environment. The acclimatization of in vitro-grown Dendrobium plantlets to an ex vitro environment by gradually weaning them towards ambient relative humidity and light levels facilitates better survival of young and physiologically sensitive plantlets. Dendrobium plantlets raised in vitro must thus undergo a period of acclimatization or transitional development to correct anatomical abnormalities and to enhance their physiological performance to ensure survival under ex vitro conditions. The most common approach to improve the survival of Dendrobium plantlets upon transfer to an ex vitro environment is their gradual adaptation to that environment. Under such conditions, plants convert rapidly from a heterotrophic or photomixotrophic state to an autotrophic growth, develop a fully functional root system, and better control their stomatal and cuticular transpiration. Gradual adaptation is carried out in a greenhouse by decreasing relative humidity using fog or mist chambers and by increasing light intensity using shading techniques. This review details the acclimatization and ex vitro survival of Dendrobium plants produced in vitro. This advice is also useful for other orchids.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

NARCIS (Netherlands)

Park, M.V.; Annema, W.; Salvati, A.; Lesniak, A.; Elsaesser, A.; Barnes, C.; McKerr, G.; Howard, C.; Lynch, I.; Dawson, K.; Piersma, A.H.; de Jong, W.H.

2009-01-01

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining

Evaluation of Green IT services with Fuzzy Screening approach

Directory of Open Access Journals (Sweden)

Sajjad Shokouhyar‎

2017-11-01

Full Text Available Regarding development of Information Technology, the world of industry has inordinately benefited, albeit that has some losses. Unless the losses are considered, advanced losses will be seen after progress with which is more difficult to cope. Neglecting the future and the risk involved in the industry, not to mention the lack of knowledge in dealing with sudden alterations, compel irrecoverable loss. In this context, information technology services in organizations are aimed to be cost-effective and have minimum environmental impact, according to green information technology strategies. Concerning significance of the issue, purpose of this research is assessment of information technology services with respect to greenness level in a general contractor organization by combination of Fuzzy Analytic Hierarchy Process and Fuzzy Screening Procedure to enhance the greenness level of IT services. The effectiveness of using this approach is including qualitative, quantitative, and uncertainty nature of the problem. In this paper, to consider the Green IT services criteria, literatures have been studied by meta-synthesis method, then the importance of the criteria has been determined by questionnaires so as to rank Green IT criteria. Eventually, the organization level has been concluded in terms of the greenness level of IT services. As a case study, IT experts and managers of KAYSON Inc. organization are considered as statistical population of this research. The reduction had the highest weight among other criteria- recycling and reusing - in KAYSON Inc. organization. Finally, the organization greenness level was determined moderate in terms of IT services.

In vitro fertilization–induced hypertriglyceridemia with secondary acute pancreatitis and diabetic ketoacidosis

Directory of Open Access Journals (Sweden)

Claire Michael Issa

2017-01-01

Full Text Available Introduction: In vitro fertilization is becoming more and more popular lately, as such light is to be shed on any possible related complication. One of these complications is the possible hormonal effect on the lipid profile of the patients. Case presentation: We present a case of a 39-year-old woman with no prior or family history of dyslipidemia, who presented with post in vitro fertilization severe hypertriglyceridemia and secondary acute pancreatitis and diabetic ketoacidosis. Discussion of the case is followed by a brief review of the literature related to in vitro fertilization–induced hypertriglyceridemia. Conclusion: This is, up to our knowledge, the sixth reported case of in vitro fertilization–induced hypertriglyceridemia with secondary acute pancreatitis. This is a serious and life-threatening complication. As such, it might be wise at least in high-risk patients (such as patients with diabetes mellitus, polycystic ovaries syndrome, obesity, and family and personal history of dyslipidemia to screen for lipid abnormalities before initiating in vitro fertilization and monitor these levels afterward.

Using lessons from breast, cervical, and colorectal cancer screening to inform the development of lung cancer screening programs.

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Armstrong, Katrina; Kim, Jane J; Halm, Ethan A; Ballard, Rachel M; Schnall, Mitchell D

2016-05-01

Multiple advisory groups now recommend that high-risk smokers be screened for lung cancer by low-dose computed tomography. Given that the development of lung cancer screening programs will face many of the same issues that have challenged other cancer screening programs, the National Cancer Institute-funded Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) consortium was used to identify lessons learned from the implementation of breast, cervical, and colorectal cancer screening that should inform the introduction of lung cancer screening. These lessons include the importance of developing systems for identifying and recruiting eligible individuals in primary care, ensuring that screening centers are qualified and performance is monitored, creating clear communication standards for reporting screening results to referring physicians and patients, ensuring follow-up is available for individuals with abnormal test results, avoiding overscreening, remembering primary prevention, and leveraging advances in cancer genetics and immunology. Overall, this experience emphasizes that effective cancer screening is a multistep activity that requires robust strategies to initiate, report, follow up, and track each step as well as a dynamic and ongoing oversight process to revise current screening practices as new evidence regarding screening is created, new screening technologies are developed, new biological markers are identified, and new approaches to health care delivery are disseminated. Cancer 2016;122:1338-1342. © 2016 American Cancer Society. © 2016 American Cancer Society.

Pharmacological screening technologies for venom peptide discovery.

Science.gov (United States)

Prashanth, Jutty Rajan; Hasaballah, Nojod; Vetter, Irina

2017-12-01

Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets - including ion channels, G-protein coupled receptors, transporters and enzymes - with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches - supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques - promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.' Copyright © 2017 Elsevier Ltd. All rights reserved.

Electron screening and its effects on big-bang nucleosynthesis

International Nuclear Information System (INIS)

Wang Biao; Bertulani, C. A.; Balantekin, A. B.

2011-01-01

We study the effects of electron screening on nuclear reaction rates occurring during the big-bang nucleosynthesis epoch. The sensitivity of the predicted elemental abundances on electron screening is studied in detail. It is shown that electron screening does not produce noticeable results in the abundances unless the traditional Debye-Hueckel model for the treatment of electron screening in stellar environments is enhanced by several orders of magnitude. This work rules out electron screening as a relevant ingredient to big-bang nucleosynthesis, confirming a previous study [see Itoh et al., Astrophys. J. 488, 507 (1997)] and ruling out exotic possibilities for the treatment of screening beyond the mean-field theoretical approach.

In Vitro Exposure Systems and Dosimetry Assessment Tools ...

Science.gov (United States)

In 2009, the passing of The Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP) and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed “modified risk”. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference titled “In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products” to bring together stakeholders representing regulatory agencies, academia, and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapor exposure systems, as well as the various approaches and challenges to quantifying the complex exposures, in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were, 1) Tobacco Smoke And E-Cigarette Aerosols, 2) Air-Liquid Interface-In Vitro Exposure Systems, 3) Dosimetry Approaches For Particles And Vapors; In Vitro Dosimetry Determinations and 4) Exposure Microenvironment/Physiology Of Cells. The two and a half day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will re

Korean Screen Cultures

DEFF Research Database (Denmark)

Screen Cultures sets out to redress this imbalance with a broad selection of essays spanning both North and South as well as different methodological approaches, from ethnographic and audience studies to cultural materialist readings. The first section of the book, «The South», highlights popular media...... new approaches to Korean popular culture beyond national borders and includes work on K-pop and Korean television drama. This book is a vital addition to existing scholarship on Korean popular culture, offering a unique view by providing an imaginary unification of the two Koreas negotiated through...