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Sample records for antileishmanial high-throughput drug

  1. Strategies to Overcome Antileishmanial Drugs Unresponsiveness

    OpenAIRE

    Shyam Sundar; Anup Singh; Om Prakash Singh

    2014-01-01

    In the absence of effective vector control measures and vaccines against leishmaniasis, effective chemotherapy remains the mainstay of treatment. As the armoury of antileishmanial drugs is limited, strategies should be made to target the emergence of drug resistance. The loss of efficacy of antimonials such as sodium stibogluconate in the Indian subcontinent which has been the mainstay of treatment for more than six decades has raised concern to save the other drugs. In the current review, we...

  2. High throughput electrophysiology: new perspectives for ion channel drug discovery

    DEFF Research Database (Denmark)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter;

    2003-01-01

    Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A...... introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery....... cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct...... characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion...

  3. High throughput electrophysiology: new perspectives for ion channel drug discovery.

    Science.gov (United States)

    Willumsen, Niels J; Bech, Morten; Olesen, Søren-Peter; Jensen, Bo Skaaning; Korsgaard, Mads P G; Christophersen, Palle

    2003-01-01

    Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

  4. Berberine derivatives as antileishmanial drugs.

    OpenAIRE

    Vennerstrom, J L; Lovelace, J K; Waits, V B; Hanson, W L; Klayman, D L

    1990-01-01

    Berberine, a quaternary alkaloid, and several of its derivatives were tested for efficacy against Leishmania donovani and Leishmania braziliensis panamensis in golden hamsters. Tetrahydroberberine was less toxic and more potent than berberine against L. donovani but was not as potent as meglumine antimonate (Glucantime), a standard drug for the treatment of leishmaniasis. Only berberine and 8-cyanodihydroberberine showed significant activity (greater than 50% suppression of lesion size) again...

  5. Strategies to Overcome Antileishmanial Drugs Unresponsiveness

    Directory of Open Access Journals (Sweden)

    Shyam Sundar

    2014-01-01

    Full Text Available In the absence of effective vector control measures and vaccines against leishmaniasis, effective chemotherapy remains the mainstay of treatment. As the armoury of antileishmanial drugs is limited, strategies should be made to target the emergence of drug resistance. The loss of efficacy of antimonials such as sodium stibogluconate in the Indian subcontinent which has been the mainstay of treatment for more than six decades has raised concern to save the other drugs. In the current review, we highlight various steps which could be implemented to halt the increasing unresponsiveness of drugs such as monitoring of therapy in the form of rational dosing and duration of treatment, understanding the mechanism of action of the drugs and drug resistance, identification of markers of resistance, distribution of drugs free of cost, evolution of effective combination therapy and immunotherapy, and proper management of HIV/VL coinfection and post-kala-azar dermal leishmaniasis (PKDL. Strong support from governmental agencies and local communities in the form of education and orientation programmes for feasibility of implementing these strategies and affordability within the context of their health systems is needed in controlling and preventing leishmaniasis.

  6. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    Science.gov (United States)

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  7. High-throughput screening technologies for drug glucuronidation profiling.

    Science.gov (United States)

    Trubetskoy, Olga; Finel, Moshe; Trubetskoy, Vladimir

    2008-08-01

    A significant number of endogenous and exogenous compounds, including many therapeutic agents, are metabolized in humans via glucuronidation, catalysed by uridine diphosphoglucuronosyltransferases (UGTs). The study of the UGTs is a growing field of research, with constantly accumulated and updated information regarding UGT structure, purification, substrate specificity and inhibition, including clinically relevant drug interactions. Development of reliable UGT assays for the assessment of individual isoform substrate specificity and for the discovery of novel isoform-specific substrates and inhibitors is crucial for understanding the function and regulation of the UGT enzyme family and its clinical and pharmacological relevance. High-throughput screening (HTS) is a powerful technology used to search for novel substrates and inhibitors for a wide variety of targets. However, application of HTS in the context of UGTs is complicated because of the poor stability, low levels of expression, low affinity and broad substrate specificity of the enzymes, combined with difficulties in obtaining individual UGT isoforms in purified format, and insufficient information regarding isoform-specific substrates and inhibitors. This review examines the current status of HTS assays used in the search for novel UGT substrates and inhibitors, emphasizing advancements and challenges in HTS technologies for drug glucuronidation profiling, and discusses possible avenues for future advancement of the field.

  8. Developments in Diagnosis and Antileishmanial Drugs

    Directory of Open Access Journals (Sweden)

    Prachi Bhargava

    2012-01-01

    Full Text Available Leishmaniasis ranks the third in disease burden in disability-adjusted life years caused by neglected tropical diseases and is the second cause of parasite-related deaths after malaria; but for a variety of reasons, it is not receiving the attention that would be justified seeing its importance. Leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus Leishmania. It is estimated that 350 million people are at risk in 88 countries, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Improvements in diagnostic methods for early case detection and latest combitorial chemotherapeutic methods have given a new hope for combating this deadly disease. The cell biology of Leishmania and mammalian cells differs considerably and this distinctness extends to the biochemical level. This provides the promise that many of the parasite’s proteins should be sufficiently different from hosts and can be successfully exploited as drug targets. This paper gives a brief overview of recent developments in the diagnosis and approaches in antileishmanial drug discovery and development.

  9. Developments in diagnosis and antileishmanial drugs.

    Science.gov (United States)

    Bhargava, Prachi; Singh, Rajni

    2012-01-01

    Leishmaniasis ranks the third in disease burden in disability-adjusted life years caused by neglected tropical diseases and is the second cause of parasite-related deaths after malaria; but for a variety of reasons, it is not receiving the attention that would be justified seeing its importance. Leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus Leishmania. It is estimated that 350 million people are at risk in 88 countries, with a global incidence of 1-1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Improvements in diagnostic methods for early case detection and latest combitorial chemotherapeutic methods have given a new hope for combating this deadly disease. The cell biology of Leishmania and mammalian cells differs considerably and this distinctness extends to the biochemical level. This provides the promise that many of the parasite's proteins should be sufficiently different from hosts and can be successfully exploited as drug targets. This paper gives a brief overview of recent developments in the diagnosis and approaches in antileishmanial drug discovery and development. PMID:23118748

  10. New approach for high-throughput screening of drug activity on Plasmodium liver stages.

    NARCIS (Netherlands)

    Gego, A.; Silvie, O.; Franetich, J.F.; Farhati, K.; Hannoun, L.; Luty, A.J.F.; Sauerwein, R.W.; Boucheix, C.; Rubinstein, E.; Mazier, D.

    2006-01-01

    Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrare

  11. A non-commercial approach for the generation of transgenic Leishmania tarentolae and its application in antileishmanial drug discovery.

    Science.gov (United States)

    Pineda, Tatiana; Valencia, Yesenia; Flórez, María F; Pulido, Sergio A; Vélez, Iván D; Robledo, Sara M

    2016-08-01

    Leishmaniasis is a parasitic infection caused by several species of the genus Leishmania that is considered as a neglected disease. Drug development process requires a robust and updated high-throughput technology to the evaluation of candidate compounds that imply the manipulation of the pathogenic species of the parasite in the laboratory. Therefore, it is restricted to trained personal and level II biosafety environments. However, it has been established the utility of Leishmania tarentolae as a model for in vitro screening of antileishmanial agents without the necessity of level II biosafety setups. In parallel the transfection of Leishmania parasites with reporter genes as the eGFP using non-commercial integration vectors like the pIRmcs3(-) has proved to be a powerful tool for the implementation of semi automatized high-throughput platforms for the evaluation of antileishmanial compounds. Here we report the generation of a new L. tarentolae strain overexpressing the eGFP gene harboured by the non-commercial vector pIR3(-). We also demonstrate its utility for the semi-automatized screening of antileshmanial compounds in intracellular forms of the L. tarentolae parasite. PMID:27174193

  12. New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages

    OpenAIRE

    Gego, Audrey; Silvie, Olivier; Franetich, Jean-François; Farhati, Khemaïs; Hannoun, Laurent; Luty, Adrian J. F.; Robert W Sauerwein; Boucheix, Claude; Rubinstein, Eric; Mazier, Dominique

    2006-01-01

    Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including ...

  13. Targeting of antileishmanial drugs produced by nanotechnologies

    OpenAIRE

    Pujals Naranjo, Georgina

    2007-01-01

    The aim of this work is to develop an effective new MGA delivery system by means of nanotechnology for the treatment of leishmaniosis which could be administered by parenteral or oral route in a future. Moreover, for ensuring the effectiveness of the formulations developed, their in vitro activities will be assessed against L. infantum. The intention is to prepare a target drug delivery system by means of different technological strategies like micro-nanoparticles by spray drying. These formu...

  14. Four clinically utilized drugs were identified and validated for treatment of adrenocortical cancer using quantitative high-throughput screening

    OpenAIRE

    Nilubol Naris; Zhang Lisa; Shen Min; Zhang Ya-Qin; He Mei; Austin Christopher P; Kebebew Electron

    2012-01-01

    Abstract Background Drug repurposing for cancer treatment is an emerging approach to discover clinically approved drugs that demonstrate antineoplastic effect. The effective therapeutics for patients with advanced adrenocortical carcinoma(ACC) are greatly needed. The objective of this study was to identify and validate drugs with antineoplastic effect in ACC cells using a novel quantitative high-throughput drug screening (qHTS) technique. Methods A quantitative high-throughput proliferation a...

  15. Perfect high throughput screening assay: a crucial technique for drug discovery

    Institute of Scientific and Technical Information of China (English)

    Guan-hua DU

    2005-01-01

    @@ Since being developed approximately 20 years ago, high throughput screening (HTS) has become one of the key techniques used in drug discovery[1]. However, three main problems are recognized with the use of HTS; namely, with the compound library, drug targets, and assay methods. Until now, the compound library has evolved based on the techniques of combinatorial chemistry and modern phytochemistry. Several functional proteins have emerged following the advance of genomics and proteomics. However,although many functional proteins have been discovered recently, they are not, as sometimes claimed, real drug targets;at best, they might be potential drug targets. The ideal targets selected for drug screening should qualify as drug targets[2]. The selection of targets for drug screening is a crucial procedure in drug screening.

  16. Engineering a Brain Cancer Chip for High-throughput Drug Screening

    Science.gov (United States)

    Fan, Yantao; Nguyen, Duong Thanh; Akay, Yasemin; Xu, Feng; Akay, Metin

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and malignant of all human primary brain cancers, in which drug treatment is still one of the most effective treatments. However, existing drug discovery and development methods rely on the use of conventional two-dimensional (2D) cell cultures, which have been proven to be poor representatives of native physiology. Here, we developed a novel three-dimensional (3D) brain cancer chip composed of photo-polymerizable poly(ethylene) glycol diacrylate (PEGDA) hydrogel for drug screening. This chip can be produced after a few seconds of photolithography and requires no silicon wafer, replica molding, and plasma bonding like microfluidic devices made of poly(dimethylsiloxane) (PDMS). We then cultured glioblastoma cells (U87), which formed 3D brain cancer tissues on the chip, and used the GBM chip to perform combinatorial treatment of Pitavastatin and Irinotecan. The results indicate that this chip is capable of high-throughput GBM cancer spheroids formation, multiple-simultaneous drug administration, and a massive parallel testing of drug response. Our approach is easily reproducible, and this chip has the potential to be a powerful platform in cases such as high-throughput drug screening and prolonged drug release. The chip is also commercially promising for other clinical applications, including 3D cell culture and micro-scale tissue engineering. PMID:27151082

  17. Engineering a Brain Cancer Chip for High-throughput Drug Screening.

    Science.gov (United States)

    Fan, Yantao; Nguyen, Duong Thanh; Akay, Yasemin; Xu, Feng; Akay, Metin

    2016-05-06

    Glioblastoma multiforme (GBM) is the most common and malignant of all human primary brain cancers, in which drug treatment is still one of the most effective treatments. However, existing drug discovery and development methods rely on the use of conventional two-dimensional (2D) cell cultures, which have been proven to be poor representatives of native physiology. Here, we developed a novel three-dimensional (3D) brain cancer chip composed of photo-polymerizable poly(ethylene) glycol diacrylate (PEGDA) hydrogel for drug screening. This chip can be produced after a few seconds of photolithography and requires no silicon wafer, replica molding, and plasma bonding like microfluidic devices made of poly(dimethylsiloxane) (PDMS). We then cultured glioblastoma cells (U87), which formed 3D brain cancer tissues on the chip, and used the GBM chip to perform combinatorial treatment of Pitavastatin and Irinotecan. The results indicate that this chip is capable of high-throughput GBM cancer spheroids formation, multiple-simultaneous drug administration, and a massive parallel testing of drug response. Our approach is easily reproducible, and this chip has the potential to be a powerful platform in cases such as high-throughput drug screening and prolonged drug release. The chip is also commercially promising for other clinical applications, including 3D cell culture and micro-scale tissue engineering.

  18. Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry.

    Science.gov (United States)

    Hopfgartner, Gérard; Bourgogne, Emmanuel

    2003-01-01

    To support pharmacokinetic and drug metabolism studies, LC-MS/MS plays more and more an essential role for the quantitation of drugs and their metabolites in biological matrices. With the new challenges encountered in drug discovery and drug development, new strategies are put in place to achieve high-throughput analysis, using serial and parallel approaches. To speed-up method development and validation, generic approaches with the direct injection of biological fluids is highly desirable. Column-switching, using various packing materials for the extraction columns, is widely applied. Improvement of mass spectrometers performance, and in particular triple quadrupoles, also strongly influences sample preparation strategies, which remain a key element in the bioanalytical process. PMID:12838545

  19. Moderate to high throughput in vitro binding kinetics for drug discovery.

    Science.gov (United States)

    Zhang, Rumin; Barbieri, Christopher M; Garcia-Calvo, Margarita; Myers, Robert W; McLaren, David; Kavana, Michael

    2016-01-01

    This review provides a concise summary for state of the art, moderate to high throughput in vitro technologies being employed to study drug-target binding kinetics. These technologies cover a wide kinetic timescale spanning up to nine orders of magnitude from milliseconds to days. Automated stopped flow measures transient and (pre)steady state kinetics from milliseconds to seconds. For seconds to hours timescale kinetics we discuss surface plasmon resonance-based biosensor, global progress curve analysis for high throughput kinetic profiling of enzyme inhibitors and activators, and filtration plate-based radioligand or fluorescent binding assays for receptor binding kinetics. Jump dilution after pre-incubation is the preferred method for very slow kinetics lasting for days. The basic principles, best practices and simulated data for these technologies are described. Finally, the application of a universal label-free technology, liquid chromatography coupled tandem mass spectrometry (LC/MS/MS), is briefly reviewed. Select literature references are highlighted for in-depth understanding. A new reality is dawning wherein binding kinetics is an integral and routine part of mechanism of action elucidation and translational, quantitative pharmacology for drug discovery. PMID:27100706

  20. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor

    Science.gov (United States)

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L.; Xu, Zheli; Ezzat, Shereen

    2016-01-01

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  1. Drug-excipient compatibility testing using a high-throughput approach and statistical design.

    Science.gov (United States)

    Wyttenbach, Nicole; Birringer, Christian; Alsenz, Jochem; Kuentz, Martin

    2005-01-01

    The aim of our research was to develop a miniaturized high throughput drug-excipient compatibility test. Experiments were planned and evaluated using statistical experimental design. Binary mixtures of a drug, acetylsalicylic acid, or fluoxetine hydrochloride, and of excipients commonly used in solid dosage forms were prepared at a ratio of approximately 1:100 in 96-well microtiter plates. Samples were exposed to different temperature (40 degrees C/ 50 degrees C) and humidity (10%/75%) for different time (1 week/4 weeks), and chemical drug degradation was analyzed using a fast gradient high pressure liquid chromatography (HPLC). Categorical statistical design was applied to identify the effects and interactions of time, temperature, humidity, and excipient on drug degradation. Acetylsalicylic acid was least stable in the presence of magnesium stearate, dibasic calcium phosphate, or sodium starch glycolate. Fluoxetine hydrochloride exhibited a marked degradation only with lactose. Factor-interaction plots revealed that the relative humidity had the strongest effect on the drug excipient blends tested. In conclusion, the developed technique enables fast drug-excipient compatibility testing and identification of interactions. Since only 0.1 mg of drug is needed per data point, fast rational preselection of the pharmaceutical additives can be performed early in solid dosage form development.

  2. Rapid identification of antifungal compounds against Exserohilum rostratum using high throughput drug repurposing screens.

    Directory of Open Access Journals (Sweden)

    Wei Sun

    Full Text Available A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug, tacrolimus (an immunosuppressive agent and floxuridine (an antimetabolite were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.

  3. Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery.

    Science.gov (United States)

    Asmild, Margit; Oswald, Nicholas; Krzywkowski, Karen M; Friis, Søren; Jacobsen, Rasmus B; Reuter, Dirk; Taboryski, Rafael; Kutchinsky, Jonathan; Vestergaard, Ras K; Schrøder, Rikke L; Sørensen, Claus B; Bech, Morten; Korsgaard, Mads P G; Willumsen, Niels J

    2003-01-01

    Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

  4. Novel Phenotypic Fluorescent Three-Dimensional Platforms for High-throughput Drug Screening and Personalized Chemotherapy.

    Science.gov (United States)

    Fang, Changge; Avis, Ingalill; Salomon, David; Cuttitta, Frank

    2013-01-01

    We have developed novel phenotypic fluorescent three-dimensional co-culture platforms that efficiently and economically screen anti-angiogenic/anti-metastatic drugs on a high-throughput scale. Individual cell populations can be identified and isolated for protein/gene expression profiling studies and cellular movement/interactions can be tracked by time-lapse cinematography. More importantly, these platforms closely parallel the in vivo angiogenic and metastatic outcomes of a given tumor xenograft in the nude mouse model but, unlike in vivo models, our co-culture platforms produce comparable results in five to nine days. Potentially, by incorporating cancer patient biopsies, the co-culture platforms should greatly improve the effectiveness and efficiency of personalized chemotherapy. PMID:23833685

  5. Unlocking the Potential of High-Throughput Drug Combination Assays Using Acoustic Dispensing.

    Science.gov (United States)

    Chan, Grace Ka Yan; Wilson, Stacy; Schmidt, Stephen; Moffat, John G

    2016-02-01

    Assessment of synergistic effects of drug combinations in vitro is a critical part of anticancer drug research. However, the complexities of dosing and analyzing two drugs over the appropriate range of doses have generally led to compromises in experimental design that restrict the quality and robustness of the data. In particular, the use of a single dose response of combined drugs, rather than a full two-way matrix of varying doses, has predominated in higher-throughput studies. Acoustic dispensing unlocks the potential of high-throughput dose matrix analysis. We have developed acoustic dispensing protocols that enable compound synergy assays in a 384-well format. This experimental design is considerably more efficient and flexible with respect to time, reagent usage, and labware than is achievable using traditional serial-dilution approaches. Data analysis tools integrated in Genedata Screener were used to efficiently deconvolute the combination compound mapping scheme and calculate compound potency and synergy metrics. We have applied this workflow to evaluate interactions among drugs targeting different nodes of the mitogen-activated protein kinase pathway in a panel of cancer cell lines. PMID:26160862

  6. Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

    Science.gov (United States)

    Debnath, Anjan; Tunac, Josefino B; Galindo-Gómez, Silvia; Silva-Olivares, Angélica; Shibayama, Mineko; McKerrow, James H

    2012-11-01

    Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

  7. Open access high throughput drug discovery in the public domain: a Mount Everest in the making.

    Science.gov (United States)

    Roy, Anuradha; McDonald, Peter R; Sittampalam, Sitta; Chaguturu, Rathnam

    2010-11-01

    High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry's best practices for a successful outcome.

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

    International Nuclear Information System (INIS)

    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.

  9. Cyanobacteria, Lyngbya aestuarii and Aphanothece bullosa as antifungal and antileishmanial drug resources

    Institute of Scientific and Technical Information of China (English)

    Maheep Kumar; Manoj Kumar Tripathi; Akanksha Srivastava; Jalaj Kumar Gour; Rakesh Kumar Singh; Ragini Tilak; Ravi Kumar Asthana

    2013-01-01

    To investigate two cyanobacteria isolated from different origins i.e. Lyngbya aestuarii(L. aestuarii) from brackish water and Aphanothece bullosa (A. bullosa) from fresh water paddy fields for antifungal and antileishmanila activity taking Candida albicans and Leishmaniadonovain as targets. Methods: Biomass of L. aestuarii and A. bullosa were harvested after 40 and 60 d respectively and lyophilized twice in methanol (100%) and redissolved in methanol (5%) for bioassay. Antifungal bioassay was done by agar well diffusion method while antileishmanial, by counting cell numbers and flageller motility observation of promastigotes and amastigotes fromL. donovani . Fluconazole and 5% methanol were used as control. Results: Both the cyanobacteria were found to be potent source of antifungal activity keeping fluconazole as positive control, however, methanolic crude extract (15 mg/mL) of A. bullosa was found more potent (larger inhibition zone) over that of methanolic crude extract of L. aestuarii. Similarly antileishmanial activity of crude extract (24.0 mg/mL) of A. bullosa was superior over that of methanolic crude extract of L. aestuarii (25.6 mg/mL). Conclusions: Antifungal and antileishmanial drugs are still limited in the market. Screening of microbes possessing antifungal and antileishmanial activity drug is of prime importance. Cyanobacteria are little explored in this context because most of the drugs in human therapy are derived from microorganisms, mainly bacterial, fungal and actinomycetes. Thus in the present study two cyanobacterial strains from different origins showed potent source of antifungal and antileishmanial biomolecules.

  10. Thermo-responsive polymer aided spheroid culture in cryogel based platform for high throughput drug screening.

    Science.gov (United States)

    Sarkar, J; Kumar, A

    2016-04-21

    In high throughput cell culture, the paradigm is now shifting from 2D to 3D systems. However, in 3D cell culture systems, it is important that the cells form spheroids with robust cell-cell interactions. We fabricated poly(N-isopropylacrylamide-co-gelatin) cryogel scaffolds for cell culture and inserted them into open-ended 96-well plates that formed a drainage and leakage protected, easy to handle high throughput platform. This platform was used to screen for the optimal concentration of poly(N-isopropylacrylamide) (pNIPAAm) as an external aid to the formation of cellular spheroids. It was found that hepatic cells (Hep G2) seeded in the presence of 0.03% pNIPAAm formed better hepatic spheroids in terms of morphology (as assessed by microscopic analysis and formation of bile canaliculi-like structures) and functionality by day 5 of culture. An increase of 44.22%, 15.75%, 36.44%, 32.05% and 27.02% was observed in glucose consumption (1.925 mM per day per 10(4) cells), albumin synthesis (164.18 ng per day per 10(4) cells), CYP1A1 (304.92 pg per min per 10(4) cells), CYP2A6 (441.23 nM per min per 10(4) cells) and phase II metabolic activity (386.18 nM per min(-1) per 10(4) cells), respectively, upon using 0.03% pNIPAAm, as compared to the 3D control. The platform was tested with other cells such as breast and lung cancer cells and found to be compatible. The cell spheroids were subjected to drug toxicity screening in cryogel based open-ended platforms. It was observed that the spheroids were more resistant to anticancer drugs, as compared to 2D and 3D controls, with approximately 11%-67% increase in the IC50 values of tamoxifen and paclitaxel. The platform also showed dose dependent and reproducible responses to drugs. PMID:27027476

  11. High-Throughput Screening for Drugs that Modulate Intermediate Filament Proteins.

    Science.gov (United States)

    Sun, Jingyuan; Groppi, Vincent E; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li; Omary, M Bishr

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high-throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green fluorescent protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug "hits" that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wild-type-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. "Hits" of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients.

  12. High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.

    Science.gov (United States)

    Lin, Xudong; Wang, Shiqi; Yu, Xudong; Liu, Zhuguo; Wang, Fei; Li, Wai Tsun; Cheng, Shuk Han; Dai, Qiuyun; Shi, Peng

    2015-02-01

    The reconstruction of neural activity across complete neural circuits, or brain activity mapping, has great potential in both fundamental and translational neuroscience research. Larval zebrafish, a vertebrate model, has recently been demonstrated to be amenable to whole brain activity mapping in behaving animals. Here we demonstrate a microfluidic array system ("Fish-Trap") that enables high-throughput mapping of brain-wide activity in awake larval zebrafish. Unlike the commonly practiced larva-processing methods using a rigid gel or a capillary tube, which are laborious and time-consuming, the hydrodynamic design of our microfluidic chip allows automatic, gel-free, and anesthetic-free processing of tens of larvae for microscopic imaging with single-cell resolution. Notably, this system provides the capability to directly couple pharmaceutical stimuli with real-time recording of neural activity in a large number of animals, and the local and global effects of pharmacoactive drugs on the nervous system can be directly visualized and evaluated by analyzing drug-induced functional perturbation within or across different brain regions. Using this technology, we tested a set of neurotoxin peptides and obtained new insights into how to exploit neurotoxin derivatives as therapeutic agents. The novel and versatile "Fish-Trap" technology can be readily unitized to study other stimulus (optical, acoustic, or physical) associated functional brain circuits using similar experimental strategies.

  13. Development of Drugs for Epstein - Barr virus using High-Throughput in silico Virtual Screening

    Science.gov (United States)

    Li, Ning; Thompson, Scott; Jiang, Hualiang; Lieberman, Paul M.; Luo, Cheng

    2010-01-01

    Importance of the field Epstein-Barr virus (EBV) is a ubiquitious human herpesvirus that is causally associated with endemic forms of Burkitt’s lymphoma (BL), nasopharyngeal carcinoma, and lymphoproliferative disease in immunosuppressed individuals. On a global scale, EBV infects over 90% of the adult population and is responsible for ~1% of all human cancers. To date, there is no efficacious drug or therapy for the treatment of EBV infection and EBV-related diseases. Areas covered in this review In this review, we discuss the existing anti-EBV inhibitors and those under development. We discuss the value of different molecular targets, including EBV lytic DNA replication enzymes, as well as proteins that are expressed exclusively during latent infection, like EBNA1 and LMP1. Since the atomic structure of the EBNA1 DNA binding domain has been described, it is an attractive target for in silico methods of drug design and small molecule screening. We discuss the use of computational methods that can greatly facilitate the development of novel inhibitors and how in silico screening methods can be applied to target proteins with known structures, like EBNA1, to treat EBV infection and disease. What the reader will gain The reader will be familiarized with the problems in targeting of EBV for inhibition by small molecules and how computational methods can greatly facilitate this process. Take home message Despite the impressive efficacy of nucleoside analogues for the treatment of herpesvirus lytic infection, there remain few effective treatments for latent infections. Since EBV-latent infection persists within and contributes to the formation of EBV-associated cancers, targeting EBV latent proteins is an unmet medical need. High throughput in silico screening can accelerate the process of drug discovery for novel and selective agents that inhibit EBV latent infection and associated disease. PMID:22822721

  14. Fluorescence polarization assays in high-throughput screening and drug discovery: a review

    Science.gov (United States)

    Hall, Matthew D.; Yasgar, Adam; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

    2016-06-01

    The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.

  15. High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

    Directory of Open Access Journals (Sweden)

    Thomas J.J. Gintjee

    2014-11-01

    Full Text Available Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD, the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD.

  16. Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

    OpenAIRE

    Samanta Etel Treiger Borborema; João Alberto Osso Junior; Heitor Franco de Andrade Junior; Nanci do Nascimento

    2016-01-01

    Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine l...

  17. A high-throughput cell-based method to predict the unbound drug fraction in the brain.

    Science.gov (United States)

    Mateus, André; Matsson, Pär; Artursson, Per

    2014-04-10

    Optimization of drug efficacy in the brain requires understanding of the local exposure to unbound drug at the site of action. This relies on measurements of the unbound drug fraction (fu,brain), which currently requires access to brain tissue. Here, we present a novel methodology using homogenates of cultured cells for rapid estimation of fu,brain. In our setup, drug binding to human embryonic kidney cell (HEK293) homogenate was measured in a small-scale dialysis apparatus. To increase throughput, we combined drugs into cassettes for simultaneous measurement of multiple compounds. Our method estimated fu,brain with an average error of 1.9-fold. We propose that our simple method can be used as an inexpensive, easily available and high-throughput alternative to brain tissues excised from laboratory animals. Thereby, estimates of unbound drug exposure can now be implemented at a much earlier stage of the drug discovery process, when molecular property changes are easier to make.

  18. Four clinically utilized drugs were identified and validated for treatment of adrenocortical cancer using quantitative high-throughput screening

    Directory of Open Access Journals (Sweden)

    Nilubol Naris

    2012-09-01

    Full Text Available Abstract Background Drug repurposing for cancer treatment is an emerging approach to discover clinically approved drugs that demonstrate antineoplastic effect. The effective therapeutics for patients with advanced adrenocortical carcinoma(ACC are greatly needed. The objective of this study was to identify and validate drugs with antineoplastic effect in ACC cells using a novel quantitative high-throughput drug screening (qHTS technique. Methods A quantitative high-throughput proliferation assay of 2,816 clinically approved drugs was performed in the NCI-H295R ACC cell line. We validated the antiproliferative effect of candidate compounds in NCI-H295R cells. Further validation was performed in 3-dimensional multicellular aggregates (MCA of NCI-H295R and SW-13 cell lines. Results We identified 79 active compounds against ACC cells; 21 had an efficacy ≥60% and IC50 50. Methotrexate inhibited growth and caused disintegration of MCA in both cell lines at concentrations well below the maximum serum level (10 to 100 fold of IC50. Pyrimethamine caused growth inhibition in both cell lines at 10 fold of IC50 concentration. Conclusions qHTS of previously approved compounds is an effective and efficient method to identify anticancer drugs for a rare cancer such as ACC. We have validated the antineoplastic effect of Bortezomib, ouabain, Methotrexate and pyrimethamine, which could be translated into clinical trials in patients with locally advanced and/or metastatic ACC.

  19. High Throughput and High Speed Organic Synthesis in Drug Discovery:An Integration of Chemistry and Technology

    Institute of Scientific and Technical Information of China (English)

    Li Chen

    2004-01-01

    Drug discovery is a complicated process that involves multiple synthetic chemistry tasks.Among them, lead generation and optimization is the core business in the discovery research.During the stage of lead generation, a large library of many thousands individual compounds will be screened against a biological target to identify a set of hits that showed desirable activity. Once a hit has been identified, analog synthesis and development of SAR around this hit and establishment of relationship between targeted protein and its cellular function become the key activity in the drug discovery project. During this process, focused libraries of a few hundred compounds (X00) will be synthesized and tested. For synthetic chemistry tasks, the efficiency can be enhanced through "parallel processing or high throughput chemisay" using automation technology with which a large number of compounds can be synthesized, purified and plated at same time for biological and pharmaceutical screenings. To accelerate the lead optimization high speed synthesis is conducted when necessary and to improve "single process" efficiency using automation technology, when the rate limiting step become developing synthetic method to produce large amount of compounds (in gram quantity) that can be used in vivo pharmacology study. With these in mind, we designed integrated HTC technology platform which supports the key chemical synthesis activities in discovery research. In this presentation, several examples will be presented to illustrate the applications of high throughput and high speed organic synthesis in drug discovery.

  20. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Jian-Bo [Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China); Ji, Nan; Pan, Wen; Hong, Ru [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102 (China); Wang, Hao [Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China); Ji, Zhi-Liang, E-mail: appo@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102 (China); Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China)

    2014-01-01

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified.

  1. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    International Nuclear Information System (INIS)

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified

  2. High throughput ratio imaging to profile caspase activity: potential application in multiparameter high content apoptosis analysis and drug screening.

    Directory of Open Access Journals (Sweden)

    Jeena Joseph

    Full Text Available Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non

  3. Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

    OpenAIRE

    Veneman, Wouter J.; Marín-Juez, Rubén; de Sonneville, Jan; Ordas, Anita; Jong-Raadsen, Susanne; Meijer, Annemarie H.; Spaink, Herman P.

    2014-01-01

    Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebr...

  4. Precision multidimensional assay for high-throughput microRNA drug discovery

    OpenAIRE

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modul...

  5. Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Gang Li

    2013-05-01

    Full Text Available Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA, there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9. Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9, a finding corroborated by expression quantitative trait loci (eQTL analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2 and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65, a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel

  6. Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

    Science.gov (United States)

    Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J.; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L.; Siminovitch, Katherine A.; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K.; Worthington, Jane; Gupta, Namrata; Clemons, Paul A.; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M.

    2013-01-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10−9). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10−9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in

  7. Statistical Methods for Analysis of High Throughput Experiments in Early Drug Development

    OpenAIRE

    Khamiakova, Tatsiana

    2013-01-01

    Introduction: Advances in biotechnology and the ability to obtain molecular profiles of biological samples, and in particular, the transcriptomic data, have been transforming the way biomedical research and early drug development are carried out for more than a decade (Clarke et al., 2004; Chengalvala et al., 2007; Hughes et al., 2011). In view of increasing costs of the drug development and nevertheless a large number of drugs which fail the clinical trials either due to the lack of efficacy...

  8. Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

    Science.gov (United States)

    Axerio-Cilies, Peter; Castañeda, Ivan P; Mirza, Amin; Reynisson, Jóhannes

    2009-03-01

    A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate. PMID:18692938

  9. poolHiTS: A Shifted Transversal Design based pooling strategy for high-throughput drug screening

    Directory of Open Access Journals (Sweden)

    Woolf Peter J

    2008-05-01

    Full Text Available Abstract Background A key goal of drug discovery is to increase the throughput of small molecule screens without sacrificing screening accuracy. High-throughput screening (HTS in drug discovery involves testing a large number of compounds in a biological assay to identify active compounds. Normally, molecules from a large compound library are tested individually to identify the activity of each molecule. Usually a small number of compounds are found to be active, however the presence of false positive and negative testing errors suggests that this one-drug one-assay screening strategy can be significantly improved. Pooling designs are testing schemes that test mixtures of compounds in each assay, thereby generating a screen of the whole compound library in fewer tests. By repeatedly testing compounds in different combinations, pooling designs also allow for error-correction. These pooled designs, for specific experiment parameters, can be simply and efficiently created using the Shifted Transversal Design (STD pooling algorithm. However, drug screening contains a number of key constraints that require specific modifications if this pooling approach is to be useful for practical screen designs. Results In this paper, we introduce a pooling strategy called poolHiTS (Pooled High-Throughput Screening which is based on the STD algorithm. In poolHiTS, we implement a limit on the number of compounds that can be mixed in a single assay. In addition, we show that the STD-based pooling strategy is limited in the error-correction that it can achieve. Due to the mixing constraint, we show that it is more efficient to split a large library into smaller blocks of compounds, which are then tested using an optimized strategy repeated for each block. We package the optimal block selection algorithm into poolHiTS. The MATLAB codes for the poolHiTS algorithm and the corresponding decoding strategy are also provided. Conclusion We have produced a practical version

  10. An Assay Suitable for High Throughput Screening of Anti-Influenza Drugs

    OpenAIRE

    Lili Mao; Jun Wang; DeGrado, William F.; Masayori Inouye

    2013-01-01

    We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production) system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple measurement of cell growth was used as readouts for drug screening. Two potential inhibitors for M2 (V27A) mutant were verified using this method, which inhibit both the mutant and wild-type M2 channels.

  11. An assay suitable for high throughput screening of anti-influenza drugs.

    Directory of Open Access Journals (Sweden)

    Lili Mao

    Full Text Available We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple measurement of cell growth was used as readouts for drug screening. Two potential inhibitors for M2 (V27A mutant were verified using this method, which inhibit both the mutant and wild-type M2 channels.

  12. Elucidating Hyperconjugation from Electronegativity to Predict Drug Conformational Energy in a High Throughput Manner.

    Science.gov (United States)

    Liu, Zhaomin; Pottel, Joshua; Shahamat, Moeed; Tomberg, Anna; Labute, Paul; Moitessier, Nicolas

    2016-04-25

    Computational chemists use structure-based drug design and molecular dynamics of drug/protein complexes which require an accurate description of the conformational space of drugs. Organic chemists use qualitative chemical principles such as the effect of electronegativity on hyperconjugation, the impact of steric clashes on stereochemical outcome of reactions, and the consequence of resonance on the shape of molecules to rationalize experimental observations. While computational chemists speak about electron densities and molecular orbitals, organic chemists speak about partial charges and localized molecular orbitals. Attempts to reconcile these two parallel approaches such as programs for natural bond orbitals and intrinsic atomic orbitals computing Lewis structures-like orbitals and reaction mechanism have appeared. In the past, we have shown that encoding and quantifying chemistry knowledge and qualitative principles can lead to predictive methods. In the same vein, we thought to understand the conformational behaviors of molecules and to encode this knowledge back into a molecular mechanics tool computing conformational potential energy and to develop an alternative to atom types and training of force fields on large sets of molecules. Herein, we describe a conceptually new approach to model torsion energies based on fundamental chemistry principles. To demonstrate our approach, torsional energy parameters were derived on-the-fly from atomic properties. When the torsional energy terms implemented in GAFF, Parm@Frosst, and MMFF94 were substituted by our method, the accuracy of these force fields to reproduce MP2-derived torsional energy profiles and their transferability to a variety of functional groups and drug fragments were overall improved. In addition, our method did not rely on atom types and consequently did not suffer from poor automated atom type assignments. PMID:27028941

  13. High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation†,‡

    OpenAIRE

    Wu, Angela R.; Tiara L A Kawahara; Rapicavoli, Nicole A; van Riggelen, Jan; Shroff, Emelyn H.; Xu, Liwen; Felsher, Dean W.; Chang, Howard Y.; Quake, Stephen R.

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein–DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP,...

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

    OpenAIRE

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

    2011-01-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumpti...

  15. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.

    Science.gov (United States)

    Gao, Hui; Korn, Joshua M; Ferretti, Stéphane; Monahan, John E; Wang, Youzhen; Singh, Mallika; Zhang, Chao; Schnell, Christian; Yang, Guizhi; Zhang, Yun; Balbin, O Alejandro; Barbe, Stéphanie; Cai, Hongbo; Casey, Fergal; Chatterjee, Susmita; Chiang, Derek Y; Chuai, Shannon; Cogan, Shawn M; Collins, Scott D; Dammassa, Ernesta; Ebel, Nicolas; Embry, Millicent; Green, John; Kauffmann, Audrey; Kowal, Colleen; Leary, Rebecca J; Lehar, Joseph; Liang, Ying; Loo, Alice; Lorenzana, Edward; Robert McDonald, E; McLaughlin, Margaret E; Merkin, Jason; Meyer, Ronald; Naylor, Tara L; Patawaran, Montesa; Reddy, Anupama; Röelli, Claudia; Ruddy, David A; Salangsang, Fernando; Santacroce, Francesca; Singh, Angad P; Tang, Yan; Tinetto, Walter; Tobler, Sonja; Velazquez, Roberto; Venkatesan, Kavitha; Von Arx, Fabian; Wang, Hui Qin; Wang, Zongyao; Wiesmann, Marion; Wyss, Daniel; Xu, Fiona; Bitter, Hans; Atadja, Peter; Lees, Emma; Hofmann, Francesco; Li, En; Keen, Nicholas; Cozens, Robert; Jensen, Michael Rugaard; Pryer, Nancy K; Williams, Juliet A; Sellers, William R

    2015-11-01

    Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

  16. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.

    Science.gov (United States)

    Astashkina, Anna; Mann, Brenda; Grainger, David W

    2012-04-01

    Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent essential aspects of in vivo pharmacology and toxicology. Several in vitro designs are optimized for high throughput to benefit screening efficiencies, allowing the entire libraries of potential pharmacologically relevant or possible toxin molecules to be screened for different types of cell signals relevant to tissue damage or to therapeutic goals. Creative approaches to multiplexed cell-based assay designs that select specific cell types, signaling pathways and reporters are routine. However, substantial percentages of new chemical and biological entities (NCEs/NBEs) that fail late-stage human drug testing, or receive regulatory "black box" warnings, or that are removed from the market for safety reasons after regulatory approvals all provide strong evidence that in vitro cell-based assays and subsequent preclinical in vivo studies do not yet provide sufficient pharmacological and toxicity data or reliable predictive capacity for understanding drug candidate performance in vivo. Without a reliable translational assay tool kit for pharmacology and toxicology, the drug development process is costly and inefficient in taking initial in vitro cell-based screens to in vivo testing and subsequent clinical approvals. Commonly employed methods of in vitro testing, including dissociated, organotypic, organ/explant, and 3-D cultures, are reviewed here with specific focus on retaining cell and molecular interactions and physiological parameters that determine cell phenotypes and their corresponding responses to bioactive agents. Distinct advantages and performance challenges for these models pertinent to cell-based assay and their predictive capabilities required for accurate correlations to in vivo mechanisms of drug toxicity are compared. PMID:22252140

  17. High throughput screening for small molecule enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery.

    Directory of Open Access Journals (Sweden)

    Dhara A Patel

    Full Text Available Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE activity in a fully automated and robust format (Z'>0.7. Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV. The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify

  18. A high-throughput lab-on-a-chip interface for zebrafish embryo tests in drug discovery and ecotoxicology

    Science.gov (United States)

    Zhu, Feng; Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Delaage, Pierre; Wlodkowic, Donald

    2013-12-01

    Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system's design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.

  19. Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

    Directory of Open Access Journals (Sweden)

    Samanta Etel Treiger Borborema

    2016-04-01

    Full Text Available Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA or pentavalent antimony salt (Sb were obtained through filter extrusion (FEL and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay. The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50 of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

  20. High Throughput Facility

    Data.gov (United States)

    Federal Laboratory Consortium — Argonne?s high throughput facility provides highly automated and parallel approaches to material and materials chemistry development. The facility allows scientists...

  1. A concentration gradient generator on a paper-based microfluidic chip coupled with cell culture microarray for high-throughput drug screening.

    Science.gov (United States)

    Hong, Bo; Xue, Peng; Wu, Yafeng; Bao, Jingnan; Chuah, Yon Jin; Kang, Yuejun

    2016-02-01

    Inspired by the paper platforms for 3-D cell culture, a paper-based microfluidic device containing drug concentration gradient was designed and constructed for investigating cell response to drugs based on high throughput analysis. This drug gradient generator was applied to generate concentration gradients of doxorubicin (DOX) as the model drug. HeLa cells encapsulated in collagen hydrogel were incubated in the device reservoirs to evaluate the cell viability based on the controlled release of DOX spatially. It was demonstrated that drug diffusion through the paper fibers created a gradient of drug concentration, which influenced cell viability. This drug screening platform has a great opportunity to be applied for drug discovery and diagnostic studies with simultaneous and parallel tests of drugs under various gradient concentrations.

  2. Co-administration of glycyrrhizic acid with the antileishmanial drug sodium antimony gluconate (SAG) cures SAG-resistant visceral leishmaniasis.

    Science.gov (United States)

    Bhattacharjee, Amrita; Majumder, Saikat; Majumdar, Suchandra Bhattacharyya; Choudhuri, Soumitra Kumar; Roy, Syamal; Majumdar, Subrata

    2015-03-01

    Since there are very few affordable antileishmanial drugs available, antimonial resistance has crippled antileishmanial therapy, thereby emphasising the need for development of novel therapeutic strategies. This study aimed to evaluate the antileishmanial role of combined therapy with sodium antimony gluconate (SAG) and the triterpenoid glycyrrhizic acid (GA) against infection with SAG-resistant Leishmania (GE1F8R). Combination therapy with GA and SAG successfully limited infection with SAG-resistant Leishmania in a synergistic manner (fractional inhibitory concentration index resistant Leishmania and co-treated with GA and SAG exhibited a significant reduction in hepatic and splenic parasite burden. In probing the mechanism, it was observed that GA treatment suppressed the expression and efflux activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), two host ABC transporters responsible for antimony efflux from host cells infected with SAG-resistant parasites. This suppression correlated with greater intracellular antimony retention during SAG therapy both in vitro and in vivo, which was reflected in the reduced parasite load. Furthermore, co-administration of GA and SAG induced a shift in the cytokine balance towards a Th1 phenotype by augmenting pro-inflammatory cytokines (such as IL-12, IFNγ and TNFα) and inducing nitric oxide generation in GE1F8R-infected macrophages as well as GE1F8R-infected mice. This study aims to provide an affordable leishmanicidal alternative to expensive antileishmanial drugs such as miltefosine and amphotericin B. Furthermore, this report explores the role of GA as a resistance modulator in MRP1- and P-gp-overexpressing conditions. PMID:25600891

  3. High-Throughput Melanin-Binding Affinity and In Silico Methods to Aid in the Prediction of Drug Exposure in Ocular Tissue.

    Science.gov (United States)

    Reilly, John; Williams, Sarah L; Forster, Cornelia J; Kansara, Viral; End, Peter; Serrano-Wu, Michael H

    2015-12-01

    Drugs possessing the ability to bind to melanin-rich tissue, such as the eye, are linked with higher ocular exposure, and therefore have the potential to affect the efficacy and safety profiles of therapeutics. A high-throughput melanin chromatographic affinity assay has been developed and validated, which has allowed the rapid melanin affinity assessment for a large number of compounds. Melanin affinity of compounds can be quickly assigned as low, medium, or high melanin binders. A high-throughput chromatographic method has been developed and fully validated to assess melanin affinity of pharmaceuticals and has been useful in predicting ocular tissue distribution in vivo studies. The high-throughput experimental approach has also allowed for a specific training set of 263 molecules for a quantitative structure-affinity relationships (QSAR) method to be developed, which has also been shown to be a predictor of ocular tissue exposure. Previous studies have reported the development of in silico QSAR models based on training sets of relatively small and mostly similar compounds; this model covers a broader range of melanin-binding affinities than what has been previously published and identified several physiochemical descriptors to be considered in the design of compounds where melanin-binding modulation is desired.

  4. Development of a high-throughput in vitro intestinal lipolysis model for rapid screening of lipid-based drug delivery systems

    DEFF Research Database (Denmark)

    Mosgaard, Mette D; Sassene, Philip; Mu, Huiling;

    2015-01-01

    PURPOSE: To develop a high-throughput in vitro intestinal lipolysis (HTP) model, without any means of pH-stat-titration, to enable a fast evaluation of lipid-based drug delivery systems (LbDDS). MATERIAL AND METHOD: The HTP model was compared to the traditionally used dynamic in vitro lipolysis...... (DIVL) model with regard to the extent of lipid digestion and drug distribution of two poorly soluble model drugs (cinnarizine and danazol), during digestion of three LbDDS (LbDDS I-III). RESULT: The HTP model was able to maintain pH around 6.5 during digestion, without the addition of Na......: The HTP model is able to predict drug distribution during digestion of LbDDS containing poorly water soluble drugs in the same manner as the DIVL model. Thus the HTP model might prove applicable for high-throughput evaluation of LbDDS in e.g. 96 well plates or small scale dissolution equipment....

  5. High-throughput transfection and engineering of primary cells and cultured cell lines - an invaluable tool for research as well as drug development.

    Science.gov (United States)

    Müller-Hartmann, Herbert; Faust, Nicole; Kazinski, Michael; Kretzschmar, Titus

    2007-11-01

    The manipulation of eukaryotic cells by introducing nucleic acids and other substrates using chemical, physical or viral methods is one of the ground-breaking tools in the life sciences. Changes in the molecular equipment of a cell induced by introducing different molecules not only enable the dissection of signal transduction and metabolic pathways, but also allow the exploitation of engineered cells as bio-factories for the production of proteins in the processes of target research and drug development. In addition to the application of engineered cells for modern cell-based assays, medically relevant engineered cells can be used in clinical settings for adoptive immunotherapy or gene therapy. With the advent of methods exploiting RNA interference (RNAi), gene identification and functional validation in eukaryotic cells have clearly become one of the most exciting methods in life sciences during the past few years. To accelerate research and development in these areas, high-quality, high-throughput approaches (i.e., using sample formats of at least 96 wells) for cell engineering are needed with increasing demand. Recent developments, especially in the field of electroporation, now allow the efficient, high-throughput engineering of virtually any cell type, including primary cells, many of which were previously considered difficult or even impossible to transfect. Primary cells freshly isolated from native tissues are gaining more and more interest, as data obtained with these cells are considered to be of higher physiological relevance than data obtained with immortalized cell lines that have been cultured for extensive periods. In this review, the various methods for cell engineering (with focus on higher eukaryotic cells) are summarized and their impact for high-throughput applications in research and drug development is discussed.

  6. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    Science.gov (United States)

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.

  7. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.

    Directory of Open Access Journals (Sweden)

    Yasuhito Shimada

    Full Text Available The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish. This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf, knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1, and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.

  8. A new high-throughput method utilizing porous silica-based nano-composites for the determination of partition coefficients of drug candidates.

    Science.gov (United States)

    Yu, Chih H; Tam, Kin; Tsang, Shik C

    2011-09-01

    We show that highly porous silica-based nanoparticles prepared via micro-emulsion and sol-gel techniques are stable colloids in aqueous solution. By incorporating a magnetic core into the porous silica nano-composite, it is found that the material can be rapidly separated (precipitated) upon exposure to an external magnetic field. Alternatively, the porous silica nanoparticles without magnetic cores can be equally separated from solution by applying a high-speed centrifugation. Using these silica-based nanostructures a new high-throughput method for the determination of partition coefficient for water/n-octanol is hereby described. First, a tiny quantity of n-octanol phase is pre-absorbed in the porous silica nano-composite colloids, which allows an establishment of interface at nano-scale between the adsorbed n-octanol with the bulk aqueous phase. Organic compounds added to the mixture can therefore undergo a rapid partition between the two phases. The concentration of drug compound in the supernatant in a small vial can be determined by UV-visible absorption spectroscopy. With the adaptation of a robotic liquid handler, a high-throughput technology for the determination of partition coefficients of drug candidates can be employed for drug screening in the industry based on these nano-separation skills. The experimental results clearly suggest that this new method can provide partition coefficient values of potential drug candidates comparable to the conventional shake-flask method but requires much shorter analytical time and lesser quantity of chemicals. PMID:21780284

  9. High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

    Directory of Open Access Journals (Sweden)

    Nathalie Aulner

    Full Text Available BACKGROUND/OBJECTIVES: Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline. CONCLUSIONS/SIGNIFICANCE: Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage

  10. Determination of drug-like properties of a novel antileishmanial compound: In vitro absorption, distribution, metabolism, and excretion studies

    Directory of Open Access Journals (Sweden)

    Mondal Susanta

    2009-01-01

    Full Text Available In drug discovery research, the compounds should not only to be potent and selective but also must possess acceptable pharmacokinetic properties such as absorption, distribution, metabolism, and excretion (ADME to increase success rate in clinical studies. Objective: Exploration of drug-like properties of 2-(2-methylquinolin-4-ylamino-N-phenyl acetamide, a potent antileishmanial compound by performing some in vitro ADME experiments along with validation of such studies. Materials and Methods: Experimental protocols were established and validated for stability (in PBS pH7.4, simulated gastric and intestinal fluid, solubility, permeability, distribution coefficient (Log D, plasma protein binding and metabolism by rat liver microsomes by using spectrophotometer or HPLC. Methods were considered valid if the results of the standard compounds matched with reported results or within acceptable range or with proper ranking (high-medium-low. Results: The compound was found to be stable (>95% remaining in all stability studies and aqueous solubility was 299.7 ± 6.42 μM. The parallel artificial membrane permeability assay (PAMPA indicated its medium permeability (Log Pe = -5.53 ± 0.01. The distribution coefficients (Log D in octanol/PBS and cyclohexane/PBS systems were found to be 0.54 and -1.33, respectively. The plasma protein binding study by the equilibrium dialysis method was observed to be 78.82 ± 0.13% while metabolism by Phase-I enzymes for 1 hour at 37°C revealed that 36.07 ± 4.15% of the compound remained after metabolism. Conclusion: The methods were found to be very useful for day-to-day ADME studies. All the studies with the antileishmanial compound ascertained that the compound bears optimum pharmacokinetic properties to be used orally as a potential drug for the treatment of leishmaniasis.

  11. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    Science.gov (United States)

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug. PMID:26851087

  12. Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway

    NARCIS (Netherlands)

    Li, Gang; Diogo, Dorothee; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J.; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L.; Siminovitch, Katherine A.; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K.; Worthington, Jane; Gupta, Namrata; Clemons, Paul A.; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M.

    2013-01-01

    Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant

  13. INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    李元

    2001-01-01

    In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.``

  14. INTRODUCTION OF THE HIGH THROUGHPUT SCREENING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    李元

    2001-01-01

    In this article, we introduce the system of high throughput screening (HTS). Its role in new drug study and current development is described. The relationship between research achievements of genome study and new type screening model of new drugs is emphasized. The personal opinions of current problems about HTS study in China are raised.

  15. A Review: The Current In Vivo Models for the Discovery and Utility of New Anti-leishmanial Drugs Targeting Cutaneous Leishmaniasis.

    Science.gov (United States)

    Mears, Emily Rose; Modabber, Farrokh; Don, Robert; Johnson, George E

    2015-01-01

    The current in vivo models for the utility and discovery of new potential anti-leishmanial drugs targeting Cutaneous Leishmaniasis (CL) differ vastly in their immunological responses to the disease and clinical presentation of symptoms. Animal models that show similarities to the human form of CL after infection with Leishmania should be more representative as to the effect of the parasite within a human. Thus, these models are used to evaluate the efficacy of new anti-leishmanial compounds before human clinical trials. Current animal models aim to investigate (i) host-parasite interactions, (ii) pathogenesis, (iii) biochemical changes/pathways, (iv) in vivo maintenance of parasites, and (v) clinical evaluation of drug candidates. This review focuses on the trends of infection observed between Leishmania parasites, the predictability of different strains, and the determination of parasite load. These factors were used to investigate the overall effectiveness of the current animal models. The main aim was to assess the efficacy and limitations of the various CL models and their potential for drug discovery and evaluation. In conclusion, we found that the following models are the most suitable for the assessment of anti-leishmanial drugs: L. major-C57BL/6 mice (or-vervet monkey, or-rhesus monkeys), L. tropica-CsS-16 mice, L. amazonensis-CBA mice, L. braziliensis-golden hamster (or-rhesus monkey). We also provide in-depth guidance for which models are not suitable for these investigations.

  16. High-throughput behavioral phenotyping of drug and alcohol susceptibility traits in the expanded panel of BXD recombinant inbred strains

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Vivek M [ORNL; Ansah, T [University of Tennessee Health Science Center, Memphis; Blaha, C, [University of Tennessee Health Science Center, Memphis; Cook, Melloni N. [University of Memphis; Hamre, Kristin M. [University of Tennessee Health Science Center, Memphis; Lariviere, William R [University of Pittsburgh; Matthews, Douglas B [Baylor University; Goldowitz, Daniel [University of British Columbia, Vancouver; Chesler, Elissa J [ORNL

    2010-01-01

    Genetic reference populations, particularly the BXD recombinant inbred strains, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and co- ariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic co-regulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium have obtained behavioral phenotype data from 260 measures related to multiple behavioral assays across several domains: self-administration, response to, and withdrawal from cocaine, MDMA, morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity; and sleep/wake cycles. All traits have been measured in both sexes and the recently expanded panel of 69 additional BXD recombinant inbred strains (N=69). Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent BXD RI lines was performed. Primary data is publicly available for heritability, sex difference and genetic analyses using www.GeneNetwork.org. These analyses include QTL detection and genetic analysis of gene expression. Stored results from these analyses are available at http://ontologicaldiscovery.org for comparison to other genomic analysis results. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

  17. High-Throughput Proteomics

    Science.gov (United States)

    Zhang, Zhaorui; Wu, Si; Stenoien, David L.; Paša-Tolić, Ljiljana

    2014-06-01

    Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

  18. Simultaneous detection for three kinds of veterinary drugs: Chloramphenicol, clenbuterol and 17-beta-estradiol by high-throughput suspension array technology

    Energy Technology Data Exchange (ETDEWEB)

    Liu Nan; Su Pu [Institute of Hygiene and Environmental Medicine, Tianjin 300050 (China); Gao Zhixian [Institute of Hygiene and Environmental Medicine, Tianjin 300050 (China)], E-mail: gaozhx@163.com; Zhu Maoxiang; Yang Zhihua; Pan Xiujie [Institute of Radiation Medicine, Beijing 100850 (China); Fang Yanjun; Chao Fuhuan [Institute of Hygiene and Environmental Medicine, Tianjin 300050 (China)

    2009-01-19

    Suspension array technology for simultaneous detection of three kinds of veterinary drugs, chloramphenicol (CAP), clenbuterol and 17-beta-estradiol has been developed. Conjugates of chloramphenicol and clenbuterol coupled with bovine serum albumin were synthesized and purified. Probes of suspension array were constituted by coupling the three conjugates on the fluorescent microspheres/beads and the microstructures of the beads' surface were observed by scanning electron microscopy which was a direct confirmation for the successful conjugates' coupling. The optimal addition of conjugates and the amounts of antibodies were optimized and selected, respectively. Standard curves were plotted and the coefficient of determination-R{sup 2} was greater than 0.989 which suggested good logistic correlation. The detection ranges for the three veterinary drugs are 40-6.25 x 10{sup 5} ng L{sup -1}, 50-7.81 x 10{sup 5} ng L{sup -1} and 1 x 10{sup 3-}7.29 x 10{sup 5} ng L{sup -1}, respectively and the lowest detection limits (LDLs) of them are 40, 50 and 1000 ng L{sup -1}, respectively. The suspension array is specific and has no significant cross-reactivity with other chemicals. Meanwhile, unknown samples were detected by suspension array and ELISA in comparison with each other. The errors between found and real for the detection of the unknown samples were relatively small to both of the two methods, whereas, the detection ranges of suspension array are broader and sensitive than that of the traditional ELISA. The high-throughput suspension array is proved to be a novel method for multi-analysis of veterinary drugs with simple operation, high sensitivity and low cost.

  19. High-throughput quantification of drugs and their metabolites in biosamples by LC-MS/MS and CE-MS/MS: possibilities and limitations.

    Science.gov (United States)

    Hopfgartner, G; Husser, C; Zell, M

    2002-02-01

    Off-line solid phase extraction with C18 disk plates and turbulent flow chromatography were evaluated versus on-line solid phase extraction using column-switching HPLC as sample preparation techniques for high-throughput analysis of pharmaceutical compounds and their metabolites by LC-MS/MS. Turbulent flow chromatography was found to be very straightforward in its applicaton, but the LOQs were more than fivefold higher compared with off-line or other on-line solid phase extraction methods. Solid phase extraction (SPE) on disk was found to be fast and sufficient efficient to minimize matrix effects and therefore an apprach to provide sensitive and reliable LC-MS/MS methods. Column-switching HPLC with microbore columns (0.5 mm i.d.) were used for fast analysis of a parent drug and four of its metabolites utilizing steep gradients in 1 minute. The application of CZE-MS/MS for bionalysis of pharamaceutical compounds is also discussed. PMID:11805734

  20. Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

    Science.gov (United States)

    Castilhos, Tamara S; Barreto, Fabiano; Meneghini, Leonardo; Bergold, Ana Maria

    2016-07-01

    A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply. PMID:27268755

  1. The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

    Science.gov (United States)

    Buse, Joshua; Purves, Randy W; Verrall, Ronald E; Badea, Ildiko; Zhang, Haixia; Mulligan, Christopher C; Peru, Kerry M; Bailey, Jonathan; Headley, John V; El-Aneed, Anas

    2014-11-01

    The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells. PMID:25395133

  2. The development and assessment of high-throughput mass spectrometry-based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate.

    Science.gov (United States)

    Buse, Joshua; Purves, Randy W; Verrall, Ronald E; Badea, Ildiko; Zhang, Haixia; Mulligan, Christopher C; Peru, Kerry M; Bailey, Jonathan; Headley, John V; El-Aneed, Anas

    2014-11-01

    The safe use of lipid-based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high-throughput MS-based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3). Analysis utilized MS instruments that detect analytes using low-resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high-resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)-QTRAP-MS/MS, FC- LTQ-Orbitrap-MS, desorption electrospray ionization-collision-induced dissociation (CID)-MS/MS and matrix assisted laser desorption ionization-ToF/ToF-MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)-CID-MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS-based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC-CID-MS/MS, the new MS-based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high-throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC-CID-MS/MS for the quantification of the G16-3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16-3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells.

  3. Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

    International Nuclear Information System (INIS)

    component in the high-throughput assessment of demethylation and risk of chromatin reorganization in epigenetic-drug screening tasks.

  4. Spectroscopic study of antileishmanial drug incubated in the promastigotes of Leishmania mexicana

    Science.gov (United States)

    Hung, J.; Castillo, J.; Jiménez, G.; Hasegawa, M.; Rodriguez, M.

    2003-11-01

    In this work we present spectroscopic study of Boldine (aporphine alkaloid) that possesses important biological activities, in particular, in interaction with the promastigotes of Leishmania mexicana. The results show the applicability of autofluorescence of this drug to determinate the possible mechanism of its biological action. The blue shift and hyperchromic effect in the emission spectrum of the drug in interaction with the parasite cells indicate an energy transference process between them. The morphological change of cell shape of the promastigotes treated with the drug is observed using confocal microscopy. This morphological cell-shape transformation evidences an important interaction between the drug studied and some protein of the parasite cell. Here we describe for the first time the fluorescence properties of the Boldine in the promastigotes of L. mexicana.

  5. Current applications of high-throughput DNA sequencing technology in antibody drug research%高通量DNA测序技术在抗体新药研发中的应用

    Institute of Scientific and Technical Information of China (English)

    余馨; 刘启刚; 王明蓉

    2012-01-01

    Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.%自2005年首次报道了一种基于微乳液PCR技术的高通量DNA测序技术(high-throughput DNA sequencing technology)以来,高通量DNA测序平台已经发展为基因组和各种基因文库序列检测的强大工具.大容量的抗体基因库是日前获得抗体新药的基础,高通量DNA测序技术为从海量的抗体基因库中快速发现功能抗体分子提供了可能.本文就近几年高通量DNA测序技术在抗体基因库的多样性分析,抗体CDR3区的高通量测序、频率分析、功能基因发现及各种展示技术与高通量DNA测序技术的对接应用等方面进行了综述,以期为抗体新药的研发提供一条新的技术路线.

  6. Trypanothione Reductase: A Viable Chemotherapeutic Target for Antitrypanosomal and Antileishmanial Drug Design

    Directory of Open Access Journals (Sweden)

    M. Omar F. Khan

    2007-01-01

    Full Text Available Trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of Trypanosoma and Leishmania spp. and affecting millions of people worldwide. A brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. Trypanothione reductase was identified as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. The biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. The process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefl y revealed.

  7. Flow analysis-hydride generation-gas phase derivative molecular absorption spectrophotometric determination of antimony in antileishmanial drugs

    Directory of Open Access Journals (Sweden)

    Máximo Gallignani

    2009-01-01

    Full Text Available In the present work, the development of a method based on the coupling of flow analysis (FA, hydride generation (HG, and derivative molecular absorption spectrophotometry (D-EAM in gas phase (GP, is described in order to determine total antimony in antileishmanial products. Second derivative order (D²224nm of the absorption spectrum (190 - 300 nm is utilized as measurement criterion. Each one of the parameters involved in the development of the proposed method was examined and optimized. The utilization of the EAM in GP as detection system in a continuous mode instead of atomic absorption spectrometry represents the great potential of the analytic proposal.

  8. The involvement of TLR2 and TLR4 in cytokine and nitric oxide production in visceral leishmaniasis patients before and after treatment with anti-leishmanial drugs.

    Directory of Open Access Journals (Sweden)

    Mariana Gatto

    Full Text Available Toll-like receptors (TLRs have significant involvement in Leishmania infection, although little is known about the relationship between these receptors, cytokines and nitric oxide (NO in patients with visceral leishmaniasis (VL before or after treatment with anti-leishmanial drugs. The goal of this study was to evaluate the expression of TLR2 and TLR4 in CD3+ and CD14+ cells and the production of TNF-α, IFN-γ, IL-17, IL-10, TGF-β and NO in peripheral blood mononuclear cells (PBMCs from VL patients pre- and post-treatment with anti-leishmanial drugs. In addition, we investigated whether these receptors were involved in the production of these cytokines and NO. In the active VL patients, increased TLR2 and TLR4 expression in lymphocytes and monocytes, increased production of TNF-α, IL-10 and TGF-β and decreased production of IFN-γ, IL-17 and NO were observed. After treatment, TLR2 and TLR4 were still expressed in lymphocytes and monocytes, the TNF-α and IL-10 levels were lower, the production of IFN-γ, IL-17 and NO was higher, and the TGF-β level remained high. Before treatment, the production of TNF-α and NO was associated with TLR2 and TLR4 expression, while IL-10 production was only associated with TLR2 expression. After treatment, both receptors were associated with the production of TNF-α, IFN-γ, IL-10 and NO, while the production of IL-17 was associated only with TLR4 expression. The results presented in this study suggest that both TLR2 and TLR4 participate in the modulation of cytokine and NO production in VL patients, contributing to the pathogenesis of VL prior to treatment and the protective immune response after treatment.

  9. 靶向G蛋白偶联受体高通量药物筛选技术研究进展%Advance in high-throughput drug screening techniques targeting G protein-coupled receptors

    Institute of Scientific and Technical Information of China (English)

    杨秀颖; 张莉; 杜冠华

    2012-01-01

    G蛋白偶联受体(GPCR)是临床广泛应用药物的主要靶点之一.靶向GPCR的高通量药物筛选新技术不断涌现,为发现高效、高选择性、低毒的药物提供可能,并使选择合理的GPCR药物筛选策略显得尤为重要.本文综述了靶向GPCR药物高通量筛选相关技术及策略的研究进展,主要包括基于GPCR受体配体结合、第二信使、β-arrestin、调控元件报告基因、内化、聚合体及变构调节剂等方面的高通量药物筛选技术,以及相关技术的代表性商品化产品以供参考.%G protein-coupled receptors (GPCRs) are important drug targets, which have been widely used in clinical practice. New assay techniques for high-throughput screening of drugs targeting GPCRs are emerging in large numbers nowadays, providing the possibility to discover the more efficient, higher selective, lower toxic GPCR drugs. The techniques make the rational design and implementation for GPCR drug screening particularly important. In this paper, we reviewed the progress of techniques and strategies for high-throughput screening of drugs targeting GPCRs, mainly including the strategies on receptor-ligand binding, second messenger detection, (3-arrestin, reporter gene, internalization, oligomerizations and allosteric modulators. We also summarized some of the representative commercial techniques for reference.

  10. Fast Gradient Elution Reversed-Phase HPLC with Diode-Array Detection as a High Throughput Screening Method for Drugs of Abuse

    Energy Technology Data Exchange (ETDEWEB)

    Peter W. Carr; K.M. Fuller; D.R. Stoll; L.D. Steinkraus; M.S. Pasha; Glenn G. Hardin

    2005-12-30

    A new approach has been developed by modifying a conventional gradient elution liquid chromatograph for the high throughput screening of biological samples to detect the presence of regulated intoxicants. The goal of this work was to improve the speed of a gradient elution screening method over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which are the basis for the identification. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between gradient runs, thereby reducing the total time for each analysis. The total analysis time for each gradient elution run is only 2.8 minutes, including 0.3 minutes for column reequilibration between analyses. Retention times standard calibration solutes are reproducible to better than 0.002 minutes in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. The discriminating power and mean list length were calculated for a library of 47 intoxicants and compared with previous work from other laboratories to evaluate fast gradient elution HPLC as a screening tool.

  11. High-Throughput Cell Toxicity Assays.

    Science.gov (United States)

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  12. High throughput protein production screening

    Science.gov (United States)

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  13. in Silico analysis of Escherichia coli polyphosphate kinase (PPK) as a novel antimicrobial drug target and its high throughput virtual screening against PubChem library

    OpenAIRE

    Saha, Saurav Bhaskar; Verma, Vivek

    2013-01-01

    Multiple drug resistance (MDR) in bacteria is a global health challenge that needs urgent attention. The 2011 outbreak caused by Escherichia coli O104:H4 in Europe has exposed the inability of present antibiotic arsenal to tackle the problem of antimicrobial infections. It has further posed a tremendous burden on entire pharmaceutical industry to find novel drugs and/or drug targets. Polyphosphate kinase (PPK) in bacteria plays a crucial role in helping latter to adapt to stringent conditions...

  14. High throughput assays for analyzing transcription factors.

    Science.gov (United States)

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed. PMID:16834538

  15. High-throughput analysis of drugs in biological fluids by desorption electrospray ionization mass spectrometry coupled with thin liquid membrane extraction

    DEFF Research Database (Denmark)

    Rosting, Cecilie; Pedersen-Bjergaard, Stig; Hansen, Steen Honore';

    2013-01-01

    Biological fluids such as urine, saliva and whole blood were analyzed for contents of drugs by a new combination of desorption electrospray ionization mass spectrometry (DESI-MS) and thin liquid membrane extraction (TLME). Analytes from the sample were extracted into a thin liquid membrane...

  16. Development and validation of a high-throughput anti-Wolbachia whole-cell screen: a route to macrofilaricidal drugs against onchocerciasis and lymphatic filariasis.

    Science.gov (United States)

    Clare, Rachel H; Cook, Darren A N; Johnston, Kelly L; Ford, Louise; Ward, Stephen A; Taylor, Mark J

    2015-01-01

    There is an urgent need to develop new, safe, and affordable macrofilaricidal drugs for onchocerciasis and lymphatic filariasis treatment and control. The Anti-Wolbachia Consortium (A·WOL) aims to provide a novel treatment with macrofilaricidal activity by targeting the essential bacterial symbiont Wolbachia. The consortium is currently screening a diverse range of compounds to find new chemical space to drive this drug discovery initiative and address this unmet demand. To increase the throughput and capacity of the A·WOL cell-based screen, we have developed a 384-well format assay using a high-content imaging system (Operetta) in conjunction with optimized Wolbachia growth dynamics in the C6/36 Aedes albopictus mosquito cell line. This assay uses texture analysis of cells stained with SYTO 11 as a direct measure of bacterial load. This validated assay has dramatically increased the capacity and throughput of the A·WOL compound library screening program 25-fold, enriching the number of new anti-Wolbachia hits identified for further development as potential macrofilaricides for onchocerciasis and lymphatic filariasis. PMID:25278497

  17. High-throughput methods for electron crystallography.

    Science.gov (United States)

    Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

    2013-01-01

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

  18. 动物源食品中兽药残留高通量快速分析检测技术%High-throughput and fast analysis detection technology of veterinary drug residues in food products of animal origin

    Institute of Scientific and Technical Information of China (English)

    孙兴权; 董振霖; 李一尘; 代弟; 苏明明; 曹际娟

    2014-01-01

    随着养殖业的迅猛发展,动物源食品兽药残留问题日益成为食品安全领域的重要内容。动物源食品基质复杂,而其中残留的兽药含量甚微,传统的样品制备及检测方法大多存在检测样品基质种类单一、检测兽药种类范围小、耗时长、重现性差等问题,缺乏一定的通用性和准确性,已不能满足当前社会发展的需要。近年来,随着QuChERS法和高分辨质谱等先进样品制备和检测技术在兽药残留检测分析领域的应用,一批高通量、自动化乃至可视化的快速高效分析检测方法也随之而起,该文即对这些高通量快速样品制备和检测方法进行综述,同时对动物源食品中兽药残留的检测和监控等工作提出建议并进行了展望。%With the rapid development of animal culture, the increasing problem of veterinary drug residues in food products of animal origin has become an important content in the field of food safety. Some problems existed in the traditional sample pretreatment and test methods in animal origin food, which contains complex matrix and trace veterinary drug residues, such as single sample matrix type, limited veterinary drugs, time consumption, poor reproducibility, etc. and mostly, lack of generality and accuracy. Therefore, those methods could no longer meet the needs of the current social development. In recent years, with the applications of the QuChERS (quick, easy, cheap, effective, rugged, safe) method and high resolution mass spectrometry (HRMS) in the field of the detection and analysis of veterinary drug residues in animal origin food, a batch of high-throughput, automation and visualization advanced sample preparation and detection technology has appeared and become a fast and efficient analysis method. In this paper, the high-throughput and fast sample preparation and detection methods are introduced and the detection and monitoring work of veterinary drug residues in

  19. 靶向G蛋白偶联受体的高通量药物筛选方法%High-throughput screening assays for G-protein-coupled receptors-targeted drug discovery

    Institute of Scientific and Technical Information of China (English)

    李静; 谢欣

    2012-01-01

    G-protein-coupled receptors (GPCR) , also known as 7 trans-membrane receptors, are the largest family of cell surface receptors. GPCR mediate many important physiological functions and are among the most successful therapeutic targets for a broad spectrum of diseases. These receptors are the targets of > 50% of the current therapeutic agents on the market. Therefore, GPCR assay development and GPCR ligand screening remain the major focus of drug discovery research worldwide. In this review, we summarize the most widely used GPCR assays and recent advances in high-throughput screening technology for GPCR drug discovery.%G蛋白偶联受体( G-protein-coupled receptors,GPCR)是一类具有7次跨膜结构的膜蛋白.GPCR介导多种重要的生理功能,与很多疾病密切相关,是最重要的现代药物靶点家族.目前市场上有近50%的药物是以GPCR为靶点的.因此,GPCR分析方法和GPCR配体筛选方法的研究是当今世界新药研究的重点和热点.本文归纳介绍了近年来被广泛使用的GPCR药物发现方法,以及靶向GPCR高通量筛选技术的最新研究进展.

  20. Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system.

    Science.gov (United States)

    Ribeiro, Tatiana G; Chávez-Fumagalli, Miguel A; Valadares, Diogo G; França, Juçara R; Rodrigues, Lívia B; Duarte, Mariana C; Lage, Paula S; Andrade, Pedro H R; Lage, Daniela P; Arruda, Leonardo V; Abánades, Daniel R; Costa, Lourena E; Martins, Vivian T; Tavares, Carlos A P; Castilho, Rachel O; Coelho, Eduardo A F; Faraco, André A G

    2014-01-01

    The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB) using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity - chitosan (Cs) and chondroitin sulfate (ChS) - were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ) nanoparticles, chitosan-chondroitin sulfate (NQC) nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB) nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and a polydispersity index of 0.2. The measured zeta potential of the nanoparticles indicated a positive charge in their surface, while scanning and transmission electron microscopy revealed spherical nanoparticles with a smooth surface. Attenuated total reflectance-Fourier transform infrared spectroscopy analysis showed an electrostatic interaction between the polymers, whereas the release profile of AmpB from the NQC-AmpB nanoparticles showed a controlled release. In addition, the Cs; ChS; and NQ, NQC, and NQC-AmpB nanoparticles proved to be effective against promastigotes of Leishmania amazonensis and Leishmania chagasi, with a synergistic effect observed between Cs and ChS. Moreover, the applied NQ, NQC, and NQC-AmpB compounds demonstrated low toxicity in murine macrophages, as well as null hemolytic activity in type O(+) human red blood cells. Pure AmpB demonstrated high toxicity in the macrophages. The results show that cells infected with L. amazonensis and later treated with Cs, ChS, NQ, NQC, NQC-AmpB nanoparticles, or pure AmpB presented with a significant reduction in parasite number in the order of 24%, 31%, 55%, 66%, 90%, and 89%, respectively. The data presented indicate that the engineered NQC-AmpB nanoparticles could potentially be used as an alternative therapy to treat leishmaniasis, mainly due its low toxicity to mammals' cells.

  1. High-throughput screening for modulators of cellular contractile force

    CERN Document Server

    Park, Chan Young; Tambe, Dhananjay; Chen, Bohao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J; Marinkovic, Aleksandar; Tschumperlin, Daniel J; Burger, Stephanie; Frykenberg, Matthew; Butler, James P; Stamer, W Daniel; Johnson, Mark; Solway, Julian; Fredberg, Jeffrey J; Krishnan, Ramaswamy

    2014-01-01

    When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signaling intermediates with poorly defined relationship to such a physiological endpoint. Using cellular force as the target, here we screened libraries to identify novel drug candidates in the case of human airway smooth muscle cells in the context of asthma, and also in the case of Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery.

  2. Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system

    Directory of Open Access Journals (Sweden)

    Ribeiro TG

    2014-02-01

    Full Text Available Tatiana G Ribeiro,1 Miguel A Chávez-Fumagalli,2 Diogo G Valadares,3 Juçara R França,1 Lívia B Rodrigues,1 Mariana C Duarte,2 Paula S Lage,2 Pedro H R Andrade,4 Daniela P Lage,4 Leonardo V Arruda,5,6 Daniel R Abánades,5,6 Lourena E Costa,2 Vivian T Martins,3 Carlos AP Tavares,3 Rachel O Castilho,1,7,* Eduardo AF Coelho,2,4,* André AG Faraco1,7,*1Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 2Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 3Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 4Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 5Programa de Pós-Graduação em Patologia Humana, Universidade Federal da Bahia, Salvador, Bahia, Brazil; 6Centro de Pesquisas Gonçalo Moniz (CPqGM, Fundação Oswaldo Cruz (FIOCRUZ, Salvador, Bahia, Brazil; 7Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil*These authors contributed equally to this workAbstract: The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity – chitosan (Cs and chondroitin sulfate (ChS – were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ nanoparticles, chitosan-chondroitin sulfate (NQC nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and

  3. High-throughput screening for modulators of cellular contractile force

    OpenAIRE

    Park, Chan Young; Zhou, Enhua H; Tambe, Dhananjay; Chen, Bohao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J.; Marinkovic, Aleksandar; Tschumperlin, Daniel J.; Burger, Stephanie; Frykenberg, Matthew; Butler, James P.; Stamer, W. Daniel; Johnson, Mark

    2014-01-01

    When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signaling intermediates with poorly defined relationship to such a physiological endpoint. Using cellular force as the target, here we screened libraries to identify novel drug candidates in the case of human airway smooth muscle cells in the context of asthma, and also in the case of Schlemm's canal endothel...

  4. A Multidisciplinary Approach to High Throughput Nuclear Magnetic Resonance Spectroscopy.

    Science.gov (United States)

    Pourmodheji, Hossein; Ghafar-Zadeh, Ebrahim; Magierowski, Sebastian

    2016-01-01

    Nuclear Magnetic Resonance (NMR) is a non-contact, powerful structure-elucidation technique for biochemical analysis. NMR spectroscopy is used extensively in a variety of life science applications including drug discovery. However, existing NMR technology is limited in that it cannot run a large number of experiments simultaneously in one unit. Recent advances in micro-fabrication technologies have attracted the attention of researchers to overcome these limitations and significantly accelerate the drug discovery process by developing the next generation of high-throughput NMR spectrometers using Complementary Metal Oxide Semiconductor (CMOS). In this paper, we examine this paradigm shift and explore new design strategies for the development of the next generation of high-throughput NMR spectrometers using CMOS technology. A CMOS NMR system consists of an array of high sensitivity micro-coils integrated with interfacing radio-frequency circuits on the same chip. Herein, we first discuss the key challenges and recent advances in the field of CMOS NMR technology, and then a new design strategy is put forward for the design and implementation of highly sensitive and high-throughput CMOS NMR spectrometers. We thereafter discuss the functionality and applicability of the proposed techniques by demonstrating the results. For microelectronic researchers starting to work in the field of CMOS NMR technology, this paper serves as a tutorial with comprehensive review of state-of-the-art technologies and their performance levels. Based on these levels, the CMOS NMR approach offers unique advantages for high resolution, time-sensitive and high-throughput bimolecular analysis required in a variety of life science applications including drug discovery.

  5. A Multidisciplinary Approach to High Throughput Nuclear Magnetic Resonance Spectroscopy.

    Science.gov (United States)

    Pourmodheji, Hossein; Ghafar-Zadeh, Ebrahim; Magierowski, Sebastian

    2016-01-01

    Nuclear Magnetic Resonance (NMR) is a non-contact, powerful structure-elucidation technique for biochemical analysis. NMR spectroscopy is used extensively in a variety of life science applications including drug discovery. However, existing NMR technology is limited in that it cannot run a large number of experiments simultaneously in one unit. Recent advances in micro-fabrication technologies have attracted the attention of researchers to overcome these limitations and significantly accelerate the drug discovery process by developing the next generation of high-throughput NMR spectrometers using Complementary Metal Oxide Semiconductor (CMOS). In this paper, we examine this paradigm shift and explore new design strategies for the development of the next generation of high-throughput NMR spectrometers using CMOS technology. A CMOS NMR system consists of an array of high sensitivity micro-coils integrated with interfacing radio-frequency circuits on the same chip. Herein, we first discuss the key challenges and recent advances in the field of CMOS NMR technology, and then a new design strategy is put forward for the design and implementation of highly sensitive and high-throughput CMOS NMR spectrometers. We thereafter discuss the functionality and applicability of the proposed techniques by demonstrating the results. For microelectronic researchers starting to work in the field of CMOS NMR technology, this paper serves as a tutorial with comprehensive review of state-of-the-art technologies and their performance levels. Based on these levels, the CMOS NMR approach offers unique advantages for high resolution, time-sensitive and high-throughput bimolecular analysis required in a variety of life science applications including drug discovery. PMID:27294925

  6. High-throughput titration of luciferase-expressing recombinant viruses.

    Science.gov (United States)

    Garcia, Vanessa; Krishnan, Ramya; Davis, Colin; Batenchuk, Cory; Le Boeuf, Fabrice; Abdelbary, Hesham; Diallo, Jean-Simon

    2014-01-01

    Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

  7. Data Management for High-Throughput Genomics

    CERN Document Server

    Roehm, Uwe

    2009-01-01

    Today's sequencing technology allows sequencing an individual genome within a few weeks for a fraction of the costs of the original Human Genome project. Genomics labs are faced with dozens of TB of data per week that have to be automatically processed and made available to scientists for further analysis. This paper explores the potential and the limitations of using relational database systems as the data processing platform for high-throughput genomics. In particular, we are interested in the storage management for high-throughput sequence data and in leveraging SQL and user-defined functions for data analysis inside a database system. We give an overview of a database design for high-throughput genomics, how we used a SQL Server database in some unconventional ways to prototype this scenario, and we will discuss some initial findings about the scalability and performance of such a more database-centric approach.

  8. The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

    Science.gov (United States)

    Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

    2016-08-01

    Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive. PMID:27476333

  9. The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

    Science.gov (United States)

    Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

    2016-08-01

    Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.

  10. High Throughput Analysis of Photocatalytic Water Purification

    NARCIS (Netherlands)

    Romao, Joana; Barata, David; Habibovic, Pamela; Mul, Guido; Baltrusaitis, Jonas

    2014-01-01

    We present a novel high throughput photocatalyst efficiency assessment method based on 96-well microplates and UV-Vis spectroscopy. We demonstrate the reproducibility of the method using methyl orange (MO) decomposition, and compare kinetic data obtained with those provided in the literature for lar

  11. Experimental study of searching for new antidepressant drugs by applying high throughput screening method%应用高通量筛选体系寻找抗抑郁新药的实验研究

    Institute of Scientific and Technical Information of China (English)

    张莉; 尚念勇; 杜冠华

    2004-01-01

    increasing. It is of great significance to use new technologies and new methods to develop antidepressant drugs with high efficacy and low toxicity.OBJECTIVE: To establish research system for finding new antidepressant leading compounds by the combination of high throughput screening method and in vivo experiment.DESIGN: A randomized and controlled experiment.SETTING, MATERIALS and INTERVENTIONS: The experiment was carried out in the Drug Institute of Chinese Academy of Medical Science. Seventy Kunming male mice weighing(20 ±2) g, and 10 male Wistar rats weighing(200 ± 20) g were used. Wister rats were killed with decollation, and the 5-Hydroxythryptamine (5-HT) reuptake in rat brain synaptosomes was measured. The mice were randomly divided into control group, fluoxetine group(2.6 mg/kg), J01113 group(30 mg/kg), J01114 group(30 mg/kg)and J10745 group(30 mg/Kg). Continuous gastric lavage and administration lasted for 5 days, once per day, and mice hanging experiment was performed every other day since the second day. The mice were administrated and compared separately in 6 minutes after self-hanging in the administration and control group. Forced swimming test was made every other day and compared in 4 minutes of rats in the administration and control group.MAIN OUTCOME MEASURE: The 5-HT reuptake inhibition in synaptosomes and the immobility time in depression models.RESULTS: The effects of more than 5 000 compounds on 5-HT reuptake were tested with high throughput screening(HTS) in vitro. Three active compounds, J01113, J01114 and J10745 were selected to test their antidepressant effects in the forced swimming test and the tail suspension test in mice. J01113, J01114 and J10745 strongly inhibited 5-HT reuptake. The IC50 of J01113, J01114 and J10745 was 1. 304 × 10-10, 9. 036 × 10-10, and 1. 447 × 10-7 mol/L respectively. After the J01113 was administrated for 3 and 5 days, the immobility time of the hanging mice[(67.2 ±47.33),(95.2 ± 47.5 ) s] was significantly less

  12. Viral detection by high-throughput sequencing.

    Science.gov (United States)

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals. PMID:25287501

  13. Viral detection by high-throughput sequencing.

    Science.gov (United States)

    Motooka, Daisuke; Nakamura, Shota; Hagiwara, Katsuro; Nakaya, Takaaki

    2015-01-01

    We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals.

  14. Bipolar electrochemistry for high throughput screening applications

    OpenAIRE

    Munktell, Sara

    2016-01-01

    Bipolar electrochemistry is an interesting concept for high throughput screening techniques due to the ability to induce gradients in a range of materials and their properties, such as composition, particle size, or dopant levels, among many others. One of the key advantages of the method is the ability to test, create or modify materials without the need for a direct electrical connection. In this thesis, the viability of this method has been explored for a range of possible applications, su...

  15. High Throughput Neuro-Imaging Informatics

    Directory of Open Access Journals (Sweden)

    Michael I Miller

    2013-12-01

    Full Text Available This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high dimensional neuroinformatic representations index containing O(E3-E4 discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high throughput machine learning methods for supporting (i cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii integration of image and non-image information for diagnosis and prognosis.

  16. High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

    Science.gov (United States)

    Bompiani, Kristin M; Caglič, Dejan; Krutein, Michelle C; Benoni, Galit; Hrones, Morgan; Lairson, Luke L; Bian, Haiyan; Smith, Garry R; Dickerson, Tobin J

    2016-08-01

    Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified. PMID:27314875

  17. Preliminary High-Throughput Metagenome Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

    2007-03-26

    Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

  18. Computational analysis of high-throughput flow cytometry data

    Science.gov (United States)

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  19. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  20. A high-throughput neutron spectrometer

    Science.gov (United States)

    Stampfl, Anton; Noakes, Terry; Bartsch, Friedl; Bertinshaw, Joel; Veliscek-Carolan, Jessica; Nateghi, Ebrahim; Raeside, Tyler; Yethiraj, Mohana; Danilkin, Sergey; Kearley, Gordon

    2010-03-01

    A cross-disciplinary high-throughput neutron spectrometer is currently under construction at OPAL, ANSTO's open pool light-water research reactor. The spectrometer is based on the design of a Be-filter spectrometer (FANS) that is operating at the National Institute of Standards research reactor in the USA. The ANSTO filter-spectrometer will be switched in and out with another neutron spectrometer, the triple-axis spectrometer, Taipan. Thus two distinct types of neutron spectrometers will be accessible: one specialised to perform phonon dispersion analysis and the other, the filter-spectrometer, designed specifically to measure vibrational density of states. A summary of the design will be given along with a detailed ray-tracing analysis. Some preliminary results will be presented from the spectrometer.

  1. Development of a membrane impregnated with a poly(dimethylsiloxane)/poly(ethylene glycol) copolymer for a high-throughput screening of the permeability of drugs, cosmetics, and other chemicals across the human skin.

    Science.gov (United States)

    Miki, Ryotaro; Ichitsuka, Yasuna; Yamada, Takumi; Kimura, Soichiro; Egawa, Yuya; Seki, Toshinobu; Juni, Kazuhiko; Ueda, Hideo; Morimoto, Yasunori

    2015-01-23

    We aimed to develop a high-throughput screening (HTS) system for preliminary predictions of human skin permeability by using an artificial membrane that can mimic the permeation behaviour of lipophilic and hydrophilic compounds across the human skin. In this study, we synthesized a copolymer containing poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) (PEG) 6000 and impregnated it onto a supportive membrane filter to prepare a PDMS/PEG 6000 copolymer-impregnated membrane. In addition, we synthesized another polymer without PEG units and used it to prepare an impregnated membrane for determining the role of PEG 6000 units in the PDMS/PEG 6000 copolymer-impregnated membrane. The permeation characteristics of the impregnated membranes were evaluated on the basis of the permeability coefficients of 12 model compounds with different lipophilicities, by using a 2-chamber diffusion cell, and these permeability coefficients were compared with those across the human skin. We obtained a good correlation between the permeability coefficients across the PDMS/PEG 6000 copolymer-impregnated membrane and human skin. Further, we evaluated the permeation characteristics of a 96-well plate model of the PDMS/PEG 6000 copolymer by using 6 model compounds. We obtained an ideal correlation between the permeability coefficients across the PDMS/PEG 6000 copolymer using a 96-well plate and those across the human skin. Thus, the PDMS/PEG 6000 copolymer would be a good candidate for preliminary evaluation of the permeability of lipophilic and hydrophilic compounds across the human skin.

  2. Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

    Science.gov (United States)

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

    2016-01-01

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

  3. High-throughput approaches to understanding gene function and mapping network architecture in bacteria.

    Science.gov (United States)

    Brochado, Ana Rita; Typas, Athanasios

    2013-04-01

    Advances in sequencing technology have provided an unprecedented view of bacterial diversity, along with a daunting number of novel genes. Within this new reality lies the challenge of developing large-scale approaches to assign function to the new genes and place them in pathways. Here, we highlight recent advances on this front, focusing on how high-throughput gene-gene, gene-drug and drug-drug interactions can yield functional and mechanistic inferences in bacteria. PMID:23403119

  4. Orthogonal NGS for High Throughput Clinical Diagnostics.

    Science.gov (United States)

    Chennagiri, Niru; White, Eric J; Frieden, Alexander; Lopez, Edgardo; Lieber, Daniel S; Nikiforov, Anastasia; Ross, Tristen; Batorsky, Rebecca; Hansen, Sherry; Lip, Va; Luquette, Lovelace J; Mauceli, Evan; Margulies, David; Milos, Patrice M; Napolitano, Nichole; Nizzari, Marcia M; Yu, Timothy; Thompson, John F

    2016-04-19

    Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

  5. Antileishmanial polyphenols from Corymbia maculata

    Indian Academy of Sciences (India)

    Jasmeen Sidana; Dinesh Neeradi; Alka Choudhary; Sushma Singh; William J Foley; Inder Pal Singh

    2013-07-01

    An activity-guided fractionation was used to identify the antileishmanial compounds of Corymbia maculata. The hexane, ethyl acetate and methanol extracts were active in in vitro antileishmanial assay. Twelve polyphenols including 8-demethyl eucalyptin (1), eucalyptin (2), myrciaphenone A (3), myrciaphenone B (4), quercetin-3---D-xylopyranoside (5), myricetin-3---L-rhamnopyranoside (6), quercetin-3---D-galactopyranoside (7), quercetin-3---D-glucopyranoside (8), quercetin-3---L-rhamnopyranoside (9), syringic acid (10), gallic acid-3-methyl ether (11), gallic acid-4-methyl ether (12) and gallic acid (13) were isolated from the active extracts. All the tested compounds except 8-demethyleucalyptin and myrciaphenone B showed strong to moderate (6.9-24.5 M) antileishmanial activity against Leishmania donovani promastigotes. An HPLC-PDA method has been developed to detect/quantify 29 compounds in the extracts of C. maculata leaves. This validated method allows simultaneous quantitation of seven flavonoids, fourteen phloroglucinols and eight other polyphenols and can be applied for qualitative as well as quantitative determination of phytoconstituents in Eucalyptus matrices.

  6. A robust robotic high-throughput antibody purification platform.

    Science.gov (United States)

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

    2016-07-15

    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. PMID:27283099

  7. A robust robotic high-throughput antibody purification platform.

    Science.gov (United States)

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

    2016-07-15

    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.

  8. In vitro antileishmanial activity of acetogenins from Annonaceae.

    Science.gov (United States)

    Raynaud-Le Grandic, S; Fourneau, C; Laurens, A; Bories, C; Hocquemiller, R; Loiseau, P M

    2004-01-01

    Twelve acetogenins from Annonaceae were evaluated in vitro for their antileishmanial activities in order to search for new lead-compounds having antileishmanial properties. The compounds were comparatively evaluated by the 50% inhibitory concentrations (IC50) determination on promastigote forms of wild-type and four drug-resistant lines of Leishmania donovani. In addition, after testing the toxicity on mouse peritoneal macrophages, the compounds were evaluated on amastigote infected macrophages and a therapeutic index was calculated. The IC50 of the acetogenins against promastigote forms of L. donovani was in a range 4.7-47.3 microM. The most active compound was Rolliniastatin 1 (IC50 at 4.7 microM). On the intramacrophage amastigote in vitro model, only seven compounds exhibited measurable antileishmanial activity with IC50 values in a range 2.5-29.7 microM. Rollinistatin 1 was the most interesting compound with IC50 of 2.5 microM and it appears as the most promising one on the basis of therapeutic index (18.08). Isoannonacin, which is active against intramacrophagic amastigotes (IC50 of 6.2 microM) with a therapeutic index of 2.05, exhibited a strong action on drug-resistant strains (IC50 from 5.1 to 9.8 microM). Acetogenins are a new chemical series with interesting in vitro antileishmanial activity and further studies will be focused on the understanding of this selectivity in regard to the membrane and mitochondrial action using specific probes.

  9. Fluorescent Approaches to High Throughput Crystallography

    Science.gov (United States)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider

  10. Ultraspecific probes for high throughput HLA typing

    Directory of Open Access Journals (Sweden)

    Eggers Rick

    2009-02-01

    Full Text Available Abstract Background The variations within an individual's HLA (Human Leukocyte Antigen genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. Results We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. Conclusion The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

  11. Validation of a High-Throughput Multiplex Genetic Detection System for Helicobacter pylori Identification, Quantification, Virulence, and Resistance Analysis

    OpenAIRE

    Zhang, Yanmei; Zhao, Fuju; Kong, Mimi; Wang, Shiwen; Nan, Li; Hu, Binjie; Olszewski, Michal A.; Miao, Yingxin; Ji, Danian; Jiang, Wenrong; Fang, Yi; Zhang, Jinghao; Chen, Fei; Xiang, Ping; Wu, Yong

    2016-01-01

    Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multip...

  12. Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy.

    Science.gov (United States)

    Ding, Yuzhe; Li, Jiannan; Xiao, Wenwu; Xiao, Kai; Lee, Joyce; Bhardwaj, Urvashi; Zhu, Zijie; Digiglio, Philip; Yang, Gaomai; Lam, Kit S; Pan, Tingrui

    2015-10-20

    Since the 1960s, combination chemotherapy has been widely utilized as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low throughput screening, laborious and time-consuming procedures, and ethical concerns. In this paper, we present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform, which integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, which can potentially speed up the entire discovery cycle of potent drug combinations. Microfluidic impact printing utilizing plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment which can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery. PMID:26334956

  13. A High-Throughput Yeast Halo Assay for Bioactive Compounds.

    Science.gov (United States)

    Bray, Walter; Lokey, R Scott

    2016-01-01

    When a disk of filter paper is impregnated with a cytotoxic or cytostatic drug and added to solid medium seeded with yeast, a visible clear zone forms around the disk whose size depends on the concentration and potency of the drug. This is the traditional "halo" assay and provides a convenient, if low-throughput, read-out of biological activity that has been the mainstay of antifungal and antibiotic testing for decades. Here, we describe a protocol for a high-throughput version of the halo assay, which uses an array of 384 pins to deliver ∼200 nL of stock solutions from compound plates onto single-well plates seeded with yeast. Using a plate reader in the absorbance mode, the resulting halos can be quantified and the data archived in the form of flat files that can be connected to compound databases with standard software. This assay has the convenience associated with the visual readout of the traditional halo assay but uses far less material and can be automated to screen thousands of compounds per day. PMID:27587777

  14. High throughput LSPR and SERS analysis of aminoglycoside antibiotics.

    Science.gov (United States)

    McKeating, Kristy S; Couture, Maxime; Dinel, Marie-Pier; Garneau-Tsodikova, Sylvie; Masson, Jean-Francois

    2016-08-15

    Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects. Patients undergoing treatment with these antibiotics are therefore commonly subjected to therapeutic drug monitoring (TDM) to ensure a safe and effective personalised dosage. The ability to detect these antibiotics in a rapid and sensitive manner in human fluids is therefore of the utmost importance in order to provide effective monitoring of these drugs, which could potentially allow for a more widespread use of this class of antibiotics. Herein, we report on the detection of various aminoglycosides, by exploiting their ability to aggregate gold nanoparticles. The number and position of the amino groups of aminoglycoside antibiotics controlled the aggregation process. We investigated the complementary techniques of surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) for dual detection of these aminoglycoside antibiotics and performed an in-depth study of the feasibility of carrying out TDM of tobramycin using a platform amenable to high throughput analysis. Herein, we also demonstrate dual detection of tobramycin using both LSPR and SERS in a single platform and within the clinically relevant concentration range needed for TDM of this particular aminoglycoside. Additionally we provide evidence that tobramycin can be detected in spiked human serum using only functionalised nanoparticles and SERS analysis. PMID:27412506

  15. A Recombinant Human Pluripotent Stem Cell Line Stably Expressing Halide-Sensitive YFP-I152L for GABAAR and GlyR-Targeted High-Throughput Drug Screening and Toxicity Testing

    Science.gov (United States)

    Kuenzel, Katharina; Friedrich, Oliver; Gilbert, Daniel F.

    2016-01-01

    GABAARs and GlyRs are considered attractive drug targets for therapeutic intervention and are also increasingly recognized in the context of in vitro neurotoxicity (NT) and developmental neurotoxicity (DNT) testing. However, systematic human-specific GABAAR and GlyR-targeted drug screening and toxicity testing is hampered due to lack of appropriate in vitro models that express native GABAARs and GlyRs. We have established a human pluripotent stem cell line (NT2) stably expressing YFP-I152L, a halide-sensitive variant of yellow fluorescent protein (YFP), allowing for fluorescence-based functional analysis of chloride channels. Upon stimulation with retinoic acid, NT2 cells undergo neuronal differentiation and allow pharmacological and toxicological evaluation of native GABAARs and GlyRs at different stages of brain maturation. We applied the cell line in concentration-response experiments with the neurotransmitters GABA and glycine as well as with the drugs strychnine, picrotoxin, fipronil, lindane, bicuculline, and zinc and demonstrate that the established in vitro model is applicable to GABAAR and GlyR-targeted pharmacological and toxicological profiling. We quantified the proportion of GABAAR and GlyR-sensitive cells, respectively, and identified percentages of approximately 20% each within the overall populations, rendering the cells a suitable model for systematic in vitro GABAAR and GlyR-targeted screening in the context of drug development and NT/DNT testing. PMID:27445687

  16. High Throughput Hall Thruster for Small Spacecraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Busek is developing a high throughput nominal 100-W Hall Effect Thruster. This device is well sized for spacecraft ranging in size from several tens of kilograms to...

  17. HIGH THROUGHPUT OF MAP PROCESSOR USING PIPELINE WINDOW DECODING

    Directory of Open Access Journals (Sweden)

    P. Nithya

    2012-11-01

    Full Text Available Turbo codes are one of the most efficient error correcting code which approaches the Shannon limit.The high throughput in turbo decoder can be achieved by parallelizing several soft Input Soft Output(SISOunits together.In this way,multiple SISO decoders work on the same data frame at the same values and delievers soft outputs can be split into three terms like the soft channel and a priori input and the extrinsic value.The extrinsic value is used for the next iteration.The high throughput of Max-Log-MAP processor tha supports both single Binary(SBand Double-binary(DB convolutional turbo codes.Decoding of these codes however an iterative processing is requires high computation rate and latency.Thus in order to achieve high throughput and to reduce latency by using serial processing techniques.The pipeline window(PWdecoding is introduced to support arbitrary frame sizes with high throughput.

  18. High Throughput Hall Thruster for Small Spacecraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Busek Co. Inc. proposes to develop a high throughput, nominal 100 W Hall Effect Thruster (HET). This HET will be sized for small spacecraft (< 180 kg), including...

  19. High Throughput Profiling of Molecular Shapes in Crystals

    Science.gov (United States)

    Spackman, Peter R.; Thomas, Sajesh P.; Jayatilaka, Dylan

    2016-02-01

    Molecular shape is important in both crystallisation and supramolecular assembly, yet its role is not completely understood. We present a computationally efficient scheme to describe and classify the molecular shapes in crystals. The method involves rotation invariant description of Hirshfeld surfaces in terms of of spherical harmonic functions. Hirshfeld surfaces represent the boundaries of a molecule in the crystalline environment, and are widely used to visualise and interpret crystalline interactions. The spherical harmonic description of molecular shapes are compared and classified by means of principal component analysis and cluster analysis. When applied to a series of metals, the method results in a clear classification based on their lattice type. When applied to around 300 crystal structures comprising of series of substituted benzenes, naphthalenes and phenylbenzamide it shows the capacity to classify structures based on chemical scaffolds, chemical isosterism, and conformational similarity. The computational efficiency of the method is demonstrated with an application to over 14 thousand crystal structures. High throughput screening of molecular shapes and interaction surfaces in the Cambridge Structural Database (CSD) using this method has direct applications in drug discovery, supramolecular chemistry and materials design.

  20. Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications

    Directory of Open Access Journals (Sweden)

    Amin Abbaszadeh Banaeiyan

    2013-12-01

    Full Text Available The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III uptake in yeast. Redistribution of a green fluorescent protein (GFP-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min−1, moderate = 50 nL min−1, and high = 100 nL min−1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.

  1. High Throughput Profiling of Molecular Shapes in Crystals.

    Science.gov (United States)

    Spackman, Peter R; Thomas, Sajesh P; Jayatilaka, Dylan

    2016-01-01

    Molecular shape is important in both crystallisation and supramolecular assembly, yet its role is not completely understood. We present a computationally efficient scheme to describe and classify the molecular shapes in crystals. The method involves rotation invariant description of Hirshfeld surfaces in terms of of spherical harmonic functions. Hirshfeld surfaces represent the boundaries of a molecule in the crystalline environment, and are widely used to visualise and interpret crystalline interactions. The spherical harmonic description of molecular shapes are compared and classified by means of principal component analysis and cluster analysis. When applied to a series of metals, the method results in a clear classification based on their lattice type. When applied to around 300 crystal structures comprising of series of substituted benzenes, naphthalenes and phenylbenzamide it shows the capacity to classify structures based on chemical scaffolds, chemical isosterism, and conformational similarity. The computational efficiency of the method is demonstrated with an application to over 14 thousand crystal structures. High throughput screening of molecular shapes and interaction surfaces in the Cambridge Structural Database (CSD) using this method has direct applications in drug discovery, supramolecular chemistry and materials design. PMID:26908351

  2. FLASH Assembly of TALENs Enables High-Throughput Genome Editing

    OpenAIRE

    Reyon, Deepak; Tsai, Shengdar Q.; Khayter, Cyd; Foden, Jennifer A.; Sander, Jeffry D.; Joung, J. Keith

    2012-01-01

    Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable ...

  3. Potential antileishmanial effect of three medicinal plants

    Directory of Open Access Journals (Sweden)

    A Eltayeb

    2012-01-01

    Full Text Available The antileishmanial activity of three organic solvent extracts and water residue of the plants: Acacia nilotica (Mimosaceae (husk, Ambrosia miratima (Astraceae (aerial shoot and Azadarichta indica (Meliaceae (leaves were tested in vitro against Leishmania donovani promastigotes. The study revealed that the extracts of A. nilotica and A. miratima have effectious antileishmanial activity at concentrations (IC 50 less than 8 μg/ml, while the extracts of A. indica lack antileishmanial activity. The chromatographic analysis of the ethyl acetate extract of A. nilotica, the most potent extract, resulted in four TLC fractions. Three of these fractions possessed antileishmanial activity. Phytochemical study of the potent fractions revealed the presence of poly hydroxyl compounds.

  4. Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

    OpenAIRE

    Yiyi Sun; Zhihe Zang; Ling Zhong; Min Wu; Qing Su; Xiurong Gao; Wang Zan; Dong Lin; Yan Zhao; Zhonglin Zhang

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the deve...

  5. SPIM-fluid: open source light-sheet based platform for high-throughput imaging.

    Science.gov (United States)

    Gualda, Emilio J; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

    2015-11-01

    Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

  6. Experimentally Validated Novel Inhibitors of Helicobacter pylori Phosphopantetheine Adenylyltransferase Discovered by Virtual High-Throughput Screening

    OpenAIRE

    Chao-Sheng Cheng; Kai-Fan Jia; Ting Chen; Shun-Ya Chang; Ming-Shen Lin; Hsien-Sheng Yin

    2013-01-01

    Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori , and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput sc...

  7. Antileishmanial activity of the estrogen receptor modulator raloxifene.

    Directory of Open Access Journals (Sweden)

    Juliana Q Reimão

    2014-05-01

    Full Text Available BACKGROUND: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3, rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. CONCLUSIONS/SIGNIFICANCE: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.

  8. NATURAL PRODUCTS AND THEIR ANTILEISHMANIAL ACTIVITY A CRITICAL REVIEW

    Directory of Open Access Journals (Sweden)

    Byadgi P.S

    2011-04-01

    Full Text Available Ayurveda described certain diseases which mimics Indian Kala-azar namely Visamajwara (Satatajwara, Krimi (Raktaja and Plihodara/Pliha roga are seems to be suitable correlation depending on their etiology, symptomatology, prognosis and treatment. Raktaja Krimi is also responsible for the manifestation Raktaja vyadhi, out of which Pliha roga is one. Plihodara is a syndrome characterized by splenomegally, debility, anorexia, indigestion, retention of stool and urine, thirst, bodyache, lassitude, cough, mild fever, emaciation, pain in belly, reddish or abnormal tinge or appearance of blue, green or yellow streaks on abdomen, severe anaemia etc.Visceral leishmaniasis patient presents at a late stage, with persistent but fluctuating low-grade fever, weight loss giving the appearance of severe starvation, spleno-or hepatosplenomegally. The skin is some times said to be muddy, pale or dark. Non-specific laboratory tests will show marked leucopenia (Pancytopenia, mainly neutropenia, anemia, and raised serum proteins, with reversal of albumin/globulin ratio. Morphology of subtypes of raktaja Krimi namely Jantumataraha mimics the leishmania donovani. Other than fever no other points supports the concept of Krimi with leishmaniasis. Besides this raktaja Krimi also manifests the diseases of blood, in which Pliha roga / Plihodara is one. Most of the clinical features mentioned for Plihodara / Pliha roga are similar with Indian Kala-azar. Besides this epidemiological incidence, etiology, pathogenesis and treatment support strongly to correlate the concept of Kala-azar vis-à-vis Plihodara / Pliha roga. So it may be summarized that Krimighna drugs as well as plihaghna and plihodara management principles are useful in the management of kala-azar. This article gives glimpses of natural products and their antileishmanial activity. It also provides the clue to select drugs from Ayurveda to develop antileishmanial drug. This article also provides the information

  9. High Throughput Danio Rerio Energy Expenditure Assay.

    Science.gov (United States)

    Williams, Savannah Y; Renquist, Benjamin J

    2016-01-01

    Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers). PMID:26863590

  10. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman

    2015-12-14

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  11. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    Science.gov (United States)

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver.

  12. Substrate independent ATPase activity may complicate high throughput screening.

    Science.gov (United States)

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  13. Screening and synthesis: high throughput technologies applied to parasitology.

    Science.gov (United States)

    Morgan, R E; Westwood, N J

    2004-01-01

    High throughput technologies continue to develop in response to the challenges set by the genome projects. This article discusses how the techniques of both high throughput screening (HTS) and synthesis can influence research in parasitology. Examples of the use of targeted and phenotype-based HTS using unbiased compound collections are provided. The important issue of identifying the protein target(s) of bioactive compounds is discussed from the synthetic chemist's perspective. This article concludes by reviewing recent examples of successful target identification studies in parasitology.

  14. High-throughput Binary Vectors for Plant Gene Function Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Yong Lei; Ping Zhao; Min-Jie Cao; Rong Cui; Xi Chen; Li-Zhong Xiong; Qi-Fa Zhang; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing,and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

  15. Perspective: Data infrastructure for high throughput materials discovery

    Science.gov (United States)

    Pfeif, E. A.; Kroenlein, K.

    2016-05-01

    Computational capability has enabled materials design to evolve from trial-and-error towards more informed methodologies that require large amounts of data. Expert-designed tools and their underlying databases facilitate modern-day high throughput computational methods. Standard data formats and communication standards increase the impact of traditional data, and applying these technologies to a high throughput experimental design provides dense, targeted materials data that are valuable for material discovery. Integrated computational materials engineering requires both experimentally and computationally derived data. Harvesting these comprehensively requires different methods of varying degrees of automation to accommodate variety and volume. Issues of data quality persist independent of type.

  16. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  17. Workflow for High Throughput Screening of Gas Sensing Materials

    Directory of Open Access Journals (Sweden)

    Ulrich Simon

    2006-04-01

    Full Text Available The workflow of a high throughput screening setup for the rapid identification ofnew and improved sensor materials is presented. The polyol method was applied to preparenanoparticular metal oxides as base materials, which were functionalised by surface doping.Using multi-electrode substrates and high throughput impedance spectroscopy (HT-IS awide range of materials could be screened in a short time. Applying HT-IS in search of newselective gas sensing materials a NO2-tolerant NO sensing material with reducedsensitivities towards other test gases was identified based on iridium doped zinc oxide.Analogous behaviour was observed for iridium doped indium oxide.

  18. A high throughput platform for understanding the influence of excipients on physical and chemical stability.

    Science.gov (United States)

    Raijada, Dhara; Cornett, Claus; Rantanen, Jukka

    2013-08-30

    The present study puts forward a miniaturized high-throughput platform to understand influence of excipient selection and processing on the stability of a given drug compound. Four model drugs (sodium naproxen, theophylline, amlodipine besylate and nitrofurantoin) and ten different excipients were selected. Binary physical mixtures of drug and excipient were transferred to a 96-well plate followed by addition of water to simulate aqueous granulation environment. The plate was subjected for XRPD measurements followed by drying and subsequent XRPD and HPLC measurements of the dried samples. Excipients with different water sorbing potential were found to influence distinctly on the phase transformation behaviour of each drug. Moreover, the amount of water addition was also a critical factor affecting phase transformation behaviour. HPLC analysis revealed one of the drug:excipient pairs with a tendency for chemical degradation. The proposed high-throughput platform can be used during early drug development to simulate typical processing induced stress in a small scale and to understand possible phase transformation behaviour and influence of excipients on this. PMID:22944300

  19. Antileishmanial Effect of 5,3′-Hydroxy-7,4′-dimethoxyflavanone of Picramnia gracilis Tul. (Picramniaceae) Fruit: In Vitro and In Vivo Studies

    OpenAIRE

    Sara M. Robledo; Wilson Cardona; Karen Ligardo; Jéssica Henao; Natalia Arbeláez; Andrés Montoya; Fernando Alzate; Pérez, Juan M.; Victor Arango; Vélez, Iván D.; Jairo Sáez

    2015-01-01

    Species of Picramnia genus are used in folk medicine to treat or prevent skin disorders, but only few species have been studied for biological activity and chemical composition. P. gracilis Tul. is a native species from Central and South America and although its fruits are edible, phytochemical analysis or medicinal uses of this species are not known. In the search of candidates to antileishmanial drugs, this work aimed to evaluate the antileishmanial activity of P. gracilis Tul. in in vitro ...

  20. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    Science.gov (United States)

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner. PMID:27424906

  1. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    Science.gov (United States)

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

  2. High-throughput bioinformatics with the Cyrille2 pipeline system.

    NARCIS (Netherlands)

    Fiers, M.W.E.J.; Burgt, van der A.; Datema, E.; Groot, de J.C.W.; Ham, van R.C.H.J.

    2008-01-01

    Background - Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses a

  3. Algorithms for mapping high-throughput DNA sequences

    DEFF Research Database (Denmark)

    Frellsen, Jes; Menzel, Peter; Krogh, Anders

    2014-01-01

    Abstract High-throughput sequencing (HTS) technologies revolutionized the field of molecular biology by enabling large scale whole genome sequencing as well as a broad range of experiments for studying the cell's inner workings directly on DNA or RNA level. Given the dramatically increased rate o...

  4. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    Science.gov (United States)

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  5. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification...

  6. High throughput defect detection with multiple parallel electron beams

    NARCIS (Netherlands)

    Himbergen, H.M.P. van; Nijkerk, M.D.; Jager, P.W.H. de; Hosman, T.C.; Kruit, P.

    2007-01-01

    A new concept for high throughput defect detection with multiple parallel electron beams is described. As many as 30 000 beams can be placed on a footprint of a in.2, each beam having its own microcolumn and detection system without cross-talk. Based on the International Technology Roadmap for Semic

  7. High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

    OpenAIRE

    Li CHEN; Wang, Yemin; Guo, Hang; Xu, Min; Deng, Zixin; Tao, Meifeng

    2012-01-01

    A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators.

  8. High-throughput screening, predictive modeling and computational embryology - Abstract

    Science.gov (United States)

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to chemical profiling to address sensitivity and specificity of molecular targets, biological pathways, cellular and developmental processes. EPA’s ToxCast project is testing 960 uniq...

  9. High-throughput screening, predictive modeling and computational embryology

    Science.gov (United States)

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to profile thousands of chemical compounds for biological activity and potential toxicity. EPA’s ToxCast™ project, and the broader Tox21 consortium, in addition to projects worldwide,...

  10. Chemometric Optimization Studies in Catalysis Employing High-Throughput Experimentation

    NARCIS (Netherlands)

    Pereira, S.R.M.

    2008-01-01

    The main topic of this thesis is the investigation of the synergies between High-Throughput Experimentation (HTE) and Chemometric Optimization methodologies in Catalysis research and of the use of such methodologies to maximize the advantages of using HTE methods. Several case studies were analysed

  11. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  12. Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

    Directory of Open Access Journals (Sweden)

    Izabel Cristina Piloto Ferreira

    2004-05-01

    Full Text Available Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV, which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae, against the extracellular forms promastigotes of L. (L. amazonensis and L. (V. braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L. amazonensis (LD50 < 47 µg/ml than L. (V. braziliensis. Based on these in vitro results against L. (L. amazonensis new studies should be made to find the compounds with anti-leishmanial activity.

  13. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  14. OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

    Science.gov (United States)

    Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

    2016-02-01

    In the last two decades, heart's normal electrical function. Consequently, all new drugs must undergo preclinical testing for cardiac liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

  15. A high throughput platform for understanding the influence of excipients on physical and chemical stability

    DEFF Research Database (Denmark)

    Raijada, Dhara; Cornett, Claus; Rantanen, Jukka;

    2013-01-01

    The present study puts forward a miniaturized high-throughput platform to understand influence of excipient selection and processing on the stability of a given drug compound. Four model drugs (sodium naproxen, theophylline, amlodipine besylate and nitrofurantoin) and ten different excipients were...... selected. Binary physical mixtures of drug and excipient were transferred to a 96-well plate followed by addition of water to simulate aqueous granulation environment. The plate was subjected for XRPD measurements followed by drying and subsequent XRPD and HPLC measurements of the dried samples. Excipients...... with different water sorbing potential were found to influence distinctly on the phase transformation behaviour of each drug. Moreover, the amount of water addition was also a critical factor affecting phase transformation behaviour. HPLC analysis revealed one of the drug:excipient pairs with a tendency...

  16. Thymol and eugenol derivatives as potential antileishmanial agents.

    Science.gov (United States)

    de Morais, Selene Maia; Vila-Nova, Nadja Soares; Bevilaqua, Claudia Maria Leal; Rondon, Fernanda Cristina; Lobo, Carlos Henrique; de Alencar Araripe Noronha Moura, Arlindo; Sales, Antônia Débora; Rodrigues, Ana Paula Ribeiro; de Figuereido, José Ricardo; Campello, Claudio Cabral; Wilson, Mary E; de Andrade, Heitor Franco

    2014-11-01

    In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.

  17. Antileishmanial activity of the essential oil from Bixa orellana.

    Science.gov (United States)

    Monzote, Lianet; García, Marley; Scull, Ramón; Cuellar, Armando; Setzer, William N

    2014-05-01

    Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. There is currently no vaccine against leishmaniasis, and chemotherapy remains the only effective control. However, conventional drugs are toxic, expensive, and require long periods of treatment, and resistance to clinical chemotherapeutic agents is emerging. Recent research on plants has shown a successful approach to obtain new antileishmanial alternatives. Herein, the in vitro and in vivo effects of the essential oil from Bixa orellana seeds against Leishmania amazonensis were evaluated. A total of 73 compounds were detected by gas chromatography-mass spectrometry analysis, of which ishwarane (18.6%) and geranylgeraniol (9.1%) were the major components. The oil showed activity against intracellular amastigote form (IC50  = 8.5 µg/mL), while the cytotoxic concentration was sevenfold higher for the host cells. The ability of Bixa oil to control disease progression of established cutaneous leishmaniasis in BALB/c mice was demonstrated, after a treatment with 30 mg/kg by intraperitoneal administration over 14 days. The present study reports for the first time the antileishmanial potentialities of the essential oil from B. orellana. PMID:23983115

  18. Antileishmanial activity of diterpene acids in copaiba oil

    Directory of Open Access Journals (Sweden)

    Adriana Oliveira dos Santos

    2013-02-01

    Full Text Available Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50 values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.

  19. Antileishmanial activity of diterpene acids in copaiba oil

    Science.gov (United States)

    dos Santos, Adriana Oliveira; Izumi, Erika; Ueda-Nakamura, Tânia; Dias-Filho, Benedito Prado; da Veiga-Júnior, Valdir Florêncio; Nakamura, Celso Vataru

    2013-01-01

    Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs. PMID:23440116

  20. Current trends in virtual high throughput screening using ligand-based and structure-based methods.

    Science.gov (United States)

    Sukumar, Nagamani; Das, Sourav

    2011-12-01

    High throughput in silico methods have offered the tantalizing potential to drastically accelerate the drug discovery process. Yet despite significant efforts expended by academia, national labs and industry over the years, many of these methods have not lived up to their initial promise of reducing the time and costs associated with the drug discovery enterprise, a process that can typically take over a decade and cost hundreds of millions of dollars from conception to final approval and marketing of a drug. Nevertheless structure-based modeling has become a mainstay of computational biology and medicinal chemistry, helping to leverage our knowledge of the biological target and the chemistry of protein-ligand interactions. While ligand-based methods utilize the chemistry of molecules that are known to bind to the biological target, structure-based drug design methods rely on knowledge of the three-dimensional structure of the target, as obtained through crystallographic, spectroscopic or bioinformatics techniques. Here we review recent developments in the methodology and applications of structure-based and ligand-based methods and target-based chemogenomics in Virtual High Throughput Screening (VHTS), highlighting some case studies of recent applications, as well as current research in further development of these methods. The limitations of these approaches will also be discussed, to give the reader an indication of what might be expected in years to come. PMID:21843144

  1. Natural Products as a Source for Antileishmanial and Antitrypanosomal Agents.

    Science.gov (United States)

    Scotti, Marcus Tullius; Scotti, Luciana; Ishiki, Hamilton; Ribeiro, Frederico Fávaro; Cruz, Rayssa Marques Duarte da; Oliveira, Michelle Pedrosa de; Mendonça, Francisco Jaime Bezerra

    2016-01-01

    Natural products are compounds extracted from plants, marine organisms, fungi or bacteria. Many researches for new drugs are based on these natural molecules, mainly by beneficial effects on health, health, efficacy, and therapeutic safety. Leishmaniosis, Chagas disease and African sleeping sickness are neglected diseases caused by the Leishmania and Trypanosoma ssp. parasites. These infections mainly affect population of developing countries; they have different symptoms, and may often lead to death. The therapeutic drugs available to treat these diseases are either obsolete, toxic, or have questionable efficacy, possibly through encountering resistance. Discovery of new, safe, effective, and affordable molecules is urgently needed. Natural organisms, as marine metabolites, alkaloids, flavonoids, steroids, terpene and coumarins provide innumerable molecules with the potential to treat these diseases. This study examines studies of natural bioactive compounds as antileishmanial and antitrypanosomal agents. PMID:27682867

  2. OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

    Science.gov (United States)

    Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

    2016-02-01

    In the last two decades, liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

  3. An antileishmanial chalcone from Chinese licorice roots

    DEFF Research Database (Denmark)

    Christensen, S B; Ming, C; Andersen, L;

    1994-01-01

    A bioassay guided fractionation of an extract of Chinese licorice roots led to the isolation of (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-[4- hydroxy-3-(3-methyl-2-butenyl]phenyl-2-propen-1-one, which in vitro showed potent antileishmanial activity. In addition, the novel chalcone (E)-1...

  4. A high-throughput multiplex method adapted for GMO detection.

    Science.gov (United States)

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  5. A high-throughput label-free nanoparticle analyser

    Science.gov (United States)

    Fraikin, Jean-Luc; Teesalu, Tambet; McKenney, Christopher M.; Ruoslahti, Erkki; Cleland, Andrew N.

    2011-05-01

    Synthetic nanoparticles and genetically modified viruses are used in a range of applications, but high-throughput analytical tools for the physical characterization of these objects are needed. Here we present a microfluidic analyser that detects individual nanoparticles and characterizes complex, unlabelled nanoparticle suspensions. We demonstrate the detection, concentration analysis and sizing of individual synthetic nanoparticles in a multicomponent mixture with sufficient throughput to analyse 500,000 particles per second. We also report the rapid size and titre analysis of unlabelled bacteriophage T7 in both salt solution and mouse blood plasma, using just ~1 × 10-6 l of analyte. Unexpectedly, in the native blood plasma we discover a large background of naturally occurring nanoparticles with a power-law size distribution. The high-throughput detection capability, scalable fabrication and simple electronics of this instrument make it well suited for diverse applications.

  6. Multiple column high-throughput e-beam inspection (EBI)

    Science.gov (United States)

    Lam, David K.; Monahan, Kevin M.; Liu, Enden D.; Tran, Cong; Prescop, Ted

    2012-03-01

    Single-column e-beam systems are used in production for the detection of electrical defects, but are too slow to be used for the detection of small physical defects, and can't meet future inspection requirements. This paper presents a multiplecolumn e-beam technology for high throughput wafer inspection. Multibeam has developed all-electrostatic columns for high-resolution imaging. The elimination of magnetic coils enables the columns to be small; e-beam deflection is faster in the absence of magnetic hysteresis. Multiple miniaturecolumns are assembled in an array. An array of 100 columns covers the entire surface of a 300mm wafer, affording simultaneous cross-wafer sampling. Column performance simulations and system architecture are presented. Also provided are examples of high throughput, more efficient, multiple-column wafer inspection.

  7. High-Throughput Transaction Executions on Graphics Processors

    CERN Document Server

    He, Bingsheng

    2011-01-01

    OLTP (On-Line Transaction Processing) is an important business system sector in various traditional and emerging online services. Due to the increasing number of users, OLTP systems require high throughput for executing tens of thousands of transactions in a short time period. Encouraged by the recent success of GPGPU (General-Purpose computation on Graphics Processors), we propose GPUTx, an OLTP engine performing high-throughput transaction executions on the GPU for in-memory databases. Compared with existing GPGPU studies usually optimizing a single task, transaction executions require handling many small tasks concurrently. Specifically, we propose the bulk execution model to group multiple transactions into a bulk and to execute the bulk on the GPU as a single task. The transactions within the bulk are executed concurrently on the GPU. We study three basic execution strategies (one with locks and the other two lock-free), and optimize them with the GPU features including the hardware support of atomic ope...

  8. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

    Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard;

    Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families.......Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individual...

  9. A CRISPR CASe for High-Throughput Silencing

    Directory of Open Access Journals (Sweden)

    Jacob eHeintze

    2013-10-01

    Full Text Available Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

  10. High-throughput theoretical design of lithium battery materials

    Science.gov (United States)

    Shi-Gang, Ling; Jian, Gao; Rui-Juan, Xiao; Li-Quan, Chen

    2016-01-01

    The rapid evolution of high-throughput theoretical design schemes to discover new lithium battery materials is reviewed, including high-capacity cathodes, low-strain cathodes, anodes, solid state electrolytes, and electrolyte additives. With the development of efficient theoretical methods and inexpensive computers, high-throughput theoretical calculations have played an increasingly important role in the discovery of new materials. With the help of automatic simulation flow, many types of materials can be screened, optimized and designed from a structural database according to specific search criteria. In advanced cell technology, new materials for next generation lithium batteries are of great significance to achieve performance, and some representative criteria are: higher energy density, better safety, and faster charge/discharge speed. Project supported by the National Natural Science Foundation of China (Grant Nos. 11234013 and 51172274) and the National High Technology Research and Development Program of China (Grant No. 2015AA034201).

  11. A High-Throughput Screen Identifies a New Natural Product with Broad-Spectrum Antibacterial Activity

    OpenAIRE

    Patrick Ymele-Leki; Shugeng Cao; Jared Sharp; Kathleen G Lambert; McAdam, Alexander J.; Husson, Robert N.; Giselle Tamayo; Jon Clardy; Watnick, Paula I.

    2012-01-01

    Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms t...

  12. NanoLuc luciferase - A multifunctional tool for high throughput antibody screening

    Directory of Open Access Journals (Sweden)

    Nicolas eBoute

    2016-02-01

    Full Text Available Based on the recent development of NanoLuc Luciferase a small (19 kDa, highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct western blotting. The new NanoLuc Luciferase protein fusion represents a swiss army knife solution for today and future high throughput antibody drug screenings.

  13. Filtration improves the performance of a high-throughput screen for anti-mycobacterial compounds.

    Directory of Open Access Journals (Sweden)

    Nancy Cheng

    Full Text Available The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.

  14. Human transcriptome array for high-throughput clinical studies

    OpenAIRE

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N.; Schweitzer, Anthony C.; Jiang, Hui; WILHELMY, JULIE; Clark, Tyson A.; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D.; Moldawer, Lyle L; Ronald V Maier; Tompkins, Ronald G.; Wong, Wing Hung

    2011-01-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple ind...

  15. High-Throughput ab-initio Dilute Solute Diffusion Database

    OpenAIRE

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

    We demonstrate automated generation of diffusion databases from high-throughput density functional theory (DFT) calculations. A total of more than 230 dilute solute diffusion systems in Mg, Al, Cu, Ni, Pd, and Pt host lattices have been determined using multi-frequency diffusion models. We apply a correction method for solute diffusion in alloys using experimental and simulated values of host self-diffusivity. We find good agreement with experimental solute diffusion data, obtaining a weighte...

  16. SNP-PHAGE – High throughput SNP discovery pipeline

    OpenAIRE

    Cregan Perry B; Choi Ik-Young; Hyten David L; Grefenstette John J; Matukumalli Lakshmi K; Van Tassell Curtis P

    2006-01-01

    Abstract Background Single nucleotide polymorphisms (SNPs) as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs or microsatellit...

  17. High throughput Single-cell Cultivation on Microfluidic Streak Plates

    OpenAIRE

    Jiang, Cheng-Ying; Dong, Libing; Zhao, Jian-Kang; Hu, Xiaofang; Shen, Chaohua; Qiao, Yuxin; Zhang, Xinyue; Wang, Yapei; Ismagilov, Rustem F.; Liu, Shuang-Jiang; Du, Wenbin

    2016-01-01

    This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for compr...

  18. Learning robust cell signalling models from high throughput proteomic data

    OpenAIRE

    Koch, Mitchell; Broom, Bradley M.; Subramanian, Devika

    2009-01-01

    We propose a framework for learning robust Bayesian network models of cell signalling from high-throughput proteomic data. We show that model averaging using Bayesian bootstrap resampling generates more robust structures than procedures that learn structures using all of the data. We also develop an algorithm for ranking the importance of network features using bootstrap resample data. We apply our algorithms to derive the T-cell signalling network from the flow cytometry data of Sachs et al....

  19. Noise and nonlinearities in high-throughput data

    OpenAIRE

    Nguyen, Viet-Anh; Koukolikova-Nicola, Zdena; Bagnoli, Franco; Lio, Pietro

    2010-01-01

    High-throughput data analyses are becoming common in biology, communications, economics and sociology. The vast amounts of data are usually represented in the form of matrices and can be considered as knowledge networks. Spectra-based approaches have proved useful in extracting hidden information within such networks and for estimating missing data, but these methods are based essentially on linear assumptions. The physical models of matching, when applicable, often suggest non-linear mechani...

  20. High-throughput sequence alignment using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    Trapnell Cole

    2007-12-01

    Full Text Available Abstract Background The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. Results This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. Conclusion MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

  1. Mass spectrometry for high-throughput metabolomics analysis of urine

    OpenAIRE

    Abdelrazig, Salah M.A.

    2015-01-01

    Direct electrospray ionisation-mass spectrometry (direct ESI-MS), by omitting the chromatographic step, has great potential for application as a high-throughput approach for untargeted urine metabolomics analysis compared to liquid chromatography-mass spectrometry (LC-MS). The rapid development and technical innovations revealed in the field of ambient ionisation MS such as nanoelectrospray ionisation (nanoESI) chip-based infusion and liquid extraction surface analysis mass spectrometry (LESA...

  2. High-Throughput FPGA Implementation of QR Decomposition

    OpenAIRE

    Muñoz, Sergio D.; Hormigo, Javier

    2014-01-01

    This brief presents a hardware design to achieve high-throughput QR decomposition, using Givens Rotation Method. It utilizes a new two-dimensional systolic array architecture with pipelined processing elements, which are based on the COordinate Rotation DIgital Computer (CORDIC) algorithm. CORDIC computes vector rotations through shifts and additions. This approach allows a continuous computation of QR factorizations with simple hardware. A fixed-point FPGA architecture for 4 x 4 mat...

  3. SSFinder: High Throughput CRISPR-Cas Target Sites Prediction Tool

    OpenAIRE

    Santosh Kumar Upadhyay; Shailesh Sharma

    2014-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system facilitates targeted genome editing in organisms. Despite high demand of this system, finding a reliable tool for the determination of specific target sites in large genomic data remained challenging. Here, we report SSFinder, a python script to perform high throughput detection of specific target sites in large nucleotide datasets. The SSFinder is a user-friendly tool, compatible wit...

  4. High-Throughput Screening of Bacterial Protein Localization

    OpenAIRE

    Werner, John N.; Gitai, Zemer

    2010-01-01

    The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where e...

  5. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

  6. High-throughput bioinformatics with the Cyrille2 pipeline system

    Directory of Open Access Journals (Sweden)

    de Groot Joost CW

    2008-02-01

    Full Text Available Abstract Background Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses are often interdependent and chained together to form complex workflows or pipelines. Given the volume of the data used and the multitude of computational resources available, specialized pipeline software is required to make high-throughput analysis of large-scale omics datasets feasible. Results We have developed a generic pipeline system called Cyrille2. The system is modular in design and consists of three functionally distinct parts: 1 a web based, graphical user interface (GUI that enables a pipeline operator to manage the system; 2 the Scheduler, which forms the functional core of the system and which tracks what data enters the system and determines what jobs must be scheduled for execution, and; 3 the Executor, which searches for scheduled jobs and executes these on a compute cluster. Conclusion The Cyrille2 system is an extensible, modular system, implementing the stated requirements. Cyrille2 enables easy creation and execution of high throughput, flexible bioinformatics pipelines.

  7. High-throughput analysis of growth differences among phage strains.

    Science.gov (United States)

    Turner, Paul E; Draghi, Jeremy A; Wilpiszeski, Regina

    2012-01-01

    Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments. PMID:22101310

  8. High throughput biotechnology in traditional fermented food industry.

    Science.gov (United States)

    Yang, Yong; Xu, Rong-man; Song, Jia; Wang, Wei-min

    2010-11-01

    Traditional fermented food is not only the staple food for most of developing countries but also the key healthy food for developed countries. As the healthy function of these foods are gradually discovered, more and more high throughput biotechnologies are being used to promote the old and new industry. As a result, the microflora, manufacturing processes and product healthy function of these foods were pushed forward either in the respect of profundity or extensiveness nowadays. The application and progress of the high throughput biotechnologies into traditional fermented food industries were different from each other, which was reviewed and detailed by the catalogues of fermented milk products (yogurt, cheese), fermented sausages, fermented vegetables (kimchi, sauerkraut), fermented cereals (sourdough) and fermented beans (tempeh, natto). Given the further promotion by high throughput biotechnologies, the middle and/or down-stream process of traditional fermented foods would be optimized and the process of industrialization of local traditional fermented food having many functional factors but in small quantity would be accelerated. The article presents some promising patents on traditional fermented food industry. PMID:20863273

  9. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    Science.gov (United States)

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  10. High-Throughput Optical Sensing Immunoassays on Smartphone.

    Science.gov (United States)

    Wang, Li-Ju; Sun, Rongrong; Vasile, Tina; Chang, Yu-Chung; Li, Lei

    2016-08-16

    We present an optical sensing platform on a smartphone for high-throughput screening immunoassays. For the first time, a designed microprism array is utilized to achieve a one-time screening of 64 samples. To demonstrate the capability and the reliability of this optical sensing platform on smartphone, human interleukin 6 (IL-6) protein and six types of plant viruses are immunoassayed. The ability of quantification is shown by a sigmoidal dose-response curve fitting to analyze IL-6 protein. The accuracy in measuring the concentrations of IL-6 protein achieves 99.1%. On the other hand, to validate on-field immunoassays by our device, a total of 1030 samples are assayed using three immunoassay methods to detect six types of plant viruses. The accuracy is up to 96.2-99.9%; in addition, there is a high degree of agreement with lab instruments. The total cost for this high-throughput optical screening platform is ∼$50 USD. The reading time is only 2 s for 64 samples. The size is just as big as a portable hard drive. Our optical sensing platform on the smartphone offers a route toward in situ high-throughput screening immunoassays for viruses, pathogens, biomarkers, and toxins by decentralizing laboratory tests. With this mobile point-of-care optical platform, the spread of disease can be timely stopped within a very short turnaround time. PMID:27434250

  11. Establishment and optimization of a high throughput phenotypic test for the detection of drug resist-ance in human immunodeficiency virus(HIV)strains%基于假病毒的高通量人类免疫缺陷病毒表型耐药性检测方法的建立和优化

    Institute of Scientific and Technical Information of China (English)

    聂建辉; 许四宏; 宋爱京; 赵娟; 陈晴晴; 马建; 黄维金; 王佑春

    2014-01-01

    目的:建立一种高通量人类免疫缺陷病毒表型耐药性检测方法。方法通过酶切连接方式失活荧光素酶基因,用 LacZ 基因替代原有的蛋白酶和逆转录酶基因。通过 PCR 方法从含耐药基因的 pSG3△env 质粒中扩增 pol 基因,用 infusion 定向克隆技术连接入酶切后的 pNL4-3. Lac,对该表型耐药性检测方法的主要影响因素进行优化。结果构建了用于高通量耐药性检测的 pNL4-3. Lac,确定了检测方法中细胞加入量为10000细胞/孔(96孔板),病毒加入量为200 TCID50/孔, DEAE-dextran 的最佳工作浓度为15μg/ ml。用12种抗病毒药物检测两种假病毒8次,确定方法具有良好的重复性(CV 值在4.32%~28.46%之间)。构建了6种不同的假病毒与基于 pSG3△env 的耐药性检测方法进行比较,两者之间具有良好的一致性。结论基于 pNL4-3. Lac 的表型耐药性检测方法结合了 pSG3△env 和 pNL4-3检测系统的优点,能高通量评价 HIV 病毒对药物的耐受性,可用于感染者耐药性分析和抗病毒药物的筛选。%Objective To establish a high throughput phenotypic test for the detection of drug re-sistance in human immunodeficiency virus(HIV)strains. Methods The gene encoding luciferase was in-activated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned in-to a new backbone plasmid through infusion. The factors that might affect the results of the test were opti-mized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse tran-scriptase. Several influential factors including cell numbers(10 000 / well),virus inoculation(200 TCID50 /well)and the concentration of DEAE-dextran(15

  12. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus

    Science.gov (United States)

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  13. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus.

    Science.gov (United States)

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  14. High Throughput Screening of Valganciclovir in Acidic Microenvironments of Polyester Thin Films

    Directory of Open Access Journals (Sweden)

    Teilo Schaller

    2015-04-01

    Full Text Available Ganciclovir and valganciclor are antiviral agents used for the treatment of cytomegalovirus retinitis. The conventional method for administering ganciclovir in cytomegalovirus retinitis patients is repeated intravitreal injections. In order to obviate the possible detrimental effects of repeated intraocular injections, to improve compliance and to eliminate systemic side-effects, we investigated the tuning of the ganciclovir pro-drug valganciclovir and the release from thin films of poly(lactic-co-glycolic acid (PLGA, polycaprolactone (PCL, or mixtures of both, as a step towards prototyping periocular valganciclovir implants. To investigate the drug release, we established and evaluated a high throughput fluorescence-based quantification screening assay for the detection of valganciclovir. Our protocol allows quantifying as little as 20 ng of valganciclovir in 96-well polypropylene plates and a 50× faster analysis compared to traditional HPLC measurements. This improvement can hence be extrapolated to other polyester matrix thin film formulations using a high-throughput approach. The acidic microenvironment within the polyester matrix was found to protect valganciclovir from degradation with resultant increases in the half-life of the drug in the periocular implant to 100 days. Linear release profiles were obtained using the pure polyester polymers for 10 days and 60 days formulations; however, gross phase separations of PCL and acid-terminated PLGA prevented tuning within these timeframes due to the phase separation of the polymer, valganciclovir, or both.

  15. Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light

    OpenAIRE

    Allahverdiyev, Adil M; Abamor, Emrah Sefik; Bagirova, Malahat; Ustundag, Cem B; Kaya, Cengiz; Kaya, Figen; Rafailovich, Miriam

    2011-01-01

    Leishmaniasis is a protozoan vector-borne disease and is one of the biggest health problems of the world. Antileishmanial drugs have disadvantages such as toxicity and the recent development of resistance. One of the best-known mechanisms of the antibacterial effects of silver nanoparticles (Ag-NPs) is the production of reactive oxygen species to which Leishmania parasites are very sensitive. So far no information about the effects of Ag-NPs on Leishmania tropica parasites, the causative agen...

  16. Synthesis and antileishmanial activity of new 1-Aryl-1H-Pyrazole-4- carboximidamides derivatives

    International Nuclear Information System (INIS)

    Chemotherapy for leishmaniasis, diseases caused by protozoa of the genus Leishmania, remains inefficient in several treatments. So there is a need to search for new drugs. In this work, we have synthesized 1-aryl-1H-pyrazole-4-carboximidamides derivatives and evaluated antileishmanial activities in vitro, as well as cytotoxic effects. Structure-activity relationship (SAR) studies were carried out with all the compounds of the series. Compound 2 showed an activity profile that can be improved through medicinal chemistry strategies. (author)

  17. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major

    OpenAIRE

    Behrouz Ezatpour; Ebrahim Saedi Dezaki; Hossein Mahmoudvand; Mojgan Azadpour; Fatemeh Ezzatkhah

    2015-01-01

    The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0–100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with ...

  18. Synthesis and antileishmanial activity of new 1-Aryl-1H-Pyrazole-4- carboximidamides derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Mauricio S. dos; Gomes, Adriana O.; Bernardino, Alice M.R.; Souza, Marcos C. de, E-mail: alicerolim@globo.co [Universidade Federal Fluminense (UFF), Niteroi, RJ (Brazil). Programa de Pos-Graduacao em Quimica Organica; Khan, Misbahul A. [The Islamia University of Bahawalpur (Pakistan). Chemistry Dept.; Brito, Monique A. de [Universidade Federal Fluminense (UFF), Niteroi, RJ (Brazil). Fac. de Farmacia. Lab. de Quimica Medicinal Computacional; Castro, Helena C.; Abreu, Paula A. [Universidade Federal Fluminense (LABioMol/GCM/UFF), Niteroi, RJ (Brazil). Inst. de Biologia. Lab. de Antibioticos, Bioquimica e Modelagem Molecular; Rodrigues, Carlos R. [Universidade Federal do Rio de Janeiro (ModMol/UFRJ), RJ (Brazil). Fac. de Farmacia. Lab. de Modelagem Molecular e QSAR; Leo, Rosa M.M. de; Leon, Leonor L.; Canto-Cavalheiro, Marilene M. [Fundacao Oswaldo Cruz (IOC/FIOCRUZ), Rio de Janeiro, RJ (Brazil). Instituto Oswaldo Cruz. Lab. de Bioquimica de Tripanosomatideos

    2011-07-01

    Chemotherapy for leishmaniasis, diseases caused by protozoa of the genus Leishmania, remains inefficient in several treatments. So there is a need to search for new drugs. In this work, we have synthesized 1-aryl-1H-pyrazole-4-carboximidamides derivatives and evaluated antileishmanial activities in vitro, as well as cytotoxic effects. Structure-activity relationship (SAR) studies were carried out with all the compounds of the series. Compound 2 showed an activity profile that can be improved through medicinal chemistry strategies. (author)

  19. Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-12-01

    Full Text Available A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1 plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET assay for high-throughput screening (HTS of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z’ factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS.

  20. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Science.gov (United States)

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  1. Novel Acoustic Loading of a Mass Spectrometer: Toward Next-Generation High-Throughput MS Screening.

    Science.gov (United States)

    Sinclair, Ian; Stearns, Rick; Pringle, Steven; Wingfield, Jonathan; Datwani, Sammy; Hall, Eric; Ghislain, Luke; Majlof, Lars; Bachman, Martin

    2016-02-01

    High-throughput, direct measurement of substrate-to-product conversion by label-free detection, without the need for engineered substrates or secondary assays, could be considered the "holy grail" of drug discovery screening. Mass spectrometry (MS) has the potential to be part of this ultimate screening solution, but is constrained by the limitations of existing MS sample introduction modes that cannot meet the throughput requirements of high-throughput screening (HTS). Here we report data from a prototype system (Echo-MS) that uses acoustic droplet ejection (ADE) to transfer femtoliter-scale droplets in a rapid, precise, and accurate fashion directly into the MS. The acoustic source can load samples into the MS from a microtiter plate at a rate of up to three samples per second. The resulting MS signal displays a very sharp attack profile and ions are detected within 50 ms of activation of the acoustic transducer. Additionally, we show that the system is capable of generating multiply charged ion species from simple peptides and large proteins. The combination of high speed and low sample volume has significant potential within not only drug discovery, but also other areas of the industry.

  2. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

    Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

  3. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  4. Computational Proteomics: High-throughput Analysis for Systems Biology

    Energy Technology Data Exchange (ETDEWEB)

    Cannon, William R.; Webb-Robertson, Bobbie-Jo M.

    2007-01-03

    High-throughput (HTP) proteomics is a rapidly developing field that offers the global profiling of proteins from a biological system. The HTP technological advances are fueling a revolution in biology, enabling analyses at the scales of entire systems (e.g., whole cells, tumors, or environmental communities). However, simply identifying the proteins in a cell is insufficient for understanding the underlying complexity and operating mechanisms of the overall system. Systems level investigations are relying more and more on computational analyses, especially in the field of proteomics generating large-scale global data.

  5. High Throughput WAN Data Transfer with Hadoop-based Storage

    International Nuclear Information System (INIS)

    Hadoop distributed file system (HDFS) is becoming more popular in recent years as a key building block of integrated grid storage solution in the field of scientific computing. Wide Area Network (WAN) data transfer is one of the important data operations for large high energy physics experiments to manage, share and process datasets of PetaBytes scale in a highly distributed grid computing environment. In this paper, we present the experience of high throughput WAN data transfer with HDFS-based Storage Element. Two protocols, GridFTP and fast data transfer (FDT), are used to characterize the network performance of WAN data transfer.

  6. High-throughput DNA sequencing: a genomic data manufacturing process.

    Science.gov (United States)

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  7. High-throughput Titration of Luciferase-expressing Recombinant Viruses

    OpenAIRE

    Garcia, Vanessa; Krishnan, Ramya; Davis, Colin; Batenchuk, Cory; Le Boeuf, Fabrice; Abdelbary, Hesham; Diallo, Jean-Simon

    2014-01-01

    Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appr...

  8. A Colloidal Stability Assay Suitable for High-Throughput Screening.

    Science.gov (United States)

    Abarca, Carla; Ali, M Monsur; Yang, Songtao; Dong, Xiaofei; Pelton, Robert H

    2016-03-01

    A library of 32 polystyrene copolymer latexes, with diameters ranging between 53 and 387 nm, was used to develop and demonstrate a high-throughput assay using a 96-well microplate platform to measure critical coagulation concentrations, a measure of colloidal stability. The most robust assay involved an automated centrifugation-decantation step to remove latex aggregates before absorbance measurements, eliminating aggregate interference with optical measurements made through the base of the multiwell plates. For smaller nanoparticles (diameter aggregation; however, the results were less sensitive than the absorbance measurements. PMID:26857643

  9. An improved high throughput sequencing method for studying oomycete communities

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Nicolaisen, Mogens

    2015-01-01

    Culture-independent studies using next generation sequencing have revolutionizedmicrobial ecology, however, oomycete ecology in soils is severely lagging behind. The aimof this study was to improve and validate standard techniques for using high throughput sequencing as a tool for studying oomyce...... usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification....

  10. Adaptive Sampling for High Throughput Data Using Similarity Measures

    Energy Technology Data Exchange (ETDEWEB)

    Bulaevskaya, V. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sales, A. P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-05-06

    The need for adaptive sampling arises in the context of high throughput data because the rates of data arrival are many orders of magnitude larger than the rates at which they can be analyzed. A very fast decision must therefore be made regarding the value of each incoming observation and its inclusion in the analysis. In this report we discuss one approach to adaptive sampling, based on the new data point’s similarity to the other data points being considered for inclusion. We present preliminary results for one real and one synthetic data set.

  11. 用于氯霉素、克伦特罗和雌二醇三种兽药残留检测的高通量悬浮芯片技术研究%Development of a high-throughput suspension microarray technology for detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol

    Institute of Scientific and Technical Information of China (English)

    刘楠; 苏璞; 高志贤; 朱茂祥; 杨陟华; 潘秀颉; 晁福寰

    2009-01-01

    . Conclusion The high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.%目的 建立一种氯霉素、克伦特罗和雌二醇(17-beta-estradiol,E2)的3种兽药残留的新型高通量悬浮芯片检测技术.方法 合成3种兽药的牛血清白蛋白(bovine serum albumin,BSA)结合物,并进行紫外和质谱鉴定.将3种蛋白结合物偶联于聚苯乙烯荧光微球上,在液相反应体系中3种小分子兽药抗原和微球上的结合物共同竞争液相中各自特异性的生物素化单抗,优化和筛选出微球上偶联BSA结合物和反应抗体的最适加入量.绘制出3种兽药残留检测的标准曲线;对不同浓度的干扰物和待测物分组,以此进行特异性检测和盲样测定.并用扫描电子显微镜(简称电镜)进行微球表面微观结构观察.结果 3种小分子兽药可与BSA成功偶联;3种结合物的加入量和抗体的加入量分别做了优化;悬浮芯片检测的标准曲线方程和方程相应的决定系数(R2)表现良好,R2>0.99;3种兽药悬浮芯片的检测区间分别为(40.00~6.25)×105ns/L,(50.00~7.81)×105ng/L和1.00×103~7.29×105ng/L;最低检出限为:40 ng/L、50 ng/L和1 μg/L;同时,悬浮芯片的特异度测试良好,与其他药物无明显交叉反应;对盲样测定的检测浓度值与实际浓度偏差在8.09%~17.03%,可认为偏差较小.电镜对微球表面微观结构的观察也直观地确证了蛋白在微球上的成功偶联.结论 高通量悬浮芯片技术操作简单,灵敏快速,成本低廉,为多种兽药残留的快速检测提供了新方法,具有广阔的应用和发展前景.

  12. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Xiaoming; Fu, Afu [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Luo, Kathy Qian, E-mail: kluo@ntu.edu.sg [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer An endothelial cell apoptosis assay using FRET-based biosensor was developed. Black-Right-Pointing-Pointer The fluorescence of the cells changed from green to blue during apoptosis. Black-Right-Pointing-Pointer This method was developed into a high-throughput assay in 96-well plates. Black-Right-Pointing-Pointer This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z Prime factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  13. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  14. High-throughput characterization for solar fuels materials discovery

    Science.gov (United States)

    Mitrovic, Slobodan; Becerra, Natalie; Cornell, Earl; Guevarra, Dan; Haber, Joel; Jin, Jian; Jones, Ryan; Kan, Kevin; Marcin, Martin; Newhouse, Paul; Soedarmadji, Edwin; Suram, Santosh; Xiang, Chengxiang; Gregoire, John; High-Throughput Experimentation Team

    2014-03-01

    In this talk I will present the status of the High-Throughput Experimentation (HTE) project of the Joint Center for Artificial Photosynthesis (JCAP). JCAP is an Energy Innovation Hub of the U.S. Department of Energy with a mandate to deliver a solar fuel generator based on an integrated photoelectrochemical cell (PEC). However, efficient and commercially viable catalysts or light absorbers for the PEC do not exist. The mission of HTE is to provide the accelerated discovery through combinatorial synthesis and rapid screening of material properties. The HTE pipeline also features high-throughput material characterization using x-ray diffraction and x-ray photoemission spectroscopy (XPS). In this talk I present the currently operating pipeline and focus on our combinatorial XPS efforts to build the largest free database of spectra from mixed-metal oxides, nitrides, sulfides and alloys. This work was performed at Joint Center for Artificial Photosynthesis, a DOE Energy Innovation Hub, supported through the Office of Science of the U.S. Department of Energy under Award No. DE-SC0004993.

  15. Human transcriptome array for high-throughput clinical studies

    Science.gov (United States)

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N.; Schweitzer, Anthony C.; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A.; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D.; Moldawer, Lyle L.; Maier, Ronald V.; Tompkins, Ronald G.; Wong, Wing Hung; Davis, Ronald W.; Xiao, Wenzhong; Toner, Mehmet; Warren, H. Shaw; Schoenfeld, David A.; Rahme, Laurence; McDonald-Smith, Grace P.; Hayden, Douglas; Mason, Philip; Fagan, Shawn; Yu, Yong-Ming; Cobb, J. Perren; Remick, Daniel G.; Mannick, John A.; Lederer, James A.; Gamelli, Richard L.; Silver, Geoffrey M.; West, Michael A.; Shapiro, Michael B.; Smith, Richard; Camp, David G.; Qian, Weijun; Tibshirani, Rob; Lowry, Stephen; Calvano, Steven; Chaudry, Irshad; Cohen, Mitchell; Moore, Ernest E.; Johnson, Jeffrey; Baker, Henry V.; Efron, Philip A.; Balis, Ulysses G. J.; Billiar, Timothy R.; Ochoa, Juan B.; Sperry, Jason L.; Miller-Graziano, Carol L.; De, Asit K.; Bankey, Paul E.; Herndon, David N.; Finnerty, Celeste C.; Jeschke, Marc G.; Minei, Joseph P.; Arnoldo, Brett D.; Hunt, John L.; Horton, Jureta; Cobb, J. Perren; Brownstein, Bernard; Freeman, Bradley; Nathens, Avery B.; Cuschieri, Joseph; Gibran, Nicole; Klein, Matthew; O'Keefe, Grant

    2011-01-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays. PMID:21317363

  16. High resolution hyperspectral imaging with a high throughput virtual slit

    Science.gov (United States)

    Gooding, Edward A.; Gunn, Thomas; Cenko, Andrew T.; Hajian, Arsen R.

    2016-05-01

    Hyperspectral imaging (HSI) device users often require both high spectral resolution, on the order of 1 nm, and high light-gathering power. A wide entrance slit assures reasonable étendue but degrades spectral resolution. Spectrometers built using High Throughput Virtual Slit™ (HTVS) technology optimize both parameters simultaneously. Two remote sensing use cases that require high spectral resolution are discussed. First, detection of atmospheric gases with intrinsically narrow absorption lines, such as hydrocarbon vapors or combustion exhaust gases such as NOx and CO2. Detecting exhaust gas species with high precision has become increasingly important in the light of recent events in the automobile industry. Second, distinguishing reflected daylight from emission spectra in the visible and NIR (VNIR) regions is most easily accomplished using the Fraunhofer absorption lines in solar spectra. While ground reflectance spectral features in the VNIR are generally quite broad, the Fraunhofer lines are narrow and provide a signature of intrinsic vs. extrinsic illumination. The High Throughput Virtual Slit enables higher spectral resolution than is achievable with conventional spectrometers by manipulating the beam profile in pupil space. By reshaping the instrument pupil with reflective optics, HTVS-equipped instruments create a tall, narrow image profile at the exit focal plane, typically delivering 5X or better the spectral resolution achievable with a conventional design.

  17. COMPUTER APPROACHES TO WHEAT HIGH-THROUGHPUT PHENOTYPING

    Directory of Open Access Journals (Sweden)

    Afonnikov D.

    2012-08-01

    Full Text Available The growing need for rapid and accurate approaches for large-scale assessment of phenotypic characters in plants becomes more and more obvious in the studies looking into relationships between genotype and phenotype. This need is due to the advent of high throughput methods for analysis of genomes. Nowadays, any genetic experiment involves data on thousands and dozens of thousands of plants. Traditional ways of assessing most phenotypic characteristics (those with reliance on the eye, the touch, the ruler are little effective on samples of such sizes. Modern approaches seek to take advantage of automated phenotyping, which warrants a much more rapid data acquisition, higher accuracy of the assessment of phenotypic features, measurement of new parameters of these features and exclusion of human subjectivity from the process. Additionally, automation allows measurement data to be rapidly loaded into computer databases, which reduces data processing time.In this work, we present the WheatPGE information system designed to solve the problem of integration of genotypic and phenotypic data and parameters of the environment, as well as to analyze the relationships between the genotype and phenotype in wheat. The system is used to consolidate miscellaneous data on a plant for storing and processing various morphological traits and genotypes of wheat plants as well as data on various environmental factors. The system is available at www.wheatdb.org. Its potential in genetic experiments has been demonstrated in high-throughput phenotyping of wheat leaf pubescence.

  18. Methods of high throughput biophysical characterization in biopharmaceutical development.

    Science.gov (United States)

    Razinkov, Vladimir I; Treuheit, Michael J; Becker, Gerald W

    2013-03-01

    Discovery and successful development of biopharmaceutical products depend on a thorough characterization of the molecule both before and after formulation. Characterization of a formulated biotherapeutic, typically a protein or large peptide, requires a rigorous assessment of the molecule's physical stability. Stability of a biotherapeutic includes not only chemical stability, i.e., degradation of the molecule to form undesired modifications, but also structural stability, including the formation of aggregates. In this review, high throughput biophysical characterization techniques are described according to their specific applications during biopharmaceutical discovery, development and manufacturing. The methods presented here are classified according to these attributes, and include spectroscopic assays based on absorbance, polarization, intrinsic and extrinsic fluorescence, surface plasmon resonance instrumentation, calorimetric methods, dynamic and static light scattering techniques, several visible particle counting and sizing methods, new viscosity assay, based on light scattering and mass spectrometry. Several techniques presented here are already implemented in industry; but, many high throughput biophysical methods are still in the initial stages of implementation or even in the prototype stage. Each technique in this report is judged by the specific application of the method through the biopharmaceutical development process. PMID:22725690

  19. High-throughput TVD-based simulation of tracer flow

    Energy Technology Data Exchange (ETDEWEB)

    Wattenbarger, R.C.; Aziz, K.; Orr, F.M. Jr. [Stanford Univ., CA (United States)

    1995-12-31

    High-throughput, total-variation-diminishing based, numerical techniques for simulating tracer flow are developed. High-throughput (HT) timestepping enables explicit differencing techniques to use timesteps that are larger than allowed by normal stability constraints. Total-variation-diminishing (TVD) based techniques are a means of controlling the solution oscillations associated with high-order differencing schemes. The concept of `upstream stability` is introduced to help combine HT timestepping with TVD-based differencing. Additionally, an improved HT timestepping algorithm for solving physically dispersive flow problems is developed. Numerical experiments indicate that HT timestepping is most beneficial for solving highly convective flows that have both a physical Peclet number and a diffusion-only numerical Peclet number greater than one. Experiments comparing standard and alternating direction TVD-based techniques with both the Superbee (SB) and third-order based (L3) limiters indicate that the TVD-SB scheme is the most accurate for purely convective problems. For physically dispersive problems, the TVD-SB scheme may be too compressive and the TVD-L3 is recommended. The alternating direction TVD scheme is the least sensitive to timestepping and the most sensitive to grid-orientation.

  20. Human transcriptome array for high-throughput clinical studies.

    Science.gov (United States)

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N; Schweitzer, Anthony C; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D; Moldawer, Lyle L; Maier, Ronald V; Tompkins, Ronald G; Wong, Wing Hung; Davis, Ronald W; Xiao, Wenzhong

    2011-03-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

  1. High throughput instruments, methods, and informatics for systems biology.

    Energy Technology Data Exchange (ETDEWEB)

    Sinclair, Michael B.; Cowie, Jim R. (New Mexico State University, Las Cruces, NM); Van Benthem, Mark Hilary; Wylie, Brian Neil; Davidson, George S.; Haaland, David Michael; Timlin, Jerilyn Ann; Aragon, Anthony D. (University of New Mexico, Albuquerque, NM); Keenan, Michael Robert; Boyack, Kevin W.; Thomas, Edward Victor; Werner-Washburne, Margaret C. (University of New Mexico, Albuquerque, NM); Mosquera-Caro, Monica P. (University of New Mexico, Albuquerque, NM); Martinez, M. Juanita (University of New Mexico, Albuquerque, NM); Martin, Shawn Bryan; Willman, Cheryl L. (University of New Mexico, Albuquerque, NM)

    2003-12-01

    High throughput instruments and analysis techniques are required in order to make good use of the genomic sequences that have recently become available for many species, including humans. These instruments and methods must work with tens of thousands of genes simultaneously, and must be able to identify the small subsets of those genes that are implicated in the observed phenotypes, or, for instance, in responses to therapies. Microarrays represent one such high throughput method, which continue to find increasingly broad application. This project has improved microarray technology in several important areas. First, we developed the hyperspectral scanner, which has discovered and diagnosed numerous flaws in techniques broadly employed by microarray researchers. Second, we used a series of statistically designed experiments to identify and correct errors in our microarray data to dramatically improve the accuracy, precision, and repeatability of the microarray gene expression data. Third, our research developed new informatics techniques to identify genes with significantly different expression levels. Finally, natural language processing techniques were applied to improve our ability to make use of online literature annotating the important genes. In combination, this research has improved the reliability and precision of laboratory methods and instruments, while also enabling substantially faster analysis and discovery.

  2. A high throughput mechanical screening device for cartilage tissue engineering.

    Science.gov (United States)

    Mohanraj, Bhavana; Hou, Chieh; Meloni, Gregory R; Cosgrove, Brian D; Dodge, George R; Mauck, Robert L

    2014-06-27

    Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying 'hits', or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.

  3. Fluorescent foci quantitation for high-throughput analysis

    Science.gov (United States)

    Ledesma-Fernández, Elena; Thorpe, Peter H.

    2015-01-01

    A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells. PMID:26290880

  4. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major.

    Science.gov (United States)

    Ezatpour, Behrouz; Saedi Dezaki, Ebrahim; Mahmoudvand, Hossein; Azadpour, Mojgan; Ezzatkhah, Fatemeh

    2015-01-01

    The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0-100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL. PMID:25815025

  5. N-(4-((E)-3-arylacryloyl)phenyl)acetamide derivatives and their antileishmanial activity

    Energy Technology Data Exchange (ETDEWEB)

    Pacheco, Dency J.; Trilleras, Jorge; Prent, Luis; Coaves, Tobinson, E-mail: jorgetrilleras@mail.uniatlantico.edu.co [Universidad del Atlantico, Barranquilla-Atlantico (Colombia). Facultad de Ciencias Basicas. Grupo de Investigacion en Compuestos Heterociclicos; Quiroga, Jairo [Universidad del Valle, Cali (Colombia). Dept. de Quimica. Grupo de Investigacion de Compuestos Heterociclicos; Gutierrez, Jennifer; Delgado, Gabriela [Universidad Nacional de Colombia, Bogota, D.C. (Colombia). Facultad de Ciencias. Departamento de Farmacia. Grupo de Investigacion en Inmunotoxicologia; Marin, Juan C. [Universidad Nacional de Colombia, Bogota, D.C. (Colombia). Facultad de Ciencias. Departamento de Farmacia. Grupo de Investigacion Farmacognosia y Fitoquimica

    2013-10-15

    The antileishmanial activity of a series of enonic derivatives (chalcones) synthesized via Claisen-Schmidt condensation reactions assisted by ultrasonic radiation was characterized by analyzing their cytotoxicity against Leishmania (Viannia) panamensis promastigotes, a species responsible for over 90% of Leishmania cases in Colombia. Two compounds were active against Leishmania with selectivity indexes of LC{sub 50} EC{sub 50} {sup -1} (lethal concentration 50 and effective concentration 50) higher than 27 and 3, respectively. These results suggest that a substitution on one of the two chalcone rings (aromatic ring A) with oxygen is convenient. Compound 3g should be further investigated for its antileishmanial activity, especially for being easy to obtain in high yields, making it possible to produce drugs for the treatment of cutaneous leishmaniasis. (author)

  6. Antileishmanial, Toxicity, and Phytochemical Evaluation of Medicinal Plants Collected from Pakistan

    Directory of Open Access Journals (Sweden)

    Naseer Ali Shah

    2014-01-01

    Full Text Available Leishmaniasis is an important parasitic problem and is in focus for development of new drugs all over the world. Objective of the present study was to evaluate phytochemical, toxicity, and antileishmanial potential of Jurinea dolomiaea, Asparagus gracilis, Sida cordata, and Stellaria media collected from different areas of Pakistan. Dry powder of plants was extracted with crude methanol and fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, and water solvents in escalating polarity order. Qualitative phytochemical analysis of different class of compounds, that is, alkaloids, saponins, terpenoids, anthraquinones, cardiac glycosides, coumarins, phlobatannins, flavonoids, phenolics, and tannins, was tested. Its appearance was observed varying with polarity of solvent used for fractionation. Antileishmanial activity was performed against Leishmania tropica KWH23 promastigote. Potent antileishmanial activity was observed for J. dolomiaea methanol extract (IC50=10.9±1.1 μg/mL in comparison to other plant extracts. However, J. dolomiaea “ethyl acetate fraction” was more active (IC50=5.3±0.2 μg/mL against Leishmania tropica KWH23 among all plant fractions as well as standard Glucantime drug (6.0±0.1 μg/mL. All the plants extract and its derived fraction exhibited toxicity in safety range (LC50 >100 in brine shrimp toxicity evaluation assay.

  7. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    OpenAIRE

    Isobe Masaharu; Yoshioka Megumi; Kurosawa Nobuyuki

    2011-01-01

    Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-am...

  8. Mutation Scanning Using MUT-MAP, a High-Throughput, Microfluidic Chip-Based, Multi-Analyte Panel

    OpenAIRE

    Patel, Rajesh; Tsan, Alison; Tam, Rachel; Desai, Rupal; Schoenbrunner, Nancy; Myers, Thomas W.; Bauer, Keith; Smith, Edward; Raja, Rajiv

    2012-01-01

    Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific P...

  9. Gold nanoparticles for high-throughput genotyping of long-range haplotypes

    Science.gov (United States)

    Chen, Peng; Pan, Dun; Fan, Chunhai; Chen, Jianhua; Huang, Ke; Wang, Dongfang; Zhang, Honglu; Li, You; Feng, Guoyin; Liang, Peiji; He, Lin; Shi, Yongyong

    2011-10-01

    Completion of the Human Genome Project and the HapMap Project has led to increasing demands for mapping complex traits in humans to understand the aetiology of diseases. Identifying variations in the DNA sequence, which affect how we develop disease and respond to pathogens and drugs, is important for this purpose, but it is difficult to identify these variations in large sample sets. Here we show that through a combination of capillary sequencing and polymerase chain reaction assisted by gold nanoparticles, it is possible to identify several DNA variations that are associated with age-related macular degeneration and psoriasis on significant regions of human genomic DNA. Our method is accurate and promising for large-scale and high-throughput genetic analysis of susceptibility towards disease and drug resistance.

  10. Accessible high-throughput virtual screening molecular docking software for students and educators.

    Directory of Open Access Journals (Sweden)

    Reed B Jacob

    2012-05-01

    Full Text Available We survey low cost high-throughput virtual screening (HTVS computer programs for instructors who wish to demonstrate molecular docking in their courses. Since HTVS programs are a useful adjunct to the time consuming and expensive wet bench experiments necessary to discover new drug therapies, the topic of molecular docking is core to the instruction of biochemistry and molecular biology. The availability of HTVS programs coupled with decreasing costs and advances in computer hardware have made computational approaches to drug discovery possible at institutional and non-profit budgets. This paper focuses on HTVS programs with graphical user interfaces (GUIs that use either DOCK or AutoDock for the prediction of DockoMatic, PyRx, DockingServer, and MOLA since their utility has been proven by the research community, they are free or affordable, and the programs operate on a range of computer platforms.

  11. Metabolic network analysis predicts efficacy of FDA-approved drugs targeting the causative agent of a neglected tropical disease

    Directory of Open Access Journals (Sweden)

    Chavali Arvind K

    2012-04-01

    Full Text Available Abstract Background Systems biology holds promise as a new approach to drug target identification and drug discovery against neglected tropical diseases. Genome-scale metabolic reconstructions, assembled from annotated genomes and a vast array of bioinformatics/biochemical resources, provide a framework for the interrogation of human pathogens and serve as a platform for generation of future experimental hypotheses. In this article, with the application of selection criteria for both Leishmania major targets (e.g. in silico gene lethality and drugs (e.g. toxicity, a method (MetDP to rationally focus on a subset of low-toxic Food and Drug Administration (FDA-approved drugs is introduced. Results This metabolic network-driven approach identified 15 L. major genes as high-priority targets, 8 high-priority synthetic lethal targets, and 254 FDA-approved drugs. Results were compared to previous literature findings and existing high-throughput screens. Halofantrine, an antimalarial agent that was prioritized using MetDP, showed noticeable antileishmanial activity when experimentally evaluated in vitro against L. major promastigotes. Furthermore, synthetic lethality predictions also aided in the prediction of superadditive drug combinations. For proof-of-concept, double-drug combinations were evaluated in vitro against L. major and four combinations involving the drug disulfiram that showed superadditivity are presented. Conclusions A direct metabolic network-driven method that incorporates single gene essentiality and synthetic lethality predictions is proposed that generates a set of high-priority L. major targets, which are in turn associated with a select number of FDA-approved drugs that are candidate antileishmanials. Additionally, selection of high-priority double-drug combinations might provide for an attractive and alternative avenue for drug discovery against leishmaniasis.

  12. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    International Nuclear Information System (INIS)

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication

  13. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells. PMID:26309218

  14. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  15. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control.

    Science.gov (United States)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L James; Lu, Wu

    2016-01-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

  16. High throughput generation and trapping of individual agarose microgel using microfluidic approach

    KAUST Repository

    Shi, Yang

    2013-02-28

    Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

  17. Fast high-throughput screening of temoporfin-loaded liposomal formulations prepared by ethanol injection method.

    Science.gov (United States)

    Yang, Kewei; Delaney, Joseph T; Schubert, Ulrich S; Fahr, Alfred

    2012-03-01

    A new strategy for fast, convenient high-throughput screening of liposomal formulations was developed, utilizing the automation of the so-called ethanol-injection method. This strategy was illustrated by the preparation and screening of the liposomal formulation library of a potent second-generation photosensitizer, temoporfin. Numerous liposomal formulations were efficiently prepared using a pipetting robot, followed by automated size characterization, using a dynamic light scattering plate reader. Incorporation efficiency of temoporfin and zeta potential were also detected in selected cases. To optimize the formulation, different parameters were investigated, including lipid types, lipid concentration in injected ethanol, ratio of ethanol to aqueous solution, ratio of drug to lipid, and the addition of functional phospholipid. Step-by-step small liposomes were prepared with high incorporation efficiency. At last, an optimized formulation was obtained for each lipid in the following condition: 36.4 mg·mL(-1) lipid, 13.1 mg·mL(-1) mPEG(2000)-DSPE, and 1:4 ethanol:buffer ratio. These liposomes were unilamellar spheres, with a diameter of approximately 50 nm, and were very stable for over 20 weeks. The results illustrate this approach to be promising for fast high-throughput screening of liposomal formulations.

  18. High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling.

    Science.gov (United States)

    Trubetskoy, O V; Finel, M; Kurkela, M; Fitzgerald, M; Peters, N R; Hoffman, F M; Trubetskoy, V S

    2007-06-01

    Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.

  19. High-throughput microcavitation bubble induced cellular mechanotransduction

    Science.gov (United States)

    Compton, Jonathan Lee

    inhibitor to IP 3 induced Ca2+ release. This capability opens the development of a high-throughput screening platform for molecules that modulate cellular mechanotransduction. We have applied this approach to screen the effects of a small set of small molecules, in a 96-well plate in less than an hour. These detailed studies offer a basis for the design, development, and implementation of a novel high-throughput mechanotransduction assay to rapidly screen the effect of small molecules on cellular mechanotransduction at high throughput.

  20. High-throughput rheology in a microfluidic device

    Science.gov (United States)

    Furst, Eric; Schultz, Kelly; Han, Hyejin; Kim, Chongyoup

    2011-11-01

    High-throughput rheological measurements in a microfluidic device are demonstrated. A series of microrheology samples is generated as droplets in an immiscible spacer fluid using a microfluidic T-junction. The compositions of the sample droplets are continuously varied over a wide range. Rheology measurements are made in each droplet using multiple particle tracking microrheology. We review critical design and operating parameters, including the droplet size, flow rates and rapid fabrication methods. Validation experiments are performed by measuring the solution viscosity of glycerine and the biopolymer heparin as a function of concentration. Finally, an analysis of droplet mixing is performed in order to optimize the device performance. Overall, the combination of microrheology with microfluidics maximizes the number of rheological measurements while simultaneously minimizing the sample preparation time and amount of material, and should be particularly suited to the characterization of scarce or expensive materials. We acknowledge financial support from the NSF (CBET-0730292).

  1. High-throughput ab-initio dilute solute diffusion database.

    Science.gov (United States)

    Wu, Henry; Mayeshiba, Tam; Morgan, Dane

    2016-01-01

    We demonstrate automated generation of diffusion databases from high-throughput density functional theory (DFT) calculations. A total of more than 230 dilute solute diffusion systems in Mg, Al, Cu, Ni, Pd, and Pt host lattices have been determined using multi-frequency diffusion models. We apply a correction method for solute diffusion in alloys using experimental and simulated values of host self-diffusivity. We find good agreement with experimental solute diffusion data, obtaining a weighted activation barrier RMS error of 0.176 eV when excluding magnetic solutes in non-magnetic alloys. The compiled database is the largest collection of consistently calculated ab-initio solute diffusion data in the world. PMID:27434308

  2. Interactive Visual Analysis of High Throughput Text Streams

    Energy Technology Data Exchange (ETDEWEB)

    Steed, Chad A [ORNL; Potok, Thomas E [ORNL; Patton, Robert M [ORNL; Goodall, John R [ORNL; Maness, Christopher S [ORNL; Senter, James K [ORNL; Potok, Thomas E [ORNL

    2012-01-01

    The scale, velocity, and dynamic nature of large scale social media systems like Twitter demand a new set of visual analytics techniques that support near real-time situational awareness. Social media systems are credited with escalating social protest during recent large scale riots. Virtual communities form rapidly in these online systems, and they occasionally foster violence and unrest which is conveyed in the users language. Techniques for analyzing broad trends over these networks or reconstructing conversations within small groups have been demonstrated in recent years, but state-of- the-art tools are inadequate at supporting near real-time analysis of these high throughput streams of unstructured information. In this paper, we present an adaptive system to discover and interactively explore these virtual networks, as well as detect sentiment, highlight change, and discover spatio- temporal patterns.

  3. The Principals and Practice of Distributed High Throughput Computing

    CERN Document Server

    CERN. Geneva

    2016-01-01

    The potential of Distributed Processing Systems to deliver computing capabilities with qualities ranging from high availability and reliability to easy expansion in functionality and capacity were recognized and formalized in the 1970’s. For more three decade these principals Distributed Computing guided the development of the HTCondor resource and job management system. The widely adopted suite of software tools offered by HTCondor are based on novel distributed computing technologies and are driven by the evolving needs of High Throughput scientific applications. We will review the principals that underpin our work, the distributed computing frameworks and technologies we developed and the lessons we learned from delivering effective and dependable software tools in an ever changing landscape computing technologies and needs that range today from a desktop computer to tens of thousands of cores offered by commercial clouds. About the speaker Miron Livny received a B.Sc. degree in Physics and Mat...

  4. Multiple-injection high-throughput gas chromatography analysis.

    Science.gov (United States)

    Schafer, Wes; Wang, Heather; Welch, Christopher J

    2016-08-01

    Multiple-injection techniques have been shown to be a simple way to perform high-throughput analysis where the entire experiment resides in a single chromatogram, simplifying the data analysis and interpretation. In this study, multiple-injection techniques are applied to gas chromatography with flame ionization detection and mass detection to significantly increase sample throughput. The unique issues of implementing a traditional "Fast" injection mode of multiple-injection techniques with gas chromatography and mass spectrometry are discussed. Stacked injections are also discussed as means to increase the throughput of longer methods where mass detection is unable to distinguish between analytes of the same mass and longer retentions are required to resolve components of interest. Multiple-injection techniques are shown to increase instrument throughput by up to 70% and to simplify data analysis, allowing hits in multiple parallel experiments to be identified easily. PMID:27292909

  5. Microfluidics for High-Throughput Quantitative Studies of Early Development.

    Science.gov (United States)

    Levario, Thomas J; Lim, Bomyi; Shvartsman, Stanislav Y; Lu, Hang

    2016-07-11

    Developmental biology has traditionally relied on qualitative analyses; recently, however, as in other fields of biology, researchers have become increasingly interested in acquiring quantitative knowledge about embryogenesis. Advances in fluorescence microscopy are enabling high-content imaging in live specimens. At the same time, microfluidics and automation technologies are increasing experimental throughput for studies of multicellular models of development. Furthermore, computer vision methods for processing and analyzing bioimage data are now leading the way toward quantitative biology. Here, we review advances in the areas of fluorescence microscopy, microfluidics, and data analysis that are instrumental to performing high-content, high-throughput studies in biology and specifically in development. We discuss a case study of how these techniques have allowed quantitative analysis and modeling of pattern formation in the Drosophila embryo. PMID:26928208

  6. Proposed high throughput electrorefining treatment for spent N- Reactor fuel

    International Nuclear Information System (INIS)

    A high-throughput electrorefining process is being adapted to treat spent N-Reactor fuel for ultimate disposal in a geologic repository. Anodic dissolution tests were made with unirradiated N-Reactor fuel to determine the type of fragmentation necessary to provide fuel segments suitable for this process. Based on these tests, a conceptual design was produced of a plant-scale electrorefiner. In this design, the diameter of an electrode assembly is about 1.07 m (42 in.). Three of these assemblies in an electrorefiner would accommodate a 3-metric-ton batch of N-Reactor fuel that would be processed at a rate of 42 kg of uranium per hour

  7. UAV-based high-throughput phenotyping in legume crops

    Science.gov (United States)

    Sankaran, Sindhuja; Khot, Lav R.; Quirós, Juan; Vandemark, George J.; McGee, Rebecca J.

    2016-05-01

    In plant breeding, one of the biggest obstacles in genetic improvement is the lack of proven rapid methods for measuring plant responses in field conditions. Therefore, the major objective of this research was to evaluate the feasibility of utilizing high-throughput remote sensing technology for rapid measurement of phenotyping traits in legume crops. The plant responses of several chickpea and peas varieties to the environment were assessed with an unmanned aerial vehicle (UAV) integrated with multispectral imaging sensors. Our preliminary assessment showed that the vegetation indices are strongly correlated (p<0.05) with seed yield of legume crops. Results endorse the potential of UAS-based sensing technology to rapidly measure those phenotyping traits.

  8. Dimensioning storage and computing clusters for efficient High Throughput Computing

    CERN Document Server

    CERN. Geneva

    2012-01-01

    Scientific experiments are producing huge amounts of data, and they continue increasing the size of their datasets and the total volume of data. These data are then processed by researchers belonging to large scientific collaborations, with the Large Hadron Collider being a good example. The focal point of Scientific Data Centres has shifted from coping efficiently with PetaByte scale storage to deliver quality data processing throughput. The dimensioning of the internal components in High Throughput Computing (HTC) data centers is of crucial importance to cope with all the activities demanded by the experiments, both the online (data acceptance) and the offline (data processing, simulation and user analysis). This requires a precise setup involving disk and tape storage services, a computing cluster and the internal networking to prevent bottlenecks, overloads and undesired slowness that lead to losses cpu cycles and batch jobs failures. In this paper we point out relevant features for running a successful s...

  9. A high-throughput chemically induced inflammation assay in zebrafish

    Directory of Open Access Journals (Sweden)

    Liebel Urban

    2010-12-01

    Full Text Available Abstract Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

  10. High throughput phenotyping for aphid resistance in large plant collections

    Directory of Open Access Journals (Sweden)

    Chen Xi

    2012-08-01

    Full Text Available Abstract Background Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. Results In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer and the circulative virus Turnip yellows virus (TuYV. In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. Conclusions This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.

  11. A Primer on High-Throughput Computing for Genomic Selection

    Directory of Open Access Journals (Sweden)

    Xiao-Lin eWu

    2011-02-01

    Full Text Available High-throughput computing (HTC uses computer clusters to solve advanced computational problems, with the goal of accomplishing high throughput over relatively long periods of time. In genomic selection, for example, a set of markers covering the entire genome is used to train a model based on known data, and the resulting model is used to predict the genetic merit of selection candidates. Sophisticated models are very computationally demanding and, with several traits to be evaluated sequentially, computing time is long and output is low. In this paper, we present scenarios and basic principles of how HTC can be used in genomic selection, implemented using various techniques from simple batch processing to pipelining in distributed computer clusters. Various scripting languages, such as shell scripting, Perl and R, are also very useful to devise pipelines. By pipelining, we can reduce total computing time and consequently increase throughput. In comparison to the traditional data processing pipeline residing on the central processors, performing general purpose computation on a graphics processing unit (GPU provide a new-generation approach to massive parallel computing in genomic selection. While the concept of HTC may still be new to many researchers in animal breeding, plant breeding, and genetics, HTC infrastructures have already been built in many institutions, such as the University of Wisconsin – Madison, which can be leveraged for genomic selection, in terms of central processing unit (CPU capacity, network connectivity, storage availability, and middleware connectivity. Exploring existing HTC infrastructures as well as general purpose computing environments will further expand our capability to meet increasing computing demands posed by unprecedented genomic data that we have today. We anticipate that HTC will impact genomic selection via better statistical models, faster solutions, and more competitive products (e.g., from design of

  12. High Throughput SNP Genotyping with Two Mini-sequencing Assays

    Institute of Scientific and Technical Information of China (English)

    Chunqing LUO; Libin DENG; Changqing ZENG

    2004-01-01

    Two mini-sequencing methods,FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry),were optimized.A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay,thus made the optimization work easier.At the same time,using multi-PCR technology,8-plex genotyping of MassArray assay was successfully carried out,some softwares were developed and the data process of MassArray was highly automated.Then these two methods were applied to high throughput SNP genotyping,the accuracy,efficiency and robustness were compared.The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product,as well as extension cycles,the SNPprimer length of FP-TDI should be 24-30 bp long,whereas MassArray assay prefers to be as short as only 16 bp.Altogether 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals,4792 sites by FP-TDI assay and 1648 sites by MassArray assay,the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively.The throughput of MassArray was higher than FP-TDI,and the cost of MassArray was lower,MassArray was more suitable for high throughput SNP genotyping.

  13. A novel function for kojic acid, a secondary metabolite from Aspergillus fungi, as antileishmanial agent.

    Directory of Open Access Journals (Sweden)

    Ana Paula D Rodrigues

    Full Text Available Kojic acid (KA is a fungal metabolite used as a topical treatment skin-whitening cosmetic agent for melasma in humans; however its potential as an anti-leishmanial agent is unknown. Chemotherapy is one of the most effective treatments for Leishmaniasis. However, the drugs available are expensive, invasive, require long-term treatment and have severe side effects. Thus, the development of new effective leishmanicidal agents is a necessity. In this study we investigated the anti-leishmanial effect of KA on L. amazonensis, following in vitro and in vivo infections. KA (50 μg/mL was found to decrease the growth by 62% (IC50 34 μg/mL and 79% (IC50 27.84 μg/mL of promastigotes and amastigotes in vitro, respectively. Ultrastructural analysis of KA-treated amastigotes showed the presence of vesicles bodies into the flagellar pocket, and an intense intracellular vacuolization and swelling of the mitochondrion. During the in vitro interaction of parasites and the host cell, KA reverses the superoxide anions (O2- inhibitory mechanism promoted by parasite. In addition, 4 weeks after KA-topical formulation treatment of infected animals, a healing process was observed with a high production of collagen fibers and a decrease in parasite burden. Thus, these results demonstrated the great potential of KA as an anti-leishmanial compound.

  14. Statistical removal of background signals from high-throughput 1H NMR line-broadening ligand-affinity screens

    International Nuclear Information System (INIS)

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction

  15. Statistical removal of background signals from high-throughput {sup 1}H NMR line-broadening ligand-affinity screens

    Energy Technology Data Exchange (ETDEWEB)

    Worley, Bradley; Sisco, Nicholas J.; Powers, Robert, E-mail: rpowers3@unl.edu [University of Nebraska-Lincoln, Department of Chemistry (United States)

    2015-09-15

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional {sup 1}H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein {sup 1}H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction.

  16. Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Elizabeth Bilsland

    2016-01-01

    Full Text Available Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7-15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts, and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds, we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident

  17. Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light

    Directory of Open Access Journals (Sweden)

    Allahverdiyev AM

    2011-11-01

    Full Text Available Adil M Allahverdiyev1, Emrah Sefik Abamor1, Malahat Bagirova1, Cem B Ustundag2, Cengiz Kaya2, Figen Kaya2, Miriam Rafailovich3 1Department of Bioengineering; 2Department of Metallurgical and Materials Engineering, Yildiz Technical University, Esenler, Istanbul, Turkey; 3Department of Materials Science and Engineering, Stony Brook University, Stony Brook, NY, USA Abstract: Leishmaniasis is a protozoan vector-borne disease and is one of the biggest health problems of the world. Antileishmanial drugs have disadvantages such as toxicity and the recent development of resistance. One of the best-known mechanisms of the antibacterial effects of silver nanoparticles (Ag-NPs is the production of reactive oxygen species to which Leishmania parasites are very sensitive. So far no information about the effects of Ag-NPs on Leishmania tropica parasites, the causative agent of leishmaniasis, exists in the literature. The aim of this study was to investigate the effects of Ag-NPs on biological parameters of L. tropica such as morphology, metabolic activity, proliferation, infectivity, and survival in host cells, in vitro. Consequently, parasite morphology and infectivity were impaired in comparison with the control. Also, enhanced effects of Ag-NPs were demonstrated on the morphology and infectivity of parasites under ultraviolet (UV light. Ag-NPs demonstrated significant antileishmanial effects by inhibiting the proliferation and metabolic activity of promastigotes by 1.5- to threefold, respectively, in the dark, and 2- to 6.5-fold, respectively, under UV light. Of note, Ag-NPs inhibited the survival of amastigotes in host cells, and this effect was more significant in the presence of UV light. Thus, for the first time the antileishmanial effects of Ag-NPs on L. tropica parasites were demonstrated along with the enhanced antimicrobial activity of Ag-NPs under UV light. Determination of the antileishmanial effects of Ag-NPs is very important for the further

  18. Antileishmanial effect of silver nanoparticles and their enhanced antiparasitic activity under ultraviolet light.

    Science.gov (United States)

    Allahverdiyev, Adil M; Abamor, Emrah Sefik; Bagirova, Malahat; Ustundag, Cem B; Kaya, Cengiz; Kaya, Figen; Rafailovich, Miriam

    2011-01-01

    Leishmaniasis is a protozoan vector-borne disease and is one of the biggest health problems of the world. Antileishmanial drugs have disadvantages such as toxicity and the recent development of resistance. One of the best-known mechanisms of the antibacterial effects of silver nanoparticles (Ag-NPs) is the production of reactive oxygen species to which Leishmania parasites are very sensitive. So far no information about the effects of Ag-NPs on Leishmania tropica parasites, the causative agent of leishmaniasis, exists in the literature. The aim of this study was to investigate the effects of Ag-NPs on biological parameters of L. tropica such as morphology, metabolic activity, proliferation, infectivity, and survival in host cells, in vitro. Consequently, parasite morphology and infectivity were impaired in comparison with the control. Also, enhanced effects of Ag-NPs were demonstrated on the morphology and infectivity of parasites under ultraviolet (UV) light. Ag-NPs demonstrated significant antileishmanial effects by inhibiting the proliferation and metabolic activity of promastigotes by 1.5- to threefold, respectively, in the dark, and 2- to 6.5-fold, respectively, under UV light. Of note, Ag-NPs inhibited the survival of amastigotes in host cells, and this effect was more significant in the presence of UV light. Thus, for the first time the antileishmanial effects of Ag-NPs on L. tropica parasites were demonstrated along with the enhanced antimicrobial activity of Ag-NPs under UV light. Determination of the antileishmanial effects of Ag-NPs is very important for the further development of new compounds containing nanoparticles in leishmaniasis treatment. PMID:22114501

  19. Assessment of network perturbation amplitudes by applying high-throughput data to causal biological networks

    Directory of Open Access Journals (Sweden)

    Martin Florian

    2012-05-01

    Full Text Available Abstract Background High-throughput measurement technologies produce data sets that have the potential to elucidate the biological impact of disease, drug treatment, and environmental agents on humans. The scientific community faces an ongoing challenge in the analysis of these rich data sources to more accurately characterize biological processes that have been perturbed at the mechanistic level. Here, a new approach is built on previous methodologies in which high-throughput data was interpreted using prior biological knowledge of cause and effect relationships. These relationships are structured into network models that describe specific biological processes, such as inflammatory signaling or cell cycle progression. This enables quantitative assessment of network perturbation in response to a given stimulus. Results Four complementary methods were devised to quantify treatment-induced activity changes in processes described by network models. In addition, companion statistics were developed to qualify significance and specificity of the results. This approach is called Network Perturbation Amplitude (NPA scoring because the amplitudes of treatment-induced perturbations are computed for biological network models. The NPA methods were tested on two transcriptomic data sets: normal human bronchial epithelial (NHBE cells treated with the pro-inflammatory signaling mediator TNFα, and HCT116 colon cancer cells treated with the CDK cell cycle inhibitor R547. Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach. The NPA scoring method successfully quantified the amplitude of TNFα-induced perturbation for each network model when compared against NF-κB nuclear localization and cell number. In addition, the degree and specificity to which CDK

  20. Robust ridge regression estimators for nonlinear models with applications to high throughput screening assay data.

    Science.gov (United States)

    Lim, Changwon

    2015-03-30

    Nonlinear regression is often used to evaluate the toxicity of a chemical or a drug by fitting data from a dose-response study. Toxicologists and pharmacologists may draw a conclusion about whether a chemical is toxic by testing the significance of the estimated parameters. However, sometimes the null hypothesis cannot be rejected even though the fit is quite good. One possible reason for such cases is that the estimated standard errors of the parameter estimates are extremely large. In this paper, we propose robust ridge regression estimation procedures for nonlinear models to solve this problem. The asymptotic properties of the proposed estimators are investigated; in particular, their mean squared errors are derived. The performances of the proposed estimators are compared with several standard estimators using simulation studies. The proposed methodology is also illustrated using high throughput screening assay data obtained from the National Toxicology Program. PMID:25490981

  1. Towards automated high-throughput screening of C. elegans on agar

    CERN Document Server

    Kabra, Mayank; O'Rourke, Eyleen J; Xie, Xin; Ljosa, Vebjorn; Jones, Thouis R; Ausubel, Frederick M; Ruvkun, Gary; Carpenter, Anne E; Freund, Yoav

    2010-01-01

    High-throughput screening (HTS) using model organisms is a promising method to identify a small number of genes or drugs potentially relevant to human biology or disease. In HTS experiments, robots and computers do a significant portion of the experimental work. However, one remaining major bottleneck is the manual analysis of experimental results, which is commonly in the form of microscopy images. This manual inspection is labor intensive, slow and subjective. Here we report our progress towards applying computer vision and machine learning methods to analyze HTS experiments that use Caenorhabditis elegans (C. elegans) worms grown on agar. Our main contribution is a robust segmentation algorithm for separating the worms from the background using brightfield images. We also show that by combining the output of this segmentation algorithm with an algorithm to detect the fluorescent dye, Nile Red, we can reliably distinguish different fluorescence-based phenotypes even though the visual differences are subtle....

  2. NETTAB 2014: From high-throughput structural bioinformatics to integrative systems biology.

    Science.gov (United States)

    Romano, Paolo; Cordero, Francesca

    2016-03-02

    The fourteenth NETTAB workshop, NETTAB 2014, was devoted to a range of disciplines going from structural bioinformatics, to proteomics and to integrative systems biology. The topics of the workshop were centred around bioinformatics methods, tools, applications, and perspectives for models, standards and management of high-throughput biological data, structural bioinformatics, functional proteomics, mass spectrometry, drug discovery, and systems biology.43 scientific contributions were presented at NETTAB 2014, including keynote, special guest and tutorial talks, oral communications, and posters. Full papers from some of the best contributions presented at the workshop were later submitted to a special Call for this Supplement.Here, we provide an overview of the workshop and introduce manuscripts that have been accepted for publication in this Supplement.

  3. Surface modification of thermoplastics--towards the plastic biochip for high throughput screening devices.

    Science.gov (United States)

    Diaz-Quijada, Gerardo A; Peytavi, Régis; Nantel, André; Roy, Emmanuel; Bergeron, Michel G; Dumoulin, Michel M; Veres, Teodor

    2007-07-01

    Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is great interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) Zeonor 1060R and Zeonex E48R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotides were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.

  4. A high-throughput approach for identification of novel general anesthetics.

    Directory of Open Access Journals (Sweden)

    Wendy A Lea

    Full Text Available Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC(1280 library of drug-like compounds. A raw hit rate of approximately 15% was reduced through the use of computational filters to yield an overall hit rate of approximately 1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.

  5. Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

    Science.gov (United States)

    Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

    2016-02-01

    Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio.

  6. Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro.In this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction.IN exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in disintegration.Baicalein,a known IN inhibitor,was found to be an IN-CCD inhibitor.The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.

  7. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting.

    Science.gov (United States)

    Tseng, Hubert; Gage, Jacob A; Haisler, William L; Neeley, Shane K; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G; Wagoner, Matthew; Souza, Glauco R

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  8. Antileishmanial activity and cytotoxicity of Brazilian plants.

    Science.gov (United States)

    Ribeiro, Tatiana G; Chávez-Fumagalli, Miguel A; Valadares, Diogo G; Franca, Juçara R; Lage, Paula S; Duarte, Mariana C; Andrade, Pedro H R; Martins, Vivian T; Costa, Lourena E; Arruda, Ana L A; Faraco, André A G; Coelho, Eduardo A F; Castilho, Rachel O

    2014-08-01

    Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance has increased the importance of discovering new therapeutic products. The present study aimed to investigate the in vitro leishmanicidal activity from 16 different Brazilian medicinal plants. Stationary-phase promastigotes of Leishmania amazonensis and murine macrophages were exposed to 44 plant extracts or fractions for 48 h at 37°C, in order to evaluate their antileishmanial activity and cytotoxicity, respectively. The most potent extracts against L. amazonensis were the hexanic extract of Dipteryx alata (IC50 of 0.08 μg/mL), the hexanic extract of Syzygium cumini (IC50 of 31.64 μg/mL), the ethanolic and hexanic extracts of leaves of Hymenaea courbaril (IC50 of 44.10 μg/mL and 35.84 μg/mL, respectively), the ethanolic extract of H. stignocarpa (IC50 of 4.69 μg/mL), the ethanolic extract of Jacaranda caroba (IC50 of 13.22 μg/mL), and the ethanolic extract of J. cuspidifolia leaves (IC50 of 10.96 μg/mL). Extracts of D. alata and J. cuspidifolia presented higher selectivity index, with high leishmanicidal activity and low cytotoxicity in the mammalian cells. The capacity in treated infected macrophages using the extracts and/or fractions of D. alata and J. cuspidifolia was also analyzed, and reductions of 95.80%, 98.31%, and 97.16%, respectively, in the parasite burden, were observed. No nitric oxide (NO) production could be observed in the treated macrophages, after stimulation with the extracts and/or fractions of D. alata and J. cuspidifolia, suggesting that the biological activity could be due to mechanisms other than macrophage activation mediated by NO production. Based on phytochemistry studies, the classes of compounds that could contribute to the observed activities are also discussed. In conclusion, the data presented in this study indicated that traditional medicinal plant extracts present effective antileishmanial activity. Future studies could focus on

  9. Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

    Science.gov (United States)

    Rice, Christopher A; Colon, Beatrice L; Alp, Mehmet; Göker, Hakan; Boykin, David W; Kyle, Dennis E

    2015-04-01

    Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of 500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri.

  10. A high-throughput screen for teratogens using human pluripotent stem cells.

    Science.gov (United States)

    Kameoka, Sei; Babiarz, Joshua; Kolaja, Kyle; Chiao, Eric

    2014-01-01

    There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.

  11. High throughput optoelectronic smart pixel systems using diffractive optics

    Science.gov (United States)

    Chen, Chih-Hao

    1999-12-01

    Recent developments in digital video, multimedia technology and data networks have greatly increased the demand for high bandwidth communication channels and high throughput data processing. Electronics is particularly suited for switching, amplification and logic functions, while optics is more suitable for interconnections and communications with lower energy and crosstalk. In this research, we present the design, testing, integration and demonstration of several optoelectronic smart pixel devices and system architectures. These systems integrate electronic switching/processing capability with parallel optical interconnections to provide high throughput network communication and pipeline data processing. The Smart Pixel Array Cellular Logic processor (SPARCL) is designed in 0.8 m m CMOS and hybrid integrated with Multiple-Quantum-Well (MQW) devices for pipeline image processing. The Smart Pixel Network Interface (SAPIENT) is designed in 0.6 m m GaAs and monolithically integrated with LEDs to implement a highly parallel optical interconnection network. The Translucent Smart Pixel Array (TRANSPAR) design is implemented in two different versions. The first version, TRANSPAR-MQW, is designed in 0.5 m m CMOS and flip-chip integrated with MQW devices to provide 2-D pipeline processing and translucent networking using the Carrier- Sense-MultipleAccess/Collision-Detection (CSMA/CD) protocol. The other version, TRANSPAR-VM, is designed in 1.2 m m CMOS and discretely integrated with VCSEL-MSM (Vertical-Cavity-Surface- Emitting-Laser and Metal-Semiconductor-Metal detectors) chips and driver/receiver chips on a printed circuit board. The TRANSPAR-VM provides an option of using the token ring network protocol in addition to the embedded functions of TRANSPAR-MQW. These optoelectronic smart pixel systems also require micro-optics devices to provide high resolution, high quality optical interconnections and external source arrays. In this research, we describe an innovative

  12. A primer on high-throughput computing for genomic selection.

    Science.gov (United States)

    Wu, Xiao-Lin; Beissinger, Timothy M; Bauck, Stewart; Woodward, Brent; Rosa, Guilherme J M; Weigel, Kent A; Gatti, Natalia de Leon; Gianola, Daniel

    2011-01-01

    High-throughput computing (HTC) uses computer clusters to solve advanced computational problems, with the goal of accomplishing high-throughput over relatively long periods of time. In genomic selection, for example, a set of markers covering the entire genome is used to train a model based on known data, and the resulting model is used to predict the genetic merit of selection candidates. Sophisticated models are very computationally demanding and, with several traits to be evaluated sequentially, computing time is long, and output is low. In this paper, we present scenarios and basic principles of how HTC can be used in genomic selection, implemented using various techniques from simple batch processing to pipelining in distributed computer clusters. Various scripting languages, such as shell scripting, Perl, and R, are also very useful to devise pipelines. By pipelining, we can reduce total computing time and consequently increase throughput. In comparison to the traditional data processing pipeline residing on the central processors, performing general-purpose computation on a graphics processing unit provide a new-generation approach to massive parallel computing in genomic selection. While the concept of HTC may still be new to many researchers in animal breeding, plant breeding, and genetics, HTC infrastructures have already been built in many institutions, such as the University of Wisconsin-Madison, which can be leveraged for genomic selection, in terms of central processing unit capacity, network connectivity, storage availability, and middleware connectivity. Exploring existing HTC infrastructures as well as general-purpose computing environments will further expand our capability to meet increasing computing demands posed by unprecedented genomic data that we have today. We anticipate that HTC will impact genomic selection via better statistical models, faster solutions, and more competitive products (e.g., from design of marker panels to realized

  13. Parallel tools in HEVC for high-throughput processing

    Science.gov (United States)

    Zhou, Minhua; Sze, Vivienne; Budagavi, Madhukar

    2012-10-01

    HEVC (High Efficiency Video Coding) is the next-generation video coding standard being jointly developed by the ITU-T VCEG and ISO/IEC MPEG JCT-VC team. In addition to the high coding efficiency, which is expected to provide 50% more bit-rate reduction when compared to H.264/AVC, HEVC has built-in parallel processing tools to address bitrate, pixel-rate and motion estimation (ME) throughput requirements. This paper describes how CABAC, which is also used in H.264/AVC, has been redesigned for improved throughput, and how parallel merge/skip and tiles, which are new tools introduced for HEVC, enable high-throughput processing. CABAC has data dependencies which make it difficult to parallelize and thus limit its throughput. The prediction error/residual, represented as quantized transform coefficients, accounts for the majority of the CABAC workload. Various improvements have been made to the context selection and scans in transform coefficient coding that enable CABAC in HEVC to potentially achieve higher throughput and increased coding gains relative to H.264/AVC. The merge/skip mode is a coding efficiency enhancement tool in HEVC; the parallel merge/skip breaks dependency between the regular and merge/skip ME, which provides flexibility for high throughput and high efficiency HEVC encoder designs. For ultra high definition (UHD) video, such as 4kx2k and 8kx4k resolutions, low-latency and real-time processing may be beyond the capability of a single core codec. Tiles are an effective tool which enables pixel-rate balancing among the cores to achieve parallel processing with a throughput scalable implementation of multi-core UHD video codec. With the evenly divided tiles, a multi-core video codec can be realized by simply replicating single core codec and adding a tile boundary processing core on top of that. These tools illustrate that accounting for implementation cost when designing video coding algorithms can enable higher processing speed and reduce

  14. DOSE RESPONSE FROM HIGH THROUGHPUT GENE EXPRESSION STUDIES AND THE INFLUENCE OF TIME AND CELL LINE ON INFERRED MODE OF ACTION BY ONTOLOGIC ENRICHMENT (SOT)

    Science.gov (United States)

    Gene expression with ontologic enrichment and connectivity mapping tools is widely used to infer modes of action (MOA) for therapeutic drugs. Despite progress in high-throughput (HT) genomic systems, strategies suitable to identify industrial chemical MOA are needed. The L1000 is...

  15. Brazilian Propolis Antileishmanial and Immunomodulatory Effects

    Directory of Open Access Journals (Sweden)

    Suelen Santos da Silva

    2013-01-01

    Full Text Available The antileishmanial and immunomodulatory effects of propolis collected in Botucatu, São Paulo State, Brazil, were evaluated in Leishmania (Viannia braziliensis experimental infection. The antileishmanial effect of propolis on promastigote forms was verified by reducing growth and by promoting morphologic alterations observed by scanning electron microscopy. In in vitro immunomodulatory assays, macrophages were pretreated with propolis and then infected with L. (V. braziliensis. In vivo, supernatants from liver cells and peritoneal exudate of BALB/c mice pretreated with propolis and infected with Leishmania (107/mL promastigotes were collected, and TNF-α and IL-12 were measured by ELISA. Macrophages incubated with propolis showed a significant increase in interiorization and further killing of parasites. An increased TNF-α production was seen in mice pretreated with propolis, whereas IL-12 was downregulated during the infection. In conclusion, Brazilian propolis showed a direct action on the parasite and displayed immunomodulatory effects on murine macrophages, even though the parasite has been reported to affect the activation pathways of the cell. The observed effects could be associated with the presence of phenolic compounds (flavonoids, aromatic acids, and benzopyranes, di- and triterpenes, and essential oils found in our propolis sample.

  16. High Throughput Multispectral Image Processing with Applications in Food Science.

    Directory of Open Access Journals (Sweden)

    Panagiotis Tsakanikas

    Full Text Available Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing's outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples.

  17. A microfluidic, high throughput protein crystal growth method for microgravity.

    Directory of Open Access Journals (Sweden)

    Carl W Carruthers

    Full Text Available The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS. The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD, as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3 cm.. After 70 days on the ISS, our samples were returned with 16 of 25 (64% microgravity cards having crystals, compared to 12 of 25 (48% of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

  18. High-throughput single-cell manipulation in brain tissue.

    Directory of Open Access Journals (Sweden)

    Joseph D Steinmeyer

    Full Text Available The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.

  19. Adaptation to high throughput batch chromatography enhances multivariate screening.

    Science.gov (United States)

    Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

    2015-09-01

    High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. PMID:25914370

  20. High-Throughput Preparation of New Photoactive Nanocomposites.

    Science.gov (United States)

    Conterosito, Eleonora; Benesperi, Iacopo; Toson, Valentina; Saccone, Davide; Barbero, Nadia; Palin, Luca; Barolo, Claudia; Gianotti, Valentina; Milanesio, Marco

    2016-06-01

    New low-cost photoactive hybrid materials based on organic luminescent molecules inserted into hydrotalcite (layered double hydroxides; LDH) were produced, which exploit the high-throughput liquid-assisted grinding (LAG) method. These materials are conceived for applications in dye-sensitized solar cells (DSSCs) as a co-absorbers and in silicon photovoltaic (PV) panels to improve their efficiency as they are able to emit where PV modules show the maximum efficiency. A molecule that shows a large Stokes' shift was designed, synthesized, and intercalated into LDH. Two dyes already used in DSSCs were also intercalated to produce two new nanocomposites. LDH intercalation allows the stability of organic dyes to be improved and their direct use in polymer melt blending. The prepared nanocomposites absorb sunlight from UV to visible and emit from blue to near-IR and thus can be exploited for light-energy management. Finally one nanocomposite was dispersed by melt blending into a poly(methyl methacrylate)-block-poly(n-butyl acrylate) copolymer to obtain a photoactive film.

  1. A fully automated high-throughput training system for rodents.

    Directory of Open Access Journals (Sweden)

    Rajesh Poddar

    Full Text Available Addressing the neural mechanisms underlying complex learned behaviors requires training animals in well-controlled tasks, an often time-consuming and labor-intensive process that can severely limit the feasibility of such studies. To overcome this constraint, we developed a fully computer-controlled general purpose system for high-throughput training of rodents. By standardizing and automating the implementation of predefined training protocols within the animal's home-cage our system dramatically reduces the efforts involved in animal training while also removing human errors and biases from the process. We deployed this system to train rats in a variety of sensorimotor tasks, achieving learning rates comparable to existing, but more laborious, methods. By incrementally and systematically increasing the difficulty of the task over weeks of training, rats were able to master motor tasks that, in complexity and structure, resemble ones used in primate studies of motor sequence learning. By enabling fully automated training of rodents in a home-cage setting this low-cost and modular system increases the utility of rodents for studying the neural underpinnings of a variety of complex behaviors.

  2. Probabilistic Assessment of High-Throughput Wireless Sensor Networks.

    Science.gov (United States)

    Kim, Robin E; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved. PMID:27258270

  3. The JCSG high-throughput structural biology pipeline.

    Science.gov (United States)

    Elsliger, Marc André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wooley, John; Wüthrich, Kurt; Wilson, Ian A

    2010-10-01

    The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.

  4. High Throughput T Epitope Mapping and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Giuseppina Li Pira

    2010-01-01

    Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

  5. Probabilistic Assessment of High-Throughput Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Robin E. Kim

    2016-05-01

    Full Text Available Structural health monitoring (SHM using wireless smart sensors (WSS has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved.

  6. Probabilistic Assessment of High-Throughput Wireless Sensor Networks.

    Science.gov (United States)

    Kim, Robin E; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved.

  7. High Throughput Multispectral Image Processing with Applications in Food Science.

    Science.gov (United States)

    Tsakanikas, Panagiotis; Pavlidis, Dimitris; Nychas, George-John

    2015-01-01

    Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT) in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing's outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models) and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples.

  8. A reproducible, high throughput method for fabricating fibrin gels

    Directory of Open Access Journals (Sweden)

    Murphy Kaitlin C

    2012-08-01

    Full Text Available Abstract Background Fibrin gels are a promising biomaterial for tissue engineering. However, current fabrication methods are time intensive with inherent variation. There is a pressing need to develop new and consistent approaches for producing fibrin-based hydrogels for examination. Findings We developed a high throughput method for creating fibrin gels using molds fabricated from polydimethylsiloxane (PDMS. Fibrin gels were produced by adding solutions of fibrinogen and thrombin to cylindrical defects in a PDMS sheet. Undisturbed gels were collected by removing the sheet, and fibrin gels were characterized. The characteristics of resulting gels were compared to published data by measuring compressive stiffness and osteogenic response of entrapped human mesenchymal stem cells (MSCs. Gels exhibited compressive moduli nearly identical to our previously reported fabrication method. Trends in alkaline phosphatase activity, an early marker of osteogenic differentiation in MSCs, were also consistent with previous data. Conclusions These findings demonstrate a streamlined approach to fibrin gel production that drastically reduces the time required to make fibrin gels, while also reducing variability between gel batches. This fabrication technique provides a valuable tool for generating large numbers of gels in a cost-effective manner.

  9. High-Throughput Preparation of New Photoactive Nanocomposites.

    Science.gov (United States)

    Conterosito, Eleonora; Benesperi, Iacopo; Toson, Valentina; Saccone, Davide; Barbero, Nadia; Palin, Luca; Barolo, Claudia; Gianotti, Valentina; Milanesio, Marco

    2016-06-01

    New low-cost photoactive hybrid materials based on organic luminescent molecules inserted into hydrotalcite (layered double hydroxides; LDH) were produced, which exploit the high-throughput liquid-assisted grinding (LAG) method. These materials are conceived for applications in dye-sensitized solar cells (DSSCs) as a co-absorbers and in silicon photovoltaic (PV) panels to improve their efficiency as they are able to emit where PV modules show the maximum efficiency. A molecule that shows a large Stokes' shift was designed, synthesized, and intercalated into LDH. Two dyes already used in DSSCs were also intercalated to produce two new nanocomposites. LDH intercalation allows the stability of organic dyes to be improved and their direct use in polymer melt blending. The prepared nanocomposites absorb sunlight from UV to visible and emit from blue to near-IR and thus can be exploited for light-energy management. Finally one nanocomposite was dispersed by melt blending into a poly(methyl methacrylate)-block-poly(n-butyl acrylate) copolymer to obtain a photoactive film. PMID:27137753

  10. Assessing the utility and limitations of high throughput virtual screening

    Directory of Open Access Journals (Sweden)

    Paul Daniel Phillips

    2016-05-01

    Full Text Available Due to low cost, speed, and unmatched ability to explore large numbers of compounds, high throughput virtual screening and molecular docking engines have become widely utilized by computational scientists. It is generally accepted that docking engines, such as AutoDock, produce reliable qualitative results for ligand-macromolecular receptor binding, and molecular docking results are commonly reported in literature in the absence of complementary wet lab experimental data. In this investigation, three variants of the sixteen amino acid peptide, α-conotoxin MII, were docked to a homology model of the a3β2-nicotinic acetylcholine receptor. DockoMatic version 2.0 was used to perform a virtual screen of each peptide ligand to the receptor for ten docking trials consisting of 100 AutoDock cycles per trial. The results were analyzed for both variation in the calculated binding energy obtained from AutoDock, and the orientation of bound peptide within the receptor. The results show that, while no clear correlation exists between consistent ligand binding pose and the calculated binding energy, AutoDock is able to determine a consistent positioning of bound peptide in the majority of trials when at least ten trials were evaluated.

  11. High-Throughput Network Communication with NetIO

    CERN Document Server

    Schumacher, J\\"orn; The ATLAS collaboration

    2016-01-01

    HPC network technologies like Infiniband, TrueScale or OmniPath provide low-latency and high-throughput communication between hosts, which makes them attractive options for data-acquisition systems in large-scale high-energy physics experiments. Like HPC networks, DAQ networks are local and include a well specified number of systems. Unfortunately traditional network communication APIs for HPC clusters like MPI or PGAS target exclusively the HPC community and are not suited well for DAQ applications. It is possible to build distributed DAQ applications using low-level system APIs like Infiniband Verbs (and this has been done), but it requires a non negligible effort and expert knowledge. On the other hand, message services like 0MQ have gained popularity in the HEP community. Such APIs allow to build distributed applications with a high-level approach and provide good performance. Unfortunately their usage usually limits developers to TCP/IP-based networks. While it is possible to operate a TCP/IP stack on to...

  12. High Throughput Interrogation of Behavioral Transitions in C. elegans

    Science.gov (United States)

    Liu, Mochi; Shaevitz, Joshua; Leifer, Andrew

    We present a high-throughput method to probe transformations from neural activity to behavior in Caenorhabditis elegans to better understand how organisms change behavioral states. We optogenetically deliver white-noise stimuli to target sensory or inter neurons while simultaneously recording the movement of a population of worms. Using all the postural movement data collected, we computationally classify stereotyped behaviors in C. elegans by clustering based on the spectral properties of the instantaneous posture. (Berman et al., 2014) Transitions between these behavioral clusters indicate discrete behavioral changes. To study the neural correlates dictating these transitions, we perform model-driven experiments and employ Linear-Nonlinear-Poisson cascades that take the white-noise stimulus as the input. The parameters of these models are fitted by reverse-correlation from our measurements. The parameterized models of behavioral transitions predict the worm's response to novel stimuli and reveal the internal computations the animal makes before carrying out behavioral decisions. Preliminary results are shown that describe the neural-behavioral transformation between neural activity in mechanosensory neurons and reversal behavior.

  13. High-throughput analysis of protein-DNA binding affinity.

    Science.gov (United States)

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  14. Management of High-Throughput DNA Sequencing Projects: Alpheus.

    Science.gov (United States)

    Miller, Neil A; Kingsmore, Stephen F; Farmer, Andrew; Langley, Raymond J; Mudge, Joann; Crow, John A; Gonzalez, Alvaro J; Schilkey, Faye D; Kim, Ryan J; van Velkinburgh, Jennifer; May, Gregory D; Black, C Forrest; Myers, M Kathy; Utsey, John P; Frost, Nicholas S; Sugarbaker, David J; Bueno, Raphael; Gullans, Stephen R; Baxter, Susan M; Day, Steve W; Retzel, Ernest F

    2008-12-26

    High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem's SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis. PMID:20151039

  15. Advances in High Throughput Screening of Biomass Recalcitrance (Poster)

    Energy Technology Data Exchange (ETDEWEB)

    Turner, G. B.; Decker, S. R.; Tucker, M. P.; Law, C.; Doeppke, C.; Sykes, R. W.; Davis, M. F.; Ziebell, A.

    2012-06-01

    This was a poster displayed at the Symposium. Advances on previous high throughput screening of biomass recalcitrance methods have resulted in improved conversion and replicate precision. Changes in plate reactor metallurgy, improved preparation of control biomass, species-specific pretreatment conditions, and enzymatic hydrolysis parameters have reduced overall coefficients of variation to an average of 6% for sample replicates. These method changes have improved plate-to-plate variation of control biomass recalcitrance and improved confidence in sugar release differences between samples. With smaller errors plant researchers can have a higher degree of assurance more low recalcitrance candidates can be identified. Significant changes in plate reactor, control biomass preparation, pretreatment conditions and enzyme have significantly reduced sample and control replicate variability. Reactor plate metallurgy significantly impacts sugar release aluminum leaching into reaction during pretreatment degrades sugars and inhibits enzyme activity. Removal of starch and extractives significantly decreases control biomass variability. New enzyme formulations give more consistent and higher conversion levels, however required re-optimization for switchgrass. Pretreatment time and temperature (severity) should be adjusted to specific biomass types i.e. woody vs. herbaceous. Desalting of enzyme preps to remove low molecular weight stabilizers and improved conversion levels likely due to water activity impacts on enzyme structure and substrate interactions not attempted here due to need to continually desalt and validate precise enzyme concentration and activity.

  16. Probabilistic Assessment of High-Throughput Wireless Sensor Networks

    Science.gov (United States)

    Kim, Robin E.; Mechitov, Kirill; Sim, Sung-Han; Spencer, Billie F.; Song, Junho

    2016-01-01

    Structural health monitoring (SHM) using wireless smart sensors (WSS) has the potential to provide rich information on the state of a structure. However, because of their distributed nature, maintaining highly robust and reliable networks can be challenging. Assessing WSS network communication quality before and after finalizing a deployment is critical to achieve a successful WSS network for SHM purposes. Early studies on WSS network reliability mostly used temporal signal indicators, composed of a smaller number of packets, to assess the network reliability. However, because the WSS networks for SHM purpose often require high data throughput, i.e., a larger number of packets are delivered within the communication, such an approach is not sufficient. Instead, in this study, a model that can assess, probabilistically, the long-term performance of the network is proposed. The proposed model is based on readily-available measured data sets that represent communication quality during high-throughput data transfer. Then, an empirical limit-state function is determined, which is further used to estimate the probability of network communication failure. Monte Carlo simulation is adopted in this paper and applied to a small and a full-bridge wireless networks. By performing the proposed analysis in complex sensor networks, an optimized sensor topology can be achieved. PMID:27258270

  17. High Throughput, Continuous, Mass Production of Photovoltaic Modules

    Energy Technology Data Exchange (ETDEWEB)

    Kurt Barth

    2008-02-06

    AVA Solar has developed a very low cost solar photovoltaic (PV) manufacturing process and has demonstrated the significant economic and commercial potential of this technology. This I & I Category 3 project provided significant assistance toward accomplishing these milestones. The original goals of this project were to design, construct and test a production prototype system, fabricate PV modules and test the module performance. The original module manufacturing costs in the proposal were estimated at $2/Watt. The objectives of this project have been exceeded. An advanced processing line was designed, fabricated and installed. Using this automated, high throughput system, high efficiency devices and fully encapsulated modules were manufactured. AVA Solar has obtained 2 rounds of private equity funding, expand to 50 people and initiated the development of a large scale factory for 100+ megawatts of annual production. Modules will be manufactured at an industry leading cost which will enable AVA Solar's modules to produce power that is cost-competitive with traditional energy resources. With low manufacturing costs and the ability to scale manufacturing, AVA Solar has been contacted by some of the largest customers in the PV industry to negotiate long-term supply contracts. The current market for PV has continued to grow at 40%+ per year for nearly a decade and is projected to reach $40-$60 Billion by 2012. Currently, a crystalline silicon raw material supply shortage is limiting growth and raising costs. Our process does not use silicon, eliminating these limitations.

  18. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  19. High-Throughput Neuroimaging-Genetics Computational Infrastructure

    Directory of Open Access Journals (Sweden)

    Ivo D Dinov

    2014-04-01

    Full Text Available Many contemporary neuroscientific investigations face significant challenges in terms of data management, computational processing, data mining and results interpretation. These four pillars define the core infrastructure necessary to plan, organize, orchestrate, validate and disseminate novel scientific methods, computational resources and translational healthcare findings. Data management includes protocols for data acquisition, archival, query, transfer, retrieval and aggregation. Computational processing involves the necessary software, hardware and networking infrastructure required to handle large amounts of heterogeneous neuroimaging, genetics, clinical and phenotypic data and meta-data. In this manuscript we describe the novel high-throughput neuroimaging-genetics computational infrastructure available at the Institute for Neuroimaging and Informatics (INI and the Laboratory of Neuro Imaging (LONI at University of Southern California (USC. INI and LONI include ultra-high-field and standard-field MRI brain scanners along with an imaging-genetics database for storing the complete provenance of the raw and derived data and meta-data. A unique feature of this architecture is the Pipeline environment, which integrates the data management, processing, transfer and visualization. Through its client-server architecture, the Pipeline environment provides a graphical user interface for designing, executing, monitoring validating, and disseminating of complex protocols that utilize diverse suites of software tools and web-services. These pipeline workflows are represented as portable XML objects which transfer the execution instructions and user specifications from the client user machine to remote pipeline servers for distributed computing. Using Alzheimer’s and Parkinson’s data, we provide several examples of translational applications using this infrastructure.

  20. Scanning fluorescence detector for high-throughput DNA genotyping

    Science.gov (United States)

    Rusch, Terry L.; Petsinger, Jeremy; Christensen, Carl; Vaske, David A.; Brumley, Robert L., Jr.; Luckey, John A.; Weber, James L.

    1996-04-01

    A new scanning fluorescence detector (SCAFUD) was developed for high-throughput genotyping of short tandem repeat polymorphisms (STRPs). Fluorescent dyes are incorporated into relatively short DNA fragments via polymerase chain reaction (PCR) and are separated by electrophoresis in short, wide polyacrylamide gels (144 lanes with well to read distances of 14 cm). Excitation light from an argon laser with primary lines at 488 and 514 nm is introduced into the gel through a fiber optic cable, dichroic mirror, and 40X microscope objective. Emitted fluorescent light is collected confocally through a second fiber. The confocal head is translated across the bottom of the gel at 0.5 Hz. The detection unit utilizes dichroic mirrors and band pass filters to direct light with 10 - 20 nm bandwidths to four photomultiplier tubes (PMTs). PMT signals are independently amplified with variable gain and then sampled at a rate of 2500 points per scan using a computer based A/D board. LabView software (National Instruments) is used for instrument operation. Currently, three fluorescent dyes (Fam, Hex and Rox) are simultaneously detected with peak detection wavelengths of 543, 567, and 613 nm, respectively. The detection limit for fluorescein-labeled primers is about 100 attomoles. Planned SCAFUD upgrades include rearrangement of laser head geometry, use of additional excitation lasers for simultaneous detection of more dyes, and the use of detector arrays instead of individual PMTs. Extensive software has been written for automatic analysis of SCAFUD images. The software enables background subtraction, band identification, multiple- dye signal resolution, lane finding, band sizing and allele calling. Whole genome screens are currently underway to search for loci influencing such complex diseases as diabetes, asthma, and hypertension. Seven production SCAFUDs are currently in operation. Genotyping output for the coming year is projected to be about one million total genotypes (DNA

  1. High-throughput screening method for lipases/esterases.

    Science.gov (United States)

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

  2. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    Energy Technology Data Exchange (ETDEWEB)

    Ni, J.

    2000-09-21

    This work describes several research projects aimed towards developing new instruments and novel methods for high throughput chemical and biological analysis. Approaches are taken in two directions. The first direction takes advantage of well-established semiconductor fabrication techniques and applies them to miniaturize instruments that are workhorses in analytical laboratories. Specifically, the first part of this work focused on the development of micropumps and microvalves for controlled fluid delivery. The mechanism of these micropumps and microvalves relies on the electrochemically-induced surface tension change at a mercury/electrolyte interface. A miniaturized flow injection analysis device was integrated and flow injection analyses were demonstrated. In the second part of this work, microfluidic chips were also designed, fabricated, and tested. Separations of two fluorescent dyes were demonstrated in microfabricated channels, based on an open-tubular liquid chromatography (OT LC) or an electrochemically-modulated liquid chromatography (EMLC) format. A reduction in instrument size can potentially increase analysis speed, and allow exceedingly small amounts of sample to be analyzed under diverse separation conditions. The second direction explores the surface enhanced Raman spectroscopy (SERS) as a signal transduction method for immunoassay analysis. It takes advantage of the improved detection sensitivity as a result of surface enhancement on colloidal gold, the narrow width of Raman band, and the stability of Raman scattering signals to distinguish several different species simultaneously without exploiting spatially-separated addresses on a biochip. By labeling gold nanoparticles with different Raman reporters in conjunction with different detection antibodies, a simultaneous detection of a dual-analyte immunoassay was demonstrated. Using this scheme for quantitative analysis was also studied and preliminary dose-response curves from an immunoassay of a

  3. Establishment of a High-Throughput Assay to Monitor Influenza A Virus RNA Transcription and Replication.

    Directory of Open Access Journals (Sweden)

    Zhen Wang

    Full Text Available Influenza A virus (IAV poses significant threats to public health because of the recent emergence of highly pathogenic strains and wide-spread resistance to available anti-influenza drugs. Therefore, new antiviral targets and new drugs to fight influenza virus infections are needed. Although IAV RNA transcription/replication represents a promising target for antiviral drug development, no assay ideal for high-throughput screening (HTS application is currently available to identify inhibitors targeting these processes. In this work, we developed a novel HTS assay to analyze the transcription and replication of IAV RNA using an A549 cell line stably expressing IAV RNA-dependent RNA polymerase (RdRp complex, NP and a viral mini-genomic RNA. Both secreted Gaussia luciferase (Gluc and blasticidin resistance gene (Bsd were encoded in the viral minigenome and expressed under the control of IAV RdRp. Gluc serves as a reporter to monitor the activity of IAV RdRp, and Bsd is used to maintain the expression of all foreign genes. Biochemical studies and the statistical analysis presented herein demonstrate the high specificity, sensitivity and reproducibility of the assay. This work provides an ideal HTS assay for the identification of inhibitors targeting the function of IAV RdRp and a convenient reporting system for mechanism study of IAV RNA transcription / replication.

  4. Experimentally validated novel inhibitors of Helicobacter pylori phosphopantetheine adenylyltransferase discovered by virtual high-throughput screening.

    Directory of Open Access Journals (Sweden)

    Chao-Sheng Cheng

    Full Text Available Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT. PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.

  5. Benchmarking Ligand-Based Virtual High-Throughput Screening with the PubChem Database

    Directory of Open Access Journals (Sweden)

    Mariusz Butkiewicz

    2013-01-01

    Full Text Available With the rapidly increasing availability of High-Throughput Screening (HTS data in the public domain, such as the PubChem database, methods for ligand-based computer-aided drug discovery (LB-CADD have the potential to accelerate and reduce the cost of probe development and drug discovery efforts in academia. We assemble nine data sets from realistic HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods. Each data set is public domain through PubChem and carefully collated through confirmation screens validating active compounds. These data sets provide the foundation for benchmarking a new cheminformatics framework BCL::ChemInfo, which is freely available for non-commercial use. Quantitative structure activity relationship (QSAR models are built using Artificial Neural Networks (ANNs, Support Vector Machines (SVMs, Decision Trees (DTs, and Kohonen networks (KNs. Problem-specific descriptor optimization protocols are assessed including Sequential Feature Forward Selection (SFFS and various information content measures. Measures of predictive power and confidence are evaluated through cross-validation, and a consensus prediction scheme is tested that combines orthogonal machine learning algorithms into a single predictor. Enrichments ranging from 15 to 101 for a TPR cutoff of 25% are observed.

  6. Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

    Science.gov (United States)

    Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

  7. Recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers

    DEFF Research Database (Denmark)

    Andersen, Lars Dyrskjøt; Zieger, Karsten; Ørntoft, Torben Falck

    2007-01-01

    individually contributed to the management of the disease. However, the development of high-throughput techniques for simultaneous assessment of a large number of markers has allowed classification of tumors into clinically relevant molecular subgroups beyond those possible by pathological classification. Here......, we review the recent advances in high-throughput molecular marker identification for superficial and invasive bladder cancers....

  8. In-vitro Evaluation of Antileishmanial Activity and Toxicity of Artemether with Focus on its Apoptotic Effect

    OpenAIRE

    Ebrahimisadr, Parisa; Ghaffarifar, Fatemeh; Mohammad Hassan, Zuhair

    2013-01-01

    Artemisinin and its derivatives are very important new class of antimalarial drugs. One of the most important artemisinin derivatives is artemether. The antiparasitic activity of artemether as a derivative of artemisinin is related to endoperoxide bridge in its structure. The aim of this study was the evaluation of antileishmanial effect of artemether, with more focus on its apoptotic effect. In this study we used artemether in concentration of 5, 10, 25, 50 and 100 μg/mL for promastigote ass...

  9. High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries.

    Science.gov (United States)

    Pérez Del Palacio, José; Díaz, Caridad; de la Cruz, Mercedes; Annang, Frederick; Martín, Jesús; Pérez-Victoria, Ignacio; González-Menéndez, Víctor; de Pedro, Nuria; Tormo, José R; Algieri, Francesca; Rodriguez-Nogales, Alba; Rodríguez-Cabezas, M Elena; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; Gálvez, Julio

    2016-07-01

    It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and a high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. In this work, we present a validated high-throughput screening approach in 96-well plate format for the discovery of new molecules with anti-inflammatory/immunomodulatory activity. The in vitro models are based on the quantitation of nitrite levels in RAW264.7 murine macrophages and interleukin-8 in Caco-2 cells. We have used this platform in a pilot project to screen a subset of 5976 noncytotoxic crude microbial extracts from the MEDINA microbial natural product collection. To our knowledge, this is the first report on an high-throughput screening of microbial natural product extracts for the discovery of immunomodulators.

  10. Antileishmanial activity of new thiophene-indole hybrids: Design, synthesis, biological and cytotoxic evaluation, and chemometric studies.

    Science.gov (United States)

    Félix, Mayara B; de Souza, Edson R; de Lima, Maria do C A; Frade, Daiana Karla G; Serafim, Vanessa de L; Rodrigues, Klinger Antonio da F; Néris, Patrícia Lima do N; Ribeiro, Frederico F; Scotti, Luciana; Scotti, Marcus T; de Aquino, Thiago M; Mendonça Junior, Francisco Jaime B; de Oliveira, Márcia R

    2016-09-15

    In the present work, thirty-two hybrid compounds containing cycloalka[b]thiophene and indole moieties (TN5, TN5 1-7, TN6, TN6 1-7, TN7, TN7 1-7, TN8, TN8 1-7) were designed, synthesized and evaluated for their cytotoxic and antileishmanial activity against Leishmania amazonensis promastigotes. More than half of the compounds (18 compounds) exhibited significant antileishmanial activity (IC50 lower than 10.0μg/L), showing better performance than the reference drugs (tri- and penta-valent antimonials). The most active compounds were TN8-7, TN6-1 and TN7 with respective IC50 values of 2.1, 2.3 and 3.2μg/mL. Demonstrating that all of the compounds were less toxic than the reference drugs, even at the highest evaluated concentration (400μg/mL), no compound tested presented human erythrocyte cytotoxicity. Compound TN8-7's effectiveness against a trivalent antimony-resistant culture was demonstrated. It was observed that TN8-7's antileishmanial activity is associated with DNA fragmentation of L. amazonensis promastigotes. Chemometric studies (CPCA, PCA, and PLS) highlight intrinsic solubility/lipophilicity, and compound size and shape as closely related to activity. Our results suggest that hybrid cycloalka[b]thiophene-indole derivatives may be considered as lead compounds for further development of new drugs for the treatment of leishmaniasis. PMID:27515718

  11. High-throughput Screening:Synthesis of a Novel Fluorescent Microspheres

    Institute of Scientific and Technical Information of China (English)

    LI Song-Jun; LIU Bai-ling

    2004-01-01

    As one of efficient analytes, fluorescent microspheres have shown much usability on many biochemical and biomedical processes. Recent applications with fluorescent microspheres have included cytokine quantitation, single nucleotide polymorphism genotyping, phosphorylated protein detection, and characterization of the molecular interaction of nuclear receptors. These,coupled with the rapid advances in molecular biology and synthesis techniques of drugs, have presented a basis for drug screening in a high-throughput format. Based on fluorescent microspheres,earlier assay formats of HTS relied mainly on proximity-dependent energy transfer including scintillation proximity assay (SPA) (Amersham Pharmacia Biotech) and FlashPlatesTM (NEN Life Science Products, Boston, MA). Indeed, drug screening-based such fluorescent emission is still accounting for about 20~50% of current content of high-throughput screening (HTS). Now, SPA is almost a standard technique in common HTS-lab. In literature, SPA microspheres is generally prepared from inorganic scintillators such as yttrium silicate and hydrophobic polymers such as polyvinyl toluene. However, in HTS research, such microspheres often show the disadvantages of strong hydrophobicity and low quantum efficiency. The strong hydrophobicity is mainly attributed to the hydrophobic monomer, vinyl toluene. The low quantum efficiency can be as a result of low transparence of the polymer, polyvinyl toluene. Thus, the subsequent treatments for such microspheres, so as coat a polyhydroxy film to decrease the hydrophobicity, are actually considerably complicated.It has been well known that poly(methyl methacrylate) (PMMA), a good biocompatible polymer with not only adequate mechanical strength but also excellent transparence, can be regarded as an ideal candidate material for fluorescent matrix. In present study, methyl methacrylate as monomer and 2,5-diphenyloxazole (DPO) as fluorescent dye were used to the fluorescent microspheres. In

  12. High-throughput Saccharification assay for lignocellulosic materials.

    Science.gov (United States)

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-01-01

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  13. High-throughput metal susceptibility testing of microbial biofilms

    Directory of Open Access Journals (Sweden)

    Turner Raymond J

    2005-10-01

    Full Text Available Abstract Background Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. Results This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32- than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic

  14. High-throughput Saccharification assay for lignocellulosic materials.

    Science.gov (United States)

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-07-03

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  15. Synthetic oxoisoaporphine alkaloids: in vitro, in vivo and in silico assessment of antileishmanial activities.

    Directory of Open Access Journals (Sweden)

    Eduardo Sobarzo-Sánchez

    Full Text Available Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one, were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05 in parasite burden in liver and spleen (99% and 78%, respectively whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH, one N-myristoyltransferase (2WUU and one type I topoisomerase (2B9S.

  16. High Throughput Extraction of Plant, Marine and Fungal Specimens for Preservation of Biologically Active Molecules

    Directory of Open Access Journals (Sweden)

    Thomas G. McCloud

    2010-06-01

    Full Text Available The Developmental Therapeutics Program (DTP of the U.S. National Cancer Institute (NCI, at its NCI-Frederick facility, has built perhaps the largest and most diverse natural products screening library in the world for drug discovery. Composed of plant, marine organism and microbial extracts, it currently contains in excess of 230,000 unique materials. From the inception of this program to identify new anticancer chemotherapeutics from natural products sources in 1987, two extracts have been sequentially prepared from each specimen: one produced by organic solvent extraction, which yields a complex material that contains non- to moderately polar small molecules, and a water-soluble extract, a milieu largely unexplored for useful drugs in earlier years, which contains polar small to medium-sized molecules. Plant specimens and microbial ferments are extracted by modified traditional methods, while the method developed to produce extracts from marine organisms is unique and very different from that used by marine natural products chemists previously, but again yields both an organic solvent soluble and a water soluble material for inclusion into the screening library. Details of high throughput extract production for preservation of biologically active molecules are presented.

  17. Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products

    Science.gov (United States)

    Pérez-Moreno, Guiomar; Cantizani, Juan; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Martín, Jesús; el Aouad, Noureddine; Pérez-Victoria, Ignacio; Tormo, José Rubén; González-Menendez, Víctor; González, Ignacio; de Pedro, Nuria; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca; González-Pacanowska, Dolores

    2016-01-01

    Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural products. PMID:26735308

  18. Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB.

    Directory of Open Access Journals (Sweden)

    Rebuma Firdessa

    Full Text Available Cutaneous leishmaniasis (CL is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide, a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN. PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.

  19. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    Science.gov (United States)

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

  20. New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Karen G Anthony

    Full Text Available BACKGROUND: In an effort to discover new drugs to treat tuberculosis (TB we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. METHODOLOGY/PRINCIPAL FINDINGS: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. CONCLUSIONS/SIGNIFICANCE: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

  1. Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

    Science.gov (United States)

    Shubhakar, Archana; Kozak, Radoslaw P; Reiding, Karli R; Royle, Louise; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

    2016-09-01

    Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies. PMID:27479043

  2. Pre-amplification in the context of high-throughput qPCR gene expression experiment

    OpenAIRE

    Korenková, Vlasta; Scott, Justin; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjöback, Robert

    2015-01-01

    Background With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-a...

  3. Isatis tinctoria mediated synthesis of amphotericin B-bound silver nanoparticles with enhanced photoinduced antileishmanial activity: A novel green approach.

    Science.gov (United States)

    Ahmad, Aftab; Wei, Yun; Syed, Fatima; Khan, Shafiullah; Khan, Gul Majid; Tahir, Kamran; Khan, Arif Ullah; Raza, Muslim; Khan, Faheem Ullah; Yuan, Qiping

    2016-08-01

    After malaria, Leishmaniasis is the most prevalent infectious disease in terms of fatality and geographical distribution. The availability of a limited number of antileishmanial agents, emerging resistance to the available drugs, and the high cost of treatment complicate the treatment of leishmaniasis. To overcome these issues, critical research for new therapeutic agents with enhanced antileishmanial potential and low treatment cost is needed. In this contribution, we developed a green protocol to prepare biogenic silver nanoparticles (AgNPs) and amphotericin B-bound biogenic silver nanoparticles (AmB-AgNPs). Phytochemicals from the aqueous extract of Isatis tinctoria were used as reducing and capping agents to prepare silver nanoparticles. Amphotericin B was successfully adsorbed on the surface of biogenic silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV-Visible spectroscopy was employed to detect the characteristic localized surface plasmon resonance peaks (LSPR) for the prepared nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies revealed the formation of spherical silver nanoparticles with an average particle size of 10-20nm. The cubic crystalline structure of the prepared nanoparticles was confirmed by X-ray diffraction (XRD) study. FTIR spectroscopic analysis revealed that plant polyphenolic compounds are mainly involved in metal reduction and capping. Under visible light irradiation, biogenic silver nanoparticles exhibited significant activity against Leishmania tropica with an IC50 value of 4.2μg/mL. The leishmanicidal activity of these nanoparticles was considerably enhanced by conjugation with amphotericin B (IC50=2.43μg/mL). In conclusion, the findings of this study reveal that adsorption of amphotericin B, an antileishmanial drug, to biogenic silver nanoparticles, could be a safe, more effective and economic alternative to the available

  4. Isatis tinctoria mediated synthesis of amphotericin B-bound silver nanoparticles with enhanced photoinduced antileishmanial activity: A novel green approach.

    Science.gov (United States)

    Ahmad, Aftab; Wei, Yun; Syed, Fatima; Khan, Shafiullah; Khan, Gul Majid; Tahir, Kamran; Khan, Arif Ullah; Raza, Muslim; Khan, Faheem Ullah; Yuan, Qiping

    2016-08-01

    After malaria, Leishmaniasis is the most prevalent infectious disease in terms of fatality and geographical distribution. The availability of a limited number of antileishmanial agents, emerging resistance to the available drugs, and the high cost of treatment complicate the treatment of leishmaniasis. To overcome these issues, critical research for new therapeutic agents with enhanced antileishmanial potential and low treatment cost is needed. In this contribution, we developed a green protocol to prepare biogenic silver nanoparticles (AgNPs) and amphotericin B-bound biogenic silver nanoparticles (AmB-AgNPs). Phytochemicals from the aqueous extract of Isatis tinctoria were used as reducing and capping agents to prepare silver nanoparticles. Amphotericin B was successfully adsorbed on the surface of biogenic silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV-Visible spectroscopy was employed to detect the characteristic localized surface plasmon resonance peaks (LSPR) for the prepared nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies revealed the formation of spherical silver nanoparticles with an average particle size of 10-20nm. The cubic crystalline structure of the prepared nanoparticles was confirmed by X-ray diffraction (XRD) study. FTIR spectroscopic analysis revealed that plant polyphenolic compounds are mainly involved in metal reduction and capping. Under visible light irradiation, biogenic silver nanoparticles exhibited significant activity against Leishmania tropica with an IC50 value of 4.2μg/mL. The leishmanicidal activity of these nanoparticles was considerably enhanced by conjugation with amphotericin B (IC50=2.43μg/mL). In conclusion, the findings of this study reveal that adsorption of amphotericin B, an antileishmanial drug, to biogenic silver nanoparticles, could be a safe, more effective and economic alternative to the available

  5. High-Throughput Industrial Coatings Research at The Dow Chemical Company.

    Science.gov (United States)

    Kuo, Tzu-Chi; Malvadkar, Niranjan A; Drumright, Ray; Cesaretti, Richard; Bishop, Matthew T

    2016-09-12

    At The Dow Chemical Company, high-throughput research is an active area for developing new industrial coatings products. Using the principles of automation (i.e., using robotic instruments), parallel processing (i.e., prepare, process, and evaluate samples in parallel), and miniaturization (i.e., reduce sample size), high-throughput tools for synthesizing, formulating, and applying coating compositions have been developed at Dow. In addition, high-throughput workflows for measuring various coating properties, such as cure speed, hardness development, scratch resistance, impact toughness, resin compatibility, pot-life, surface defects, among others have also been developed in-house. These workflows correlate well with the traditional coatings tests, but they do not necessarily mimic those tests. The use of such high-throughput workflows in combination with smart experimental designs allows accelerated discovery and commercialization.

  6. Wide Throttling, High Throughput Hall Thruster for Science and Exploration Missions Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In response to Topic S3.04 "Propulsion Systems," Busek Co. Inc. will develop a high throughput Hall effect thruster with a nominal peak power of 1-kW and wide...

  7. Wide Throttling, High Throughput Hall Thruster for Science and Exploration Missions Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In response to Topic S3-04 "Propulsion Systems," Busek proposes to develop a high throughput Hall effect thruster with a nominal peak power of 1-kW and wide...

  8. A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.

    Science.gov (United States)

    Ramm, Susanne; Adler, Melanie; Vaidya, Vishal S

    2016-01-01

    Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc. PMID:27479365

  9. Liquid marbles for high-throughput biological screening of anchorage-dependent cells.

    Science.gov (United States)

    Oliveira, Nuno M; Correia, Clara R; Reis, Rui L; Mano, João F

    2015-01-28

    Stable liquid marbles (LM) are produced by coating liquid droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liquid environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liquid on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe(3+) is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the analysis of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment.

  10. Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

    Science.gov (United States)

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

    2011-03-01

    Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

  11. The impact of computer science in molecular medicine: enabling high-throughput research.

    Science.gov (United States)

    de la Iglesia, Diana; García-Remesal, Miguel; de la Calle, Guillermo; Kulikowski, Casimir; Sanz, Ferran; Maojo, Víctor

    2013-01-01

    The Human Genome Project and the explosion of high-throughput data have transformed the areas of molecular and personalized medicine, which are producing a wide range of studies and experimental results and providing new insights for developing medical applications. Research in many interdisciplinary fields is resulting in data repositories and computational tools that support a wide diversity of tasks: genome sequencing, genome-wide association studies, analysis of genotype-phenotype interactions, drug toxicity and side effects assessment, prediction of protein interactions and diseases, development of computational models, biomarker discovery, and many others. The authors of the present paper have developed several inventories covering tools, initiatives and studies in different computational fields related to molecular medicine: medical informatics, bioinformatics, clinical informatics and nanoinformatics. With these inventories, created by mining the scientific literature, we have carried out several reviews of these fields, providing researchers with a useful framework to locate, discover, search and integrate resources. In this paper we present an analysis of the state-of-the-art as it relates to computational resources for molecular medicine, based on results compiled in our inventories, as well as results extracted from a systematic review of the literature and other scientific media. The present review is based on the impact of their related publications and the available data and software resources for molecular medicine. It aims to provide information that can be useful to support ongoing research and work to improve diagnostics and therapeutics based on molecular-level insights.

  12. High-throughput characterization of Echinococcus spp. metacestode miRNomes.

    Science.gov (United States)

    Cucher, Marcela; Macchiaroli, Natalia; Kamenetzky, Laura; Maldonado, Lucas; Brehm, Klaus; Rosenzvit, Mara Cecilia

    2015-03-01

    Echinococcosis is a worldwide zoonosis of great public health concern, considered a neglected disease by the World Health Organisation. The cestode parasites Echinococcus granulosus sensu lato (s. l.) and Echinococcus multilocularis are the main aetiological agents. In the intermediate host, these parasites display particular developmental traits that lead to different patterns of disease progression. In an attempt to understand the causes of these differences, we focused on the analysis of microRNAs (miRNAs), small non-coding regulatory RNAs with major roles in development of animals and plants. In this work, we analysed the small RNA expression pattern of the metacestode, the stage of sanitary relevance, and provide a detailed description of Echinococcus miRNAs. Using high-throughput small RNA sequencing, we believe that we have carried out the first experimental identification of miRNAs in E. multilocularis and have expanded the Echinococcus miRNA catalogue to 38 miRNA genes, including one miRNA only present in E. granulosus s. l. Our findings show that although both species share the top five highest expressed miRNAs, 13 are differentially expressed, which could be related to developmental differences. We also provide evidence that uridylation is the main miRNA processing mechanism in Echinococcus spp. These results provide detailed information on Echinococcus miRNAs, which is the first step in understanding their role in parasite biology and disease establishment and/or progression, and their future potential use as drug or diagnostic targets.

  13. High-Throughput Automated Phenotyping of Two Genetic Mouse Models of Huntington's Disease.

    Science.gov (United States)

    Balci, Fuat; Oakeshott, Stephen; Shamy, Jul Lea; El-Khodor, Bassem F; Filippov, Igor; Mushlin, Richard; Port, Russell; Connor, David; Paintdakhi, Ahmad; Menalled, Liliana; Ramboz, Sylvie; Howland, David; Kwak, Seung; Brunner, Dani

    2013-01-01

    Phenotyping with traditional behavioral assays constitutes a major bottleneck in the primary screening, characterization, and validation of genetic mouse models of disease, leading to downstream delays in drug discovery efforts. We present a novel and comprehensive one-stop approach to phenotyping, the PhenoCube™. This system simultaneously captures the cognitive performance, motor activity, and circadian patterns of group-housed mice by use of home-cage operant conditioning modules (IntelliCage) and custom-built computer vision software. We evaluated two different mouse models of Huntington's Disease (HD), the R6/2 and the BACHD in the PhenoCube™ system. Our results demonstrated that this system can efficiently capture and track alterations in both cognitive performance and locomotor activity patterns associated with these disease models. This work extends our prior demonstration that PhenoCube™ can characterize circadian dysfunction in BACHD mice and shows that this system, with the experimental protocols used, is a sensitive and efficient tool for a first pass high-throughput screening of mouse disease models in general and mouse models of neurodegeneration in particular. PMID:23863947

  14. Commentary: Roles for Pathologists in a High-throughput Image Analysis Team.

    Science.gov (United States)

    Aeffner, Famke; Wilson, Kristin; Bolon, Brad; Kanaly, Suzanne; Mahrt, Charles R; Rudmann, Dan; Charles, Elaine; Young, G David

    2016-08-01

    Historically, pathologists perform manual evaluation of H&E- or immunohistochemically-stained slides, which can be subjective, inconsistent, and, at best, semiquantitative. As the complexity of staining and demand for increased precision of manual evaluation increase, the pathologist's assessment will include automated analyses (i.e., "digital pathology") to increase the accuracy, efficiency, and speed of diagnosis and hypothesis testing and as an important biomedical research and diagnostic tool. This commentary introduces the many roles for pathologists in designing and conducting high-throughput digital image analysis. Pathology review is central to the entire course of a digital pathology study, including experimental design, sample quality verification, specimen annotation, analytical algorithm development, and report preparation. The pathologist performs these roles by reviewing work undertaken by technicians and scientists with training and expertise in image analysis instruments and software. These roles require regular, face-to-face interactions between team members and the lead pathologist. Traditional pathology training is suitable preparation for entry-level participation on image analysis teams. The future of pathology is very exciting, with the expanding utilization of digital image analysis set to expand pathology roles in research and drug development with increasing and new career opportunities for pathologists. PMID:27343178

  15. Screening for Antifibrotic Compounds Using High Throughput System Based on Fluorescence Polarization

    Directory of Open Access Journals (Sweden)

    Branko Stefanovic

    2014-04-01

    Full Text Available Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I mRNA and α2(I mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL. Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis in vivo. Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.

  16. The motivations and methodology for high-throughput PET imaging of small animals in cancer research

    International Nuclear Information System (INIS)

    Over the last decade, small-animal PET imaging has become a vital platform technology in cancer research. With the development of molecularly targeted therapies and drug combinations requiring evaluation of different schedules, the number of animals to be imaged within a PET experiment has increased. This paper describes experimental design requirements to reach statistical significance, based on the expected change in tracer uptake in treated animals as compared to the control group, the number of groups that will be imaged, and the expected intra-animal variability for a given tracer. We also review how high-throughput studies can be performed in dedicated small-animal PET, high-resolution clinical PET systems and planar positron imaging systems by imaging more than one animal simultaneously. Customized beds designed to image more than one animal in large-bore small-animal PET scanners are described. Physics issues related to the presence of several rodents within the field of view (i.e. deterioration of spatial resolution and sensitivity as the radial and the axial offsets increase, respectively, as well as a larger effect of attenuation and the number of scatter events), which can be assessed by using the NEMA NU 4 image quality phantom, are detailed. (orig.)

  17. The motivations and methodology for high-throughput PET imaging of small animals in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Aide, Nicolas [Francois Baclesse Cancer Centre, Nuclear Medicine Department, Caen Cedex (France); Caen University, BioTICLA team, EA 4656, IFR 146, Caen (France); Visser, Eric P. [Radboud University Nijmegen Medical Center, Nuclear Medicine Department, Nijmegen (Netherlands); Lheureux, Stephanie [Caen University, BioTICLA team, EA 4656, IFR 146, Caen (France); Francois Baclesse Cancer Centre, Clinical Research Unit, Caen (France); Heutte, Natacha [Francois Baclesse Cancer Centre, Clinical Research Unit, Caen (France); Szanda, Istvan [King' s College London, Division of Imaging Sciences and Biomedical Engineering, London (United Kingdom); Hicks, Rodney J. [Peter MacCallum Cancer Centre, Centre for Molecular Imaging, East Melbourne (Australia)

    2012-09-15

    Over the last decade, small-animal PET imaging has become a vital platform technology in cancer research. With the development of molecularly targeted therapies and drug combinations requiring evaluation of different schedules, the number of animals to be imaged within a PET experiment has increased. This paper describes experimental design requirements to reach statistical significance, based on the expected change in tracer uptake in treated animals as compared to the control group, the number of groups that will be imaged, and the expected intra-animal variability for a given tracer. We also review how high-throughput studies can be performed in dedicated small-animal PET, high-resolution clinical PET systems and planar positron imaging systems by imaging more than one animal simultaneously. Customized beds designed to image more than one animal in large-bore small-animal PET scanners are described. Physics issues related to the presence of several rodents within the field of view (i.e. deterioration of spatial resolution and sensitivity as the radial and the axial offsets increase, respectively, as well as a larger effect of attenuation and the number of scatter events), which can be assessed by using the NEMA NU 4 image quality phantom, are detailed. (orig.)

  18. High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission

    Science.gov (United States)

    Plouffe, David M.; Wree, Melanie; Du, Alan Y.; Meister, Stephan; Li, Fengwu; Patra, Kailash; Lubar, Aristea; Okitsu, Shinji L.; Flannery, Erika L.; Kato, Nobutaka; Tanaseichuk, Olga; Comer, Eamon; Zhou, Bin; Kuhen, Kelli; Zhou, Yingyao; Leroy, Didier; Schreiber, Stuart L.; Scherer, Christina A.; Vinetz, Joseph; Winzeler, Elizabeth A.

    2016-01-01

    Summary Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs. PMID:26749441

  19. High-throughput sorting of the highest producing cell via a transiently protein-anchored system.

    Directory of Open Access Journals (Sweden)

    Kuo-Hsiang Chuang

    Full Text Available Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR was used to join a human anti-epithelial growth factor antibody (αEGFR Ab and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7. The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165 between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

  20. High-throughput synthesis and characterization of BiMoVOX materials

    OpenAIRE

    Russu, Sergio; Tromp, Moniek; Tsapatsaris, Nikolaos; Beesley, Angela M.; Schroeder, Sven L M; Weller, Mark T.; Evans, John

    2007-01-01

    The high throughput synthesis and characterization of a particular family of ceramic materials, bismuth molybdenum vanadium oxides (BiMoVOX), suitable as inorganic yellow pigments and low temperature oxidation catalysts, is described. Samples, synthesized by calcination and peroxo sol-gel methods, are characterized by X-ray powder diffraction, UV-visible and XAFS spectroscopy. A combined high-throughput XRD/XAFS study of a 54 samples array, with simultaneous refinement of data of ...

  1. Self-encoding Functional Resin Applying for Combinatorial Chemistry and High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    DU Lei; CHEN Tong-sheng

    2004-01-01

    A novel solid phase organic synthesis resin was synthesized for combinatorial high-throughput screening,which based on FTIR spectra self-encoding functional resin technology. A new deconvolution strategy termed position encoding deconvolution had illustrated and was compared with some popular combinatorial deconvolution strategies in efficiency and information content. The mimic high throughput screening of hexapeptide library successfully proved the applying of the self-encoding functional resin technology and the position encoding deconvolution strategy.

  2. Droplet microfluidic technology for single-cell high-throughput screening

    OpenAIRE

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J. Brian; Rothberg, Jonathan M.; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-01-01

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform t...

  3. An Automated High-throughput Array Microscope for Cancer Cell Mechanics

    OpenAIRE

    Cribb, Jeremy A.; Osborne, Lukas D.; Kellie Beicker; Matthew Psioda; Jian Chen; E. Timothy O’Brien; Taylor II, Russell M.; Leandra Vicci; Joe Ping-Lin Hsiao; Chong Shao; Michael Falvo; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Richard Superfine

    2016-01-01

    Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image ...

  4. Application of high-throughput sequencing in understanding human oral microbiome related with health and disease

    OpenAIRE

    Chen, Hui; Jiang, Wen

    2014-01-01

    The oral microbiome is one of most diversity habitat in the human body and they are closely related with oral health and disease. As the technique developing, high-throughput sequencing has become a popular approach applied for oral microbial analysis. Oral bacterial profiles have been studied to explore the relationship between microbial diversity and oral diseases such as caries and periodontal disease. This review describes the application of high-throughput sequencing for characterization...

  5. Development of Asymmetric Hydrogenation Catalysts via High Throughput Experimentation

    NARCIS (Netherlands)

    Vries, J.G. de; Lefort, L.

    2013-01-01

    The dynamics of drugs discovery imposes severe time constraints on the development chemist in charge of implementing the large scale production of a new Active Pharmaceutical Ingredient (API). This results in the use of well-established and robust transformations at the expense of the cost-efficienc

  6. Lessons from high-throughput protein crystallization screening: 10 years of practical experience

    Science.gov (United States)

    JR, Luft; EH, Snell; GT, DeTitta

    2011-01-01

    Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

  7. Small-molecule activators of insulin-degrading enzyme discovered through high-throughput compound screening.

    Directory of Open Access Journals (Sweden)

    Christelle Cabrol

    Full Text Available BACKGROUND: Hypocatabolism of the amyloid beta-protein (Abeta by insulin-degrading enzyme (IDE is implicated in the pathogenesis of Alzheimer disease (AD, making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. METHODOLOGY/PRINCIPAL FINDINGS: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates. CONCLUSIONS/SIGNIFICANCE: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

  8. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.

    Directory of Open Access Journals (Sweden)

    George W Rogers

    Full Text Available Recently developed technologies have enabled multi-well measurement of O(2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 µg of mitochondrial protein per well. Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.

  9. Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

    Science.gov (United States)

    Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

    2012-10-01

    Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. PMID:22744888

  10. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major

    Directory of Open Access Journals (Sweden)

    Behrouz Ezatpour

    2015-01-01

    Full Text Available The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime, against Leishmania tropica and Leishmania major. This extract (0–100 µg/mL was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER and then tested on cutaneous leishmaniasis (CL in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P<0.05 inhibited the growth rate of promastigote (IC50 58.6±3.2 µg/mL and intramacrophage amastigotes (37.3±2.5 µg/mL of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P<0.05 reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL.

  11. Strychnos pseudoquina and Its Purified Compounds Present an Effective In Vitro Antileishmanial Activity

    Directory of Open Access Journals (Sweden)

    Paula Sousa Lage

    2013-01-01

    Full Text Available The development of new and cost-effective alternative therapeutic strategies to treat leishmaniasis has become a high priority. In the present study, the antileishmanial activity of Strychnos pseudoquina St. Hil. was investigated and pure compounds that presented this biological effect were isolated. An ethyl acetate extract was prepared, and it proved to be effective against Leishmania amazonensis. A bioactivity-guided fractionation was performed, and two flavonoids were identified, quercetin 3-O-methyl ether and strychnobiflavone, which presented an effective antileishmanial activity against L. amazonensis, and studies were extended to establish their minimum inhibitory concentrations (IC50, their leishmanicidal effects on the intra-macrophage Leishmania stage, as well as their cytotoxic effects on murine macrophages (CC50, and in O+ human red blood cells. The data presented in this study showed the potential of an ethyl acetate extract of S. pseudoquina, as well as two flavonoids purified from it, which can be used as a therapeutic alternative on its own, or in association with other drugs, to treat disease evoked by L. amazonensis.

  12. 4-Acetoxydolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis as Antileishmanial Agent

    Directory of Open Access Journals (Sweden)

    Elizandra Aparecida Britta

    2011-11-01

    Full Text Available Natural marine products have shown an interesting array of diverse and novel chemical structures with potent biological activities. Our study reports the antiproliferative assays of crude extracts, fraction and pure compound (4R,9S,14S-4α-acetoxy-9β,14α-dihydroxydolast-1(15,7-diene (1 obtained from brown alga Canistrocarpus cervicornis showing the antileishmanial activity. We showed that 1 had a dose-dependent activity during 72 h of treatment, exhibiting IC50 of 2.0 µg/mL, 12.0 µg/mL, and 4.0 µg/mL for promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis, respectively. A cytotoxicity assay showed that the action of the isolated compound 1 was 93.0 times less toxic to the macrophage than to the protozoan. Additionally, compound 1 induced ultrastructural changes, including extensive mitochondrial damage; decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential; and lipid peroxidation in parasite cells. The use of 1 from C. cervicornis against L. amazonensis parasites might be of great interest as a future alternative to the development of new antileishmanial drugs.

  13. 4-Acetoxydolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis as Antileishmanial Agent

    Science.gov (United States)

    dos Santos, Adriana Oliveira; Britta, Elizandra Aparecida; Bianco, Everson Miguel; Ueda-Nakamura, Tania; Filho, Benedito Prado Dias; Pereira, Renato Crespo; Nakamura, Celso Vataru

    2011-01-01

    Natural marine products have shown an interesting array of diverse and novel chemical structures with potent biological activities. Our study reports the antiproliferative assays of crude extracts, fraction and pure compound (4R,9S,14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene (1) obtained from brown alga Canistrocarpus cervicornis showing the antileishmanial activity. We showed that 1 had a dose-dependent activity during 72 h of treatment, exhibiting IC50 of 2.0 μg/mL, 12.0 μg/mL, and 4.0 μg/mL for promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis, respectively. A cytotoxicity assay showed that the action of the isolated compound 1 was 93.0 times less toxic to the macrophage than to the protozoan. Additionally, compound 1 induced ultrastructural changes, including extensive mitochondrial damage; decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential; and lipid peroxidation in parasite cells. The use of 1 from C. cervicornis against L. amazonensis parasites might be of great interest as a future alternative to the development of new antileishmanial drugs. PMID:22163190

  14. High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weiss, Andrew; Delproposto, James; Giroux, Craig N

    2004-04-01

    Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets. To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge. To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae. Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves. This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult. These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant. Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress. As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells. Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics. PMID:15033507

  15. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  16. Evaluation of Apoptotic and Antileishmanial Activities of Artemisinin on Promastigotes and BALB/C Mice Infected with Leishmania major.

    Directory of Open Access Journals (Sweden)

    Fatemeh Ghaffarifar

    2015-06-01

    Full Text Available In leishmaniasis, some drugs prescribed for treatment have toxic effects and there are reports about drug resistance in some countries. Due to this fact, using herbal drugs such as artemisinin with good efficacy and low toxic effect might be suitable.We evaluated the apoptotic effect of artemisinin on Leishmania major in vitro and the antileishmanial activities of artemisinin on leishmaniasis in BALB/c mice and at the end INF-γ and IL-4 cytokines levels were detected by ELISA in spleen cell culture supernatants. During treatment the lesion size and survival rate were measured each four and ten days, respectively.Percentage of early and late apoptosis in promastigotes of control group and promastigotes treated with 10, 25, 50 and 100 μg/ml of artemisinin after 48 h were 0.13, 16.04, 41.23, 49.03 and 81.83, respectively. The IFN-γ in ointment treated group were higher than those of other groups (P<0.05. The in vivo results showed that ointment compounds healed the lesions more effectively rather than intraperitoneal injection method (P<0.05. The survival rate of mice 150 days after challenge in treated group with ointment of artemisinin was 66% while all mice in control groups were died.All of in vitro results represented that this drug had antileishmanial effects and these results were confirmed by evaluation effects in vivo condition of leishmaniasis. Interestingly, according to these results it can be concluded that this drug has antileishmanial effects in vitro and in vivo conditions. Artemisinin induces cytotoxic effect on L. major via apoptosis-related mechanism.

  17. Design and Implementation of High-Throughput Screening Assays.

    Science.gov (United States)

    Powell, David J; Hertzberg, Robert P; Macarrόn, Ricardo

    2016-01-01

    HTS remains at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful consideration of many options and variables, starting with the choice of screening strategy and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:27316985

  18. High-throughput Phenotyping and Genomic Selection: The Frontiers of Crop Breeding Converge

    Institute of Scientific and Technical Information of China (English)

    Llorenc Cabrera-Bosquet; José Crossa; Jarislav von Zitzewitz; Maria Dolors Serret; José Luis Araus

    2012-01-01

    Genomic selection (GS) and high-throughput phenotyping have recently been captivating the interest of the crop breeding community from both the public and private sectors world-wide.Both approaches promise to revolutionize the prediction of complex traits,including growth,yield and adaptation to stress.Whereas high-throughput phenotyping may help to improve understanding of crop physiology,most powerful techniques for high-throughput field phenotyping are empirical rather than analytical and comparable to genomic selection.Despite the fact that the two methodological approaches represent the extremes of what is understood as the breeding process (phenotype versus genome),they both consider the targeted traits (e.g.grain yield,growth,phenology,plant adaptation to stress) as a black box instead of dissecting them as a set of secondary traits (i.e.physiological) putatively related to the target trait.Both GS and high-throughput phenotyping have in common their empirical approach enabling breeders to use genome profile or phenotype without understanding the underlying biology.This short review discusses the main aspects of both approaches and focuses on the case of genomic selection of maize flowering traits and near-infrared spectroscopy (NIRS) and plant spectral reflectance as high-throughput field phenotyping methods for complex traits such as crop growth and yield.

  19. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Directory of Open Access Journals (Sweden)

    Kyle C Wilcox

    Full Text Available Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs. AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs. This method gives a soluble membrane protein library (SMPL--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can

  20. Development of a high-throughput Candida albicans biofilm chip.

    Directory of Open Access Journals (Sweden)

    Anand Srinivasan

    Full Text Available We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B. Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.

  1. A high-throughput screen for aggregation-based inhibition in a large compound library.

    Science.gov (United States)

    Feng, Brian Y; Simeonov, Anton; Jadhav, Ajit; Babaoglu, Kerim; Inglese, James; Shoichet, Brian K; Austin, Christopher P

    2007-05-17

    High-throughput screening (HTS) is the primary technique for new lead identification in drug discovery and chemical biology. Unfortunately, it is susceptible to false-positive hits. One common mechanism for such false-positives is the congregation of organic molecules into colloidal aggregates, which nonspecifically inhibit enzymes. To both evaluate the feasibility of large-scale identification of aggregate-based inhibition and quantify its prevalence among screening hits, we tested 70,563 molecules from the National Institutes of Health Chemical Genomics Center (NCGC) library for detergent-sensitive inhibition. Each molecule was screened in at least seven concentrations, such that dose-response curves were obtained for all molecules in the library. There were 1274 inhibitors identified in total, of which 1204 were unambiguously detergent-sensitive. We identified these as aggregate-based inhibitors. Thirty-one library molecules were independently purchased and retested in secondary low-throughput experiments; 29 of these were confirmed as either aggregators or nonaggregators, as appropriate. Finally, with the dose-response information collected for every compound, we could examine the correlation between aggregate-based inhibition and steep dose-response curves. Three key results emerge from this study: first, detergent-dependent identification of aggregate-based inhibition is feasible on the large scale. Second, 95% of the actives obtained in this screen are aggregate-based inhibitors. Third, aggregate-based inhibition is correlated with steep dose-response curves, although not absolutely. The results of this screen are being released publicly via the PubChem database.

  2. Genetic profiles of cervical tumors by high-throughput sequencing for personalized medical care

    International Nuclear Information System (INIS)

    Cancer treatment is facing major evolution since the advent of targeted therapies. Building genetic profiles could predict sensitivity or resistance to these therapies and highlight disease-specific abnormalities, supporting personalized patient care. In the context of biomedical research and clinical diagnosis, our laboratory has developed an oncogenic panel comprised of 226 genes and a dedicated bioinformatic pipeline to explore somatic mutations in cervical carcinomas, using high-throughput sequencing. Twenty-nine tumors were sequenced for exons within 226 genes. The automated pipeline used includes a database and a filtration system dedicated to identifying mutations of interest and excluding false positive and germline mutations. One-hundred and seventy-six total mutational events were found among the 29 tumors. Our cervical tumor mutational landscape shows that most mutations are found in PIK3CA (E545K, E542K) and KRAS (G12D, G13D) and others in FBXW7 (R465C, R505G, R479Q). Mutations have also been found in ALK (V1149L, A1266T) and EGFR (T259M). These results showed that 48% of patients display at least one deleterious mutation in genes that have been already targeted by the Food and Drug Administration approved therapies. Considering deleterious mutations, 59% of patients could be eligible for clinical trials. Sequencing hundreds of genes in a clinical context has become feasible, in terms of time and cost. In the near future, such an analysis could be a part of a battery of examinations along the diagnosis and treatment of cancer, helping to detect sensitivity or resistance to targeted therapies and allow advancements towards personalized oncology

  3. High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

    Science.gov (United States)

    Bageshwar, Umesh K.; VerPlank, Lynn; Baker, Dwight; Dong, Wen; Hamsanathan, Shruthi; Whitaker, Neal; Sacchettini, James C.; Musser, Siegfried M.

    2016-01-01

    The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors. PMID:26901445

  4. A Perspective on the Future of High-Throughput RNAi Screening: Will CRISPR Cut Out the Competition or Can RNAi Help Guide the Way?

    Science.gov (United States)

    Taylor, Jessica; Woodcock, Simon

    2015-09-01

    For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA-Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery.

  5. Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

    Science.gov (United States)

    Wu, Meng; Coblitz, Brian; Shikano, Sojin; Long, Shunyou; Spieker, Matt; Frutos, Anthony G; Mukhopadhyay, Sunil; Li, Min

    2006-10-16

    Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection. PMID:17011553

  6. Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

    Science.gov (United States)

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

    The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens. PMID:27581289

  7. High-throughput parallel SPM for metrology, defect, and mask inspection

    Science.gov (United States)

    Sadeghian, H.; Herfst, R. W.; van den Dool, T. C.; Crowcombe, W. E.; Winters, J.; Kramer, G. F. I. J.

    2014-10-01

    Scanning probe microscopy (SPM) is a promising candidate for accurate assessment of metrology and defects on wafers and masks, however it has traditionally been too slow for high-throughput applications, although recent developments have significantly pushed the speed of SPM [1,2]. In this paper we present new results obtained with our previously presented high-throughput parallel SPM system [3,4] that showcase two key advances that are required for a successful deployment of SPM in high-throughput metrology, defect and mask inspection. The first is a very fast (up to 40 lines/s) image acquisition and a comparison of the image quality as function of speed. Secondly, a fast approach method: measurements of the scan-head approaching the sample from 0.2 and 1.0 mm distance in under 1.4 and 6 seconds respectively.

  8. PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors.

    Science.gov (United States)

    Almada, Pedro; Culley, Siân; Henriques, Ricardo

    2015-10-15

    Single Molecule Localization Microscopy (SMLM) techniques such as Photo-Activation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) enable fluorescence microscopy super-resolution: the overcoming of the resolution barrier imposed by the diffraction of light. These techniques are based on acquiring hundreds or thousands of images of single molecules, locating them and reconstructing a higher-resolution image from the high-precision localizations. These methods generally imply a considerable trade-off between imaging speed and resolution, limiting their applicability to high-throughput workflows. Recent advancements in scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera sensors and localization algorithms reduce the temporal requirements for SMLM, pushing it toward high-throughput microscopy. Here we outline the decisions researchers face when considering how to adapt hardware on a new system for sCMOS sensors with high-throughput in mind. PMID:26079924

  9. Recent Progress Using High-throughput Sequencing Technologies in Plant Molecular Breeding

    Institute of Scientific and Technical Information of China (English)

    Qiang Gao; Guidong Yue; Wenqi Li; Junyi Wang; Jiaohui Xu; Ye Yin

    2012-01-01

    High-throughput sequencing is a revolutionary technological innovation in DNA sequencing.This technology has an ultra-low cost per base of sequencing and an overwhelmingly high data output.High-throughput sequencing has brought novel research methods and solutions to the research fields of genomics and post-genomics.Furthermore,this technology is leading to a new molecular breeding revolution that has landmark significance for scientific research and enables us to launch multi-level,multifaceted,and multi-extent studies in the fields of crop genetics,genomics,and crop breeding.In this paper,we review progress in the application of high-throughput sequencing technologies to plant molecular breeding studies.

  10. Filtering high-throughput protein-protein interaction data using a combination of genomic features

    Directory of Open Access Journals (Sweden)

    Patil Ashwini

    2005-04-01

    Full Text Available Abstract Background Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. Results In this study, we use a combination of 3 genomic features – structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology – as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90% and good specificity (63%. We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. Conclusion A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

  11. Quantitative high-throughput screen identifies inhibitors of the Schistosoma mansoni redox cascade.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    Full Text Available Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR and peroxiredoxin (Prx and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50s ranging from micromolar to the assay response limit ( approximately 25 nM. This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump

  12. High-throughput exposure modeling to support prioritization of chemicals in personal care products

    DEFF Research Database (Denmark)

    Csiszar, Susan A.; Ernstoff, Alexi; Fantke, Peter;

    2016-01-01

    We demonstrate the application of a high-throughput modeling framework to estimate exposure to chemicals used in personal care products (PCPs). As a basis for estimating exposure, we use the product intake fraction (PiF), defined as the mass of chemical taken by an individual or population per mass...... intakes were associated with body lotion. Bioactive doses derived from high-throughput in vitro toxicity data were combined with the estimated PiFs to demonstrate an approach to estimate bioactive equivalent chemical content and to screen chemicals for risk....

  13. High-throughput operando Raman-quadrupole mass spectrometer (QMS) system to screen catalytic systems.

    Science.gov (United States)

    García-Casado, Manuel; Prieto, José; Vico-Ruiz, Emilio; Lozano-Diz, Enrique; Goberna-Selma, Consuelo; Bañares, Miguel A

    2014-01-01

    This paper describes the design and setup of a high-throughput Raman system for an array of eight parallel catalytic reactors during reaction conditions. The "operando" methodology combines in situ spectroscopy during catalytic reaction with a simultaneous activity measurement. The high-throughput operando Raman system, multi-operando, is a device that automates this operando methodology for several catalyst samples at the same time, all samples being in the same reaction conditions. We describe how the system is made, how Raman system positions and acquires spectra, and how each reactor outlet gas is selected and analyzed. PMID:24405956

  14. Statistical challenges in the detection of mutation and variation using high throughput sequencing

    OpenAIRE

    Pfeifer, Susanne; McVean, Gilean

    2012-01-01

    The aim of this thesis is to obtain a better understanding of mutation rates within as well as between the genomes of humans and chimpanzees using data generated by high throughput sequencers. I will start with a review of the field and an overview of the technologies and protocols used to generate and analyse high throughput sequencing data. I apply some of the discussed techniques to show that there is evidence of a selective advantage of pathogenic de novo mutations in the Fibroblast Growt...

  15. High-throughput analysis of total nitrogen content that replaces the classic Kjeldahl method.

    Science.gov (United States)

    Yasuhara, T; Nokihara, K

    2001-10-01

    A high-throughput method for determination of total nitrogen content has been developed. The method involves decomposition of samples, followed by trapping and quantitative colorimetric determination of the resulting ammonia. The present method is rapid, facile, and economical. Thus, it can replace the classic Kjeldahl method through its higher efficiency for determining multiple samples. Compared to the classic method, the present method is economical and environmentally friendly. Based on the present method, a novel reactor was constructed to realize routine high-throughput analyses of multiple samples such as those found for pharmaceutical materials, foods, and/or excrements.

  16. Perspective: Composition-structure-property mapping in high-throughput experiments: Turning data into knowledge

    Science.gov (United States)

    Hattrick-Simpers, Jason R.; Gregoire, John M.; Kusne, A. Gilad

    2016-05-01

    With their ability to rapidly elucidate composition-structure-property relationships, high-throughput experimental studies have revolutionized how materials are discovered, optimized, and commercialized. It is now possible to synthesize and characterize high-throughput libraries that systematically address thousands of individual cuts of fabrication parameter space. An unresolved issue remains transforming structural characterization data into phase mappings. This difficulty is related to the complex information present in diffraction and spectroscopic data and its variation with composition and processing. We review the field of automated phase diagram attribution and discuss the impact that emerging computational approaches will have in the generation of phase diagrams and beyond.

  17. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  18. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2014-01-01

    Full Text Available The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

  19. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    Science.gov (United States)

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  20. Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

    Directory of Open Access Journals (Sweden)

    Yu-Shih Lin

    Full Text Available Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α. The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

  1. Simultaneous determination of multiple angiotensin type 1 receptor antagonists and its application to high-throughput pharmacokinetic study

    Science.gov (United States)

    Zhu, Xiaoyan; Sun, Jianguo; Hao, Haiping; Wang, Guangji; Hu, Xiaoling; Lv, Hua; Gu, Shenghua; Wu, Xiaoming; Xu, Jinyi

    2008-05-01

    A rapid and sensitive high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) detection was developed for the simultaneous determination of multiple angiotensin type 1 receptor antagonists (AT1RAs) WX472, WX581, 1b and telmisartan in rat plasma for the purpose of high-throughout pharmacokinetic screening. The method was operated under selected reaction monitoring (SRM) mode in the positive ion mode. The analytes and the internal standard (pitavastatin) were extracted from 100 [mu]L rat plasma under acidic conditions by liquid-liquid extraction with ethyl acetate. The analytes and internal standard were baseline separated on a Gemini analytical column (3 [mu]m, 150 mm × 2.0 mm) with the adoption of a gradient elution using acetonitrile and 0.05% aqueous formic acid. The standard curves were linear in the concentration ranges of 4.5-900 ng/mL for WX472, 5-1000 ng/mL for WX581 and 0.5-100 ng/mL for 1b and telmisartan. Intra- and inter-batch precisions (R.S.D.%) were all within 15% and the method assessed a quite good accuracy (R.E.%). Recoveries were found to be >65% for all the compounds and no obvious matrix effects were found. This method has been successfully applied to the high-throughput pharmacokinetic screening study for both cassette dosing and cassette analysis of four compounds to rats. Significant drug-drug interactions were observed after cassette dosing. The study suggested that cassette analysis of pooled samples would be a better choice for the high-throughput pharmacokinetic screening of angiotensin type 1 receptor antagonists.

  2. Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis.

    Science.gov (United States)

    Lakshmi, Bhavana Sethu; Wang, Ruobing; Madhubala, Rentala

    2014-06-24

    Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform.

  3. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2008-01-01

    Aim:To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs.Methods:The donor DNA duplex,with a sequence identical to the U5 end of HIV-1 long terminal repeats,is labeled at its 5' end with biotin (BIO).The target DNA duplex is labeled at its 3' end with digoxin (DIG).IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product.Streptavidin-coated magnetic beads were used to capture the product,and the amount of DIG was measured as the ST reaction product.The assay was optimized in 96-well microplate format for high-throughput screening purpose.Moreover,the assay was applied in a ST reaction character study,and the efficiency of the assay in the identification of antiviral compounds was tested.Results:The end-point values,measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings.The ST reaction char-acter and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays.The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9.Conclusion:The assay presented here has been proven to be rapid,sensitive,and specific for the detection of IN ST activity,the reaction character study,as well as for the identification of antiviral drugs targeting IN.

  4. Antileishmanial activity of some plants growing in Algeria: Juglans regia, Lawsonia inermis and Salvia officinalis.

    Science.gov (United States)

    Serakta, M; Djerrou, Z; Mansour-Djaalab, H; Kahlouche-Riachi, F; Hamimed, S; Trifa, W; Belkhiri, A; Edikra, N; Hamdi Pacha, Y

    2013-01-01

    The current study was undertaken to evaluate in vitro the antileishmanial activity of three plants growing wild in Algeria : Juglans regia, Lawsonia inermis and Salvia officinalis. The hydroalcoholic extracts of these plants were tested on the growth of the promastigotes of Leishmania major. The plant extract effects were compared with three controls : CRL1 composed of 1 ml RPMI inoculated with 10(6) of promastigotes, CRL2 composed of 1 ml RPMI inoculated with 10(6) of promastigotes and 100 µl of hydroalcoholic solvent, CRL3 composed of 1 ml RPMI inoculated with 10(6) of promastigotes and 100 µl of Glucantim as a reference drug in the management of leishmaniasis. The results showed that both J. regia and L. inermis extracts reduced the promastigotes number significantly (Pofficinalis showed a total inhibition of the Leishmania major growth.

  5. High-throughput verification of transcriptional starting sites by Deep-RACE

    DEFF Research Database (Denmark)

    Olivarius, Signe; Plessy, Charles; Carninci, Piero

    2009-01-01

    We present a high-throughput method for investigating the transcriptional starting sites of genes of interest, which we named Deep-RACE (Deep–rapid amplification of cDNA ends). Taking advantage of the latest sequencing technology, it allows the parallel analysis of multiple genes and is free of...

  6. High-Throughput Dietary Exposure Predictions for Chemical Migrants from Food Packaging Materials

    Science.gov (United States)

    United States Environmental Protection Agency researchers have developed a Stochastic Human Exposure and Dose Simulation High -Throughput (SHEDS-HT) model for use in prioritization of chemicals under the ExpoCast program. In this research, new methods were implemented in SHEDS-HT...

  7. A high-throughput method for GMO multi-detection using a microfluidic dynamic array

    NARCIS (Netherlands)

    Brod, F.C.A.; Dijk, van J.P.; Voorhuijzen, M.M.; Dinon, A.Z.; Guimarães, L.H.S.; Scholtens, I.M.J.; Arisi, A.C.M.; Kok, E.J.

    2014-01-01

    The ever-increasing production of genetically modified crops generates a demand for high-throughput DNAbased methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the nu

  8. A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

    Science.gov (United States)

    Fujikawa, Yuuta; Morisaki, Fumika; Ogura, Asami; Morohashi, Kana; Enya, Sora; Niwa, Ryusuke; Goto, Shinji; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Inoue, Hideshi

    2015-07-21

    We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

  9. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Science.gov (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  10. A novel high-throughput irradiator for in vitro radiation sensitivity bioassays

    Science.gov (United States)

    Fowler, Tyler L.

    Given the emphasis on more personalized radiation therapy there is an ongoing and compelling need to develop high-throughput screening tools to further examine the biological effects of ionizing radiation on cells, tissues and organ systems in either the research or clinical setting. Conventional x-ray irradiators are designed to provide maximum versatility to radiobiology researchers, typically accommodating small animals, tissue or blood samples, and cellular applications. This added versatility often impedes the overall sensitivity and specificity of an experiment resulting in a trade-off between the number of absorbed doses (or dose rates) and biological endpoints that can be investigated in vitro in a reasonable amount of time. Therefore, modern irradiator designs are incompatible with current high-throughput bioassay technologies. Furthermore, important dosimetry and calibration characteristics (i.e. dose build-up region, beam attenuation, and beam scatter) of these irradiators are typically unknown to the end user, which can lead to significant deviation between delivered dose and intended dose to cells that adversely impact experimental results. Therefore, the overarching goal of this research is to design and develop a robust and fully automated high-throughput irradiator for in vitro radiation sensitivity investigations. Additionally, in vitro biological validation of this system was performed by assessing intracellular reactive oxygen species production, physical DNA double strand breaks, and activation of cellular DNA repair mechanisms. Finally, the high-throughput irradiator was used to investigate autophagic flux, a cellular adaptive response, as a potential biomarker of radiation sensitivity.

  11. High-throughput analysis of the impact of antibiotics on the human intestinal microbiota composition

    NARCIS (Netherlands)

    Ladirat, S.E.; Schols, H.A.; Nauta, A.; Schoterman, M.H.C.; Keijser, B.J.F.; Montijn, R.C.; Gruppen, H.; Schuren, F.H.J.

    2013-01-01

    Antibiotic treatments can lead to a disruption of the human microbiota. In this in-vitro study, the impact of antibiotics on adult intestinal microbiota was monitored in a new high-throughput approach: a fermentation screening-platform was coupled with a phylogenetic microarray analysis (Intestinal-

  12. Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

    Science.gov (United States)

    There are thousands of chemicals that are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The ToxCast high-throughput screening (HTS) program has evaluated over 1,800 chemicals in concentration-response across ~8...

  13. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    Science.gov (United States)

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale.

  14. High-Throughput Bubble Screening Method for Combinatorial Discovery of Electrocatalysts for Water Splitting

    OpenAIRE

    Xiang, Chengxiang; Suram, Santosh K.; Haber, Joel A.; Guevarra, Dan W.; Soedarmadji, Ed; Jin, Jian; Gregoire, John M.

    2014-01-01

    Combinatorial synthesis and screening for discovery of electrocatalysts has received increasing attention, particularly for energy-related technologies. High-throughput discovery strategies typically employ a fast, reliable initial screening technique that is able to identify active catalyst composition regions. Traditional electrochemical characterization via current–voltage measurements is inherently throughput-limited, as such measurements are most readily performed by serial screening. Pa...

  15. High-throughput Raman chemical imaging for rapid evaluation of food safety and quality

    Science.gov (United States)

    High-throughput macro-scale Raman chemical imaging was realized on a newly developed line-scan hyperspectral system. The system utilizes a custom-designed 785 nm line laser with maximum power of 5 W as an excitation source. A 24 cm × 1 mm excitation line is normally projected on the sample surface u...

  16. High-throughput screening for industrial enzyme production hosts by droplet microfluidics

    DEFF Research Database (Denmark)

    Sjostrom, Staffan L.; Bai, Yunpeng; Huang, Mingtao;

    2014-01-01

    A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α...

  17. High-throughput fluorescence assay of cytochrome P450 3A4

    OpenAIRE

    Cheng, Qian; Guengerich, F. Peter

    2013-01-01

    Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence.

  18. Cancer panomics: computational methods and infrastructure for integrative analysis of cancer high-throughput "omics" data

    DEFF Research Database (Denmark)

    Brunak, Søren; De La Vega, Francisco M.; Rätsch, Gunnar;

    2014-01-01

    Targeted cancer treatment is becoming the goal of newly developed oncology medicines and has already shown promise in some spectacular cases such as the case of BRAF kinase inhibitors in BRAF-mutant (e.g. V600E) melanoma. These developments are driven by the advent of high-throughput sequencing, ...

  19. Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus;

    Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

  20. An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments

    NARCIS (Netherlands)

    Duitama, Jorge; Quintero, Juan Camilo; Cruz, Daniel Felipe; Quintero, Constanza; Hubmann, Georg; Foulquié-Moreno, Maria R.; Verstrepen, Kevin J.; Thevelein, Johan M.; Tohme, Joe

    2014-01-01

    Recent advances in high-throughput sequencing (HTS) technologies and computing capacity have produced unprecedented amounts of genomic data that have unraveled the genetics of phenotypic variability in several species. However, operating and integrating current software tools for data analysis still

  1. High-throughput open source computational methods for genetics and genomics

    NARCIS (Netherlands)

    Prins, J.C.P.

    2015-01-01

    Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly refle

  2. A perspective on high throughput analysis of pesticide residues in foods

    Institute of Scientific and Technical Information of China (English)

    Kai ZHANG; Jon W WONG; Perry G WANG

    2011-01-01

    The screening of pesticide residues plays a vital role in food safety. Applications of high throughput analytical procedures are desirable for screening a large number of pesticides and food samples in a time-effi- cient and cost-effective manner. This review discusses how sample throughput of pesticide analysis could be improved with an emphasis on sample preparation, instrumentation and data analysis.

  3. High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry

    Science.gov (United States)

    In this work we report a high throughput mass spectrometry-based technique for rapid high resolution strain identification of Campylobacter jejuni. This method readily distinguishes C. jejuni from C. coli, has comparable resolving power to multi-locus sequence typing (MLST), is applicable to mixtur...

  4. Complementing high-throughput X-ray powder diffraction data with quantum-chemical calculations

    DEFF Research Database (Denmark)

    Naelapaa, Kaisa; van de Streek, Jacco; Rantanen, Jukka;

    2012-01-01

    High-throughput crystallisation and characterisation platforms provide an efficient means to carry out solid-form screening during the pre-formulation phase. To determine the crystal structures of identified new solid phases, however, usually requires independent crystallisation trials to produce...

  5. A high throughput DNA extraction method with high yield and quality

    Directory of Open Access Journals (Sweden)

    Xin Zhanguo

    2012-07-01

    Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

  6. High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors

    NARCIS (Netherlands)

    Koudijs, M.J.; Klijn, C.; van der Weyden, L.; Kool, J.; ten Hoeve, J.; Sie, D.; Prasetyanti, P.R.; Schut, E.; Kas, S.; Whipp, T.; Cuppen, E.; Wessels, L.; Adams, D.J.; Jonkers, J.

    2011-01-01

    Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on

  7. A high throughput electron lithography system using a field emission gun

    International Nuclear Information System (INIS)

    This paper discusses production quantities of GaAs FET's and MMIC's in demand for satellite communications and defense applications. Device requirements are enumerated and development of a high throughput submicron and nanometric lithography system based on a field emission electron gun is described. The authors present the system and performance characteristics of this machine and offers results from a recent installation

  8. The Power of High-Throughput Experimentation in Homogeneous Catalysis Research for Fine Chemicals

    NARCIS (Netherlands)

    Vries, Johannes G. de; Vries, André H.M. de

    2003-01-01

    The use of high-throughput experimentation (HTE) in homogeneous catalysis research for the production of fine chemicals is an important breakthrough. Whereas in the past stoichiometric chemistry was often preferred because of time-to-market constraints, HTE allows catalytic solutions to be found wit

  9. Improved detection of artifactual viral minority variants in high-throughput sequencing data

    NARCIS (Netherlands)

    M.R.A. Welkers (Matthijs); M. Jonges (Marcel); R.E. Jeeninga (Rienk); M.P.G. Koopmans D.V.M. (Marion); M.D. de Jong (Menno)

    2015-01-01

    textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the

  10. Improved detection of artifactual viral minority variants in high-throughput sequencing data

    NARCIS (Netherlands)

    M.R.A. Welkers (Matthijs); M. Jonges (Marcel); R.E. Jeeninga (Rienk); M.P.G. Koopmans D.V.M. (Marion); M.D. de Jong (Menno)

    2014-01-01

    textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the

  11. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure tha...

  12. Protocol: A high-throughput DNA extraction system suitable for conifers

    Directory of Open Access Journals (Sweden)

    Rajora Om P

    2008-08-01

    Full Text Available Abstract Background High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers. Results We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP and another for high-throughput (HTP DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species. Conclusion A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.

  13. Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

    Science.gov (United States)

    Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

    2013-06-01

    High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome

  14. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  15. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    Directory of Open Access Journals (Sweden)

    Delyan P Ivanov

    Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

  16. Evaluation of the antileishmanial and cytotoxic effects of various extracts of garlic (Allium sativum) on Leishmania tropica.

    Science.gov (United States)

    Mahmoudvand, Hossein; Sepahvand, Peyman; Jahanbakhsh, Sareh; Azadpour, Mozhgan

    2016-06-01

    Cutaneous leishmaniasis (CL) is a major public health problem in tropical and subtropical countries worldwide. Treatment of CL by pentavalent antimony compounds remains a challenge because of limited efficacy, toxic side effects and drug resistance. In the present study, in vitro antileishmanial and cytotoxic activity of garlic extracts against promastigote forms of Leishmania tropica and murine macrophages was evaluated by colorimetric cell viability (MTT) assay. The results revealed that the methanolic and aqueous extracts of garlic were effective in inhibiting promastigote growth of L. tropica with IC50 (50 % inhibitory concentrations) values 12.3 and 19.2 µg/ml, respectively. In addition, methanolic and aqueous extracts of garlic showed low cytotoxicity against murine macrophages with CC50 (cytotoxicity concentration for 50 % of cells) values 291.4 and 348.2 µg/ml, respectively. Findings of present study were the first step in the search for new antileishmanial drugs. However, further works are required to evaluate exact effect of these extracts in volunteer human subjects. PMID:27413315

  17. High-Throughput Synthesis and Characterization of BiMoVOX Materials

    International Nuclear Information System (INIS)

    The high throughput synthesis and characterization of a particular family of ceramic materials, bismuth molybdenum vanadium oxides (BiMoVOX), suitable as inorganic yellow pigments and low temperature oxidation catalysts, is described. Samples, synthesized by calcination and peroxo sol-gel methods, are characterized by X-ray powder diffraction, UV-visible and XAFS spectroscopy. A combined high-throughput XRD/XAFS study of a 54 samples array, with simultaneous refinement of data of both techniques, has been performed. Molybdenum doping of bismuth vanadate results in a phase transition from monoclinic BiVO4 to tetragonal Bi(V,Mo)O4, both of scheelite type. Both central metals, V5+ and Mo6+, remain in a tetrahedral coordination. UV/visible spectroscopy identifies a linear blue shift as a function of Mo6+ amount

  18. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing.

    Science.gov (United States)

    Woodruff, Kristina; Maerkl, Sebastian J

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  19. High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

    DEFF Research Database (Denmark)

    Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K;

    2014-01-01

    BACKGROUND: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent...... distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. METHODS: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45......-colour technique is rapid, cost effective and robust with comparable sensitivity to microscopy and capable of discriminating between live and dead and/or compromised parasites. Staining for CD45 improved parasitaemia estimates in ADCI assay since high numbers of leucocytes interfered with the accurate...

  20. Miniature high-throughput chemosensing of yield, ee, and absolute configuration from crude reaction mixtures.

    Science.gov (United States)

    Bentley, Keith W; Zhang, Peng; Wolf, Christian

    2016-02-01

    High-throughput experimentation (HTE) has emerged as a widely used technology that accelerates discovery and optimization processes with parallel small-scale reaction setups. A high-throughput screening (HTS) method capable of comprehensive analysis of crude asymmetric reaction mixtures (eliminating product derivatization or isolation) would provide transformative impact by matching the pace of HTE. We report how spontaneous in situ construction of stereodynamic metal probes from readily available, inexpensive starting materials can be applied to chiroptical chemosensing of the total amount, enantiomeric excess (ee), and absolute configuration of a wide variety of amines, diamines, amino alcohols, amino acids, carboxylic acids, α-hydroxy acids, and diols. This advance and HTS potential are highlighted with the analysis of 1 mg of crude reaction mixtures of a catalytic asymmetric reaction. This operationally simple assay uses a robust mix-and-measure protocol, is amenable to microscale platforms and automation, and provides critical time efficiency and sustainability advantages over traditional serial methods. PMID:26933684

  1. Development of scalable high throughput fermentation approaches for physiological characterisation of yeast and filamentous fungi

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen

    of strains generated through genetic engineering programmes, the traditionally applied methods for strain characterisation, which are typically labour intensive and time consuming, have become somewhat limited due to throughput capacity. Unfortunately, most high throughput methods only provide low levels...... producing the heterologous model polyketide, 6-methylsalicylic acid (6-MSA). An automated methodology for high throughput screening focusing on growth rates, together with a fully automated method for quantitative physiological characterisation in microtiter plates, was established for yeast. Full...... scalability was demonstrated through comparative physiological characterisation of yeasts, cultivated in both microtiter plates and bioreactors, revealing that the growth rate and yield coefficients of all non-volatile products including biomass could be correlated. The highly correlated results were taken...

  2. A High Throughput MAC Protocol for Wireless Sensor Networks in Surveillance Applications

    Directory of Open Access Journals (Sweden)

    Jian-hua WANG

    2013-09-01

    Full Text Available Monitoring a given environment is a kind of major applications in wireless sensor networks. These WSNs should often meet special requirements, such as high throughput support, service differentiation support and energy efficiency. However, related works always emphasis on one or two of them, and hardly consider comprehensively. In this paper, we propose a new-style MAC protocol, which based on the above-mentioned factors. Cluster-based multi-hop scheduling and priority-aware schedule switching are crucial technologies in the MAC protocol. Moreover, idle listening energy consumption is reduced by using a synchronized duty cycle. Experiments show that the proposed strategy achieves our goals. The energy consumption of the radio module is reduced while high throughput is provided.

  3. High throughput route selection in multi-rate wireless mesh networks

    Institute of Scientific and Technical Information of China (English)

    WEI Yi-fei; GUO Xiang-li; SONG Mei; SONG Jun-de

    2008-01-01

    Most existing Ad-hoc routing protocols use the shortest path algorithm with a hop count metric to select paths. It is appropriate in single-rate wireless networks, but has a tendency to select paths containing long-distance links that have low data rates and reduced reliability in multi-rate networks. This article introduces a high throughput routing algorithm utilizing the multi-rate capability and some mesh characteristics in wireless fidelity (WiFi) mesh networks. It uses the medium access control (MAC) transmission time as the routing metric, which is estimated by the information passed up from the physical layer. When the proposed algorithm is adopted, the Ad-hoc on-demand distance vector (AODV) routing can be improved as high throughput AODV (HT-AODV). Simulation results show that HT-AODV is capable of establishing a route that has high data-rate, short end-to-end delay and great network throughput.

  4. Lognormality and oscillations in the coverage of high-throughput transcriptomic data towards gene ends

    International Nuclear Information System (INIS)

    High-throughput transcriptomics experiments have reached the stage where the count of the number of reads alignable to a given position can be treated as an almost-continuous signal. This allows us to ask questions of biophysical/biotechnical nature, but which may still have biological implications. Here we show that when sequencing RNA fragments from one end, as is the case on most platforms, an oscillation in the read count is observed at the other end. We further show that these oscillations can be well described by Kolmogorov’s 1941 broken stick model. We investigate how the model can be used to improve predictions of gene ends (3′ transcript ends), but conclude that with present data the improvement is only marginal. The results highlight subtle effects in high-throughput transcriptomics experiments which do not have a biological origin, but which may still be used to obtain biological information. (paper)

  5. Standard finishing categories for high-throughput sequencing of viral genomes.

    Science.gov (United States)

    Ladner, J T; Kuhn, J H; Palacios, G

    2016-04-01

    Viral genome sequencing has become the cornerstone of almost all aspects of virology. In particular, high-throughput, next-generation viral genome sequencing has become an integral part of molecular epidemiological investigations into outbreaks of viral disease, such as the recent outbreaks of Middle Eastern respiratory syndrome, Ebola virus disease and Zika virus infection. Multiple institutes have acquired the expertise and necessary infrastructure to perform such investigations, as evidenced by the accumulation of thousands of novel viral sequences over progressively shorter time periods. The authors recently proposed a nomenclature comprised of five high-throughput sequencing standard categories to describe the quality of determined viral genome sequences. These five categories (standard draft, high quality, coding complete, complete and finished) cover all levels of viral genome finishing and can be applied to sequences determined by any technology platform or assembly technique.

  6. Gradient Technology for High-Throughput Screening of Interactions between Cells and Nanostructured Materials

    Directory of Open Access Journals (Sweden)

    Andrew Michelmore

    2012-01-01

    Full Text Available We present a novel substrate suitable for the high-throughput analysis of cell response to variations in surface chemistry and nanotopography. Electrochemical etching was used to produce silicon wafers with nanopores between 10 and 100 nm in diameter. Over this substrate and flat silicon wafers, a gradient film ranging from hydrocarbon to carboxylic acid plasma polymer was deposited, with the concentration of surface carboxylic acid groups varying between 0.7 and 3% as measured by XPS. MG63 osteoblast-like cells were then cultured on these substrates and showed greatest cell spreading and adhesion onto porous silicon with a carboxylic acid group concentration between 2-3%. This method has great potential for high-throughput screening of cell-material interaction with particular relevance to tissue engineering.

  7. Turning tumor-promoting copper into an anti-cancer weapon via high-throughput chemistry.

    Science.gov (United States)

    Wang, F; Jiao, P; Qi, M; Frezza, M; Dou, Q P; Yan, B

    2010-01-01

    Copper is an essential element for multiple biological processes. Its concentration is elevated to a very high level in cancer tissues for promoting cancer development through processes such as angiogenesis. Organic chelators of copper can passively reduce cellular copper and serve the role as inhibitors of angiogenesis. However, they can also actively attack cellular targets such as proteasome, which plays a critical role in cancer development and survival. The discovery of such molecules initially relied on a step by step synthesis followed by biological assays. Today high-throughput chemistry and high-throughput screening have significantly expedited the copper-binding molecules discovery to turn "cancer-promoting" copper into anti-cancer agents.

  8. Computational high-throughput screening of fluid permeability in heterogeneous fiber materials.

    Science.gov (United States)

    Röding, Magnus; Schuster, Erich; Logg, Katarina; Lundman, Malin; Bergström, Per; Hanson, Charlotta; Gebäck, Tobias; Lorén, Niklas

    2016-07-20

    We explore computational high-throughput screening as a design strategy for heterogeneous, isotropic fiber materials. Fluid permeability, a key property in the design of soft porous materials, is systematically studied using a multi-scale lattice Boltzmann framework. After characterizing microscopic permeability as a function of solid volume fraction in the microstructure, we perform high-throughput computational screening of in excess of 35 000 macrostructures consisting of a continuous bulk interrupted by spherical/elliptical domains with either lower or higher microscopic permeability (hence with two distinct microscopic solid volume fractions and therefore two distinct microscopic permeabilities) to assess which parameters determine macroscopic permeability for a fixed average solid volume fraction. We conclude that the fractions of bulk and domains and the distribution of solid volume fraction between them are the primary determinants of macroscopic permeability, and that a substantial increase in permeability compared to the corresponding homogenous material is attainable. PMID:27367292

  9. A pulse-front-tilt-compensated streaked optical spectrometer with high throughput and picosecond time resolution

    Science.gov (United States)

    Katz, J.; Boni, R.; Rivlis, R.; Muir, C.; Froula, D. H.

    2016-11-01

    A high-throughput, broadband optical spectrometer coupled to the Rochester optical streak system equipped with a Photonis P820 streak tube was designed to record time-resolved spectra with 1-ps time resolution. Spectral resolution of 0.8 nm is achieved over a wavelength coverage range of 480 to 580 nm, using a 300-groove/mm diffraction grating in conjunction with a pair of 225-mm-focal-length doublets operating at an f/2.9 aperture. Overall pulse-front tilt across the beam diameter generated by the diffraction grating is reduced by preferentially delaying discrete segments of the collimated input beam using a 34-element reflective echelon optic. The introduced delay temporally aligns the beam segments and the net pulse-front tilt is limited to the accumulation across an individual sub-element. The resulting spectrometer design balances resolving power and pulse-front tilt while maintaining high throughput.

  10. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

    Energy Technology Data Exchange (ETDEWEB)

    Hura, Greg L.; Menon, Angeli L.; Hammel, Michal; Rambo, Robert P.; Poole II, Farris L.; Tsutakawa, Susan E.; Jenney Jr, Francis E.; Classen, Scott; Frankel, Kenneth A.; Hopkins, Robert C.; Yang, Sungjae; Scott, Joseph W.; Dillard, Bret D.; Adams, Michael W. W.; Tainer, John A.

    2009-07-20

    We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

  11. Multiple and high-throughput droplet reactions via combination of microsampling technique and microfluidic chip

    KAUST Repository

    Wu, Jinbo

    2012-11-20

    Microdroplets offer unique compartments for accommodating a large number of chemical and biological reactions in tiny volume with precise control. A major concern in droplet-based microfluidics is the difficulty to address droplets individually and achieve high throughput at the same time. Here, we have combined an improved cartridge sampling technique with a microfluidic chip to perform droplet screenings and aggressive reaction with minimal (nanoliter-scale) reagent consumption. The droplet composition, distance, volume (nanoliter to subnanoliter scale), number, and sequence could be precisely and digitally programmed through the improved sampling technique, while sample evaporation and cross-contamination are effectively eliminated. Our combined device provides a simple model to utilize multiple droplets for various reactions with low reagent consumption and high throughput. © 2012 American Chemical Society.

  12. High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

    Science.gov (United States)

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  13. High-throughput engineering and analysis of peptide binding to class II MHC.

    Science.gov (United States)

    Jiang, Wei; Boder, Eric T

    2010-07-27

    Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.

  14. A Discrete Time Markov Chain Model for High Throughput Bidirectional Fano Decoders

    CERN Document Server

    Xu, Ran; Morris, Kevin; Kocak, Taskin

    2010-01-01

    The bidirectional Fano algorithm (BFA) can achieve at least two times decoding throughput compared to the conventional unidirectional Fano algorithm (UFA). In this paper, bidirectional Fano decoding is examined from the queuing theory perspective. A Discrete Time Markov Chain (DTMC) is employed to model the BFA decoder with a finite input buffer. The relationship between the input data rate, the input buffer size and the clock speed of the BFA decoder is established. The DTMC based modelling can be used in designing a high throughput parallel BFA decoding system. It is shown that there is a tradeoff between the number of BFA decoders and the input buffer size, and an optimal input buffer size can be chosen to minimize the hardware complexity for a target decoding throughput in designing a high throughput parallel BFA decoding system.

  15. Macro-to-micro structural proteomics: native source proteins for high-throughput crystallization.

    Directory of Open Access Journals (Sweden)

    Monica Totir

    Full Text Available Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

  16. A high-throughput, multi-channel photon-counting detector with picosecond timing

    CERN Document Server

    Lapington, J S; Miller, G M; Ashton, T J R; Jarron, P; Despeisse, M; Powolny, F; Howorth, J; Milnes, J

    2009-01-01

    High-throughput photon counting with high time resolution is a niche application area where vacuum tubes can still outperform solid-state devices. Applications in the life sciences utilizing time-resolved spectroscopies, particularly in the growing field of proteomics, will benefit greatly from performance enhancements in event timing and detector throughput. The HiContent project is a collaboration between the University of Leicester Space Research Centre, the Microelectronics Group at CERN, Photek Ltd., and end-users at the Gray Cancer Institute and the University of Manchester. The goal is to develop a detector system specifically designed for optical proteomics, capable of high content (multi-parametric) analysis at high throughput. The HiContent detector system is being developed to exploit this niche market. It combines multi-channel, high time resolution photon counting in a single miniaturized detector system with integrated electronics. The combination of enabling technologies; small pore microchanne...

  17. DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population

    OpenAIRE

    Colasuonno, Pasqualina; Incerti, Ornella; Lozito, Maria Luisa; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

    2016-01-01

    Background Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mi...

  18. High-Throughput Sequencing—The Key to Rapid Biodiversity Assessment of Marine Metazoa?

    OpenAIRE

    Inga Mohrbeck; Raupach, Michael J.; Pedro Martínez Arbizu; Thomas Knebelsberger; Silke Laakmann

    2015-01-01

    The applications of traditional morphological and molecular methods for species identification are greatly restricted by processing speed and on a regional or greater scale are generally considered unfeasible. In this context, high-throughput sequencing, or metagenetics, has been proposed as an efficient tool to document biodiversity. Here we evaluated the effectiveness of 454 pyrosequencing in marine metazoan community analysis using the 18S rDNA: V1-V2 region. Multiplex pyrosequencing of th...

  19. Geochip: A high throughput genomic tool for linking community structure to functions

    Energy Technology Data Exchange (ETDEWEB)

    Van Nostrand, Joy D.; Liang, Yuting; He, Zhili; Li, Guanghe; Zhou, Jizhong

    2009-01-30

    GeoChip is a comprehensive functional gene array that targets key functional genes involved in the geochemical cycling of N, C, and P, sulfate reduction, metal resistance and reduction, and contaminant degradation. Studies have shown the GeoChip to be a sensitive, specific, and high-throughput tool for microbial community analysis that has the power to link geochemical processes with microbial community structure. However, several challenges remain regarding the development and applications of microarrays for microbial community analysis.

  20. High-throughput in-volume processing in glass with isotropic spatial resolutions in three dimensions

    CERN Document Server

    Tan, Yuanxin; Chu, Wei; Liao, Yang; Qiao, Lingling; Cheng, Ya

    2016-01-01

    We report on fabrication of three dimensional (3D) microstructures in glass with isotropic spatial resolutions. To achieve high throughput fabrication, we expand the focal spot size with a low-numerical-aperture lens, which naturally results in a degraded axial resolution. We solve the problem with simultaneous spatial temporal focusing which leads to an isotropic laser-affected volume with a spatial resolution of ~100 micron.

  1. High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

    OpenAIRE

    Bae, Hyeonhu; Park, Minwoo; Jang, Byungryul; Kang, Yura; Park, Jinwoo; Lee, Hosik; Chung, Haegeun; Chung, ChiHye; Hong, Suklyun; Kwon, Yongkyung; Yakobson, Boris I.; Lee, Hoonkyung

    2016-01-01

    Nanostructured materials, such as zeolites and metal-organic frameworks, have been considered to capture CO2. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO2 capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO2 from gaseous mixtures under low CO2 pressures...

  2. High-throughput screening of metal-porphyrin-like graphenes for selective capture of carbon dioxide

    OpenAIRE

    Hyeonhu Bae; Minwoo Park; Byungryul Jang; Yura Kang; Jinwoo Park; Hosik Lee; Haegeun Chung; ChiHye Chung; Suklyun Hong; Yongkyung Kwon; Yakobson, Boris I.; Hoonkyung Lee

    2016-01-01

    Nano-materials, such as metal-organic frameworks, have been considered to capture CO$_2$. However, their application has been limited largely because they exhibit poor selectivity for flue gases and low capture capacity under low pressures. We perform a high-throughput screening for selective CO$_2$ capture from flue gases by using first principles thermodynamics. We find that elements with empty d orbitals selectively attract CO$_2$ from gaseous mixtures under low CO$_2$ pressures at 300 K a...

  3. Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs

    OpenAIRE

    Wagner, Darcy E; Fenn, Spencer L.; Bonenfant, Nicholas R.; Marks, Elliot R.; Borg, Zachary; Saunders, Patrick; Oldinski, Rachael A.; Weiss, Daniel J

    2014-01-01

    Whole organ decellularization of complex organs, such as lungs, presents a unique opportunity for use of acellular scaffolds for ex vivo tissue engineering or for studying cell-extracellular matrix interactions ex vivo. A growing body of literature investigating decellularizing and recellularizing rodent lungs has provided important proof of concept models and rodent lungs are readily available for high throughput studies. In contrast, comparable progress in large animal and human lungs has b...

  4. SNP HiTLink: a high-throughput linkage analysis system employing dense SNP data

    OpenAIRE

    Kuwano Ryozo; Miyashita Akinori; Goto Jun; Takahashi Yuji; Date Hidetoshi; Nakahara Yasuo; Fukuda Yoko; Adachi Hiroki; Nakamura Eiji; Tsuji Shoji

    2009-01-01

    Abstract Background During this recent decade, microarray-based single nucleotide polymorphism (SNP) data are becoming more widely used as markers for linkage analysis in the identification of loci for disease-associated genes. Although microarray-based SNP analyses have markedly reduced genotyping time and cost compared with microsatellite-based analyses, applying these enormous data to linkage analysis programs is a time-consuming step, thus, necessitating a high-throughput platform. Result...

  5. A high-throughput method for quantifying metabolically active yeast cells

    OpenAIRE

    Nandy, Subir Kumar; Knudsen, Peter Boldsen; Rosenkjær, Alexander; Eliasson Lantz, Anna; Thykær, Jette; Workman, Mhairi

    2015-01-01

    By redesigning the established methylene blue reduction test for bacteria and yeast, we present a cheap and efficient methodology for quantitative physiology of eukaryotic cells applicable for high-throughput systems. Validation of themethod in fermenters and highthroughput systems proved equivalent, displaying reduction curves that interrelated directly with CFU counts. For growth rate estimation, the methylene blue reduction test (MBRT) proved superior, since the discriminatory nature of th...

  6. High-sensitivity high-throughput chip based biosensor array for multiplexed detection of heavy metals

    Science.gov (United States)

    Yan, Hai; Tang, Naimei; Jairo, Grace A.; Chakravarty, Swapnajit; Blake, Diane A.; Chen, Ray T.

    2016-03-01

    Heavy metal ions released into the environment from industrial processes lead to various health hazards. We propose an on-chip label-free detection approach that allows high-sensitivity and high-throughput detection of heavy metals. The sensing device consists of 2-dimensional photonic crystal microcavities that are combined by multimode interferometer to form a sensor array. We experimentally demonstrate the detection of cadmium-chelate conjugate with concentration as low as 5 parts-per-billion (ppb).

  7. High Throughput Single-cell and Multiple-cell Micro-encapsulation

    OpenAIRE

    Lagus, Todd P.; Edd, Jon F.

    2012-01-01

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of ...

  8. Progress in high-throughput assays of MGMT and APE1 activities in cell extracts

    OpenAIRE

    Georgiadis, Panagiotis; Polychronaki, Nektaria; Kyrtopoulos, Soterios A.

    2012-01-01

    DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6...

  9. Versatile synthesis of probes for high-throughput enzyme activity screening

    OpenAIRE

    de Rond, Tristan; Peralta-Yahya, Pamela; Cheng, Xiaoliang; Northen, Trent R.; Keasling, Jay D.

    2013-01-01

    Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We ...

  10. Microfabricated magnetic sifter for high-throughput and high-gradient magnetic separation

    OpenAIRE

    Earhart, Christopher M.; Wilson, Robert J.; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2009-01-01

    A microfabricated magnetic sifter has been designed and fabricated for applications in biological sample preparation. The device enables high-throughput, high-gradient magnetic separation of magnetic nanoparticles by utilizing columnar fluid flow through a dense array (~5000/mm2) of micropatterned slots in a magnetically soft membrane. The potential of the sifter for separation of magnetic nanoparticles conjugated with capture antibodies is demonstrated through quantitative separation experim...

  11. High-throughput nucleotide sequence analysis of diverse bacterial communities in leachates of decomposing pig carcasses

    OpenAIRE

    Seung Hak Yang; Joung Soo Lim; Modabber Ahmed Khan; Bong Soo Kim; Dong Yoon Choi; Eun Young Lee; Hee Kwon Ahn

    2015-01-01

    The leachate generated by the decomposition of animal carcass has been implicated as an environmental contaminant surrounding the burial site. High-throughput nucleotide sequencing was conducted to investigate the bacterial communities in leachates from the decomposition of pig carcasses. We acquired 51,230 reads from six different samples (1, 2, 3, 4, 6 and 14 week-old carcasses) and found that sequences representing the phylum Firmicutes predominated. The diversity of bacterial 16S rRNA gen...

  12. Discovering Ce-rich oxygen evolution catalysts, from high throughput screening to water electrolysis

    OpenAIRE

    Haber, Joel A.; Cai, Yun; Jung, Suho; Xiang, Chengxiang; Mitrovic, Slobodan; Jin, Jian; Bell, Alexis T.; Gregoire, John M.

    2014-01-01

    We report a new Ce-rich family of active oxygen evolution reaction (OER) catalysts composed of earth abundant elements, discovered using high-throughput methods. High resolution inkjet printing was used to produce 5456 discrete oxide compositions containing the elements nickel, iron, cobalt and cerium. The catalytic performance of each of these compositions was measured under conditions applicable to distributed solar fuels generation using a three-electrode scanning drop electrochemical cell...

  13. On the optimal trimming of high-throughput mRNA sequence data

    OpenAIRE

    MacManes, Matthew D

    2014-01-01

    The widespread and rapid adoption of high-throughput sequencing technologies has afforded researchers the opportunity to gain a deep understanding of genome level processes that underlie evolutionary change, and perhaps more importantly, the links between genotype and phenotype. In particular, researchers interested in functional biology and adaptation have used these technologies to sequence mRNA transcriptomes of specific tissues, which in turn are often compared to other tissues, or other ...

  14. ‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

    OpenAIRE

    Kim, Hwangbeom; Han, Hyojun; Ahn, Jinwoo; Lee, Joongoo; Cho, Namjin; Jang, Hoon; Kim, Hyoki; Kwon, Sunghoon; Bang, Duhee

    2012-01-01

    We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, a...

  15. Combinatorial Method/High Throughput Strategies for Hydrogel Optimization in Tissue Engineering Applications

    OpenAIRE

    Smith Callahan, Laura A.

    2016-01-01

    Combinatorial method/high throughput strategies, which have long been used in the pharmaceutical industry, have recently been applied to hydrogel optimization for tissue engineering applications. Although many combinatorial methods have been developed, few are suitable for use in tissue engineering hydrogel optimization. Currently, only three approaches (design of experiment, arrays and continuous gradients) have been utilized. This review highlights recent work with each approach. The benefi...

  16. Evaluation of Nanofluidics Technology for High-Throughput SNP Genotyping in a Clinical Setting

    OpenAIRE

    Chan, Maurice; Chan, Mei Wen; Loh, Ting Wei; Law, Hai Yang; Yoon, Chui Sheun; Than, Sint Sint; Chua, Jia Mei; Wong, Chow Yin; Yong, Wei Sean; Yap, Yoon Sim; Ho, Gay Hui; Ang, Peter; Lee, Ann Siew Gek

    2011-01-01

    The current need for high-throughput genotyping platforms for targeted validation of disease-associated single nucleotide polymorphisms (SNPs) motivated us to evaluate a novel nanofluidics platform for genotyping DNA extracted from peripheral blood and buccal wash samples. SNP genotyping was performed using a Fluidigm 48.48 Dynamic Array biochip on the BioMark polymerase chain reaction platform and results were compared against standard TaqMan assays and DNA sequencing. Pilot runs using these...

  17. Melanin-Based High-Throughput Screen for l-Tyrosine Production in Escherichia coli▿

    OpenAIRE

    Santos, Christine Nicole S.; Stephanopoulos, Gregory

    2007-01-01

    We present the development of a simple, high-throughput screen for identifying bacterial strains capable of l-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link l-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient...

  18. Miniaturized droplets-based microarray of chemical and biological high-throughput tests

    OpenAIRE

    Neto, Ana I.; Correia, Clara R.; Custódio, Catarina A.; Mano, J.F

    2013-01-01

    Publicado em "Journal of Tissue Engineering and Regenerative Medicine, vol. 7, supp. 1 (2013) The development of high-throughput and combinatorial technologies is helping to speed up research that is applicable in many areas of chemistry, engineering and biology. We propose a simple, versatile high-efficient and new superhydrophobic platform, which permits to arrange of quasi-spherical aqueous-based droplets with the capability to support and monitor a series of chemical/biolog...

  19. Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Braga, Thiago Verano; Roepstorff, Peter

    2015-01-01

    The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines...... for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis...

  20. High Cycle Fatigue of Al and Cu Thin Films by a Novel High-Throughput Method

    OpenAIRE

    Burger, Sofie

    2013-01-01

    In the last two decades, the reliability of small electronic devices used in automotive or consumer electronics gained researchers attention. Thus, there is the need to understand the fatigue properties and damage mechanisms of thin films. In this thesis a novel high-throughput testing method for thin films on Si substrate is presented. The specialty of this method is to test one sample at different strain amplitudes at the same time and measure an entire lifetime curve with only one experiment.

  1. Differential Scanning Fluorimetry provides high throughput data on silk protein transitions

    OpenAIRE

    Vollrath, Fritz; Hawkins, Nick; Porter, David; Holland, Chris; Boulet-Audet, Maxime

    2014-01-01

    Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted ‘silk' fibroin show that DSF can provide valuable insights into details of...

  2. Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning.

    Science.gov (United States)

    Ozkumur, Ayca Yalcin; Goods, Brittany A; Love, J Christopher

    2015-09-01

    Living-cell-based screens can facilitate lead discovery of functional therapeutics of interest. A versatile and scalable method is reported that uses dense arrays of nanowells for imparting defined patterns on monolayers of cells. It is shown that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutralizing antibodies against HIV. PMID:26121321

  3. Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

    OpenAIRE

    Guo, Feng; Zhang, Tong

    2012-01-01

    Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also seque...

  4. Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

    OpenAIRE

    Nayar, Asha S.; Dougherty, Thomas J.; Ferguson, Keith E.; Granger, Brett A.; McWilliams, Lisa; Stacey, Clare; Leach, Lindsey J.; Narita, Shin-ichiro; Tokuda, Hajime; Miller, Alita A.; Brown, Dean G.; McLeod, Sarah M.

    2015-01-01

    A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. ...

  5. A High-Throughput Random Access Protocol for Multiuser MIMO Systems

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2008-05-01

    Full Text Available We propose a high-throughput random access protocol for 2×2 multiuser multiple-input multiple-output (MIMO systems. The cross-layer mechanism utilizes the packets combining technique to exploit the advantages of both spatial multiplexing and multipacket reception. Analytical result indicates that the proposed scheme achieves 0.669 per spatial degree of freedom in stable throughput, which is much higher than those in the existed studies.

  6. Computational Analysis of High-Throughput Sequencing Data in Cardiac Disease and Skeletal Muscle Development

    OpenAIRE

    Bansal, Vikas

    2016-01-01

    The advent of the high-throughput sequencing (HTS) technology has greatly accelerated research in life sciences. Due to its low cost and high efficiency, it is nowadays commonly used to answer various biological questions. In general, in HTS, the sequence of millions of DNA fragments is determined in parallel and these fragments can in turn be generated using different sequencing methods. With the rapid advancement of HTS technologies, their applications seem almost endless, for example it is...

  7. A high throughput (>90%), large compensation range, single-prism femtosecond pulse compressor

    OpenAIRE

    Kong, Lingjie; Cui, Meng

    2013-01-01

    We demonstrate a high throughput, large compensation range, single-prism femtosecond pulse compressor, using a single prism and two roof mirrors. The compressor has zero angular dispersion, zero spatial dispersion, zero pulse-front tilt, and unity magnification. The high efficiency is achieved by adopting two roof mirrors as the retroreflectors. We experimentally achieved ~ -14500 fs2 group delay dispersion (GDD) with 30 cm of prism tip-roof mirror prism separation, and ~90.7% system throughp...

  8. Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds

    OpenAIRE

    Tomasini-Johansson, Bianca R.; Johnson, Ian A.; Hoffmann, F. Michael; Mosher, Deane F.

    2012-01-01

    Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. Control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we dev...

  9. High-throughput screening of tick-borne pathogens in Europe

    DEFF Research Database (Denmark)

    Michelet, Lorraine; Delannoy, Sabine; Devillers, Elodie;

    2014-01-01

    was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia......, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia...

  10. High-throughput sequencing of frozen and paraffin-embedded tumor and normal tissue

    OpenAIRE

    Kerick, M; Timmermann, B; Schweiger, M.

    2010-01-01

    Until now high-throughput sequencing of tumor samples relied on DNA isolated from fresh frozen tissues, the preparation of which, however, is relatively laborious. The use of preserved material, i.e. from tissue banks, could help to avoid this limitation and would enable the reanalysis of diverse clinical trials. So far we have shown that formalin-fixed paraffin-embedded (FFPE) tissue samples can be used for genomic re-sequencing processes. FFPE samples are amply available from surgical tumor...

  11. Patterning cell using Si-stencil for high-throughput assay

    KAUST Repository

    Wu, Jinbo

    2011-01-01

    In this communication, we report a newly developed cell pattering methodology by a silicon-based stencil, which exhibited advantages such as easy handling, reusability, hydrophilic surface and mature fabrication technologies. Cell arrays obtained by this method were used to investigate cell growth under a temperature gradient, which demonstrated the possibility of studying cell behavior in a high-throughput assay. This journal is © The Royal Society of Chemistry 2011.

  12. High throughput screening of particle conditioning operations: I. System design and method development.

    Science.gov (United States)

    Noyes, Aaron; Huffman, Ben; Godavarti, Ranga; Titchener-Hooker, Nigel; Coffman, Jonathan; Sunasara, Khurram; Mukhopadhyay, Tarit

    2015-08-01

    The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale-down system (USD) for high throughput particle conditioning (HTPC) composed of off-the-shelf components. The apparatus is comprised of a temperature-controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot-scale performance of a non-geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale-up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale-up heuristics. PMID:25728932

  13. A computational framework for boosting confidence in high-throughput protein-protein interaction datasets

    OpenAIRE

    Hosur, R.; Peng, J.; A Vinayagam; Stelzl, U.; Xu, J.; Perrimon, N; Bienkowska, J.; Berger, B.

    2012-01-01

    Improving the quality and coverage of the protein interactome is of tantamount importance for biomedical research, particularly given the various sources of uncertainty in high-throughput techniques. We introduce a structure-based framework, Coev2Net, for computing a single confidence score that addresses both false-positive and false-negative rates. Coev2Net is easily applied to thousands of binary protein interactions and has superior predictive performance over existing methods. We experim...

  14. Applications of high-throughput genome and transcriptome analysis in human disease

    OpenAIRE

    Inkeles, Megan So

    2014-01-01

    The development of gene expression profiling technology has enabled the high-throughput discovery of the genes and pathways that underlie disease pathophysiology and phenotype. This work analyzes microarray and RNA sequencing data to identify genes and functional pathways associated with human diseases. In the first part, gene expression profiles derived from pancreatic ductal adenocarcinoma tumors are correlated to patient disease free survival time in order to find genes that confer a pro...

  15. Evolutionary Dynamics of Retrotransposons Assessed by High-Throughput Sequencing in Wild Relatives of Wheat

    OpenAIRE

    Senerchia, Natacha; Wicker, Thomas; Felber, François; Parisod, Christian

    2013-01-01

    Transposable elements (TEs) represent a major fraction of plant genomes and drive their evolution. An improved understanding of genome evolution requires the dynamics of a large number of TE families to be considered. We put forward an approach bypassing the required step of a complete reference genome to assess the evolutionary trajectories of high copy number TE families from genome snapshot with high-throughput sequencing. Low coverage sequencing of the complex genomes of Aegilops cylindri...

  16. Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms

    OpenAIRE

    Khare, Siddharth M.; Awasthi, Anjali; V. Venkataraman; Sandhya P Koushika

    2015-01-01

    Measuring forces applied by multi-cellular organisms is valuable in investigating biomechanics of their locomotion. Several technologies have been developed to measure such forces, for example, strain gauges, micro-machined sensors, and calibrated cantilevers. We introduce an innovative combination of techniques as a high throughput screening tool to assess forces applied by multiple genetic model organisms. First, we fabricated colored Polydimethylsiloxane (PDMS) micropillars where the color...

  17. High-throughput spatial light modulation two-photon microscopy for fast functional imaging

    OpenAIRE

    Pozzi, Paolo; Gandolfi, Daniela; Tognolina, Marialuisa; Chirico, Giuseppe; Mapelli, Jonathan; D’Angelo, Egidio

    2015-01-01

    The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup ...

  18. Zebrafish needle EMG: a new tool for high-throughput drug screens.

    Science.gov (United States)

    Cho, Sung-Joon; Nam, Tai-Seung; Byun, Donghak; Choi, Seok-Yong; Kim, Myeong-Kyu; Kim, Sohee

    2015-09-01

    Zebrafish models have recently been highlighted as a valuable tool in studying the molecular basis of neuromuscular diseases and developing new pharmacological treatments. Needle electromyography (EMG) is needed not only for validating transgenic zebrafish models with muscular dystrophies (MD), but also for assessing the efficacy of therapeutics. However, performing needle EMG on larval zebrafish has not been feasible due to the lack of proper EMG sensors and systems for such small animals. We introduce a new type of EMG needle electrode to measure intramuscular activities of larval zebrafish, together with a method to hold the animal in position during EMG, without anesthetization. The silicon-based needle electrode was found to be sufficiently strong and sharp to penetrate the skin and muscles of zebrafish larvae, and its shape and performance did not change after multiple insertions. With the use of the proposed needle electrode and measurement system, EMG was successfully performed on zebrafish at 30 days postfertilization (dpf) and at 5 dpf. Burst patterns and spike morphology of the recorded EMG signals were analyzed. The measured single spikes were triphasic with an initial positive deflection, which is typical for motor unit action potentials, with durations of ∼10 ms, whereas the muscle activity was silent during the anesthetized condition. These findings confirmed the capability of this system of detecting EMG signals from very small animals such as 5 dpf zebrafish. The developed EMG sensor and system are expected to become a helpful tool in validating zebrafish MD models and further developing therapeutics.

  19. A High-Throughput Screen Targeting Malaria Transmission Stages Opens New Avenues for Drug Development

    OpenAIRE

    Buchholz, Kathrin; Burke, Thomas A; Williamson, Kim C.; Wiegand, Roger C.; Wirth, Dyann F; Marti, Matthias

    2011-01-01

    A major goal of the worldwide malaria eradication program is the reduction and eventual elimination of malaria transmission. All currently available antimalarial compounds were discovered on the basis of their activity against the asexually reproducing red blood cell stages of the parasite, which are responsible for the morbidity and mortality of human malaria. Resistance against these compounds is widespread, and there is an urgent need for novel approaches to reduce the emergence of resista...

  20. Treatment for Relapsed/Refractory AML Based on a High Throughput Drug Sensitivity Assay

    Science.gov (United States)

    2016-05-10

    Adult Acute Megakaryoblastic Leukemia (M7); Adult Acute Minimally Differentiated Myeloid Leukemia (M0); Adult Acute Monoblastic Leukemia (M5a); Adult Acute Monocytic Leukemia (M5b); Adult Acute Myeloblastic Leukemia With Maturation (M2); Adult Acute Myeloblastic Leukemia Without Maturation (M1); Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Adult Acute Myelomonocytic Leukemia (M4); Adult Erythroleukemia (M6a); Adult Pure Erythroid Leukemia (M6b); Chronic Myelomonocytic Leukemia; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Myeloid Leukemia; Refractory Anemia With Excess Blasts