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Sample records for semi-automated high-throughput fluorescent

  1. Development of a semi-automated high throughput transient transfection system.

    PubMed

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody. PMID:24704608

  2. A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.

    PubMed

    Duverger, Yohann; Belougne, Jérôme; Scaglione, Sarah; Brandli, Dominique; Beclin, Christophe; Ewbank, Jonathan J

    2007-01-01

    The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1. The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism. PMID:17164286

  3. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    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

  4. Robofurnace: A semi-automated laboratory chemical vapor deposition system for high-throughput nanomaterial synthesis and process discovery

    PubMed Central

    Oliver, C. Ryan; Westrick, William; Koehler, Jeremy; Brieland-Shoultz, Anna; Anagnostopoulos-Politis, Ilias; Cruz-Gonzalez, Tizoc; Hart, A. John

    2013-01-01

    Laboratory research and development on new materials, such as nanostructured thin films, often utilizes manual equipment such as tube furnaces due to its relatively low cost and ease of setup. However, these systems can be prone to inconsistent outcomes due to variations in standard operating procedures and limitations in performance such as heating and cooling rates restrict the parameter space that can be explored. Perhaps more importantly, maximization of research throughput and the successful and efficient translation of materials processing knowledge to production-scale systems, relies on the attainment of consistent outcomes. In response to this need, we present a semi-automated lab-scale chemical vapor deposition (CVD) furnace system, called “Robofurnace.” Robofurnace is an automated CVD system built around a standard tube furnace, which automates sample insertion and removal and uses motion of the furnace to achieve rapid heating and cooling. The system has a 10-sample magazine and motorized transfer arm, which isolates the samples from the lab atmosphere and enables highly repeatable placement of the sample within the tube. The system is designed to enable continuous operation of the CVD reactor, with asynchronous loading/unloading of samples. To demonstrate its performance, Robofurnace is used to develop a rapid CVD recipe for carbon nanotube (CNT) forest growth, achieving a 10-fold improvement in CNT forest mass density compared to a benchmark recipe using a manual tube furnace. In the long run, multiple systems like Robofurnace may be linked to share data among laboratories by methods such as Twitter. Our hope is Robofurnace and like automation will enable machine learning to optimize and discover relationships in complex material synthesis processes. PMID:24289435

  5. Robofurnace: A semi-automated laboratory chemical vapor deposition system for high-throughput nanomaterial synthesis and process discovery

    SciTech Connect

    Oliver, C. Ryan; Westrick, William; Koehler, Jeremy; Brieland-Shoultz, Anna; Anagnostopoulos-Politis, Ilias; Cruz-Gonzalez, Tizoc; Hart, A. John

    2013-11-15

    Laboratory research and development on new materials, such as nanostructured thin films, often utilizes manual equipment such as tube furnaces due to its relatively low cost and ease of setup. However, these systems can be prone to inconsistent outcomes due to variations in standard operating procedures and limitations in performance such as heating and cooling rates restrict the parameter space that can be explored. Perhaps more importantly, maximization of research throughput and the successful and efficient translation of materials processing knowledge to production-scale systems, relies on the attainment of consistent outcomes. In response to this need, we present a semi-automated lab-scale chemical vapor deposition (CVD) furnace system, called “Robofurnace.” Robofurnace is an automated CVD system built around a standard tube furnace, which automates sample insertion and removal and uses motion of the furnace to achieve rapid heating and cooling. The system has a 10-sample magazine and motorized transfer arm, which isolates the samples from the lab atmosphere and enables highly repeatable placement of the sample within the tube. The system is designed to enable continuous operation of the CVD reactor, with asynchronous loading/unloading of samples. To demonstrate its performance, Robofurnace is used to develop a rapid CVD recipe for carbon nanotube (CNT) forest growth, achieving a 10-fold improvement in CNT forest mass density compared to a benchmark recipe using a manual tube furnace. In the long run, multiple systems like Robofurnace may be linked to share data among laboratories by methods such as Twitter. Our hope is Robofurnace and like automation will enable machine learning to optimize and discover relationships in complex material synthesis processes.

  6. Robofurnace: a semi-automated laboratory chemical vapor deposition system for high-throughput nanomaterial synthesis and process discovery.

    PubMed

    Oliver, C Ryan; Westrick, William; Koehler, Jeremy; Brieland-Shoultz, Anna; Anagnostopoulos-Politis, Ilias; Cruz-Gonzalez, Tizoc; Hart, A John

    2013-11-01

    Laboratory research and development on new materials, such as nanostructured thin films, often utilizes manual equipment such as tube furnaces due to its relatively low cost and ease of setup. However, these systems can be prone to inconsistent outcomes due to variations in standard operating procedures and limitations in performance such as heating and cooling rates restrict the parameter space that can be explored. Perhaps more importantly, maximization of research throughput and the successful and efficient translation of materials processing knowledge to production-scale systems, relies on the attainment of consistent outcomes. In response to this need, we present a semi-automated lab-scale chemical vapor deposition (CVD) furnace system, called "Robofurnace." Robofurnace is an automated CVD system built around a standard tube furnace, which automates sample insertion and removal and uses motion of the furnace to achieve rapid heating and cooling. The system has a 10-sample magazine and motorized transfer arm, which isolates the samples from the lab atmosphere and enables highly repeatable placement of the sample within the tube. The system is designed to enable continuous operation of the CVD reactor, with asynchronous loading∕unloading of samples. To demonstrate its performance, Robofurnace is used to develop a rapid CVD recipe for carbon nanotube (CNT) forest growth, achieving a 10-fold improvement in CNT forest mass density compared to a benchmark recipe using a manual tube furnace. In the long run, multiple systems like Robofurnace may be linked to share data among laboratories by methods such as Twitter. Our hope is Robofurnace and like automation will enable machine learning to optimize and discover relationships in complex material synthesis processes. PMID:24289435

  7. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

  8. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2005-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics

  9. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Minamitani, Elizabeth Forsythe; Pusey, Marc L.

    2004-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution 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 a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment

  10. Fluorescent Approaches to High Throughput Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth

    2004-01-01

    X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low

  11. Semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

    PubMed

    Romano, Shannon N; Gorelick, Daniel A

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  12. Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

    PubMed Central

    Romano, Shannon N.; Gorelick, Daniel A.

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  13. High-throughput screening with micro-x-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-01

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity.

  14. High-throughput screening with micro-x-ray fluorescence

    SciTech Connect

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-15

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity.

  15. Scanning fluorescence detector for high-throughput DNA genotyping

    NASA Astrophysics Data System (ADS)

    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

  16. Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas.

    PubMed

    Alexander, Nathan S; Palczewska, Grazyna; Palczewski, Krzysztof

    2015-08-01

    Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE. PMID:26309765

  17. Semi-automated Volumetric and Morphological Assessment of Glioblastoma Resection with Fluorescence-Guided Surgery

    PubMed Central

    Cordova, J. Scott; Gurbani, Saumya S.; Holder, Chad A.; Olson, Jeffrey J.; Schreibmann, Eduard; Shi, Ran; Guo, Ying; Shu, Hui-Kuo G.; Shim, Hyunsuk; Hadjipanayis, Costas G.

    2016-01-01

    Purpose Glioblastoma (GBM) neurosurgical resection relies on contrast-enhanced MRI-based neuronavigation. However, it is well-known that infiltrating tumor extends beyond contrast enhancement. Fluorescence-guided surgery (FGS) using 5-aminolevulinic acid (5-ALA) was evaluated to improve extent of resection (EOR) of GBMs. Pre-operative morphological tumor metrics were also assessed. Procedures Thirty patients from a Phase II trial evaluating 5-ALA FGS in newly diagnosed GBM were assessed. Tumors were segmented pre-operatively to assess morphological features as well as post-operatively to evaluate EOR and residual tumor volume (RTV). Results Median EOR and RTV were 94.3% and 0.821 cm3, respectively. Pre-operative surface area to volume ratio and RTV were significantly associated with overall survival, even when controlling for the known survival confounders. Conclusions This study supports claims that 5-ALA FGS is helpful at decreasing tumor burden and prolonging survival in GBM. Moreover, morphological indices are shown to impact both resection and patient survival. PMID:26463215

  18. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    SciTech Connect

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-02-20

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  19. Cosputtered composition-spread reproducibility established by high-throughput x-ray fluorescence

    SciTech Connect

    Gregoire, John M.; Dale, Darren; Kazimirov, Alexander; DiSalvo, Francis J.; Dover, R. Bruce van

    2010-09-15

    We describe the characterization of sputtered yttria-zirconia composition spread thin films by x-ray fluorescence (XRF). We also discuss our automated analysis of the XRF data, which was collected in a high throughput experiment at the Cornell High Energy Synchrotron Source. The results indicate that both the composition reproducibility of the library deposition and the composition measurements have a precision of better than 1 atomic percent.

  20. Flat field correction for high-throughput imaging of fluorescent samples.

    PubMed

    Kask, Peet; Palo, Kaupo; Hinnah, Chris; Pommerencke, Thora

    2016-09-01

    Vignetting of microscopic images impacts both the visual impression of the images and any image analysis applied to it. Especially in high-throughput screening high demands are made on an automated image analysis. In our work we focused on fluorescent samples and found that two profiles (background and foreground) for each imaging channel need to be estimated to achieve a sufficiently flat image after correction. We have developed a method which runs completely unsupervised on a wide range of assays. By adding a reliable internal quality control we mitigate the risk of introducing artefacts into sample images through correction. The method requires hundreds of images for the foreground profile, thus limiting its application to high-throughput screening where this requirement is fulfilled in routine operation. PMID:27028041

  1. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    NASA Astrophysics Data System (ADS)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  2. [Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay].

    PubMed

    Feng, Weizong; Lin, Junhan; Cai, Shaoli; Zou, Youtu; Chen, Guoren; Huang, Ping; Lin, Yajing; Wang, Bingbing; Lin, Lin

    2011-04-01

    We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement. PMID:21847993

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

    PubMed

    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

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

    PubMed Central

    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

  5. Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria.

    PubMed

    Zeder, M; Pernthaler, J

    2009-09-01

    Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrated various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in bright-field and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multispot focus measurements, and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions. PMID:19658173

  6. Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

    NASA Astrophysics Data System (ADS)

    Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

    2014-11-01

    We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications.

  7. Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

    PubMed Central

    Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

    2014-01-01

    We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

  8. Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase

    PubMed Central

    Bulfer, Stacie L.; McQuade, Thomas J.; Larsen, Martha J.; Trievel, Raymond C.

    2011-01-01

    Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism. PMID:21073853

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  11. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.

    PubMed

    Pitzler, Christian; Wirtz, Georgette; Vojcic, Ljubica; Hiltl, Stephanie; Böker, Alexander; Martinez, Ronny; Schwaneberg, Ulrich

    2014-12-18

    Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials. PMID:25525992

  12. Identification of Fluorescent Compounds with Non-Specific Binding Property via High Throughput Live Cell Microscopy

    PubMed Central

    Nath, Sangeeta; Spencer, Virginia A.; Han, Ju; Chang, Hang; Zhang, Kai; Fontenay, Gerald V.; Anderson, Charles; Hyman, Joel M.; Nilsen-Hamilton, Marit; Chang, Young-Tae; Parvin, Bahram

    2012-01-01

    Introduction Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. Method Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. Results The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties. Conclusion We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented. PMID:22242152

  13. High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis

    PubMed Central

    2013-01-01

    Background In order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity. Results Based on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of

  14. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

    PubMed

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  15. Pulsed laser activated cell sorter (PLACS) for high-throughput fluorescent mammalian cell sorting

    NASA Astrophysics Data System (ADS)

    Chen, Yue; Wu, Ting-Hsiang; Chung, Aram; Kung, Yu-Chung; Teitell, Michael A.; Di Carlo, Dino; Chiou, Pei-Yu

    2014-09-01

    We present a Pulsed Laser Activated Cell Sorter (PLACS) realized by exciting laser induced cavitation bubbles in a PDMS microfluidic channel to create high speed liquid jets to deflect detected fluorescent samples for high speed sorting. Pulse laser triggered cavitation bubbles can expand in few microseconds and provide a pressure higher than tens of MPa for fluid perturbation near the focused spot. This ultrafast switching mechanism has a complete on-off cycle less than 20 μsec. Two approaches have been utilized to achieve 3D sample focusing in PLACS. One is relying on multilayer PDMS channels to provide 3D hydrodynamic sheath flows. It offers accurate timing control of fast (2 m sec-1) passing particles so that synchronization with laser bubble excitation is possible, an critically important factor for high purity and high throughput sorting. PLACS with 3D hydrodynamic focusing is capable of sorting at 11,000 cells/sec with >95% purity, and 45,000 cells/sec with 45% purity using a single channel in a single step. We have also demonstrated 3D focusing using inertial flows in PLACS. This sheathless focusing approach requires 10 times lower initial cell concentration than that in sheath-based focusing and avoids severe sample dilution from high volume sheath flows. Inertia PLACS is capable of sorting at 10,000 particles sec-1 with >90% sort purity.

  16. Micro x-ray fluorescence as a high throughput screening method for metal chelating compounds

    NASA Astrophysics Data System (ADS)

    Minogue, Edel M.; Havrilla, George J.; Taylor, Tammy P.; Burrell, Anthony K.; Warner, Benjamin P.

    2005-06-01

    Micro X-ray Fluorescence (MXRF) has proven to be a powerful tool in the rapid and quantitative means of screening oliogpeptides. MXRF is a non-destructive method of analysis, which can detect elemental composition of a sample by measuring its characteristic X-ray emission wavelengths or energies. An effective high throughput screening technique is described for the rapid screening of bead-based libraries by MXRF in order to identify suitable chelating agents that will bind metals found in radioactive dispersive devices. It is a sensitive technique which in conjunction with the wide range of chemistry inherent in peptide libraries (e.g. varying charge, length, hydrophobicity, aromaticity etc.), provides a rapid and quantitative means for screening chelator-ion binding. The method involves the selection of a suitable library of ligands; in this case it is a bead-based library of peptides. The library is exposed to the cation of interest and immobilized on to a microarray. The array is then analyzed by MXRF enabling rapid identification of chelating agents. This enables the screening of approximately 27,500 sequences per day. Initial experiments carried out successfully identified sequences that are selective for Co under certain binding conditions. This involved the screening of 8,400 sequences in adverse environmental conditions containing possible interferences (e.g. Ca, Fe, Al, Cs, Ir), which could be encountered in our application.

  17. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors

    PubMed Central

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A.

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  18. High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.

    PubMed

    Ramlee, Muhammad Khairul; Yan, Tingdong; Cheung, Alice M S; Chuah, Charles T H; Li, Shang

    2015-01-01

    Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants. PMID:26498861

  19. High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

    PubMed Central

    Ramlee, Muhammad Khairul; Yan, Tingdong; Cheung, Alice M. S.; Chuah, Charles T. H.; Li, Shang

    2015-01-01

    Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants. PMID:26498861

  20. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.

    PubMed

    Avonto, Cristina; Chittiboyina, Amar G; Rua, Diego; Khan, Ikhlas A

    2015-12-01

    Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. PMID:26455772

  1. Microelectrophoresis devices with integrated fluorescence detectors and reactors for high-throughput DNA sequencing

    NASA Astrophysics Data System (ADS)

    Soper, Steven A.; Ford, Sean M.; Davies, Jack; Williams, Daryl C.; Cheng, Benxu; Klopf, J. Michael; Calderon, Gina M.; Saile, Volker

    1997-05-01

    This work describes the development of micro-devices for high-throughput DNA sequencing applications. Basically, two research efforts will be discussed; (1) fabrication and characterization of micro-reactors to prepare Sanger chain terminated DNA sequencing fragments on a nanoliter scale and; (2) x-ray photolithography of PMMA substrates for the high aspect ratio preparation of electrophoresis devices. The micro-reactor consisted of a 5'-biotinylated catfish olfactory gene, which was amplified by PCR, and attached to the interior wall of an aminoalkylisilane derivatized fused- silica capillary tube via a streptavidin/biotin linkage. Coverage of the interior capillary wall with biotinylated DNA averaged 77 percent. Stability of the anchored template under pressure and electroosmotic rinsing was favorable, requiring approximately 150 h of continuous rinsing to reduce the coverage by only 50 percent. The capillary micro- reactor was placed inside an air thermocycler to control temperature during Sanger ddNTP chain extension and directly coupled to a capillary separation column filled with a LPA solution via low dead volume capillary interlocks. The complimentary DNA fragments generated in the reactor were heat denatured from the immobilized template and directly injected onto a gel-filled capillary using electropumping for size fractionation and detection using NIR-LIF analysis. The total amount of termination fragments in the 31 nL reactor volume was estimated to be 5.2 X 1013 moles and sequencing was shown to produce read lengths on the order to 400 bases. Work will also be described concerning the development of micro-electrophoresis devices in x-ray sensitive photoresists using LIGA techniques. An electrophoresis device with an integrated fluorescence detector was constructed for the high resolution separation of DNA oligonucleotides. The choice of substrate for the electrophoresis was PMMA, due to its intrinsic low electroosmotic flow. Using x-ray lithography in

  2. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    SciTech Connect

    Hui Su

    2001-05-01

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm{sub 2} for 40-{micro}m wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.

  3. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    PubMed Central

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  4. Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy.

    PubMed

    Polzer, Hans; Volkmer, Elias; Saller, Maximilian M; Prall, Wolf C; Haasters, Florian; Drosse, Inga; Anz, David; Mutschler, Wolf; Schieker, Matthias

    2012-02-01

    The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC. PMID:21951128

  5. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    SciTech Connect

    Hui Su

    2001-05-25

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm{sup 2} for 40-{micro}m wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.

  6. A fluorescence-based high throughput assay for the determination of small molecule–human serum albumin protein binding

    PubMed Central

    McCallum, Megan M.; Pawlak, Alan J.; Shadrick, William R.; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David J.; Arnold, Leggy A.

    2014-01-01

    Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the LOPAC small molecule library of 1280 compounds identifying known high protein binders. The small molecule competition of HSA–Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3–24 μM. The compound affinity towards HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. PMID:24390461

  7. Fluorescence imaging technology (FI) for high-throughput screening of selenide-modified nano-TiO2 catalysts.

    PubMed

    Wang, Liping; Lee, Jianchao; Zhang, Meijuan; Duan, Qiannan; Zhang, Jiarui; Qi, Hailang

    2016-02-18

    A high-throughput screening (HTS) method based on fluorescence imaging (FI) was implemented to evaluate the catalytic performance of selenide-modified nano-TiO2. Chemical ink-jet printing (IJP) technology was reformed to fabricate a catalyst library comprising 1405 (Ni(a)Cu(b)Cd(c)Ce(d)In(e)Y(f))Se(x)/TiO2 (M6Se/Ti) composite photocatalysts. Nineteen M6Se/Tis were screened out from the 1405 candidates efficiently. PMID:26777131

  8. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

    PubMed

    Kruger, Wade; Gilbert, Daniel; Hawthorne, Rebecca; Hryciw, Deanne H; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W

    2005-06-01

    There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. PMID:15862914

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

    SciTech Connect

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    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.

  10. A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting.

    PubMed

    Liu, Yan; Xue, Zheng-Lian; Chen, Shao-Peng; Wang, Zhou; Zhang, Yong; Gong, Wei-Liang; Zheng, Zhi-Ming

    2016-06-01

    To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K2, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl2 before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains. PMID:27001261

  11. High throughput identification of promiscuous inhibitors from screening libraries with the use of a thiol-containing fluorescent probe

    PubMed Central

    McCallum, Megan M.; Nandhikonda, Premchendar; Temmer, Jonathan J.; Eyermann, Charles; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David; Arnold, Leggy A.

    2013-01-01

    Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates in respect to thiol-reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe MSTI was synthesized and used to evaluate small molecules from the LOPAC collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high throughput manner enabling the assessment of screening libraries in respect to thiol-reactive compounds. PMID:23446699

  12. High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

    PubMed

    Lokesh, G L; Rachamallu, Aparna; Kumar, G D Kishore; Natarajan, Amarnath

    2006-05-01

    The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening. PMID:16500609

  13. Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery

    PubMed Central

    Botelho, Hugo M.; Uliyakina, Inna; Awatade, Nikhil T.; Proença, Maria C.; Tischer, Christian; Sirianant, Lalida; Kunzelmann, Karl; Pepperkok, Rainer; Amaral, Margarida D.

    2015-01-01

    Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking – Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts. PMID:25762484

  14. Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery.

    PubMed

    Botelho, Hugo M; Uliyakina, Inna; Awatade, Nikhil T; Proença, Maria C; Tischer, Christian; Sirianant, Lalida; Kunzelmann, Karl; Pepperkok, Rainer; Amaral, Margarida D

    2015-01-01

    Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts. PMID:25762484

  15. Use of fluorescence for the high-throughput evaluation of synergistic thermal and photo stabilizer interactions in poly (vinyl chloride)

    SciTech Connect

    Wu Chunyong; Wicks, Douglas A.

    2005-06-15

    The selection of thermal and photo stabilizers for poly (vinyl chloride) (PVC) using conventional methods is a time-consuming process. The high-throughput screening method developed in this research demonstrates rapid and efficient ways to quantify the effectiveness of PVC stabilizers with respect to raw plastic materials, stabilizers, levels of use, and testing conditions. An experimental protocol using liquid sampling and fluorescence measurement was developed to determine the effectiveness of formulations. This was used to evaluate the performance of stabilizers based on the change of fluorescence emission at 440 nm after thermal aging or ultraviolet (UV) radiation. The performance of PVC formulations using six different types of stabilizers was successfully mapped for both PVC resin and flexible PVC.

  16. Use of fluorescence for the high-throughput evaluation of synergistic thermal and photo stabilizer interactions in poly (vinyl chloride)

    NASA Astrophysics Data System (ADS)

    Wu, Chunyong; Wicks, Douglas A.

    2005-06-01

    The selection of thermal and photo stabilizers for poly (vinyl chloride) (PVC) using conventional methods is a time-consuming process. The high-throughput screening method developed in this research demonstrates rapid and efficient ways to quantify the effectiveness of PVC stabilizers with respect to raw plastic materials, stabilizers, levels of use, and testing conditions. An experimental protocol using liquid sampling and fluorescence measurement was developed to determine the effectiveness of formulations. This was used to evaluate the performance of stabilizers based on the change of fluorescence emission at 440nm after thermal aging or ultraviolet (UV) radiation. The performance of PVC formulations using six different types of stabilizers was successfully mapped for both PVC resin and flexible PVC.

  17. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    PubMed Central

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  18. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

    PubMed

    Khalil, Jacques Y B; Robert, Stephane; Reteno, Dorine G; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  19. High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging

    PubMed Central

    Gallardo, Ignacio F.; Pasupathy, Praveenkumar; Brown, Maxwell; Manhart, Carol M.; Neikirk, Dean P.; Alani, Eric; Finkelstein, Ilya J.

    2015-01-01

    Single-molecule studies of protein–DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains—a method for organizing arrays of DNA molecules on a fluid lipid bilayer—has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900 000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3—a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments. PMID:26325477

  20. High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging

    PubMed Central

    Arpali, Serap Altay; Arpali, Caglar; Coskun, Ahmet F.; Chiang, Hsin-Hao; Ozcan, Aydogan

    2012-01-01

    Undiluted blood samples are difficult to image in large volumes since blood constitutes a highly absorbing and scattering medium. As a result of this limitation, optical imaging of rare cells (e.g., circulating tumour cells) within unprocessed whole blood remains a challenge, demanding the use of special micro-fluidic technologies. Here we demonstrate a new fluorescent on-chip imaging modality that can rapidly screen large volumes of absorbing and scattering media such as undiluted whole blood samples for detection of fluorescent micro-objects at low concentrations (for example ≤ 50–100 particles/mL). In this high-throughput imaging modality, a large area micro-fluidic device (e.g., 7–18 cm2), which contains for example ~0.3–0.7 mL of undiluted whole blood sample, is directly positioned onto a wide-field opto-electronic sensor-array such that the fluorescent emission within the micro-channel can be detected without the use of any imaging lenses. This micro-fluidic device is then illuminated and laterally scanned with an array of Gaussian excitation spots, which is generated through a spatial light modulator. For each scanning position of this excitation array, a lensfree fluorescent image of the blood sample is captured using the opto-electronic sensor-array, resulting in a sequence of images (e.g., 144 lensfree frames captured in ~36 seconds) for the same sample chip. Digitally merging these lensfree fluorescent images based on a Maximum Intensity Projection (MIP) algorithm enabled us to significantly boost the signal-to-noise ratio (SNR) and contrast of the fluorescent micro-objects within whole blood, which normally remain undetected (i.e., hidden) using conventional uniform excitation schemes, involving plane wave illumination. This high-throughput on-chip imaging platform based on structured excitation could be useful for rare cell research by enabling rapid screening of large volume micro-fluidic devices that process whole blood and other optically

  1. Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Tuvshintulga, Bumduuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-08-30

    The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (-)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. PMID:27523944

  2. Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

    PubMed

    Kováčová, Kristína; Farkaš, Vladimír

    2016-04-01

    Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated. PMID:26754421

  3. Novel inhibitors for PRMT1 discovered by high-throughput screening using activity-based fluorescence polarization.

    PubMed

    Dillon, Myles B C; Bachovchin, Daniel A; Brown, Steven J; Finn, M G; Rosen, Hugh; Cravatt, Benjamin F; Mowen, Kerri A

    2012-07-20

    Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high-throughput screening assay for PRMT1 that utilizes a hyper-reactive cysteine within the active site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine-containing cysteine-reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM-dependent methyltransferases. PMID:22506763

  4. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

    PubMed Central

    Chen, Jin; Dalal, Ravindra V.; Petrov, Alexey N.; Tsai, Albert; O’Leary, Seán E.; Chapin, Karen; Cheng, Janice; Ewan, Mark; Hsiung, Pei-Lin; Lundquist, Paul; Turner, Stephen W.; Hsu, David R.; Puglisi, Joseph D.

    2014-01-01

    Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community. PMID:24379388

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

    PubMed

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-02-24

    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. PMID:22290227

  6. Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

    PubMed Central

    Shukla, Sunita J.; Nguyen, Dac-Trung; MacArthur, Ryan; Simeonov, Anton; Frazee, William J.; Hallis, Tina M.; Marks, Bryan D.; Singh, Upinder; Eliason, Hildegard C.; Printen, John; Austin, Christopher P.; Inglese, James

    2009-01-01

    Abstract The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug–drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z′-factors of ≥0.5. Seven- to 15-point concentration–response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format. PMID:19505231

  7. High-throughput single-molecule fluorescence spectroscopy using parallel detection

    PubMed Central

    Michalet, X.; Colyer, R. A.; Scalia, G.; Kim, T.; Levi, Moran; Aharoni, Daniel; Cheng, Adrian; Guerrieri, F.; Arisaka, Katsushi; Millaud, Jacques; Rech, I.; Resnati, D.; Marangoni, S.; Gulinatti, A.; Ghioni, M.; Tisa, S.; Zappa, F.; Cova, S.; Weiss, S.

    2011-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors. PMID:21625288

  8. Fluorescence techniques for determination of the membrane potentials in high throughput screening.

    PubMed

    Przybylo, Magda; Borowik, Tomasz; Langner, Marek

    2010-11-01

    The characterization of small molecules requires identification and evaluation of several predictive parameters, when selecting compounds for pharmacological applications and/or determining their toxicity. A number of them are correlated with the compound interaction with biological membranes and/or capacity to cross them. The knowledge of the extent of adsorption, partition coefficient and permeability along with the compound ability to alter membrane properties are critical for such studies. Lipid bilayers are frequently used as the adequate experimental models of a biological membrane despite their simple structure and a limited number of components. A significant number of the biologically relevant lipid bilayer properties are related to its electrostatics. Three electrostatic potentials were defined for the lipid bilayer; the intrinsic or induced surface electrostatic potential, the dipole potential and the membrane potential. Each of them was measured with dedicated methodologies. The complex measurement protocols and technically demanding instrumentation made the development of efficient HTS approaches for complete characterization of membrane electrostatics practically impossible. However, the rapid development of fluorescence techniques accompanied by rapid growth in diversity and number of dedicated fluorescent probes enabled characterization of lipid bilayer electrostatics in a moderately simple manner. Technically advanced, compact and automated workstations, capable of measuring practically all fluorescence parameters, are now available. Therefore, the proper selection of fluorescent probes with measuring procedures can be designed to evaluate drug candidates in context of their ability to alter membrane electrostatics. In the paper we present a critical review of available fluorescence methods, useful for the membrane electrostatics evaluation and discuss the feasibility of their adaptation to HTS procedures. The significance of the presented

  9. High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe

    PubMed Central

    Riedl, Thilo; van Boxtel, Egon; Bosch, Martijn; Parren, Paul W. H. I.; Gerritsen, Arnout F.

    2016-01-01

    Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs. PMID:26518032

  10. Toward Fluorescence-Based High-Throughput Screening for Enantiomeric Excess in Amines and Amino Acid Derivatives.

    PubMed

    Shcherbakova, Elena G; Brega, Valentina; Minami, Tsuyoshi; Sheykhi, Sara; James, Tony D; Anzenbacher, Pavel

    2016-07-11

    A highly accurate and reliable screening method for enantiomeric excess of amine derivatives in the presence of water is reported. The fluorescence-based screening system has been realized by self-assembly of chiral diol-type dyes (BINOL, VANOL and VAPOL), 2-formylphenylboronic acid, and chiral amines forming iminoboronate esters. The structure and chirality of the amine analytes determine the stability of the diastereomeric iminoboronate esters, which in turn display differential fluorescence. The fluorescence signal reflects the enantiomeric purity of the chiral amines and was utilized in high-throughput arrays. The arrays were able to recognize enantiomeric excess of amines, amino esters, and amino alcohols. In addition to qualitative analysis, quantitative experiments were successfully performed. Studies of the role of additives such as water or citrate were carried out to gain insight into the stability of the iminoboronate esters. It is shown that the above additives destabilize less stable esters while the stable esters remain unchanged. Thus, the presence of water and citrate leads to increased difference between the diastereomeric iminoboronates and contributes to the enantiodiscrimination of the chiral amines. PMID:27271215

  11. Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions.

    PubMed

    Guzmán, Camilo; Oetken-Lindholm, Christina; Abankwa, Daniel

    2016-04-01

    Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM) allows precise quantification of the FRET efficiency in intact cells, as FLIM is independent of fluorophore concentration, detection efficiency, and fluorescence intensity. We have developed an automated FLIM system using a commercial frequency domain FLIM attachment (Lambert Instruments) for wide-field imaging. Our automated FLIM system is capable of imaging and analyzing up to 50 different positions of a slide in less than 4 min, or the inner 60 wells of a 96-well plate in less than 20 min. Automation is achieved using a motorized stage and controller (Prior Scientific) coupled with a Zeiss Axio Observer body and full integration into the Lambert Instruments FLIM acquisition software. As an application example, we analyze the interaction of the oncoprotein Ras and its effector Raf after drug treatment. In conclusion, our automated FLIM imaging system requires only commercial components and may therefore allow for a broader use of this technique in chemogenomics projects. PMID:26384400

  12. Fluorescent probe for high-throughput screening of membrane protein expression

    PubMed Central

    Backmark, A E; Olivier, N; Snijder, A; Gordon, E; Dekker, N; Ferguson, A D

    2013-01-01

    Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture. PMID:23776061

  13. A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction.

    PubMed

    Kimple, Adam J; Yasgar, Adam; Hughes, Mark; Jadhav, Ajit; Willard, Francis S; Muller, Robin E; Austin, Christopher P; Inglese, James; Ibeanu, Gordon C; Siderovski, David P; Simeonov, Anton

    2008-06-01

    The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively. PMID:18537560

  14. Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

    PubMed

    Kwon, Keehwan; Nagarajan, Rajesh; Stivers, James T

    2004-11-30

    Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover. PMID:15554707

  15. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes.

    PubMed

    Bachovchin, Daniel A; Brown, Steven J; Rosen, Hugh; Cravatt, Benjamin F

    2009-04-01

    High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein. PMID:19329999

  16. Development of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection.

    PubMed

    Kumagai, Kazuo; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

    2014-02-15

    Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z'-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. PMID:24299989

  17. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

    PubMed Central

    Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

    2012-01-01

    A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

  18. Fluorescent-magnetic dual-encoded nanospheres: a promising tool for fast-simultaneous-addressable high-throughput analysis.

    PubMed

    Xie, Min; Hu, Jun; Wen, Cong-Ying; Zhang, Zhi-Ling; Xie, Hai-Yan; Pang, Dai-Wen

    2012-01-27

    Bead-based optical encoding or magnetic encoding techniques are promising in high-throughput multiplexed detection and separation of numerous species under complicated conditions. Therefore, a self-assembly strategy implemented in an organic solvent is put forward to fabricate fluorescent-magnetic dual-encoded nanospheres. Briefly, hydrophobic trioctylphosphine oxide-capped CdSe/ZnS quantum dots (QDs) and oleic acid-capped nano-γ-Fe2O3 magnetic particles are directly, selectively and controllably assembled on branched poly(ethylene imine)-coated nanospheres without any pretreatment, which is crucial to keep the high quantum yield of QDs and good dispersibility of γ-Fe2O3. Owing to the tunability of coating amounts of QDs and γ-Fe2O3 as well as controllable fluorescent emissions of deposited-QDs, dual-encoded nanospheres with different photoluminescent emissions and gradient magnetic susceptibility are constructed. Using this improved layer-by-layer self-assembly approach, deposition of hydrophobic nanoparticles onto hydrophilic carriers in organic media can be easily realized; meanwhile, fluorescent-magnetic dual-functional nanospheres can be further equipped with readable optical and magnetic addresses. The resultant fluorescent-magnetic dual-encoded nanospheres possess both the unique optical properties of QDs and the superparamagnetic properties of γ-Fe2O3, exhibiting good monodispersibility, huge encoding capacity and nanoscale particle size. Compared with the encoded microbeads reported by others, the nanometre scale of the dual-encoded nanospheres gives them minimum steric hindrance and higher flexibility. PMID:22173487

  19. A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine

    PubMed Central

    Tian, H; Ip, L; Luo, H; Chang, D C; Luo, K Q

    2006-01-01

    Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called ‘caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery. PMID:17179946

  20. Ultrasensitive and High-Throughput Fluorescence Analysis of Droplet Contents with Orthogonal Line Confocal Excitation

    PubMed Central

    Jeffries, Gavin D.M.; Lorenz, Robert M.; Chiu, Daniel T.

    2010-01-01

    This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (S/N) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations, and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze pico-molar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second. PMID:21062029

  1. A HIGH-THROUGHPUT FLUORESCENCE ACTIVATED NANOSCALE SUBCELLULAR SORTER WITH SINGLE-MOLECULE SENSITIVITY

    PubMed Central

    Schiro, Perry G.; Gadd, Jennifer C.; Yen, Gloria S.; Chiu, Daniel T.

    2012-01-01

    Recent single-cell and single-molecule studies have shown that a variety of subpopulations exist within biological systems, such as synaptic vesicles, that have previously been overlooked in common bulk studies. By isolating and enriching these various subpopulations, detailed analysis with a variety of analytical techniques can be done to further understand the role that various subpopulations play in cellular dynamics and how alterations to these subpopulations affect the overall function of the biological system. Previous sorters lack the sensitivity, sorting speed, and efficiency to isolate synaptic vesicles and other nanoscale systems. This paper describes the development of a fluorescence activated nanoscale subcellular sorter that can sort nearly 10 million objects per hour with single-molecule sensitivity. Utilizing a near-nanoscale channel system, we were able to achieve upwards of 91% recovery of desired objects with a 99.7% purity. PMID:22574902

  2. A simple, rapid, and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple, rapid, and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) was developed. Three haptens were labeled with a fluorescein probe and used as tracers to develop a homogenous FPIA using a broad-specificity monoclon...

  3. Dealing with reduced data acquisition times in Fluorescence Correlation Spectroscopy (FCS) for High-Throughput Screening (HTS) applications

    NASA Astrophysics Data System (ADS)

    Davis, Lloyd M.; Ball, David A.; Williams, Peter E.; Swift, Kerry M.; Matayoshi, Edmund D.

    2003-07-01

    Fluorescence Correlation Spectroscopy (FCS) may be used to assay the binding of drug-like ligands to signaling proteins and other bio-particles. For High Throughput Screening (HTS), a competitive format is typically used in which binding of an unlabeled compound results in displacement of a fluorescently labeled ligand. Unweighted curve-fitting of the normalized autocorrelation function (ACF) to a two-diffusion-component model can resolve the fractions of free and bound ligand if the diffusion rates differ sufficiently and if the experimentally estimated ACF has adequate precision. However, for HTS (and also for intracellular FCS studies) it is desirable to minimize the experimental data collection time. In this case, the precision of the ACF is limited and it becomes important to account for the statistical features of the ACF estimate when designing an assay. The errors at different points in the estimated ACF are correlated and hence least-squares fitting methods are not strictly statistically rigorous. We compare different methods for estimating and curve-fitting the ACF from the raw data of short duration FCS measurements. The methods are applied to data from experiments to assay binding of Alexa-488-labeled Bak peptide with Bcl-xL, which is an intracellular protein that acts to protect against programmed cell death. We present results from a detailed Monte Carlo simulation of the experiment, which is useful for validating short-duration assay capabilities. We also discuss the measurement of changes in steady state fluorescence anisotropy due to restricted rotational diffusion upon binding, which provides a complementary assay method.

  4. High-Throughput Growth Prediction for Lactuca sativa L. Seedlings Using Chlorophyll Fluorescence in a Plant Factory with Artificial Lighting

    PubMed Central

    Moriyuki, Shogo; Fukuda, Hirokazu

    2016-01-01

    Poorly grown plants that result from differences in individuals lead to large profit losses for plant factories that use large electric power sources for cultivation. Thus, identifying and culling the low-grade plants at an early stage, using so-called seedlings diagnosis technology, plays an important role in avoiding large losses in plant factories. In this study, we developed a high-throughput diagnosis system using the measurement of chlorophyll fluorescence (CF) in a commercial large-scale plant factory, which produces about 5000 lettuce plants every day. At an early stage (6 days after sowing), a CF image of 7200 seedlings was captured every 4 h on the final greening day by a high-sensitivity CCD camera and an automatic transferring machine, and biological indices were extracted. Using machine learning, plant growth can be predicted with a high degree of accuracy based on biological indices including leaf size, amount of CF, and circadian rhythms in CF. Growth prediction was improved by addition of temporal information on CF. The present data also provide new insights into the relationships between growth and temporal information regulated by the inherent biological clock. PMID:27242805

  5. A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

    PubMed Central

    Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio

    2012-01-01

    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. PMID:23300705

  6. An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

    PubMed

    He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

    2016-04-18

    Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. PMID:26879206

  7. High-Throughput Growth Prediction for Lactuca sativa L. Seedlings Using Chlorophyll Fluorescence in a Plant Factory with Artificial Lighting.

    PubMed

    Moriyuki, Shogo; Fukuda, Hirokazu

    2016-01-01

    Poorly grown plants that result from differences in individuals lead to large profit losses for plant factories that use large electric power sources for cultivation. Thus, identifying and culling the low-grade plants at an early stage, using so-called seedlings diagnosis technology, plays an important role in avoiding large losses in plant factories. In this study, we developed a high-throughput diagnosis system using the measurement of chlorophyll fluorescence (CF) in a commercial large-scale plant factory, which produces about 5000 lettuce plants every day. At an early stage (6 days after sowing), a CF image of 7200 seedlings was captured every 4 h on the final greening day by a high-sensitivity CCD camera and an automatic transferring machine, and biological indices were extracted. Using machine learning, plant growth can be predicted with a high degree of accuracy based on biological indices including leaf size, amount of CF, and circadian rhythms in CF. Growth prediction was improved by addition of temporal information on CF. The present data also provide new insights into the relationships between growth and temporal information regulated by the inherent biological clock. PMID:27242805

  8. Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

    PubMed

    Yaoi, Takuro; Jiang, Xin; Li, Xianqiang

    2006-06-01

    Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor kappaB in inflammatory diseases, and peroxisome proliferator-activated receptor gamma in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multiple samples with high sensitivity. PMID:16834534

  9. High energy x-ray diffraction/x-ray fluorescence spectroscopy for high-throughput analysis of composition spread thin films

    SciTech Connect

    Gregoire, John M.; Dale, Darren; Kazimirov, Alexander; DiSalvo, Francis J.; Dover, R. Bruce van

    2009-12-15

    High-throughput crystallography is an important tool in materials research, particularly for the rapid assessment of structure-property relationships. We present a technique for simultaneous acquisition of diffraction images and fluorescence spectra on a continuous composition spread thin film using a 60 keV x-ray source. Subsequent noninteractive data processing provides maps of the diffraction profiles, thin film fiber texture, and composition. Even for highly textured films, our diffraction technique provides detection of diffraction from each family of Bragg reflections, which affords direct comparison of the measured profiles with powder patterns of known phases. These techniques are important for high throughput combinatorial studies as they provide structure and composition maps which may be correlated with performance trends within an inorganic library.

  10. High-Throughput Screening for Small Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

    PubMed Central

    Evelyn, Chris R.; Ferng, Timothy; Rojas, Rafael J.; Larsen, Martha J.; Sondek, John; Neubig, Richard R.

    2009-01-01

    Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications. PMID:19196702

  11. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    PubMed

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  12. The Design, Synthesis and Potential Utility of Fluorescence Probes that Target DFG-out Conformation of p38[alpha] for High Throughput Screening Binding Assay

    SciTech Connect

    Tecle, Haile; Feru, Frederic; Liu, Hu; Kuhn, Cyrille; Rennie, Glen; Morris, Mark; Shao, Jiangxing; Cheng, Alan C.; Gikunju, Diana; Miret, Juan; Coli, Rocco; Xi, Simon; Clugston, Susan L.; Low, Simon; Kazmirski, Steven; Ding, Yuan-Hua; Cao, Qing; Johnson, Theresa L.; Deshmukh, Gayatri D.; DiNitto, Jonathan P.; Wu, Joe C.; English, Jessie M.; Pfizer

    2010-10-18

    The design, synthesis and utility of fluorescence probes that bind to the DFG-out conformation of p38{alpha} kinase are described. Probes that demonstrate good affinity for p38{alpha}, have been identified and one of the probes, PF-04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non-classical p38{alpha} inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non-classical kinase inhibitors to the unactive form of the enzyme.

  13. Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs

    PubMed Central

    Hickey, Scott F.; Hammond, Ming C.

    2014-01-01

    Summary Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization (FP) assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class. PMID:24560607

  14. High-throughput analysis of lipid hydroperoxides in edible oils and fats using the fluorescent reagent diphenyl-1-pyrenylphosphine.

    PubMed

    Santas, Jonathan; Guzmán, Yeimmy J; Guardiola, Francesc; Rafecas, Magdalena; Bou, Ricard

    2014-11-01

    A fluorometric method for the determination of hydroperoxides (HP) in edible oils and fats using the reagent diphenyl-1-pyrenylphosphine (DPPP) was developed and validated. Two solvent media containing 100% butanol or a mixture of chloroform/methanol (2:1, v/v) can be used to solubilise lipid samples. Regardless of the solvent used to solubilise the sample, the DPPP method was precise, accurate, sensitive and easy to perform. The HP content of 43 oil and fat samples was determined and the results were compared with those obtained by means of the AOCS Official Method for the determination of peroxide value (PV) and the ferrous oxidation-xylenol orange (FOX) method. The proposed method not only correlates well with the PV and FOX methods, but also presents some advantages such as requiring low sample and solvent amounts and being suitable for high-throughput sample analysis. PMID:24874381

  15. High-Throughput Analysis With 96-Capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection

    SciTech Connect

    Gang Xue

    2001-12-31

    The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. The authors first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis. The authors further improved the fluorescence detection by step laser scanning. A computer-controlled galvanometer scanner is adapted for scanning a focused laser beam across a 96-capillary array for laser-induced fluorescence detection. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries. The limit of detection for fluorescein is 3 x 10{sup -11} M (S/N = 3) for 5-mW of total laser power scanned at 4 Hz. The observed cross-talk among capillaries is 0.2%. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components, and flexibility due to the independent paths for excitation and emission.

  16. Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

    PubMed Central

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-01-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

  17. A lipophilic fluorescent LipidGreen1-based quantification method for high-throughput screening analysis of intracellular poly-3-hydroxybutyrate.

    PubMed

    Choi, Ji Eun; Na, Hye Young; Yang, Taek Ho; Rhee, Sung-Keun; Song, Jae Kwang

    2015-12-01

    Poly-3-hydroxybutyrate (PHB), the most abundant type of polyhydroxyalkanoates (PHA) is synthesized inside a variety of microorganisms as a primary candidate for industrial PHB production. Lipophilic dyes such as Nile red and BODIPY have been used to quantify intracellular PHB, but their uses have often been limited in terms of sensitivity and accuracy. In this study, a newly developed lipophilic fluorescent dye LipidGreen1 was used to quantify intracellular PHB. LipidGreen1 stained viable colonies by adding the dye into the medium which enabled the effective selection of PHB-positive cells. Furthermore, the fluorescence intensity of LipidGreen1 maintained its fluorescence intensity much longer than that of Nile red. The fluorescence intensities of intracellular PHB stained by LipidGreen1 accurately agreed with PHB contents measured by gas chromatography. In addition, internalization of LipidGreen1 in Escherichia coli cell was not necessary to obtain quantitative measurements. PHB-synthase mutants were differentiated by fluorescence intensities with a good correlation to increased levels of PHB production. These results show that LipidGreen1 is sensitive and accurate in high-throughput screening of newly isolated and genetically modified bacteria with enhanced PHB production. PMID:26253390

  18. A high-throughput time-resolved mini-silicon photomultiplier with embedded fluorescence lifetime estimation in 0.13 μm CMOS.

    PubMed

    Tyndall, David; Rae, Bruce R; Li, David Day-Uei; Arlt, Jochen; Johnston, Abigail; Richardson, Justin A; Henderson, Robert K

    2012-12-01

    We describe a miniaturized, high-throughput, time-resolved fluorescence lifetime sensor implemented in a 0.13 m CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of fluorescence lifetime estimations on a single device. Detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with 400 ps output pulses and a 10% fill-factor. An array of time-to-digital converters (TDCs) with ≈50 ps resolution records up to 8 photon events during each excitation period. Data from the TDC array is then processed using a centre-of-mass method (CMM) pre-calculation to produce fluorescence lifetime estimations in real-time. The sensor is believed to be the first reported implementation of embedded fluorescence lifetime estimation. The system is demonstrated in a practical laboratory environment with measurements of a variety of fluorescent dyes with different single exponential lifetimes, successfully showing the sensor's ability to overcome the classic pile-up limitation of time-correlated single photon counting (TCSPC) by over an order of magnitude. PMID:23853257

  19. Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

    PubMed Central

    Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

    2016-01-01

    Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

  20. A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.

    PubMed

    Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, Neil; Truman, Caroline; Hardern, Ian M; Ogilvie, Donald J

    2016-06-15

    Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition. PMID:27036617

  1. High throughput optical scanner

    DOEpatents

    Basiji, David A.; van den Engh, Gerrit J.

    2001-01-01

    A scanning apparatus is provided to obtain automated, rapid and sensitive scanning of substrate fluorescence, optical density or phosphorescence. The scanner uses a constant path length optical train, which enables the combination of a moving beam for high speed scanning with phase-sensitive detection for noise reduction, comprising a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and reflect it to the substrate, whereby it is swept across the substrate along a scan arc, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical path length from the light source to the photodetector is substantially constant throughout the sweep across the substrate. The optical train can further include a waveguide or mirror to collect emitted or scattered light from the substrate and direct it to the photodetector. For phase-sensitive detection the light source is intensity modulated and the detector is connected to phase-sensitive detection electronics. A scanner using a substrate translator is also provided. For two dimensional imaging the substrate is translated in one dimension while the scanning mirror scans the beam in a second dimension. For a high throughput scanner, stacks of substrates are loaded onto a conveyor belt from a tray feeder.

  2. High-throughput profiling of tissue and tissue model microarrays: Combined transmitted light and 3-color fluorescence digital pathology

    PubMed Central

    Nederlof, Michel; Watanabe, Shigeo; Burnip, Bill; Taylor, D. Lansing; Critchley-Thorne, Rebecca

    2011-01-01

    For many years pathologists have used Hematoxylin and Eosin (H&E), single marker immunohistochemistry (IHC) and in situ hybridization with manual analysis by microscopy or at best simple digital imaging. There is a growing trend to update pathology to a digital workflow to improve objectivity and productivity, as has been done in radiology. There is also a need for tissue-based multivariate biomarker assays to improve the accuracy of diagnostic, prognostic, and predictive testing. Multivariate tests are not compatible with the traditional single marker, manual analysis pathology methods but instead require a digital platform with brightfield and fluorescence imaging, quantitative image analysis, and informatics. Here we describe the use of the Hamamatsu NanoZoomer Digital Pathology slide scanner with HCImage software for combined brightfield and multiplexed fluorescence biomarker analysis and highlight its applications in biomarker research and pathology testing. This combined approach will be an important aid to pathologists in making critical diagnoses. PMID:22200032

  3. Development and implementation of a miniaturized high-throughput time-resolved fluorescence energy transfer assay to identify small molecule inhibitors of polo-like kinase 1.

    PubMed

    Sharlow, Elizabeth R; Leimgruber, Stephanie; Shun, Tong Ying; Lazo, John S

    2007-12-01

    Polo-like kinase (Plk) 1 is a key enzyme involved in regulating the mammalian cell cycle that is also a validated anticancer drug target. Nonetheless, there are relatively few readily available potent and selective small molecule inhibitors of Plk1. To increase the availability of pharmacologically valuable Plk1 inhibitors, we describe herein the development, variability assessment, validation, and implementation of a 384-well automated, miniaturized high-throughput time-resolved fluorescence energy transfer screening assay designed to identify Plk1 kinase inhibitors. Using a small molecule library of pharmaceutically active compounds to gauge high-throughput assay robustness and reproducibility, we found nine general kinase inhibitors, including H-89, which was selected as the minimum control. We then interrogated a 97,101 compound library from the National Institutes of Health repository for small molecule inhibitors of Plk1 kinase activity. The initial primary hit rate in a single 10 microM concentration format was 0.21%. Hit compounds were subjected to concentration-response confirmation and interference assays. Identified in the screen were seven compounds with 50% inhibitory concentration (IC50) values below 1 microM, 20 compounds with IC50 values between 1 microM and 5 microM, and eight compounds with IC50 values between 5 and 10 microM, which could be assigned to seven distinct chemotype classes. Hit compounds were also examined for their ability to inhibit other kinases such as protein kinase D, focal adhesion kinase, rho-associated coiled coil protein kinase 2, c-jun NH2-terminal kinase 3, and protein kinase A via experimentation or data-mining. These compounds should be useful as probes for the biological activity of Plk1 and as leads for the development of new selective inhibitors of Plk1. PMID:18181689

  4. High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels.

    PubMed

    Huang, Aihua; Qiu, Zhigang; Jin, Min; Shen, Zhiqiang; Chen, Zhaoli; Wang, Xinwei; Li, Jun-Wen

    2014-08-18

    Bacterial pathogens are mostly responsible for food-borne diseases, and there is still substantial room for improvement in the effective detection of these organisms. In the present study, we explored a new method to detect target pathogens easily and rapidly with high sensitivity and specificity. This method uses an oligonucleotide microarray combined with quantum dots as fluorescent labels. Oligonucleotide probes targeting the 16SrRNA gene were synthesized to create an oligonucleotide microarray. The PCR products labeled with biotin were subsequently hybridized using an oligonucleotide microarray. Following incubation with CdSe/ZnS quantum dots coated with streptavidin, fluorescent signals were detected with a PerkinElmer Gx Microarray Scanner. The results clearly showed specific hybridization profiles corresponding to the bacterial species assessed. Two hundred and sixteen strains of food-borne bacterial pathogens, including standard strains and isolated strains from food samples, were used to test the specificity, stability, and sensitivity of the microarray system. We found that the oligonucleotide microarray combined with quantum dots used as fluorescent labels can successfully discriminate the bacterial organisms at the genera or species level, with high specificity and stability as well as a sensitivity of 10 colony forming units (CFU)/mL of pure culture. We further tested 105 mock-contaminated food samples and achieved consistent results as those obtained from traditional biochemical methods. Together, these results indicate that the quantum dot-based oligonucleotide microarray has the potential to be a powerful tool in the detection and identification of pathogenic bacteria in foods. PMID:24927399

  5. Rapid high-throughput assessment of aerobic bacteria in complex samples by fluorescence-based oxygen respirometry.

    PubMed

    O'Mahony, Fiach C; Papkovsky, Dmitri B

    2006-02-01

    A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given. PMID:16461677

  6. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species. PMID:27369550

  7. Automatic Tumor-Stroma Separation in Fluorescence TMAs Enables the Quantitative High-Throughput Analysis of Multiple Cancer Biomarkers

    PubMed Central

    Lahrmann, Bernd; Halama, Niels; Sinn, Hans-Peter; Schirmacher, Peter; Jaeger, Dirk; Grabe, Niels

    2011-01-01

    The upcoming quantification and automation in biomarker based histological tumor evaluation will require computational methods capable of automatically identifying tumor areas and differentiating them from the stroma. As no single generally applicable tumor biomarker is available, pathology routinely uses morphological criteria as a spatial reference system. We here present and evaluate a method capable of performing the classification in immunofluorescence histological slides solely using a DAPI background stain. Due to the restriction to a single color channel this is inherently challenging. We formed cell graphs based on the topological distribution of the tissue cell nuclei and extracted the corresponding graph features. By using topological, morphological and intensity based features we could systematically quantify and compare the discrimination capability individual features contribute to the overall algorithm. We here show that when classifying fluorescence tissue slides in the DAPI channel, morphological and intensity based features clearly outpace topological ones which have been used exclusively in related previous approaches. We assembled the 15 best features to train a support vector machine based on Keratin stained tumor areas. On a test set of TMAs with 210 cores of triple negative breast cancers our classifier was able to distinguish between tumor and stroma tissue with a total overall accuracy of 88%. Our method yields first results on the discrimination capability of features groups which is essential for an automated tumor diagnostics. Also, it provides an objective spatial reference system for the multiplex analysis of biomarkers in fluorescence immunohistochemistry. PMID:22164226

  8. Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format

    PubMed Central

    Chiaraviglio, Lucius

    2014-01-01

    Abstract Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening. PMID:24831788

  9. High-throughput drug profiling with voltage- and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes.

    PubMed

    Bedut, Stephane; Seminatore-Nole, Christine; Lamamy, Veronique; Caignard, Sarah; Boutin, Jean A; Nosjean, Olivier; Stephan, Jean-Philippe; Coge, Francis

    2016-07-01

    Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety. PMID:27199128

  10. High throughput protein production screening

    DOEpatents

    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.

  11. Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format

    PubMed Central

    Ayoub, Mohammed Akli; Trebaux, Julien; Vallaghe, Julie; Charrier-Savournin, Fabienne; Al-Hosaini, Khaled; Gonzalez Moya, Arturo; Pin, Jean-Philippe; Pfleger, Kevin D. G.; Trinquet, Eric

    2014-01-01

    The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose–response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z′-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling PMID:25002860

  12. High-throughput Tetrad Analysis

    PubMed Central

    Ludlow, Catherine L.; Scott, Adrian C.; Cromie, Gareth A.; Jeffery, Eric W.; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L.; Hays, Michelle; Dudley, Aimée M.

    2013-01-01

    Tetrad analysis has been a gold standard genetic technique for several decades. Unfortunately, the manual nature of the process has relegated its application to small-scale studies and limited its integration with rapidly evolving DNA sequencing technologies. We have developed a rapid, high-throughput method, called Barcode Enabled Sequencing of Tetrads (BEST), that replaces the manual processes of isolating, disrupting and spacing tetrads. BEST uses a meiosis-specific GFP fusion protein to isolate tetrads by fluorescence-activated cell sorting and molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. We demonstrate the approach in Saccharomyces cerevisiae, but BEST is readily transferable to microorganisms in which meiotic mapping is significantly more laborious. PMID:23666411

  13. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

    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.

  14. High throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryocondensation pump with a unique regeneration mechanism that allows continuous operation has been constructed and tested. The pump features a device referred to as the ''Snail'' which removes the cryofrost layer as it is moved over the pumping surfaces. A forepump pumps the sublimed gas generated inside the Snail. The compression ratio of the pump is the ratio of the cryopump speed to the leakage conductance of the Snail. Deuterium had been pumped continuously at 30 torr.L/s at a speed of 2000 L/s and a compression ratio of 100. The pump, being all metal sealed and free of lubricating fluids, has many potential applications where untraclean high throughput pumping is desirable. Since the pump regenerates on a time scale of 60 seconds, the inventory in the pump is minimized - an important consideration when pumping radioactive materials such as tritium. Test data and a videotape of the Snail removing the cryofrost will be shown.

  15. Determination of urine cofilin-1 level in acute kidney injury using a high-throughput localized surface plasmon-coupled fluorescence biosensor

    NASA Astrophysics Data System (ADS)

    Chang, Ying-Feng; Chao, Cheng-Han; Lin, Lih-Yuan; Tsai, Cheng-Han; Chou, Chien; Lee, Yi-Jang

    2014-01-01

    The actin-depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia-induced renal disorders. We examine whether cofilin-1 is associated with acute kidney injury (AKI) using human urine samples. We exploited a 96-well based high-throughput biosensor that uses gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients (n=37 from 47 cases analyzed) was twofold higher than that of healthy adults (n=21 from 29 cases analyzed). The receiver operating characteristic (ROC) curve showed that cofilin-1 was acceptable for discriminating AKI patients from healthy adults. However, an increase of the sample size is required to conclude the importance of urine cofilin-1 on AKI diagnosis, and the high-throughput ultrasensitive biosensor used in this study would greatly accelerate the measurement of urine cofilin-1 in an increased sample size.

  16. A semi-automated method for purification of milligram quantities of proteins on the QIAcube

    PubMed Central

    McGraw, J; Tatipelli, VK; Traore, MC; Feyijinmi, O; Eangoor, P; Lane, S; Stollar, EJ

    2015-01-01

    A growing number of studies require the purification of multiple proteins simultaneously and the development of simple economical high-throughput purification methods is essential. We have tested the purification of two related proteins in a variety of conditions to benchmark the semi-automated affinity chromatography method for the QIAcube that we have developed. We find that this new QIAcube method can successfully purify milligram quantities of proteins with minimal user involvement and performs as well as methods based on gravity. The method could easily be adapted to other chromatography resins and should prove to be a versatile method for optimizing protein expression or purification conditions for multiple proteins while obtaining sufficient amounts for subsequent biochemical analyses. PMID:24508590

  17. Path Planning for Semi-automated Simulated Robotic Neurosurgery

    PubMed Central

    Hu, Danying; Gong, Yuanzheng; Hannaford, Blake; Seibel, Eric J.

    2015-01-01

    This paper considers the semi-automated robotic surgical procedure for removing the brain tumor margins, where the manual operation is a tedious and time-consuming task for surgeons. We present robust path planning methods for robotic ablation of tumor residues in various shapes, which are represented in point-clouds instead of analytical geometry. Along with the path plans, corresponding metrics are also delivered to the surgeon for selecting the optimal candidate in the automated robotic ablation. The selected path plan is then executed and tested on RAVEN™ II surgical robot platform as part of the semi-automated robotic brain tumor ablation surgery in a simulated tissue phantom. PMID:26705501

  18. Accurate and semi-automated analysis of bacterial association with mammalian cells.

    PubMed

    Murphy, C M; Paré, S; Galea, G; Simpson, J C; Smith, S G J

    2016-03-01

    To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria. PMID:26769557

  19. CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

    PubMed

    Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

    2013-01-01

    : The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

  20. High throughput screening technologies for ion channels

    PubMed Central

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

    2016-01-01

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

  1. Semi-automated microplate monitoring of protein polymerization and aggregation.

    PubMed

    Garcia, Veronica M; Rowlett, Veronica W; Margolin, William; Morano, Kevin A

    2016-09-01

    Static light scattering (SLS) is a commonly used technique for monitoring dynamics of high molecular weight protein complexes such as protein oligomers or aggregates. However, traditional methods are limited to testing a single condition and typically require large amounts of protein and specialized equipment. We show that a standard microplate reader can be used to characterize the molecular dynamics of different types of protein complexes, with the multiple advantages of microscale experimental volumes, semi-automated protocols and highly parallel processing. PMID:27251433

  2. Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum

    PubMed Central

    Soelberg, Scott D.; Weigel, Kris M.; Hiraiwa, Morgan; Cairns, Andrew; Lee, Hyun-Boo; Furlong, Clement E.; Oh, Kieseok; Lee, Kyong-Hoon; Gao, Dayong; Chung, Jae-Hyun; Cangelosi, Gerard A.

    2014-01-01

    An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 106-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure. PMID:24465845

  3. Semi-Automated Digital Image Analysis of Pick's Disease and TDP-43 Proteinopathy.

    PubMed

    Irwin, David J; Byrne, Matthew D; McMillan, Corey T; Cooper, Felicia; Arnold, Steven E; Lee, Edward B; Van Deerlin, Vivianna M; Xie, Sharon X; Lee, Virginia M-Y; Grossman, Murray; Trojanowski, John Q

    2016-01-01

    Digital image analysis of histology sections provides reliable, high-throughput methods for neuropathological studies but data is scant in frontotemporal lobar degeneration (FTLD), which has an added challenge of study due to morphologically diverse pathologies. Here, we describe a novel method of semi-automated digital image analysis in FTLD subtypes including: Pick's disease (PiD, n=11) with tau-positive intracellular inclusions and neuropil threads, and TDP-43 pathology type C (FTLD-TDPC, n=10), defined by TDP-43-positive aggregates predominantly in large dystrophic neurites. To do this, we examined three FTLD-associated cortical regions: mid-frontal gyrus (MFG), superior temporal gyrus (STG) and anterior cingulate gyrus (ACG) by immunohistochemistry. We used a color deconvolution process to isolate signal from the chromogen and applied both object detection and intensity thresholding algorithms to quantify pathological burden. We found object-detection algorithms had good agreement with gold-standard manual quantification of tau- and TDP-43-positive inclusions. Our sampling method was reliable across three separate investigators and we obtained similar results in a pilot analysis using open-source software. Regional comparisons using these algorithms finds differences in regional anatomic disease burden between PiD and FTLD-TDP not detected using traditional ordinal scale data, suggesting digital image analysis is a powerful tool for clinicopathological studies in morphologically diverse FTLD syndromes. PMID:26538548

  4. High-throughput tetrad analysis.

    PubMed

    Ludlow, Catherine L; Scott, Adrian C; Cromie, Gareth A; Jeffery, Eric W; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L; Hays, Michelle; Dudley, Aimée M

    2013-07-01

    Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious. PMID:23666411

  5. High-throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible.

  6. Development of a sensitive and reliable high performance liquid chromatography method with fluorescence detection for high-throughput analysis of multi-class mycotoxins in Coix seed.

    PubMed

    Kong, Wei-Jun; Li, Jun-Yuan; Qiu, Feng; Wei, Jian-He; Xiao, Xiao-He; Zheng, Yuguo; Yang, Mei-Hua

    2013-10-17

    As an edible and medicinal plant, Coix seed is readily contaminated by more than one group of mycotoxins resulting in potential risk to human health. A reliable and sensitive method has been developed to determine seven mycotoxins (aflatoxins B1, B2, G1, G2, zearalenone, α-zearalenol, and β-zearalenol) simultaneously in 10 batches of Coix seed marketed in China. The method is based on a rapid ultrasound-assisted solid-liquid extraction (USLE) using methanol/water (80/20) followed by immunoaffinity column (IAC) clean-up, on-line photochemical derivatization (PCD), and high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). Careful optimization of extraction, clean-up, separation and detection conditions was accomplished to increase sample throughput and to attain rapid separation and sensitive detection. Method validation was performed by analyzing samples spiked at three different concentrations for the seven mycotoxins. Recoveries were from 73.5% to 107.3%, with relative standard deviations (RSDs) lower than 7.7%. The intra- and inter-day precisions, expressed as RSDs, were lower than 4% for all studied analytes. Limits of detection and quantification ranged from 0.01 to 50.2 μg kg(-1), and from 0.04 to 125.5 μg kg(-1), respectively, which were below the tolerance levels for mycotoxins set by the European Union. Samples that tested positive were further analyzed by HPLC tandem electrospray ionization mass spectrometry for confirmatory purposes. This is the first application of USLE-IAC-HPLC-PCD-FLD for detecting the occurrence of multi-class mycotoxins in Coix seed. PMID:24091376

  7. Toward high-throughput genotyping: dynamic and automatic software for manipulating large-scale genotype data using fluorescently labeled dinucleotide markers.

    PubMed

    Li, J L; Deng, H; Lai, D B; Xu, F; Chen, J; Gao, G; Recker, R R; Deng, H W

    2001-07-01

    To efficiently manipulate large amounts of genotype data generated with fluorescently labeled dinucleotide markers, we developed a Microsoft database management system, named. offers several advantages. First, it accommodates the dynamic nature of the accumulations of genotype data during the genotyping process; some data need to be confirmed or replaced by repeat lab procedures. By using, the raw genotype data can be imported easily and continuously and incorporated into the database during the genotyping process that may continue over an extended period of time in large projects. Second, almost all of the procedures are automatic, including autocomparison of the raw data read by different technicians from the same gel, autoadjustment among the allele fragment-size data from cross-runs or cross-platforms, autobinning of alleles, and autocompilation of genotype data for suitable programs to perform inheritance check in pedigrees. Third, provides functions to track electrophoresis gel files to locate gel or sample sources for any resultant genotype data, which is extremely helpful for double-checking consistency of raw and final data and for directing repeat experiments. In addition, the user-friendly graphic interface of renders processing of large amounts of data much less labor-intensive. Furthermore, has built-in mechanisms to detect some genotyping errors and to assess the quality of genotype data that then are summarized in the statistic reports automatically generated by. The can easily handle >500,000 genotype data entries, a number more than sufficient for typical whole-genome linkage studies. The modules and programs we developed for the can be extended to other database platforms, such as Microsoft SQL server, if the capability to handle still greater quantities of genotype data simultaneously is desired. PMID:11435414

  8. Semi-automated Image Processing for Preclinical Bioluminescent Imaging

    PubMed Central

    Slavine, Nikolai V; McColl, Roderick W

    2015-01-01

    Objective Bioluminescent imaging is a valuable noninvasive technique for investigating tumor dynamics and specific biological molecular events in living animals to better understand the effects of human disease in animal models. The purpose of this study was to develop and test a strategy behind automated methods for bioluminescence image processing from the data acquisition to obtaining 3D images. Methods In order to optimize this procedure a semi-automated image processing approach with multi-modality image handling environment was developed. To identify a bioluminescent source location and strength we used the light flux detected on the surface of the imaged object by CCD cameras. For phantom calibration tests and object surface reconstruction we used MLEM algorithm. For internal bioluminescent sources we used the diffusion approximation with balancing the internal and external intensities on the boundary of the media and then determined an initial order approximation for the photon fluence we subsequently applied a novel iterative deconvolution method to obtain the final reconstruction result. Results We find that the reconstruction techniques successfully used the depth-dependent light transport approach and semi-automated image processing to provide a realistic 3D model of the lung tumor. Our image processing software can optimize and decrease the time of the volumetric imaging and quantitative assessment. Conclusion The data obtained from light phantom and lung mouse tumor images demonstrate the utility of the image reconstruction algorithms and semi-automated approach for bioluminescent image processing procedure. We suggest that the developed image processing approach can be applied to preclinical imaging studies: characteristics of tumor growth, identify metastases, and potentially determine the effectiveness of cancer treatment. PMID:26618187

  9. Automated cognome construction and semi-automated hypothesis generation.

    PubMed

    Voytek, Jessica B; Voytek, Bradley

    2012-06-30

    Modern neuroscientific research stands on the shoulders of countless giants. PubMed alone contains more than 21 million peer-reviewed articles with 40-50,000 more published every month. Understanding the human brain, cognition, and disease will require integrating facts from dozens of scientific fields spread amongst millions of studies locked away in static documents, making any such integration daunting, at best. The future of scientific progress will be aided by bridging the gap between the millions of published research articles and modern databases such as the Allen brain atlas (ABA). To that end, we have analyzed the text of over 3.5 million scientific abstracts to find associations between neuroscientific concepts. From the literature alone, we show that we can blindly and algorithmically extract a "cognome": relationships between brain structure, function, and disease. We demonstrate the potential of data-mining and cross-platform data-integration with the ABA by introducing two methods for semi-automated hypothesis generation. By analyzing statistical "holes" and discrepancies in the literature we can find understudied or overlooked research paths. That is, we have added a layer of semi-automation to a part of the scientific process itself. This is an important step toward fundamentally incorporating data-mining algorithms into the scientific method in a manner that is generalizable to any scientific or medical field. PMID:22584238

  10. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  11. Semi-automated object tracking methods in biological imaging.

    PubMed

    Davis, Michael A; Praěský, Ondřej; Sysko, Laura R

    2015-01-01

    Time-lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior. Tracking and extracting measurements of objects from time-lapse datasets are challenges that result from the complexity and dynamics of each object's motion and intensity or the appearance of new objects in the field of view. A wide range of strategies for proper data sampling, object detection, image analysis, and post-analysis interpretation are available. Theory and methods for single-particle tracking, spot detection, and object linking are discussed in this unit, as well as examples with step-by-step procedures for utilizing semi-automated software and visualization tools for achieving tracking results and interpreting this output. PMID:25559222

  12. Observer performance in semi-automated microbleed detection

    NASA Astrophysics Data System (ADS)

    Kuijf, Hugo J.; Brundel, Manon; de Bresser, Jeroen; Viergever, Max A.; Biessels, Geert Jan; Geerlings, Mirjam I.; Vincken, Koen L.

    2013-03-01

    Cerebral microbleeds are small bleedings in the human brain, detectable with MRI. Microbleeds are associated with vascular disease and dementia. The number of studies involving microbleed detection is increasing rapidly. Visual rating is the current standard for detection, but is a time-consuming process, especially at high-resolution 7.0 T MR images, has limited reproducibility and is highly observer dependent. Recently, multiple techniques have been published for the semi-automated detection of microbleeds, attempting to overcome these problems. In the present study, a 7.0 T dual-echo gradient echo MR image was acquired in 18 participants with microbleeds from the SMART study. Two experienced observers identified 54 microbleeds in these participants, using a validated visual rating scale. The radial symmetry transform (RST) can be used for semi-automated detection of microbleeds in 7.0 T MR images. In the present study, the results of the RST were assessed by two observers and 47 microbleeds were identified: 35 true positives and 12 extra positives (microbleeds that were missed during visual rating). Hence, after scoring a total number of 66 microbleeds could be identified in the 18 participants. The use of the RST increased the average sensitivity of observers from 59% to 69%. More importantly, inter-observer agreement (ICC and Dice's coefficient) increased from 0.85 and 0.64 to 0.98 and 0.96, respectively. Furthermore, the required rating time was reduced from 30 to 2 minutes per participant. By fine-tuning the RST, sensitivities up to 90% can be achieved, at the cost of extra false positives.

  13. Fast semi-automated lesion demarcation in stroke

    PubMed Central

    de Haan, Bianca; Clas, Philipp; Juenger, Hendrik; Wilke, Marko; Karnath, Hans-Otto

    2015-01-01

    Lesion–behaviour mapping analyses require the demarcation of the brain lesion on each (usually transverse) slice of the individual stroke patient's brain image. To date, this is generally thought to be most precise when done manually, which is, however, both time-consuming and potentially observer-dependent. Fully automated lesion demarcation methods have been developed to address these issues, but these are often not practicable in acute stroke research where for each patient only a single image modality is available and the available image modality differs over patients. In the current study, we evaluated a semi-automated lesion demarcation approach, the so-called Clusterize algorithm, in acute stroke patients scanned in a range of common image modalities. Our results suggest that, compared to the standard of manual lesion demarcation, the semi-automated Clusterize algorithm is capable of significantly speeding up lesion demarcation in the most commonly used image modalities, without loss of either lesion demarcation precision or lesion demarcation reproducibility. For the three investigated acute datasets (CT, DWI, T2FLAIR), containing a total of 44 patient images obtained in a regular clinical setting at patient admission, the reduction in processing time was on average 17.8 min per patient and this advantage increased with increasing lesion volume (up to 60 min per patient for the largest lesion volumes in our datasets). Additionally, our results suggest that performance of the Clusterize algorithm in a chronic dataset with 11 T1 images was comparable to its performance in the acute datasets. We thus advocate the use of the Clusterize algorithm, integrated into a simple, freely available SPM toolbox, for the precise, reliable and fast preparation of imaging data for lesion–behaviour mapping analyses. PMID:26413473

  14. LeafJ: an ImageJ plugin for semi-automated leaf shape measurement.

    PubMed

    Maloof, Julin N; Nozue, Kazunari; Mumbach, Maxwell R; Palmer, Christine M

    2013-01-01

    High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review and recent examples). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS). Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices. To date, leaf shape programs (e.g. SHAPE, LAMINA, LeafAnalyzer, LEAFPROCESSOR) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping. PMID:23380664

  15. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

    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

  16. Semi-Automated Discovery of Application Session Structure

    SciTech Connect

    Kannan, J.; Jung, J.; Paxson, V.; Koksal, C.

    2006-09-07

    While the problem of analyzing network traffic at the granularity of individual connections has seen considerable previous work and tool development, understanding traffic at a higher level---the structure of user-initiated sessions comprised of groups of related connections---remains much less explored. Some types of session structure, such as the coupling between an FTP control connection and the data connections it spawns, have prespecified forms, though the specifications do not guarantee how the forms appear in practice. Other types of sessions, such as a user reading email with a browser, only manifest empirically. Still other sessions might exist without us even knowing of their presence, such as a botnet zombie receiving instructions from its master and proceeding in turn to carry them out. We present algorithms rooted in the statistics of Poisson processes that can mine a large corpus of network connection logs to extract the apparent structure of application sessions embedded in the connections. Our methods are semi-automated in that we aim to present an analyst with high-quality information (expressed as regular expressions) reflecting different possible abstractions of an application's session structure. We develop and test our methods using traces from a large Internet site, finding diversity in the number of applications that manifest, their different session structures, and the presence of abnormal behavior. Our work has applications to traffic characterization and monitoring, source models for synthesizing network traffic, and anomaly detection.

  17. Semi-Automated Segmentation of Microbes in Color Images

    NASA Astrophysics Data System (ADS)

    Reddy, Chandankumar K.; Liu, Feng-I.; Dazzo, Frank B.

    2003-01-01

    The goal of this work is to develop a system that can semi-automate the detection of multicolored foreground objects in digitized color images that also contain complex and very noisy backgrounds. Although considered a general problem of color image segmentation, our application is microbiology where various colored stains are used to reveal information on the microbes without cultivation. Instead of providing a simple threshold, the proposed system offers an interactive environment whereby the user chooses multiple sample points to define the range of color pixels comprising the foreground microbes of interest. The system then uses the color and spatial distances of these target points to segment the microbes from the confusing background of pixels whose RGB values lie outside the newly defined range and finally finds each cell's boundary using region-growing and mathematical morphology. Some other image processing methods are also applied to enhance the resultant image containing the colored microbes against a noise-free background. The prototype performs with 98% accuracy on a test set compared to ground truth data. The system described here will have many applications in image processing and analysis where one needs to segment typical pixel regions of similar but non-identical colors.

  18. A Semi-automated Abundance Survey of Ap Stars

    NASA Astrophysics Data System (ADS)

    Hall, Martin P.; Kurtz, Don; Elkin, Vladimir; Bruntt, Hans

    2015-08-01

    We have carried out an abundance analysis on the high-resolution spectra of approximately 350 Ap stars collected between 2007 and 2010 on the FEROS Echelle (Fibre-led, Extended Range, Echelle ) spectrograph housed at the 2.2-m telescope at European Southern Observatory at La Silla, Chile. We employed the VWA package (vsin I, wavelength shift, abundance analysis) for preliminary selection of spectral lines, and a semi-automated set of routines which we developed in the programming language IDL, to calculate the equivalent widths and abundances of ions of Iron and the rare earth elements Neodymium and Praseodymium using the WIDTH program and NEMO model atmospheres. Initial results are presented, which reinforce the correlation between iron abundance and effective temperature, from an over-abundance in the late Bp stars, to under-abundant in the early F stars. Results also suggest that the disequilibrium in abundances of the first and second ionisation stages of these ions in the rapidly oscillating Ap (roAp) stars may a consequence of the relatively cool temperatures of those stars, rather than a signature of pulsation.

  19. Semi-automated software service integration in virtual organisations

    NASA Astrophysics Data System (ADS)

    Afsarmanesh, Hamideh; Sargolzaei, Mahdi; Shadi, Mahdieh

    2015-08-01

    To enhance their business opportunities, organisations involved in many service industries are increasingly active in pursuit of both online provision of their business services (BSs) and collaborating with others. Collaborative Networks (CNs) in service industry sector, however, face many challenges related to sharing and integration of their collection of provided BSs and their corresponding software services. Therefore, the topic of service interoperability for which this article introduces a framework is gaining momentum in research for supporting CNs. It contributes to generation of formal machine readable specification for business processes, aimed at providing their unambiguous definitions, as needed for developing their equivalent software services. The framework provides a model and implementation architecture for discovery and composition of shared services, to support the semi-automated development of integrated value-added services. In support of service discovery, a main contribution of this research is the formal representation of services' behaviour and applying desired service behaviour specified by users for automated matchmaking with other existing services. Furthermore, to support service integration, mechanisms are developed for automated selection of the most suitable service(s) according to a number of service quality aspects. Two scenario cases are presented, which exemplify several specific features related to service discovery and service integration aspects.

  20. Literature classification for semi-automated updating of biological knowledgebases

    PubMed Central

    2013-01-01

    Background As the output of biological assays increase in resolution and volume, the body of specialized biological data, such as functional annotations of gene and protein sequences, enables extraction of higher-level knowledge needed for practical application in bioinformatics. Whereas common types of biological data, such as sequence data, are extensively stored in biological databases, functional annotations, such as immunological epitopes, are found primarily in semi-structured formats or free text embedded in primary scientific literature. Results We defined and applied a machine learning approach for literature classification to support updating of TANTIGEN, a knowledgebase of tumor T-cell antigens. Abstracts from PubMed were downloaded and classified as either "relevant" or "irrelevant" for database update. Training and five-fold cross-validation of a k-NN classifier on 310 abstracts yielded classification accuracy of 0.95, thus showing significant value in support of data extraction from the literature. Conclusion We here propose a conceptual framework for semi-automated extraction of epitope data embedded in scientific literature using principles from text mining and machine learning. The addition of such data will aid in the transition of biological databases to knowledgebases. PMID:24564403

  1. Automated Cognome Construction and Semi-automated Hypothesis Generation

    PubMed Central

    Voytek, Jessica B.; Voytek, Bradley

    2012-01-01

    Modern neuroscientific research stands on the shoulders of countless giants. PubMed alone contains more than 21 million peer-reviewed articles with 40–50,000 more published every month. Understanding the human brain, cognition, and disease will require integrating facts from dozens of scientific fields spread amongst millions of studies locked away in static documents, making any such integration daunting, at best. The future of scientific progress will be aided by bridging the gap between the millions of published research articles and modern databases such as the Allen Brain Atlas (ABA). To that end, we have analyzed the text of over 3.5 million scientific abstracts to find associations between neuroscientific concepts. From the literature alone, we show that we can blindly and algorithmically extract a “cognome”: relationships between brain structure, function, and disease. We demonstrate the potential of data-mining and cross-platform data-integration with the ABA by introducing two methods for semiautomated hypothesis generation. By analyzing statistical “holes” and discrepancies in the literature we can find understudied or overlooked research paths. That is, we have added a layer of semi-automation to a part of the scientific process itself. This is an important step toward fundamentally incorporating data-mining algorithms into the scientific method in a manner that is generalizable to any scientific or medical field. PMID:22584238

  2. High-Throughput Screening in Primary Neurons

    PubMed Central

    Sharma, Punita; Ando, D. Michael; Daub, Aaron; Kaye, Julia A.; Finkbeiner, Steven

    2013-01-01

    Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss our adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells. PMID:22341232

  3. High-throughput screening in primary neurons.

    PubMed

    Sharma, Punita; Ando, D Michael; Daub, Aaron; Kaye, Julia A; Finkbeiner, Steven

    2012-01-01

    Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss the adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells. PMID:22341232

  4. Evaluation of a semi-automated platelet-counting system.

    PubMed

    Rowan, R M; Fraser, C; Gray, J H; McDonald, G A

    1977-04-01

    Coulter Electronics Ltd have produced a semi-automated platelet-counting system. Platelet-rich plasma may be obtained either by tube sedimentation or by means of the Thrombo-fuge, the latter being an instrument designed to produce accelerated sedimentation. The instrument is linear over the entire range of platelet counts, and machine reproducibility is good. Comparison of machine-rated with visual counts satisfied statistical evaluation. The technique can be handled by one operator and platelet counts can be achieved at the rate of 30 per hour by both methods although individual counts on the Thrombo-fuge may be obtained in approximately one-quarter of the time required for tube sedimentation. The throughput using the Thrombo-fuge could certainly be doubled were two sample plates supplied. Few problems were encountered during the evaluation and most could be avoided by meticulous technique. Visual counts must be performed when the sample haematocrit is greater than 50%-Discrepant counts have been obtained in patients with white cell counts exceeding 50 X 10(9)/1 and in patients with giant platelets. ESR elevation for any reason does not lead to serious discrepancy in results. The incidence of platelet clumping due to the presence of platelet agglutinins and of microclot formation due to inadequate mixing is probably much higher than is commonly thought, and certainly peripheral blood film scrutiny should never be omitted in patients with low counts. Careful examination of peripheral blood films must be combined with instrument counting for some time lest further causes of discrepant counting emerge. PMID:856881

  5. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  6. MouseMove: an open source program for semi-automated analysis of movement and cognitive testing in rodents

    PubMed Central

    Samson, Andre L.; Ju, Lining; Ah Kim, Hyun; Zhang, Shenpeng R.; Lee, Jessica A. A.; Sturgeon, Sharelle A.; Sobey, Christopher G.; Jackson, Shaun P.; Schoenwaelder, Simone M.

    2015-01-01

    The Open Field (OF) test is one of the most commonly used assays for assessing exploratory behaviour and generalised locomotor activity in rodents. Nevertheless, the vast majority of researchers still rely upon costly commercial systems for recording and analysing OF test results. Consequently, our aim was to design a freely available program for analysing the OF test and to provide an accompanying protocol that was minimally invasive, rapid, unbiased, without the need for specialised equipment or training. Similar to commercial systems, we show that our software—called MouseMove—accurately quantifies numerous parameters of movement including travel distance, speed, turning and curvature. To assess its utility, we used MouseMove to quantify unilateral locomotor deficits in mice following the filament-induced middle cerebral artery occlusion model of acute ischemic stroke. MouseMove can also monitor movement within defined regions-of-interest and is therefore suitable for analysing the Novel Object Recognition test and other field-related cognitive tests. To the best of our knowledge, MouseMove is the first open source software capable of providing qualitative and quantitative information on mouse locomotion in a semi-automated and high-throughput fashion, and hence MouseMove represents a sound alternative to commercial movement analysis systems. PMID:26530459

  7. Semi-Automated Diagnosis, Repair, and Rework of Spacecraft Electronics

    NASA Technical Reports Server (NTRS)

    Struk, Peter M.; Oeftering, Richard C.; Easton, John W.; Anderson, Eric E.

    2008-01-01

    NASA's Constellation Program for Exploration of the Moon and Mars places human crews in extreme isolation in resource scarce environments. Near Earth, the discontinuation of Space Shuttle flights after 2010 will alter the up- and down-mass capacity for the International Space Station (ISS). NASA is considering new options for logistics support strategies for future missions. Aerospace systems are often composed of replaceable modular blocks that minimize the need for complex service operations in the field. Such a strategy however, implies a robust and responsive logistics infrastructure with relatively low transportation costs. The modular Orbital Replacement Units (ORU) used for ISS requires relatively large blocks of replacement hardware even though the actual failed component may really be three orders of magnitude smaller. The ability to perform in-situ repair of electronics circuits at the component level can dramatically reduce the scale of spares and related logistics cost. This ability also reduces mission risk, increases crew independence and improves the overall supportability of the program. The Component-Level Electronics Assembly Repair (CLEAR) task under the NASA Supportability program was established to demonstrate the practicality of repair by first investigating widely used soldering materials and processes (M&P) performed by modest manual means. The work will result in program guidelines for performing manual repairs along with design guidance for circuit reparability. The next phase of CLEAR recognizes that manual repair has its limitations and some highly integrated devices are extremely difficult to handle and demand semi-automated equipment. Further, electronics repairs require a broad range of diagnostic capability to isolate the faulty components. Finally repairs must pass functional tests to determine that the repairs are successful and the circuit can be returned to service. To prevent equipment demands from exceeding spacecraft volume

  8. Rapid Methods for High-Throughput Detection of Sulfoxides▿

    PubMed Central

    Shainsky, Janna; Derry, Netta-Lee; Leichtmann-Bardoogo, Yael; Wood, Thomas K.; Fishman, Ayelet

    2009-01-01

    Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment. PMID:19465532

  9. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  10. NeuronMetrics: Software for Semi-Automated Processing of Cultured-Neuron Images

    PubMed Central

    Narro, Martha L.; Yang, Fan; Kraft, Robert; Wenk, Carola; Efrat, Alon; Restifo, Linda L.

    2007-01-01

    Using primary cell culture to screen for changes in neuronal morphology requires specialized analysis software. We developed NeuronMetrics™ for semi-automated, quantitative analysis of two-dimensional (2D) images of fluorescently labeled cultured neurons. It skeletonizes the neuron image using two complementary image-processing techniques, capturing fine terminal neurites with high fidelity. An algorithm was devised to span wide gaps in the skeleton. NeuronMetrics uses a novel strategy based on geometric features called faces to extract a branch-number estimate from complex arbors with numerous neurite-to-neurite contacts, without creating a precise, contact-free representation of the neurite arbor. It estimates total neurite length, branch number, primary neurite number, territory (the area of the convex polygon bounding the skeleton and cell body), and Polarity Index (a measure of neuronal polarity). These parameters provide fundamental information about the size and shape of neurite arbors, which are critical factors for neuronal function. NeuronMetrics streamlines optional manual tasks such as removing noise, isolating the largest primary neurite, and correcting length for self-fasciculating neurites. Numeric data are output in a single text file, readily imported into other applications for further analysis. Written as modules for ImageJ, NeuronMetrics provides practical analysis tools that are easy to use and support batch processing. Depending on the need for manual intervention, processing time for a batch of ~60 2D images is 1.0–2.5 hours, from a folder of images to a table of numeric data. NeuronMetrics’ output accelerates the quantitative detection of mutations and chemical compounds that alter neurite morphology in vitro, and will contribute to the use of cultured neurons for drug discovery. PMID:17270152

  11. Design and development of a semi-automated module for the preparation of metallic PET radionuclides

    NASA Astrophysics Data System (ADS)

    Trejo-Ballado, F.; Lopez-Rodriguez, V.; Gaspar-Carcamo, R. E.; Hurtado-Chong, G.; Avila-Rodriguez, Miguel A.

    2012-12-01

    The method for the production of metallic radionuclides has been widely reported, and most of them share a common ion chromatography purification technique. The aim of this work is to design and develop a semi-automated remotely controlled module for the purification of metallic PET radionuclides via cation exchange chromatography.

  12. An integrated, semi-automated approach to thermochemical conversion research for sustainable farming systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An integrated, semi-automated system is presented for the rapid and efficient testing and production of research-scale quantities of biochar. This biochar, produced from agricultural waste materials, can easily be incorporated in future sustainable livestock farming systems. These farming systems wi...

  13. A high throughput system for the preparation of single stranded templates grown in microculture.

    PubMed

    Kolner, D E; Guilfoyle, R A; Smith, L M

    1994-01-01

    A high throughput system for the preparation of single stranded M13 sequencing templates is described. Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein. Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration. Individual glass fiber discs are punched out on the cell harvester and dried briefly. The DNA samples are then eluted in water by centrifugation. The processing time from 96 microcultures to sequence quality templates is approximately 1 hr. Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week. Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987). By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed. Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989). Although minimal human intervention is required, processing time is at least twice as long. Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing. These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here. PMID:7987011

  14. High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

    PubMed Central

    Džakula, Željko; Kim, Sung K.; Mazloom, Amin R.; Zhu, Zhanyang; Tynan, John; Lu, Tim; McLennan, Graham; Palomaki, Glenn E.; Canick, Jacob A.; Oeth, Paul; Deciu, Cosmin; van den Boom, Dirk; Ehrich, Mathias

    2013-01-01

    Background Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. Methods Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. Results Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. Conclusions These data suggest that the developed laboratory methods in concert with improved bioinformatic

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

    PubMed

    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

  16. Construction of biological networks from unstructured information based on a semi-automated curation workflow.

    PubMed

    Szostak, Justyna; Ansari, Sam; Madan, Sumit; Fluck, Juliane; Talikka, Marja; Iskandar, Anita; De Leon, Hector; Hofmann-Apitius, Martin; Peitsch, Manuel C; Hoeng, Julia

    2015-01-01

    Capture and representation of scientific knowledge in a structured format are essential to improve the understanding of biological mechanisms involved in complex diseases. Biological knowledge and knowledge about standardized terminologies are difficult to capture from literature in a usable form. A semi-automated knowledge extraction workflow is presented that was developed to allow users to extract causal and correlative relationships from scientific literature and to transcribe them into the computable and human readable Biological Expression Language (BEL). The workflow combines state-of-the-art linguistic tools for recognition of various entities and extraction of knowledge from literature sources. Unlike most other approaches, the workflow outputs the results to a curation interface for manual curation and converts them into BEL documents that can be compiled to form biological networks. We developed a new semi-automated knowledge extraction workflow that was designed to capture and organize scientific knowledge and reduce the required curation skills and effort for this task. The workflow was used to build a network that represents the cellular and molecular mechanisms implicated in atherosclerotic plaque destabilization in an apolipoprotein-E-deficient (ApoE(-/-)) mouse model. The network was generated using knowledge extracted from the primary literature. The resultant atherosclerotic plaque destabilization network contains 304 nodes and 743 edges supported by 33 PubMed referenced articles. A comparison between the semi-automated and conventional curation processes showed similar results, but significantly reduced curation effort for the semi-automated process. Creating structured knowledge from unstructured text is an important step for the mechanistic interpretation and reusability of knowledge. Our new semi-automated knowledge extraction workflow reduced the curation skills and effort required to capture and organize scientific knowledge. The

  17. High throughput network for multiprocessor interconnections

    NASA Astrophysics Data System (ADS)

    Raatikainen, Pertti; Zidbeck, Juha

    1993-05-01

    Multiprocessor architectures are needed to support modern broadband applications, since traditional bus structures are not capable of providing high throughput. New bus structures are needed, especially in the area of network components and terminals. A study to find an efficient and cost effective interconnection topology for the future high speed products is presented. The most common bus topologies are introduced, and their characteristics are estimated to decide which one of them offers best performance and lowest implementation cost. The ring topology is chosen to be studied in more detail. Four competing bus access schemes for the high throughput ring are introduced as well as simulation models for each of them. Using transfer delay and throughput results, as well as keeping the implementation point of view in mind, the best candidate is selected to be studied and experimented in the succeeding research project.

  18. High-throughput screening methods for nitrilases.

    PubMed

    Xue, Ya-Ping; Yang, Yue-Kai; Lv, Sheng-Zhi; Liu, Zhi-Qiang; Zheng, Yu-Guo

    2016-04-01

    Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations. PMID:26894402

  19. A Novel High-Throughput Viscometer.

    PubMed

    Deshmukh, Suraj; Bishop, Matthew T; Dermody, Daniel; Dietsche, Laura; Kuo, Tzu-Chi; Mushrush, Melissa; Harris, Keith; Zieman, Jonathan; Morabito, Paul; Orvosh, Brian; Patrick, Don

    2016-07-11

    A novel, rapid, parallel, and high-throughput system for measuring viscosity of materials under different conditions of shear rate, temperature, time, etc., has been developed. This unique system utilizes the transient flow of a complex fluid through pipettes. This approach offers significant practical advantages over microfluidic-based devices for viscosity screening: no cleanup is required, the method is high throughput (<1 h for 100 samples), and only small sample volumes (<1 mL) are used. This paper details for the first time the experimental and modeling efforts to implement this mass- and pressure-based viscosity measurement concept as a robust viscosity estimation tool. This approach is very well-suited for viscosity measurements in high-throughput formulation workflows, as it is rapid and parallel and operates directly on samples in various microtiter plate formats. We present systematic experimental observations together with numerical and analytical modeling approaches to characterize instrument capabilities and limitations. The complex transient flow of fluids through these pipettes leads to data-rich pressure profiles. Numerical and analytical modeling is then used to extract viscosity and other rheological parameters from these pressure profiles. We have successfully utilized this viscosity screening tool for a multitude of complex fluids including oils, paints, solvents, and detergents. PMID:27259016

  20. High-Throughput Investigation of Delafossite materials

    NASA Astrophysics Data System (ADS)

    Haycock, Barry; Kylee Underwood, M.; Lekse, Jonathan; Matranga, Christopher; Lewis, James P.

    2013-03-01

    We present the application of high-throughput calculations to the intriguing problem of the forbidden optical transition in the CuGa1-xFexO2 delafossites, which is prototypical of many delafossite systems. When 5% or more of the Ga sites are replaced with Fe, there is a sudden shift to an optical band gap of 1.5eV from 2.5eV. Using high-throughput calculations and data mining techniques, we show the most likely positional configurations for x = 0.00 through x = 0.10 of the Fe atoms relative to one another. Implications of this result and applications of the techniques used are discussed, including the development of candidate materials via high-throughput analysis of constituent search-space. Funded by the National Science Foundation through NSF DMR 09-03225 and a subcontract from NETL (URS RES) for Work Activity 0004000.6.600.007.002.420.000.005 ARRA ICMI Project.

  1. Image-based high-throughput screening for inhibitors of angiogenesis.

    PubMed

    Evensen, Lasse; Link, Wolfgang; Lorens, James B

    2013-01-01

    Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling. PMID:23027002

  2. Semi-automated and fully automated mammographic density measurement and breast cancer risk prediction.

    PubMed

    Llobet, Rafael; Pollán, Marina; Antón, Joaquín; Miranda-García, Josefa; Casals, María; Martínez, Inmaculada; Ruiz-Perales, Francisco; Pérez-Gómez, Beatriz; Salas-Trejo, Dolores; Pérez-Cortés, Juan-Carlos

    2014-09-01

    The task of breast density quantification is becoming increasingly relevant due to its association with breast cancer risk. In this work, a semi-automated and a fully automated tools to assess breast density from full-field digitized mammograms are presented. The first tool is based on a supervised interactive thresholding procedure for segmenting dense from fatty tissue and is used with a twofold goal: for assessing mammographic density (MD) in a more objective and accurate way than via visual-based methods and for labeling the mammograms that are later employed to train the fully automated tool. Although most automated methods rely on supervised approaches based on a global labeling of the mammogram, the proposed method relies on pixel-level labeling, allowing better tissue classification and density measurement on a continuous scale. The fully automated method presented combines a classification scheme based on local features and thresholding operations that improve the performance of the classifier. A dataset of 655 mammograms was used to test the concordance of both approaches in measuring MD. Three expert radiologists measured MD in each of the mammograms using the semi-automated tool (DM-Scan). It was then measured by the fully automated system and the correlation between both methods was computed. The relation between MD and breast cancer was then analyzed using a case-control dataset consisting of 230 mammograms. The Intraclass Correlation Coefficient (ICC) was used to compute reliability among raters and between techniques. The results obtained showed an average ICC=0.922 among raters when using the semi-automated tool, whilst the average correlation between the semi-automated and automated measures was ICC=0.838. In the case-control study, the results obtained showed Odds Ratios (OR) of 1.38 and 1.50 per 10% increase in MD when using the semi-automated and fully automated approaches respectively. It can therefore be concluded that the automated and semi-automated

  3. High Throughput Screening For Hazard and Risk of Environmental Contaminants

    EPA Science Inventory

    High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

  4. Reconfigurable microfluidic dilution for high-throughput quantitative assays.

    PubMed

    Fan, Jinzhen; Li, Baoqing; Xing, Siyuan; Pan, Tingrui

    2015-06-21

    This paper reports a reconfigurable microfluidic dilution device for high-throughput quantitative assays, which can easily produce discrete logarithmic/binary concentration profiles ranging from 1 to 100-fold dilution in parallel from a fixed sample volume (e.g., 10 μL) without any assistance of continuous fluidic pump or robotic automation. The integrated dilution generation chip consists of switchable distribution and collection channels, metering reservoirs, reaction chambers, and pressure-activatable Laplace valves. Following the sequential loading of a sample, a diluent, and a detection reagent into their individual metering chambers, the top microfluidic layer can be reconfigured to collect the metered chemicals into the reaction chambers in parallel, where detection will be conducted. To facilitate mixing and reaction in the microchambers, two acoustic microstreaming actuation mechanisms have been investigated for easy integrability and accessibility. Furthermore, the microfluidic dilution generator has been characterized by both colorimetric and fluorescent means. A further demonstration of the generic usage of the quantitative dilution chip has utilized the commonly available bicinchoninic acid (BCA) assay to analyse the protein concentrations of human tissue extracts. In brief, the microfluidic dilution generator offers a high-throughput high-efficiency quantitative analytical alternative to conventional quantitative assay platforms, by simple manipulation of a minute amount of chemicals in a compact microfluidic device with minimal equipment requirement, which can serve as a facile tool for biochemical and biological analyses in regular laboratories, point-of-care settings and low-resource environments. PMID:25994379

  5. High-throughput assays for DNA gyrase and other topoisomerases.

    PubMed

    Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

    2006-01-01

    We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

  6. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

    PubMed

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J; Sidransky, Ellen; Zheng, Wei

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  7. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

    PubMed Central

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J.; Sidransky, Ellen

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

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

    PubMed

    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

  9. Clustering of High Throughput Gene Expression Data

    PubMed Central

    Pirim, Harun; Ekşioğlu, Burak; Perkins, Andy; Yüceer, Çetin

    2012-01-01

    High throughput biological data need to be processed, analyzed, and interpreted to address problems in life sciences. Bioinformatics, computational biology, and systems biology deal with biological problems using computational methods. Clustering is one of the methods used to gain insight into biological processes, particularly at the genomics level. Clearly, clustering can be used in many areas of biological data analysis. However, this paper presents a review of the current clustering algorithms designed especially for analyzing gene expression data. It is also intended to introduce one of the main problems in bioinformatics - clustering gene expression data - to the operations research community. PMID:23144527

  10. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  11. High throughput chemical munitions treatment system

    DOEpatents

    Haroldsen, Brent L.; Stofleth, Jerome H.; Didlake, Jr., John E.; Wu, Benjamin C-P

    2011-11-01

    A new High-Throughput Explosive Destruction System is disclosed. The new system is comprised of two side-by-side detonation containment vessels each comprising first and second halves that feed into a single agent treatment vessel. Both detonation containment vessels further comprise a surrounding ventilation facility. Moreover, the detonation containment vessels are designed to separate into two half-shells, wherein one shell can be moved axially away from the fixed, second half for ease of access and loading. The vessels are closed by means of a surrounding, clam-shell type locking seal mechanisms.

  12. Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

    PubMed

    Merritt, Jason; Kuehn, Seppe

    2016-01-01

    We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation. PMID:27616752

  13. A semi-automated micro-method for the histological assessment of fat embolism.

    PubMed

    Busuttil, A; Hanley, J J

    1994-01-01

    A method of quantitatively determining the volume of fat emboli in a tissue using an image analysis system (I.B.A.S.) was developed. This procedure is an interactive, semi-automated tool allowing the quick and accurate gathering of large quantities of data from sections of different tissue samples stained by osmium tetroxide. The development of this procedure was aimed at producing a system which is reliable, reproducible and semi-automated thereby enabling epidemiological and serial studies to be made of a large number of histological sections from different tissues. The system was tested in a study of tissue sections from a series of fatalities from an aircraft crash in an attempt at correlating the incidence of fat emboli with the presence of multiple fractures and soft tissue injuries, the correlation to be made being between the quantitative presence of fat emboli and the extent and severity of injuries suffered. PMID:7529546

  14. Semi-automated continuous-flow enzyme immunoassay for antiepileptic drugs in serum.

    PubMed

    Hayashi, S; Kurooka, S; Arisue, K; Kohda, K; Hayashi, C

    1983-10-01

    We have developed a semi-automated method for measuring five kinds of antiepileptic drugs in serum by successfully adapting commercial competitive-binding enzyme immunoassay kits (MARKIT; Dainippon) for use with a continuous-flow analyzer (Technicon AutoAnalyzer II equipped with a dialyzer). The free enzyme-labeled drug is automatically separated by a microfilter from the competitive immunoreaction mixture between labeled and unlabeled drug for anti-drug immunoglobulin coupled to bacterial cell walls. The concentrations of the antiepileptic drugs in serum samples can be determined by automated measurement of enzyme activity of the enzyme-labeled drugs. Results of the semi-automated method correlated well with those obtained by manual enzyme immunoassay, gas-liquid chromatography, and "high-pressure" liquid chromatography. The correlation coefficients were all greater than 0.95, showing the practicality of this method for therapeutic monitoring of antiepileptic drugs. PMID:6352086

  15. Reducing Fuel Consumption through Semi-Automated Platooning with Class 8 Tractor Trailer Combinations (Poster)

    SciTech Connect

    Lammert, M.; Gonder, J.

    2014-07-01

    This poster describes the National Renewable Energy Laboratory's evaluation of the fuel savings potential of semi-automated truck platooning. Platooning involves reducing aerodynamic drag by grouping vehicles together and decreasing the distance between them through the use of electronic coupling, which allows multiple vehicles to accelerate or brake simultaneously. The NREL study addressed the need for data on American style line-haul sleeper cabs with modern aerodynamics and over a range of trucking speeds common in the United States.

  16. High-throughput screening of binary catalysts for oxygen electroreduction

    NASA Astrophysics Data System (ADS)

    Liu, Jing Hua; Jeon, Min Ku; Woo, Seong Ihl

    2006-01-01

    A series of Pt based and non-Pt catalysts for proton exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) have been evaluated towards oxygen reduction, by high-throughput optical screening. Fluorescein was first used as pH indicator for detecting pH change of the electrolyte in the vicinity of cathode caused by oxygen reduction. Arrays of catalyst spot comprised of binary catalysts and pure Pt were prepared by using robotic micro-dispenser. The analysis of fluorescence images has showed that some of Pt based catalysts including PtBi, PtCu, PtSe, PtTe and PtIr, as well as RuFe, as a non-Pt catalyst, exhibited higher activities and methanol tolerance than pure Pt. Moreover, acceptable stability of these catalysts at high potential in acid environment suits them to the requirements of cathode catalyst in PEMFC or DMFC.

  17. Preliminary High-Throughput Metagenome Assembly

    SciTech Connect

    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. Economic consequences of high throughput maskless lithography

    NASA Astrophysics Data System (ADS)

    Hartley, John G.; Govindaraju, Lakshmi

    2005-11-01

    Many people in the semiconductor industry bemoan the high costs of masks and view mask cost as one of the significant barriers to bringing new chip designs to market. All that is needed is a viable maskless technology and the problem will go away. Numerous sites around the world are working on maskless lithography but inevitably, the question asked is "Wouldn't a one wafer per hour maskless tool make a really good mask writer?" Of course, the answer is yes, the hesitation you hear in the answer isn't based on technology concerns, it's financial. The industry needs maskless lithography because mask costs are too high. Mask costs are too high because mask pattern generators (PG's) are slow and expensive. If mask PG's become much faster, mask costs go down, the maskless market goes away and the PG supplier is faced with an even smaller tool demand from the mask shops. Technical success becomes financial suicide - or does it? In this paper we will present the results of a model that examines some of the consequences of introducing high throughput maskless pattern generation. Specific features in the model include tool throughput for masks and wafers, market segmentation by node for masks and wafers and mask cost as an entry barrier to new chip designs. How does the availability of low cost masks and maskless tools affect the industries tool makeup and what is the ultimate potential market for high throughput maskless pattern generators?

  19. A semi-automated tool for treatment plan-quality evaluation and clinical trial quality assurance

    NASA Astrophysics Data System (ADS)

    Wang, Jiazhou; Chen, Wenzhou; Studenski, Matthew; Cui, Yunfeng; Lee, Andrew J.; Xiao, Ying

    2013-07-01

    The goal of this work is to develop a plan-quality evaluation program for clinical routine and multi-institutional clinical trials so that the overall evaluation efficiency is improved. In multi-institutional clinical trials evaluating the plan quality is a time-consuming and labor-intensive process. In this note, we present a semi-automated plan-quality evaluation program which combines MIMVista, Java/MATLAB, and extensible markup language (XML). More specifically, MIMVista is used for data visualization; Java and its powerful function library are implemented for calculating dosimetry parameters; and to improve the clarity of the index definitions, XML is applied. The accuracy and the efficiency of the program were evaluated by comparing the results of the program with the manually recorded results in two RTOG trials. A slight difference of about 0.2% in volume or 0.6 Gy in dose between the semi-automated program and manual recording was observed. According to the criteria of indices, there are minimal differences between the two methods. The evaluation time is reduced from 10-20 min to 2 min by applying the semi-automated plan-quality evaluation program.

  20. A semi-automated method of monitoring dam passage of American Eels Anguilla rostrata

    USGS Publications Warehouse

    Welsh, Stuart; Aldinger, Joni L.

    2014-01-01

    Fish passage facilities at dams have become an important focus of fishery management in riverine systems. Given the personnel and travel costs associated with physical monitoring programs, automated or semi-automated systems are an attractive alternative for monitoring fish passage facilities. We designed and tested a semi-automated system for eel ladder monitoring at Millville Dam on the lower Shenandoah River, West Virginia. A motion-activated eel ladder camera (ELC) photographed each yellow-phase American Eel Anguilla rostrata that passed through the ladder. Digital images (with date and time stamps) of American Eels allowed for total daily counts and measurements of eel TL using photogrammetric methods with digital imaging software. We compared physical counts of American Eels with camera-based counts; TLs obtained with a measuring board were compared with TLs derived from photogrammetric methods. Data from the ELC were consistent with data obtained by physical methods, thus supporting the semi-automated camera system as a viable option for monitoring American Eel passage. Time stamps on digital images allowed for the documentation of eel passage time—data that were not obtainable from physical monitoring efforts. The ELC has application to eel ladder facilities but can also be used to monitor dam passage of other taxa, such as crayfishes, lampreys, and water snakes.

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

    PubMed Central

    2010-01-01

    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. PMID:21176202

  2. High-throughput single-molecule force spectroscopy for membrane proteins

    NASA Astrophysics Data System (ADS)

    Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

    2008-09-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  3. PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors.

    PubMed

    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

  4. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  5. High-throughput cellular RNA device engineering.

    PubMed

    Townshend, Brent; Kennedy, Andrew B; Xiang, Joy S; Smolke, Christina D

    2015-10-01

    Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary-interaction RNA devices performed better in terms of gene silencing, activation ratio and ligand sensitivity than optimized RNA devices that rely on secondary-structure changes. We applied our method to build biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate the underlying sequence-structure-function relationships that empower rational design of complex biomolecules. PMID:26258292

  6. High-Throughput Cell Toxicity Assays.

    PubMed

    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

  7. High throughput assays for analyzing transcription factors.

    PubMed

    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

  8. Origin and evolution of high throughput screening

    PubMed Central

    Pereira, D A; Williams, J A

    2007-01-01

    This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100μl. A nominal 30mM source compound concentration provided high μM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for 125I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced ‘hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle. PMID:17603542

  9. High-Throughput Methods for Electron Crystallography

    PubMed Central

    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 the natural environment of a lipid membrane. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, images and diffraction can be recorded by electron microscopy. 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 and potentially complementary 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 detergent complexation by cyclodextrin; a specialized pipetting robot has been designed not only to titrate cyclodextrin, 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. PMID:23132066

  10. Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E▿ †

    PubMed Central

    Hines, Harry B.; Kim, Alexander D.; Stafford, Robert G.; Badie, Shirin S.; Brueggeman, Ernst E.; Newman, David J.; Schmidt, James J.

    2008-01-01

    The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes. PMID:18083881

  11. High-throughput microcavitation bubble induced cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    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.

  12. High Throughput Screening and Selection Methods for Directed Enzyme Evolution

    PubMed Central

    2015-01-01

    Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized. PMID:26074668

  13. Data Analysis for High-Throughput RNAi Screening.

    PubMed

    Azorsa, David O; Turnidge, Megan A; Arora, Shilpi

    2016-01-01

    High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening. PMID:27581298

  14. A semi-automated computer tool for the analysis of retinal vessel diameter dynamics.

    PubMed

    Euvrard, Guillaume; Genevois, Olivier; Rivals, Isabelle; Massin, Pascale; Collet, Amélie; Sahel, José-Alain; Paques, Michel

    2013-06-01

    Retinal vessels are directly accessible to clinical observation. This has numerous potential interests for medical investigations. Using the Retinal Vessel Analyzer, a dedicated eye fundus camera enabling dynamic, video-rate recording of micrometric changes of the diameter of retinal vessels, we developed a semi-automated computer tool that extracts the heart beat rate and pulse amplitude values from the records. The extracted data enabled us to show that there is a decreasing relationship between heart beat rate and pulse amplitude of arteries and veins. Such an approach will facilitate the modeling of hemodynamic interactions in small vessels. PMID:23566397

  15. Ultrasound measurements of carotid intima-media thickness by two semi-automated analysis systems.

    PubMed

    Ring, M; Eriksson, M J; Jogestrand, T; Caidahl, K

    2016-09-01

    Increased carotid intima-media thickness (cIMT) is associated with an increased risk of cardiac events and stroke. Several semi-automated edge-detection techniques for measuring cIMT are used for research and in clinical practice. Our aim was to compare two currently available semi-automated techniques for the measurement of cIMT. Carotid ultrasound recordings were obtained from 99 subjects (mean age 54·4 ± 8·9 years, range 33-69) without known cardiovascular diseases using a General Electric (GE) Vivid 7 ultrasound scanner, 8-MHz transducer. The far-wall cIMT was evaluated 1-2 cm proximal to the carotid bulb. Three diastolic images (ECG R-wave) from the left and three images from the right common carotid arteries were analysed using GE and Artery Measurement System (AMS) semi-automated softwares. Mean systolic and diastolic blood pressures were 120 ± 13 and 76 ± 8 mmHg, respectively. The cIMTmean (left + right)/2 by GE and cIMTmean (left + right)/2 AMS were highly correlated (r = 0·92, P<0·001). Higher values were measured by GE (0·72 ± 0·12 mm) compared with AMS (0·69 ± 0·12 mm), and this was significant (P<0·001). The coefficients of variation for the intra-observer variability of cIMTmean (left + right)/2 were 1·0% (GE) and 2·2% (AMS). cIMTmean measured by GE's semi-automated edge-detection method correlated well with that measured by AMS. However, there were small but significant systematic differences between the cIMTmean values measured by the two techniques. Thus, the use of only one type of measurement program seems favourable in follow-up studies and when evaluating treatment effects. PMID:26046377

  16. Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

    PubMed Central

    Sawetzki, Tobias; Eggleton, Charles D.; Desai, Sanjay A.; Marr, David W.M.

    2013-01-01

    The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods. PMID:24268140

  17. High-throughput analysis and protein engineering using microcapillary arrays.

    PubMed

    Chen, Bob; Lim, Sungwon; Kannan, Arvind; Alford, Spencer C; Sunden, Fanny; Herschlag, Daniel; Dimov, Ivan K; Baer, Thomas M; Cochran, Jennifer R

    2016-02-01

    We describe a multipurpose technology platform, termed μSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the μSCALE platform. PMID:26641932

  18. High throughput sample processing and automated scoring.

    PubMed

    Brunborg, Gunnar; Jackson, Petra; Shaposhnikov, Sergey; Dahl, Hildegunn; Azqueta, Amaya; Collins, Andrew R; Gutzkow, Kristine B

    2014-01-01

    The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring. PMID:25389434

  19. High throughput sample processing and automated scoring

    PubMed Central

    Brunborg, Gunnar; Jackson, Petra; Shaposhnikov, Sergey; Dahl, Hildegunn; Azqueta, Amaya; Collins, Andrew R.; Gutzkow, Kristine B.

    2014-01-01

    The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring. PMID:25389434

  20. Orthogonal NGS for High Throughput Clinical Diagnostics

    PubMed Central

    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.

    2016-01-01

    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. PMID:27090146

  1. High-throughput rod-induced electrospinning

    NASA Astrophysics Data System (ADS)

    Wu, Dezhi; Xiao, Zhiming; Teh, Kwok Siong; Han, Zhibin; Luo, Guoxi; Shi, Chuan; Sun, Daoheng; Zhao, Jinbao; Lin, Liwei

    2016-09-01

    A high throughput electrospinning process, directly from flat polymer solution surfaces induced by a moving insulating rod, has been proposed and demonstrated. Different rods made of either phenolic resin or paper with a diameter of 1–3 cm and a resistance of about 100–500 MΩ, has been successfully utilized in the process. The rod is placed approximately 10 mm above the flat polymer solution surface with a moving speed of 0.005–0.4 m s‑1 this causes the solution to generate multiple liquid jets under an applied voltage of 15–60 kV for the tip-less electrospinning process. The local electric field induced by the rod can boost electrohydrodynamic instability in order to generate Taylor cones and liquid jets. Experimentally, it is found that a large rod diameter and a small solution-to-rod distance can enhance the local electrical field to reduce the magnitude of the applied voltage. In the prototype setup with poly (ethylene oxide) polymer solution, an area of 5 cm  ×  10 cm and under an applied voltage of 60 kV, the maximum throughput of nanofibers is recorded to be approximately144 g m‑2 h‑1.

  2. High-Throughput Enzyme Kinetics Using Microarrays

    SciTech Connect

    Guoxin Lu; Edward S. Yeung

    2007-11-01

    We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)- coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K{sub m} obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner.

  3. A Novel High-Throughput Approach to Measure Hydroxyl Radicals Induced by Airborne Particulate Matter

    PubMed Central

    Son, Yeongkwon; Mishin, Vladimir; Welsh, William; Lu, Shou-En; Laskin, Jeffrey D.; Kipen, Howard; Meng, Qingyu

    2015-01-01

    Oxidative stress is one of the key mechanisms linking ambient particulate matter (PM) exposure with various adverse health effects. The oxidative potential of PM has been used to characterize the ability of PM induced oxidative stress. Hydroxyl radical (•OH) is the most destructive radical produced by PM. However, there is currently no high-throughput approach which can rapidly measure PM-induced •OH for a large number of samples with an automated system. This study evaluated four existing molecular probes (disodium terephthalate, 3′-p-(aminophenyl)fluorescein, coumarin-3-carboxylic acid, and sodium benzoate) for their applicability to measure •OH induced by PM in a high-throughput cell-free system using fluorescence techniques, based on both our experiments and on an assessment of the physicochemical properties of the probes reported in the literature. Disodium terephthalate (TPT) was the most applicable molecular probe to measure •OH induced by PM, due to its high solubility, high stability of the corresponding fluorescent product (i.e., 2-hydroxyterephthalic acid), high yield compared with the other molecular probes, and stable fluorescence intensity in a wide range of pH environments. TPT was applied in a high-throughput format to measure PM (NIST 1648a)-induced •OH, in phosphate buffered saline. The formed fluorescent product was measured at designated time points up to 2 h. The fluorescent product of TPT had a detection limit of 17.59 nM. The soluble fraction of PM contributed approximately 76.9% of the •OH induced by total PM, and the soluble metal ions of PM contributed 57.4% of the overall •OH formation. This study provides a promising cost-effective high-throughput method to measure •OH induced by PM on a routine basis. PMID:26516887

  4. Semi-automated based ground-truthing GUI for airborne imagery

    NASA Astrophysics Data System (ADS)

    Phan, Chung; Lydic, Rich; Moore, Tim; Trang, Anh; Agarwal, Sanjeev; Tiwari, Spandan

    2005-06-01

    Over the past several years, an enormous amount of airborne imagery consisting of various formats has been collected and will continue into the future to support airborne mine/minefield detection processes, improve algorithm development, and aid in imaging sensor development. The ground-truthing of imagery is a very essential part of the algorithm development process to help validate the detection performance of the sensor and improving algorithm techniques. The GUI (Graphical User Interface) called SemiTruth was developed using Matlab software incorporating signal processing, image processing, and statistics toolboxes to aid in ground-truthing imagery. The semi-automated ground-truthing GUI is made possible with the current data collection method, that is including UTM/GPS (Universal Transverse Mercator/Global Positioning System) coordinate measurements for the mine target and fiducial locations on the given minefield layout to support in identification of the targets on the raw imagery. This semi-automated ground-truthing effort has developed by the US Army RDECOM CERDEC Night Vision and Electronic Sensors Directorate (NVESD), Countermine Division, Airborne Application Branch with some support by the University of Missouri-Rolla.

  5. Validation of a New Semi-Automated Technique to Evaluate Muscle Capillarization.

    PubMed

    Ballak, Sam B; Yap, Moi H; Harding, Peter J; Degens, Hans

    2016-01-01

    The method of capillary domains has often been used to study capillarization of skeletal and heart muscle. However, the conventional data processing method using a digitizing tablet is an arduous and time-consuming task. Here we compare a new semi-automated capillary domain data collection and analysis in muscle tissue with the standard capillary domain method. The capillary density (1481±59 vs. 1447±54 caps mm(-2); R2:0.99; P<0.01) and heterogeneity of capillary spacing (0.085±0.002 vs. 0.085±0.002; R2:0.95; P<0.01) were similar in both methods. The fiber cross-sectional area correlated well between the methods (R2:0.84; P<0.01) and did not differ significantly (~8% larger in the old than new method at P=0.08). The latter was likely due to differences in outlining the contours between the two methods. In conclusion, the semi-automated method gives quantitatively and qualitatively similar data as the conventional method and saves a considerable amount of time. PMID:26782199

  6. Agile based "Semi-"Automated Data ingest process : ORNL DAAC example

    NASA Astrophysics Data System (ADS)

    Santhana Vannan, S. K.; Beaty, T.; Cook, R. B.; Devarakonda, R.; Hook, L.; Wei, Y.; Wright, D.

    2015-12-01

    The ORNL DAAC archives and publishes data and information relevant to biogeochemical, ecological, and environmental processes. The data archived at the ORNL DAAC must be well formatted, self-descriptive, and documented, as well as referenced in a peer-reviewed publication. The ORNL DAAC ingest team curates diverse data sets from multiple data providers simultaneously. To streamline the ingest process, the data set submission process at the ORNL DAAC has been recently updated to use an agile process and a semi-automated workflow system has been developed to provide a consistent data provider experience and to create a uniform data product. The goals of semi-automated agile ingest process are to: 1.Provide the ability to track a data set from acceptance to publication 2. Automate steps that can be automated to improve efficiencies and reduce redundancy 3.Update legacy ingest infrastructure 4.Provide a centralized system to manage the various aspects of ingest. This talk will cover the agile methodology, workflow, and tools developed through this system.

  7. von Willebrand Factor multimers profiling with a semi-automated system.

    PubMed

    Pasotti, Fabio; Martini, Giuliana; Caimi, Luigi; Ricotta, Doris

    2013-03-01

    Abnormalities in plasma von Willebrand factor (vWF) concentration and function result in von Willebrand disease (vWD). The diagnosis requires a battery of tests such as screening procedures, confirmatory tests, phenotypic characterization, and genotyping. The phenotypic testing (multimer pattern analysis) is important in order to subclassify the hereditary and the acquired forms of vWD. Only few laboratories are skilled to perform this analysis. The extreme range of protein size from 250 kDa monomer to over 20,000 kDa multimers requires a time-consuming procedure (3-4 days) and presents many technical difficulties. To standardize the method and to overcome technical difficulties, we developed a rapid and sensitive semi-automated method to visualize the multimeric structure of vWF. The semi-automated method we present performs the electrophoresis of patient's plasma in 120 min on a precast gel. Gels are suitable for the G26 Interlab instrumentation. After gel blotting, the method allows visualization of the vWF multimer pattern directly on the membrane. We reduced the time required from 72 to 8 h and we propose this test for the first level screening of vWF multimer deficiency. PMID:23334940

  8. High-Throughput Baculovirus Expression System for Membrane Protein Production.

    PubMed

    Kalathur, Ravi C; Panganiban, Marinela; Bruni, Renato

    2016-01-01

    The ease of use, robustness, cost-effectiveness, and posttranslational machinery make baculovirus expression system a popular choice for production of eukaryotic membrane proteins. This system can be readily adapted for high-throughput operations. This chapter outlines the techniques and procedures for cloning, transfection, small-scale production, and purification of membrane protein samples in a high-throughput manner. PMID:27485337

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

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    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

  11. High-throughput measurements of the optical redox ratio using a commercial microplate reader

    NASA Astrophysics Data System (ADS)

    Cannon, Taylor M.; Shah, Amy T.; Walsh, Alex J.; Skala, Melissa C.

    2015-01-01

    There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p<0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p<0.05) and lack of response (p>0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

  12. Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles.

    PubMed

    Ewing, Heather; Fernández-Vega, Virneliz; Spicer, Timothy P; Chase, Peter; Brown, Steven; Scampavia, Louis; Roush, William R; Riley, Sean; Rosen, Hugh; Hodder, Peter; Lambeau, Gerard; Gelb, Michael H

    2016-08-01

    There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. PMID:27146384

  13. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

    Scanlon, Thomas C.; Dostal, Sarah M.; Griswold, Karl E.

    2014-01-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  14. High-throughput microfluidic line scan imaging for cytological characterization

    NASA Astrophysics Data System (ADS)

    Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

    2015-03-01

    Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

  15. GiA Roots: software for the high throughput analysis of plant root system architecture

    PubMed Central

    2012-01-01

    Background Characterizing root system architecture (RSA) is essential to understanding the development and function of vascular plants. Identifying RSA-associated genes also represents an underexplored opportunity for crop improvement. Software tools are needed to accelerate the pace at which quantitative traits of RSA are estimated from images of root networks. Results We have developed GiA Roots (General Image Analysis of Roots), a semi-automated software tool designed specifically for the high-throughput analysis of root system images. GiA Roots includes user-assisted algorithms to distinguish root from background and a fully automated pipeline that extracts dozens of root system phenotypes. Quantitative information on each phenotype, along with intermediate steps for full reproducibility, is returned to the end-user for downstream analysis. GiA Roots has a GUI front end and a command-line interface for interweaving the software into large-scale workflows. GiA Roots can also be extended to estimate novel phenotypes specified by the end-user. Conclusions We demonstrate the use of GiA Roots on a set of 2393 images of rice roots representing 12 genotypes from the species Oryza sativa. We validate trait measurements against prior analyses of this image set that demonstrated that RSA traits are likely heritable and associated with genotypic differences. Moreover, we demonstrate that GiA Roots is extensible and an end-user can add functionality so that GiA Roots can estimate novel RSA traits. In summary, we show that the software can function as an efficient tool as part of a workflow to move from large numbers of root images to downstream analysis. PMID:22834569

  16. Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.

    PubMed

    Morado, Dustin R; Hu, Bo; Liu, Jun

    2016-01-01

    Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET. PMID:26863591

  17. Semi-automated identification of cones in the human retina using circle Hough transform

    PubMed Central

    Bukowska, Danuta M.; Chew, Avenell L.; Huynh, Emily; Kashani, Irwin; Wan, Sue Ling; Wan, Pak Ming; Chen, Fred K

    2015-01-01

    A large number of human retinal diseases are characterized by a progressive loss of cones, the photoreceptors critical for visual acuity and color perception. Adaptive Optics (AO) imaging presents a potential method to study these cells in vivo. However, AO imaging in ophthalmology is a relatively new phenomenon and quantitative analysis of these images remains difficult and tedious using manual methods. This paper illustrates a novel semi-automated quantitative technique enabling registration of AO images to macular landmarks, cone counting and its radius quantification at specified distances from the foveal center. The new cone counting approach employs the circle Hough transform (cHT) and is compared to automated counting methods, as well as arbitrated manual cone identification. We explore the impact of varying the circle detection parameter on the validity of cHT cone counting and discuss the potential role of using this algorithm in detecting both cones and rods separately. PMID:26713186

  18. Semi-automated identification of cones in the human retina using circle Hough transform.

    PubMed

    Bukowska, Danuta M; Chew, Avenell L; Huynh, Emily; Kashani, Irwin; Wan, Sue Ling; Wan, Pak Ming; Chen, Fred K

    2015-12-01

    A large number of human retinal diseases are characterized by a progressive loss of cones, the photoreceptors critical for visual acuity and color perception. Adaptive Optics (AO) imaging presents a potential method to study these cells in vivo. However, AO imaging in ophthalmology is a relatively new phenomenon and quantitative analysis of these images remains difficult and tedious using manual methods. This paper illustrates a novel semi-automated quantitative technique enabling registration of AO images to macular landmarks, cone counting and its radius quantification at specified distances from the foveal center. The new cone counting approach employs the circle Hough transform (cHT) and is compared to automated counting methods, as well as arbitrated manual cone identification. We explore the impact of varying the circle detection parameter on the validity of cHT cone counting and discuss the potential role of using this algorithm in detecting both cones and rods separately. PMID:26713186

  19. Semantic enrichment of medical forms - semi-automated coding of ODM-elements via web services.

    PubMed

    Breil, Bernhard; Watermann, Andreas; Haas, Peter; Dziuballe, Philipp; Dugas, Martin

    2012-01-01

    Semantic interoperability is an unsolved problem which occurs while working with medical forms from different information systems or institutions. Standards like ODM or CDA assure structural homogenization but in order to compare elements from different data models it is necessary to use semantic concepts and codes on an item level of those structures. We developed and implemented a web-based tool which enables a domain expert to perform semi-automated coding of ODM-files. For each item it is possible to inquire web services which result in unique concept codes without leaving the context of the document. Although it was not feasible to perform a totally automated coding we have implemented a dialog based method to perform an efficient coding of all data elements in the context of the whole document. The proportion of codable items was comparable to results from previous studies. PMID:22874367

  20. Development of a semi-automated workcell for repair of printed circuit boards

    SciTech Connect

    Bennett, D.W.; Evans, M.S.

    1990-08-01

    Printed circuit boards that comprise US Army electronic systems are repaired at Army depots. An existing automated diagnostic system determines the area of failure; either by identifying failed components or failed board traces. Currently, repairs are performed manually by trained technicians. A system is being developed for repair of through-hole printed circuit boards. It is comprised of many automated and operator-assisted functions to perform the multiple operations related to replacement of failed components. When completed, this system will demonstrate economic payback by reducing skilled labor requirements and decreasing rework. The semi-automated system integrates human operators into the process while maintaining high productivity. After several fully automated systems were conceived and modelled, it was found that the configuration that provided the best return on investment was comprised of a mix of autonomous and operator-assisted functions. 1 ref., 1 fig.

  1. Semi-Automated Atlas-based Analysis of Brain Histological Sections

    PubMed Central

    Kopec, Charles D.; Bowers, Amanda C.; Pai, Shraddha; Brody, Carlos D.

    2011-01-01

    Quantifying the location and/or number of features in a histological section of the brain currently requires one to first, manually register a corresponding section from a tissue atlas onto the experimental section and second, count the features. No automated method exists for the first process (registering), and most automated methods for the second process (feature counting) operate reliably only in a high signal-to-noise regime. To reduce experimenter bias and inconsistencies and increase the speed of these analyses, we developed Atlas Fitter, a semi-automated, open-source MatLab-based software package that assists in rapidly registering atlas panels onto histological sections. We also developed CellCounter, a novel fully-automated cell counting algorithm that is designed to operate on images with non-uniform background intensities and low signal-to-noise ratios. PMID:21194546

  2. Improving Perceived and Actual Text Difficulty for Health Information Consumers using Semi-Automated Methods

    PubMed Central

    Leroy, Gondy; Endicott, James E.; Mouradi, Obay; Kauchak, David; Just, Melissa L.

    2012-01-01

    We are developing algorithms for semi-automated simplification of medical text. Based on lexical and grammatical corpus analysis, we identified a new metric, term familiarity, to help estimate text difficulty. We developed an algorithm that uses term familiarity to identify difficult text and select easier alternatives from lexical resources such as WordNet, UMLS and Wiktionary. Twelve sentences were simplified to measure perceived difficulty using a 5-point Likert scale. Two documents were simplified to measure actual difficulty by posing questions with and without the text present (information understanding and retention). We conducted a user study by inviting participants (N=84) via Amazon Mechanical Turk. There was a significant effect of simplification on perceived difficulty (p<.001). We also saw slightly improved understanding with better question-answering for simplified documents but the effect was not significant (p=.097). Our results show how term familiarity is a valuable component in simplifying text in an efficient and scalable manner. PMID:23304324

  3. SCOUSE: Semi-automated multi-COmponent Universal Spectral-line fitting Engine

    NASA Astrophysics Data System (ADS)

    Henshaw, J. D.; Longmore, S. N.; Kruijssen, J. M. D.; Davies, B.; Bally, J.; Barnes, A.; Battersby, C.; Burton, M.; Cunningham, M. R.; Dale, J. E.; Ginsburg, A.; Immer, K.; Jones, P. A.; Kendrew, S.; Mills, E. A. C.; Molinari, S.; Moore, T. J. T.; Ott, J.; Pillai, T.; Rathborne, J.; Schilke, P.; Schmiedeke, A.; Testi, L.; Walker, D.; Walsh, A.; Zhang, Q.

    2016-01-01

    The Semi-automated multi-COmponent Universal Spectral-line fitting Engine (SCOUSE) is a spectral line fitting algorithm that fits Gaussian files to spectral line emission. It identifies the spatial area over which to fit the data and generates a grid of spectral averaging areas (SAAs). The spatially averaged spectra are fitted according to user-provided tolerance levels, and the best fit is selected using the Akaike Information Criterion, which weights the chisq of a best-fitting solution according to the number of free-parameters. A more detailed inspection of the spectra can be performed to improve the fit through an iterative process, after which SCOUSE integrates the new solutions into the solution file.

  4. Using tomoauto – a protocol for high-throughput automated cryo-electron tomography

    PubMed Central

    Morado, Dustin R.; Hu, Bo; Liu, Jun

    2016-01-01

    We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions. Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III

  5. Performance evaluation of the PROCLEIX West Nile virus assay on semi-automated and automated systems.

    PubMed

    Linnen, Jeffrey M; Deras, Michael L; Cline, Janice; Wu, Wen; Broulik, Amy S; Cory, Robin E; Knight, James L; Cass, Michelle M J; Collins, Cynthia S; Giachetti, Cristina

    2007-09-01

    The PROCLEIX West Nile virus assay (WNV assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA). The assay was used under an investigational protocol in the United States to screen blood donations for West Nile virus (WNV) RNA starting in the summer of 2003, and was licensed by the FDA in December 2005 for use on the PROCLEIX System, also known as the enhanced semi-automated system (eSAS). Performance characteristics for the assay were determined on both eSAS and the fully automated PROCLEIX TIGRIS (TIGRIS) System. Detection of both lineage 1 and lineage 2 WNV was demonstrated on both systems. For lineage 1, the 95% detection limit was 8.2 copies/ml for eSAS and 9.8 copies/ml for the TIGRIS system. For lineage 2, > or =95% detection was seen at > or =30 copies/ml on both systems. The overall specificity of the assay was >99.9% in fresh and frozen plasma specimens. Reproducibility studies on the TIGRIS system yielded > or =99.1% agreement with expected results for the 3-member panel tested (0, 30, and 100 copies/ml). The WNV assay exhibited robust performance in cadaveric specimens and specimens representing various donor and donation conditions, including specimens from different plasma collection tubes that were subjected to multiple freeze/thaw cycles; specimens with elevated levels of endogenous substances; specimens containing other viruses and microorganisms; and specimens from patients with autoimmune and other diseases. Overall, these studies demonstrate high sensitivity, specificity, and reproducibility of the WNV assay on both the semi-automated and automated systems. PMID:17607786

  6. An Interactive Tool For Semi-automated Statistical Prediction Using Earth Observations and Models

    NASA Astrophysics Data System (ADS)

    Zaitchik, B. F.; Berhane, F.; Tadesse, T.

    2015-12-01

    We developed a semi-automated statistical prediction tool applicable to concurrent analysis or seasonal prediction of any time series variable in any geographic location. The tool was developed using Shiny, JavaScript, HTML and CSS. A user can extract a predictand by drawing a polygon over a region of interest on the provided user interface (global map). The user can select the Climatic Research Unit (CRU) precipitation or Climate Hazards Group InfraRed Precipitation with Station data (CHIRPS) as predictand. They can also upload their own predictand time series. Predictors can be extracted from sea surface temperature, sea level pressure, winds at different pressure levels, air temperature at various pressure levels, and geopotential height at different pressure levels. By default, reanalysis fields are applied as predictors, but the user can also upload their own predictors, including a wide range of compatible satellite-derived datasets. The package generates correlations of the variables selected with the predictand. The user also has the option to generate composites of the variables based on the predictand. Next, the user can extract predictors by drawing polygons over the regions that show strong correlations (composites). Then, the user can select some or all of the statistical prediction models provided. Provided models include Linear Regression models (GLM, SGLM), Tree-based models (bagging, random forest, boosting), Artificial Neural Network, and other non-linear models such as Generalized Additive Model (GAM) and Multivariate Adaptive Regression Splines (MARS). Finally, the user can download the analysis steps they used, such as the region they selected, the time period they specified, the predictand and predictors they chose and preprocessing options they used, and the model results in PDF or HTML format. Key words: Semi-automated prediction, Shiny, R, GLM, ANN, RF, GAM, MARS

  7. Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator.

    PubMed

    Williams, Diana L; Adams, Linda B; Lahiri, Ramanuj

    2014-10-01

    Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue. PMID:25019518

  8. High-Throughput Pharmacokinetics for Environmental Chemicals (SOT)

    EPA Science Inventory

    High throughput screening (HTS) promises to allow prioritization of thousands of environmental chemicals with little or no in vivo information. For bioactivity identified by HTS, toxicokinetic (TK) models are essential to predict exposure thresholds below which no significant bio...

  9. Evaluating Rapid Models for High-Throughput Exposure Forecasting (SOT)

    EPA Science Inventory

    High throughput exposure screening models can provide quantitative predictions for thousands of chemicals; however these predictions must be systematically evaluated for predictive ability. Without the capability to make quantitative, albeit uncertain, forecasts of exposure, the ...

  10. HIGH THROUGHPUT ASSESSMENTS OF CONVENTIONAL AND ALTERNATIVE COMPOUNDS

    EPA Science Inventory

    High throughput approaches for quantifying chemical hazard, exposure, and sustainability have the potential to dramatically impact the pace and nature of risk assessments. Integrated evaluation strategies developed at the US EPA incorporate inherency,bioactivity,bioavailability, ...

  11. MIPHENO: Data normalization for high throughput metabolic analysis.

    EPA Science Inventory

    High throughput methodologies such as microarrays, mass spectrometry and plate-based small molecule screens are increasingly used to facilitate discoveries from gene function to drug candidate identification. These large-scale experiments are typically carried out over the course...

  12. High throughput growth and characterization of thin film materials

    NASA Astrophysics Data System (ADS)

    Mao, Samuel S.

    2013-09-01

    It usually takes more than 10 years for a new material from initial research to its first commercial application. Therefore, accelerating the pace of discovery of new materials is critical to tackling challenges in areas ranging from clean energy to national security. As discovery of new materials has not kept pace with the product design cycles in many sectors of industry, there is a pressing need to develop and utilize high throughput screening and discovery technologies for the growth and characterization of new materials. This article presents two distinctive types of high throughput thin film material growth approaches, along with a number of high throughput characterization techniques, established in the author's group. These approaches include a second-generation "discrete" combinatorial semiconductor discovery technology that enables the creation of arrays of individually separated thin film semiconductor materials of different compositions, and a "continuous" high throughput thin film material screening technology that enables the realization of ternary alloy libraries with continuously varying elemental ratios.

  13. High-throughput detection method for influenza virus.

    PubMed

    Kumar, Pawan; Bartoszek, Allison E; Moran, Thomas M; Gorski, Jack; Bhattacharyya, Sanjib; Navidad, Jose F; Thakar, Monica S; Malarkannan, Subramaniam

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture (1,2). Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus (3) to test the efficacy of this technique using MDCK cells (4). MDCK cells (10(4) or 5 x 10(3) per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 (5) and hemagglutinin (1) are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 10(2)-10(5) PFU could be consistently observed. Minimal but detectable positivity consistently seen with 10(2)-10(3) PFU PR8 viral titers demonstrated the high

  14. High-throughput Detection Method for Influenza Virus

    PubMed Central

    Kumar, Pawan; Bartoszek, Allison E.; Moran, Thomas M.; Gorski, Jack; Bhattacharyya, Sanjib; Navidad, Jose F.; Thakar, Monica S.; Malarkannan, Subramaniam

    2012-01-01

    Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2. Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near

  15. High-throughput sequencing of cytosine methylation in plant DNA

    PubMed Central

    2013-01-01

    Cytosine methylation is a significant and widespread regulatory factor in plant systems. Methods for the high-throughput sequencing of methylation have allowed a greatly improved characterisation of the methylome. Here we discuss currently available methods for generation and analysis of high-throughput sequencing of methylation data. We also discuss the results previously acquired through sequencing plant methylomes, and highlight remaining challenges in this field. PMID:23758782

  16. Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

    SciTech Connect

    Pinzon, NM; Aukema, KG; Gralnick, JA; Wackett, LP

    2011-06-28

    A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput

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

    PubMed Central

    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

  18. Towards Chip Scale Liquid Chromatography and High Throughput Immunosensing

    SciTech Connect

    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

  19. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.

    PubMed

    Binder, Stephan; Schendzielorz, Georg; Stäbler, Norma; Krumbach, Karin; Hoffmann, Kristina; Bott, Michael; Eggeling, Lothar

    2012-01-01

    We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. PMID:22640862

  20. A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level

    PubMed Central

    2012-01-01

    We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. PMID:22640862

  1. Quantifying cellular interaction dynamics in 3-D fluorescence microscopy data

    PubMed Central

    Klauschen, Frederick; Ishii, Masaru; Qi, Hai; Bajénoff, Marc; Egen, Jackson G.; Germain, Ronald N.; Meier-Schellersheim, Martin

    2012-01-01

    The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3-D fluorescence microscopy images. As example application we present the analysis of drug-induced and S1P1-knock-out-related changes in bone-osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data about T-B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets. PMID:19696749

  2. Semi-automated DIRSIG scene modeling from three-dimensional lidar and passive imagery

    NASA Astrophysics Data System (ADS)

    Lach, Stephen R.

    The Digital Imaging and Remote Sensing Image Generation (DIRSIG) model is an established, first-principles based scene simulation tool that produces synthetic multispectral and hyperspectral images from the visible to long wave infrared (0.4 to 20 microns). Over the last few years, significant enhancements such as spectral polarimetric and active Light Detection and Ranging (lidar) models have also been incorporated into the software, providing an extremely powerful tool for multi-sensor algorithm testing and sensor evaluation. However, the extensive time required to create large-scale scenes has limited DIRSIG's ability to generate scenes "on demand." To date, scene generation has been a laborious, time-intensive process, as the terrain model, CAD objects and background maps have to be created and attributed manually. To shorten the time required for this process, this research developed an approach to reduce the man-in-the-loop requirements for several aspects of synthetic scene construction. Through a fusion of 3D lidar data with passive imagery, we were able to semi-automate several of the required tasks in the DIRSIG scene creation process. Additionally, many of the remaining tasks realized a shortened implementation time through this application of multi-modal imagery. Lidar data is exploited to identify ground and object features as well as to define initial tree location and building parameter estimates. These estimates are then refined by analyzing high-resolution frame array imagery using the concepts of projective geometry in lieu of the more common Euclidean approach found in most traditional photogrammetric references. Spectral imagery is also used to assign material characteristics to the modeled geometric objects. This is achieved through a modified atmospheric compensation applied to raw hyperspectral imagery. These techniques have been successfully applied to imagery collected over the RIT campus and the greater Rochester area. The data used

  3. High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.

    PubMed

    Green, Jon M; Metz, Jeremy; Lee, Okhyun; Trznadel, Maciej; Takesono, Aya; Brown, A Ross; Owen, Stewart F; Kudoh, Tetsuhiro; Tyler, Charles R

    2016-06-21

    Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates. PMID:27227508

  4. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    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

  5. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

    PubMed Central

    Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  6. A Protocol for a High-Throughput Multiplex Cell Viability Assay.

    PubMed

    Gilbert, Daniel F; Boutros, Michael

    2016-01-01

    High-throughput cell viability assays are broadly used in RNAi and small molecule screening experiments to identify compounds that selectively kill cancer cells or as counter screens to exclude the compounds that have a generic effect on cell growth. While there are several assaying techniques available, cellular fitness is often assessed on the basis of one single and often rather indirect physiological indicator. This can lead to inconsistencies and poor correspondence between cell viability screening experiments, conducted under comparable conditions but with different viability indicators. Multiplexing, i.e., the combination of different individual assaying techniques in one experiment and subsequent comparative analysis of multiparametric data can decrease inter-assay variability and increase dataset concordance. Here, we describe a protocol for a multiplexing approach for high-throughput cell viability screening to address the issues encountered in the classical strategy using a single fitness indicator described above. The method combines a biochemical, luminescence-based approach and two fluorescence-based assay types. The biochemical method assesses cellular fitness by quantifying intracellular ATP concentration. Calcein labeling reflects cell fitness through membrane integrity and indirect measurement of ATP-dependent enzymatic esterase activity. Hoechst DNA stain correlates cell fitness with cellular DNA content. The presented multiplexing approach is suitable for low, medium and high-throughput screening and has the potential to decrease inter-assay variability and increase dataset concordance as well as reproducibility of experimental results. PMID:27581285

  7. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway. PMID:25331704

  8. A high throughput array microscope for the mechanical characterization of biomaterials

    NASA Astrophysics Data System (ADS)

    Cribb, Jeremy; Osborne, Lukas D.; Hsiao, Joe Ping-Lin; Vicci, Leandra; Meshram, Alok; O'Brien, E. Tim; Spero, Richard Chasen; Taylor, Russell; Superfine, Richard

    2015-02-01

    In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

  9. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

    PubMed

    Stowe, Cassandra; Pizzey, Arnold; Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  10. A high throughput array microscope for the mechanical characterization of biomaterials

    PubMed Central

    Cribb, Jeremy; Osborne, Lukas D.; Hsiao, Joe Ping-Lin; Vicci, Leandra; Meshram, Alok; O’Brien, E. Tim; Spero, Richard Chasen; Taylor, Russell; Superfine, Richard

    2015-01-01

    In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression. PMID:25725856

  11. A Semi-Automated Single Day Image Differencing Technique to Identify Animals in Aerial Imagery

    PubMed Central

    Terletzky, Pat; Ramsey, Robert Douglas

    2014-01-01

    Our research presents a proof-of-concept that explores a new and innovative method to identify large animals in aerial imagery with single day image differencing. We acquired two aerial images of eight fenced pastures and conducted a principal component analysis of each image. We then subtracted the first principal component of the two pasture images followed by heuristic thresholding to generate polygons. The number of polygons represented the number of potential cattle (Bos taurus) and horses (Equus caballus) in the pasture. The process was considered semi-automated because we were not able to automate the identification of spatial or spectral thresholding values. Imagery was acquired concurrently with ground counts of animal numbers. Across the eight pastures, 82% of the animals were correctly identified, mean percent commission was 53%, and mean percent omission was 18%. The high commission error was due to small mis-alignments generated from image-to-image registration, misidentified shadows, and grouping behavior of animals. The high probability of correctly identifying animals suggests short time interval image differencing could provide a new technique to enumerate wild ungulates occupying grassland ecosystems, especially in isolated or difficult to access areas. To our knowledge, this was the first attempt to use standard change detection techniques to identify and enumerate large ungulates. PMID:24454827

  12. Semi-Automated Cloud/shadow Removal and Land Cover Change Detection Using Satellite Imagery

    NASA Astrophysics Data System (ADS)

    Sah, A. K.; Sah, B. P.; Honji, K.; Kubo, N.; Senthil, S.

    2012-08-01

    Multi-platform/sensor and multi-temporal satellite data facilitates analysis of successive change/monitoring over the longer period and there by forest biomass helping REDD mechanism. The historical archive satellite imagery, specifically Landsat, can play an important role for historical trend analysis of forest cover change at national level. Whereas the fresh high resolution satellite, such as ALOS, imagery can be used for detailed analysis of present forest cover status. ALOS satellite imagery is most suitable as it offers data with optical (AVNIR-2) as well as SAR (PALSAR) sensors. AVNIR-2 providing data in multispectral modes play due role in extracting forest information. In this study, a semi-automated approach has been devised for cloud/shadow and haze removal and land cover change detection. Cloud/shadow pixels are replaced by free pixels of same image with the help of PALSAR image. The tracking of pixel based land cover change for the 1995-2009 period in combination of Landsat and latest ALOS data from its AVNIR-2 for the tropical rain forest area has been carried out using Decision Tree Classifiers followed by un-supervised classification. As threshold for tree classifier, criteria of NDVI refined by reflectance value has been employed. The result shows all pixels have been successfully registered to the pre-defined 6 categories; in accordance with IPCC definition; of land cover types with an overall accuracy 80 percent.

  13. Automated and semi-automated field testing of night vision goggles

    NASA Astrophysics Data System (ADS)

    Scopatz, Stephen; Paszkeicz, Dominic; Langsdorf, Brent

    2016-05-01

    This paper will discuss the development and results of a new field portable test set for Gen 2 and Gen 3 night vision goggles that automates many of the tests supported by currently available NVG test products. The major innovation is the use of MTF testing with a knife edge target. MTF testing is established in the laboratory environment and well suited to replace the operator's interpretation of the USAF 1951 resolution chart. Results will be presented to show the more consistent performance of the MTF approach as compared to the known operator variations when humans determine resolution. Other standard tests are semi-automated and/or video-assisted, such as infinity focus, spot defects, and distortion. The presentation will show repeatability across test units and operators on the key tests. The presentation will include automatically generated examples of the report files for each test run on each goggle. All of these capabilities are provided in a package that matches the form factor of other products in use to test NVG's. A discussion of the user interface and the ease of use of the system will be included as well as the improvement in the test time for each goggle type.

  14. SwarmPS: rapid, semi-automated single particle selection software.

    PubMed

    Woolford, David; Ericksson, Geoffery; Rothnagel, Rosalba; Muller, David; Landsberg, Michael J; Pantelic, Radosav S; McDowall, Alasdair; Pailthorpe, Bernard; Young, Paul R; Hankamer, Ben; Banks, Jasmine

    2007-01-01

    Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites. PMID:16774837

  15. CLustre: semi-automated lineament clustering for palaeo-glacial reconstruction

    NASA Astrophysics Data System (ADS)

    Smith, Mike; Anders, Niels; Keesstra, Saskia

    2016-04-01

    Palaeo glacial reconstructions, or "inversions", using evidence from the palimpsest landscape are increasingly being undertaken with larger and larger databases. Predominant in landform evidence is the lineament (or drumlin) where the biggest datasets number in excess of 50,000 individual forms. One stage in the inversion process requires the identification of lineaments that are generically similar and then their subsequent interpretation in to a coherent chronology of events. Here we present CLustre, a semi-authomated algorithm that clusters lineaments using a locally adaptive, region growing, method. This is initially tested using 1,500 model runs on a synthetic dataset, before application to two case studies (where manual clustering has been undertaken by independent researchers): (1) Dubawnt Lake, Canada and (2) Victoria island, Canada. Results using the synthetic data show that classifications are robust in most scenarios, although specific cases of cross-cutting lineaments may lead to incorrect clusters. Application to the case studies showed a very good match to existing published work, with differences related to limited numbers of unclassified lineaments and parallel cross-cutting lineaments. The value in CLustre comes from the semi-automated, objective, application of a classification method that is repeatable. Once classified, summary statistics of lineament groups can be calculated and then used in the inversion.

  16. Practical Implementation of Semi-Automated As-Built Bim Creation for Complex Indoor Environments

    NASA Astrophysics Data System (ADS)

    Yoon, S.; Jung, J.; Heo, J.

    2015-05-01

    In recent days, for efficient management and operation of existing buildings, the importance of as-built BIM is emphasized in AEC/FM domain. However, fully automated as-built BIM creation is a tough issue since newly-constructed buildings are becoming more complex. To manage this problem, our research group has developed a semi-automated approach, focusing on productive 3D as-built BIM creation for complex indoor environments. In order to test its feasibility for a variety of complex indoor environments, we applied the developed approach to model the `Charlotte stairs' in Lotte World Mall, Korea. The approach includes 4 main phases: data acquisition, data pre-processing, geometric drawing, and as-built BIM creation. In the data acquisition phase, due to its complex structure, we moved the scanner location several times to obtain the entire point clouds of the test site. After which, data pre-processing phase entailing point-cloud registration, noise removal, and coordinate transformation was followed. The 3D geometric drawing was created using the RANSAC-based plane detection and boundary tracing methods. Finally, in order to create a semantically-rich BIM, the geometric drawing was imported into the commercial BIM software. The final as-built BIM confirmed that the feasibility of the proposed approach in the complex indoor environment.

  17. Semi-Automated Detection of Surface Degradation on Bridges Based on a Level Set Method

    NASA Astrophysics Data System (ADS)

    Masiero, A.; Guarnieri, A.; Pirotti, F.; Vettore, A.

    2015-08-01

    Due to the effect of climate factors, natural phenomena and human usage, buildings and infrastructures are subject of progressive degradation. The deterioration of these structures has to be monitored in order to avoid hazards for human beings and for the natural environment in their neighborhood. Hence, on the one hand, monitoring such infrastructures is of primarily importance. On the other hand, unfortunately, nowadays this monitoring effort is mostly done by expert and skilled personnel, which follow the overall data acquisition, analysis and result reporting process, making the whole monitoring procedure quite expensive for the public (and private, as well) agencies. This paper proposes the use of a partially user-assisted procedure in order to reduce the monitoring cost and to make the obtained result less subjective as well. The developed method relies on the use of images acquired with standard cameras by even inexperienced personnel. The deterioration on the infrastructure surface is detected by image segmentation based on a level sets method. The results of the semi-automated analysis procedure are remapped on a 3D model of the infrastructure obtained by means of a terrestrial laser scanning acquisition. The proposed method has been successfully tested on a portion of a road bridge in Perarolo di Cadore (BL), Italy.

  18. a Semi-Automated Point Cloud Processing Methodology for 3d Cultural Heritage Documentation

    NASA Astrophysics Data System (ADS)

    Kıvılcım, C. Ö.; Duran, Z.

    2016-06-01

    The preliminary phase in any architectural heritage project is to obtain metric measurements and documentation of the building and its individual elements. On the other hand, conventional measurement techniques require tremendous resources and lengthy project completion times for architectural surveys and 3D model production. Over the past two decades, the widespread use of laser scanning and digital photogrammetry have significantly altered the heritage documentation process. Furthermore, advances in these technologies have enabled robust data collection and reduced user workload for generating various levels of products, from single buildings to expansive cityscapes. More recently, the use of procedural modelling methods and BIM relevant applications for historic building documentation purposes has become an active area of research, however fully automated systems in cultural heritage documentation still remains open. In this paper, we present a semi-automated methodology, for 3D façade modelling of cultural heritage assets based on parametric and procedural modelling techniques and using airborne and terrestrial laser scanning data. We present the contribution of our methodology, which we implemented in an open source software environment using the example project of a 16th century early classical era Ottoman structure, Sinan the Architect's Şehzade Mosque in Istanbul, Turkey.

  19. Semi-automated query construction for content-based endomicroscopy video retrieval.

    PubMed

    Tafreshi, Marzieh Kohandani; Linard, Nicolas; André, Barbara; Ayache, Nicholas; Vercauteren, Tom

    2014-01-01

    Content-based video retrieval has shown promising results to help physicians in their interpretation of medical videos in general and endomicroscopic ones in particular. Defining a relevant query for CBVR can however be a complex and time-consuming task for non-expert and even expert users. Indeed, uncut endomicroscopy videos may very well contain images corresponding to a variety of different tissue types. Using such uncut videos as queries may lead to drastic performance degradations for the system. In this study, we propose a semi-automated methodology that allows the physician to create meaningful and relevant queries in a simple and efficient manner. We believe that this will lead to more reproducible and more consistent results. The validation of our method is divided into two approaches. The first one is an indirect validation based on per video classification results with histopathological ground-truth. The second one is more direct and relies on perceived inter-video visual similarity ground-truth. We demonstrate that our proposed method significantly outperforms the approach with uncut videos and approaches the performance of a tedious manual query construction by an expert. Finally, we show that the similarity perceived between videos by experts is significantly correlated with the inter-video similarity distance computed by our retrieval system. PMID:25333105

  20. The Israel DNA database--the establishment of a rapid, semi-automated analysis system.

    PubMed

    Zamir, Ashira; Dell'Ariccia-Carmon, Aviva; Zaken, Neomi; Oz, Carla

    2012-03-01

    The Israel Police DNA database, also known as IPDIS (Israel Police DNA Index System), has been operating since February 2007. During that time more than 135,000 reference samples have been uploaded and more than 2000 hits reported. We have developed an effective semi-automated system that includes two automated punchers, three liquid handler robots and four genetic analyzers. An inhouse LIMS program enables full tracking of every sample through the entire process of registration, pre-PCR handling, analysis of profiles, uploading to the database, hit reports and ultimately storage. The LIMS is also responsible for the future tracking of samples and their profiles to be expunged from the database according to the Israeli DNA legislation. The database is administered by an in-house developed software program, where reference and evidentiary profiles are uploaded, stored, searched and matched. The DNA database has proven to be an effective investigative tool which has gained the confidence of the Israeli public and on which the Israel National Police force has grown to rely. PMID:21727053

  1. Optimizing Drosophila Olfactory Learning with a Semi-automated Training Device

    PubMed Central

    Murakami, Satoshi; Dan, Chuntao; Zagaeski, Brendan; Maeyama, Yuko; Kunes, Sam; Tabata, Tetsuya

    2010-01-01

    Drosophila olfactory aversive conditioning has served as a powerful model system with which to elucidate the molecular and neuronal mechanisms underlying memory formation. In the typical protocol, flies are exposed to a constant odor stream while receiving a pulsed electric shock in the conditioning tube of a manual apparatus. We have devised a simple, low-cost semi-automated conditioning apparatus that computationally controls the delivery of odor and shock. A semiconductor-based odor sensor is employed to monitor the change of odor concentration in the training tube. The system thus allows electric shocks to be precisely matched with odor concentration in the training tube. We found that short-term memory performance was improved with a pulsed odor flow protocol, in which odor is presented in short pulses, each paired with electric shock, rather than as a constant flow. The effect of pulsed odor flow might be ascribed to the phenomenon of ‘conditioned approach’, where approach toward an odor is induced when the electric shock is presented before odor pulse ends. Our data shows that the system is applicable to the study of olfactory memory formation and to the examination of conditioning parameters at a level of detail not practical with a manual apparatus. PMID:20153774

  2. Advances in High-Throughput Single-Cell Microtechnologies

    PubMed Central

    Weaver, Westbrook M.; Tseng, Peter; Kunze, Anja; Masaeli, Mahdohkht; Chung, Aram J.; Dudani, Jaideep S.; Kittur, Harsha; Kulkarni, Rajan P.; Di Carlo, Dino

    2013-01-01

    Micro-scale biological tools that have allowed probing of individual cells - from the genetic, to proteomic, to phenotypic level - have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics. PMID:24484889

  3. Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

    PubMed Central

    2014-01-01

    Background Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex. Results In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay. Conclusion Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system. PMID:25126113

  4. Perspective: Data infrastructure for high throughput materials discovery

    NASA Astrophysics Data System (ADS)

    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.

  5. Implementation of high throughput experimentation techniques for kinetic reaction testing.

    PubMed

    Nagy, Anton J

    2012-02-01

    Successful implementation of High throughput Experimentation (EE) tools has resulted in their increased acceptance as essential tools in chemical, petrochemical and polymer R&D laboratories. This article provides a number of concrete examples of EE systems, which have been designed and successfully implemented in studies, which focus on deriving reaction kinetic data. The implementation of high throughput EE tools for performing kinetic studies of both catalytic and non-catalytic systems results in a significantly faster acquisition of high-quality kinetic modeling data, required to quantitatively predict the behavior of complex, multistep reactions. PMID:21902639

  6. High-throughput screening for modulators of cellular contractile force†

    PubMed Central

    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; Solway, Julian; Fredberg, Jeffrey J.

    2015-01-01

    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 signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and 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. PMID:25953078

  7. Workflow for High Throughput Screening of Gas Sensing Materials

    PubMed Central

    Koplin, Tobias J.; Siemons, Maike; Océn-Valéntin, César; Sanders, Daniel; Simon, Ulrich

    2006-01-01

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

  8. Advances in high throughput DNA sequence data compression.

    PubMed

    Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz

    2016-06-01

    Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted. PMID:26846812

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

    PubMed

    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

  10. C. elegans in high-throughput drug discovery

    PubMed Central

    O’Reilly, Linda P.; Luke, Cliff J.; Perlmutter, David H.; Silverman, Gary A.; Pak, Stephen C.

    2014-01-01

    C. elegans has proven to be a useful model organism for investigating molecular and cellular aspects of numerous human diseases. More recently, investigators have explored the use of this organism as a tool for drug discovery. Although earlier drug screens were labor-intensive and low in throughput, recent advances in high-throughput liquid workflows, imaging platforms and data analysis software have made C. elegans a viable option for automated high-throughput drug screens. This review will outline the evolution of C. elegans-based drug screening, discuss the inherent challenges of using C. elegans, and highlight recent technological advances that have paved the way for future drug screens. PMID:24333896

  11. High throughput drug discovery with ESI-FTICR

    NASA Astrophysics Data System (ADS)

    Sannes-Lowery, Kristin A.; Cummins, Lendell L.; Chen, Shuo; Drader, Jared J.; Hofstadler, Steven A.

    2004-11-01

    Ribonucleic acids (RNA) are an attractive target for drug discovery since they play critical roles in cellular functions. Because small structured subdomains are known to mimic the behavior of the entire RNA, it is possible to design RNA drug targets that are amenable to interrogation by high performance mass spectrometry. We have developed a high throughput drug discovery platform that uses electrospray ionization Fourier transform ion cyclotron mass spectrometry to investigate ligand binding to structured RNA drug targets. This assay is called multitarget affinity/specificity screening (MASS). Using MASS, we show that it is possible to screen synthetic and natural product libraries in a high throughput and robust manner.

  12. ClinicalTrials.gov as a Data Source for Semi-Automated Point-Of-Care Trial Eligibility Screening

    PubMed Central

    Pfiffner, Pascal B.; Oh, JiWon; Miller, Timothy A.; Mandl, Kenneth D.

    2014-01-01

    Background Implementing semi-automated processes to efficiently match patients to clinical trials at the point of care requires both detailed patient data and authoritative information about open studies. Objective To evaluate the utility of the ClinicalTrials.gov registry as a data source for semi-automated trial eligibility screening. Methods Eligibility criteria and metadata for 437 trials open for recruitment in four different clinical domains were identified in ClinicalTrials.gov. Trials were evaluated for up to date recruitment status and eligibility criteria were evaluated for obstacles to automated interpretation. Finally, phone or email outreach to coordinators at a subset of the trials was made to assess the accuracy of contact details and recruitment status. Results 24% (104 of 437) of trials declaring on open recruitment status list a study completion date in the past, indicating out of date records. Substantial barriers to automated eligibility interpretation in free form text are present in 81% to up to 94% of all trials. We were unable to contact coordinators at 31% (45 of 146) of the trials in the subset, either by phone or by email. Only 53% (74 of 146) would confirm that they were still recruiting patients. Conclusion Because ClinicalTrials.gov has entries on most US and many international trials, the registry could be repurposed as a comprehensive trial matching data source. Semi-automated point of care recruitment would be facilitated by matching the registry's eligibility criteria against clinical data from electronic health records. But the current entries fall short. Ultimately, improved techniques in natural language processing will facilitate semi-automated complex matching. As immediate next steps, we recommend augmenting ClinicalTrials.gov data entry forms to capture key eligibility criteria in a simple, structured format. PMID:25334031

  13. Rapid and Semi-Automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks

    PubMed Central

    Holcomb, Paul S.; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics—the study of how neurons wire together in the brain—is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes. PMID:27259933

  14. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    PubMed Central

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  15. Nano-bio Hybrid Materials for a New Generation of High-throughput Diagnostic Systems

    NASA Astrophysics Data System (ADS)

    Nabiev, Igor

    Nano-bio hybrid materials obtained by conjugation of capture molecules and plasmonic (metal) or excitonic (semiconductor) nanocrystals or microspheres encoded with fluorescent semiconductor nanocrystals of different colors are the basis for development of a new generation of high-throughput diagnostic systems. Here, the general principles of development of "ideal" diagnostic nanoprobes based on oriented conjugates of capture molecules with the nanoparticles of different chemical compositions or with optically encoded microspheres are summarized and the basic requirements for individual components of the photonic nanoprobes being developed are discussed in the context of ensuring their advantages over the existing photonic diagnostic systems.

  16. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization.

    PubMed

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  17. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    PubMed Central

    Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

    2007-01-01

    Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content. PMID:17381841

  18. Is a semi-automated approach indicated in the application of the automated micronucleus assay for triage purposes?

    PubMed

    Thierens, H; Vral, A; Vandevoorde, C; Vandersickel, V; de Gelder, V; Romm, H; Oestreicher, U; Rothkamm, K; Barnard, S; Ainsbury, E; Sommer, S; Beinke, C; Wojcik, A

    2014-06-01

    Within the EU MULTIBIODOSE project, the automated micronucleus (MN) assay was optimised for population triage in large-scale radiological emergencies. For MN scoring, two approaches were applied using the Metafer4 platform (MetaSystems, Germany): fully automated scoring and semi-automated scoring with visual inspection of the gallery of MN-positive objects. Dose-response curves were established for acute and protracted whole-body and partial-body exposures. A database of background MN yields was set up, allowing determination of the dose detection threshold in both scoring modes. An analysis of the overdispersion of the MN frequency distribution σ(2)/µ obtained by semi-automated scoring showed that the value of this parameter represents a reliability check of the calculated equivalent total body dose in case the accident overexposure is a partial-body exposure. The elaborated methodology was validated in an accident training exercise. Overall, the semi-automated scoring procedure represents important added value to the automated MN assay. PMID:24743767

  19. Semi-automated recognition of planation surfaces and other flat landforms: a case study from the Aggtelek Karst, Hungary

    NASA Astrophysics Data System (ADS)

    Veselský, Michal; Bandura, Peter; Burian, Libor; Harciníková, Tatiana; Bella, Pavel

    2015-11-01

    This study deals with the possibilities of expertdriven semi-automated recognition of planation surfaces and other flat landforms in the area of the Aggtelek Karst, Hungary. Planation surfaces are the most debatable and vague landforms and can be defined as parts of terrain formed by long-lasting erosion-denudation processes under the stagnant erosion base conditions. In terms of denudation chronology they can be considered as morphological indicators of different evolution stages of area. In karst areas planation surfaces and river terraces are mostly correlated with cave levels, which originated in relation to the same stagnant erosion base. Because there is no general method of delineation of planation surfaces, the main objective of the study was to find a suitable method for semi-automated recognition of flat landforms in the Aggtelek Karst, which should correspond to different phases of the Jósva River incision and therefore could be correlated to the multilevel cave system of the study area. Several methods for semi-automated landform classification were tested for recognition of flat surfaces in a relatively objective way. Slope gradient thresholding tool, and r.param.scale and r.geomorphon modules implemented in GRASS GIS were tested. As a result, the r.geomorphon module was proven as the most suitable method for delineation of relatively flat surfaces. Findings of the presented work can be used as a morphological indicator of the comprehensive reconstruction of evolution of the Aggtelek Karst and the Slovak Karst.

  20. High-Throughput Single-Molecule Studies of Protein-DNA Interactions

    PubMed Central

    Robison, Aaron D.; Finkelstein, Ilya J.

    2014-01-01

    Fluorescence and force-based single-molecule studies of protein-nucleic acid interactions continue to shed critical insights into many aspects of DNA and RNA processing. As single-molecule assays are inherently low-throughput, obtaining statistically relevant datasets remains a major challenge. Additionally, most fluorescence-based single-molecule particle-tracking assays are limited to observing fluorescent proteins that are in the low-nanomolar range, as spurious background signals predominate at higher fluorophore concentrations. These technical limitations have traditionally limited the types of questions that could be addressed via single-molecule methods. In this review, we describe new approaches for high-throughput and high-concentration single-molecule biochemical studies. We conclude with a discussion of outstanding challenges for the single-molecule biologist and how these challenges can be tackled to further approach the biochemical complexity of the cell. PMID:24859086

  1. Aspirator Gun for High-Throughput Mosquito Bioassays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe an innovative aspirator gun designed to transfer anaesthetized mosquitoes directly into glass bioassay tubes. The gun has been used for thousands of transfers with extremely low associated mortality and is the central component of a high-throughput bioassay system. The gun is constructed...

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

    EPA Science Inventory

    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,...

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

    EPA Science Inventory

    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...

  4. Aspirator gun for high-throughput mosquito bioassays.

    PubMed

    Aldridge, Robert L; Wynn, W Wayne; Britch, Seth C; Linthicum, Kenneth J

    2012-03-01

    We describe an innovative aspirator gun designed to transfer individual anesthetized mosquitoes directly into glass bioassay tubes. The gun has been used for thousands of transfers with extremely low associated mortality and is the central component of a high-throughput bioassay system. The gun is constructed using readily obtainable materials and can be modified for a range of insects. PMID:22533090

  5. High Throughput Assays and Exposure Science (ISES annual meeting)

    EPA Science Inventory

    High throughput screening (HTS) data characterizing chemical-induced biological activity has been generated for thousands of environmentally-relevant chemicals by the US inter-agency Tox21 and the US EPA ToxCast programs. For a limited set of chemicals, bioactive concentrations r...

  6. High Throughput Exposure Estimation Using NHANES Data (SOT)

    EPA Science Inventory

    In the ExpoCast project, high throughput (HT) exposure models enable rapid screening of large numbers of chemicals for exposure potential. Evaluation of these models requires empirical exposure data and due to the paucity of human metabolism/exposure data such evaluations includ...

  7. High Throughput Sequence Analysis for Disease Resistance in Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preliminary results of a computational analysis of high throughput sequencing data from Zea mays and the fungus Aspergillus are reported. The Illumina Genome Analyzer was used to sequence RNA samples from two strains of Z. mays (Va35 and Mp313) collected over a time course as well as several specie...

  8. Accounting For Uncertainty in The Application Of High Throughput Datasets

    EPA Science Inventory

    The use of high throughput screening (HTS) datasets will need to adequately account for uncertainties in the data generation process and propagate these uncertainties through to ultimate use. Uncertainty arises at multiple levels in the construction of predictors using in vitro ...

  9. Environmental Impact on Vascular Development Predicted by High Throughput Screening

    EPA Science Inventory

    Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High throughput screening (HTS) in EPA’s ToxCastTM project provides vast d...

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

    EPA Science Inventory

    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...

  11. GENO PROFILER: BATCH PROCESSING OF HIGH THROUGHPUT CAPILLARY FINGERPRINTING DATA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput fingerprinting techniques employing capillary electrophoresis place new demands on the editing of fingerprint files for the downstream contig assembly program, FPC. A cross-platform software application, GenoProfiler, was developed for automated editing of sized fingerprinting profil...

  12. New High Throughput Methods to Estimate Chemical Exposure

    EPA Science Inventory

    EPA has made many recent advances in high throughput bioactivity testing. However, concurrent advances in rapid, quantitative prediction of human and ecological exposures have been lacking, despite the clear importance of both measures for a risk-based approach to prioritizing an...

  13. Semi-automated filtering of data outliers to improve spatial analysis of piezometric data

    NASA Astrophysics Data System (ADS)

    Tremblay, Yohann; Lemieux, Jean-Michel; Fortier, Richard; Molson, John; Therrien, René; Therrien, Pierre; Comeau, Guillaume; Talbot Poulin, Marie-Catherine

    2015-08-01

    The identification and removal of data outliers remains a major challenge for spatial analysis of piezometric data. In this context, a simple semi-automated procedure for filtering outliers of depth to static water level was developed and used as a part of a regional groundwater-mapping project in the Québec Metropolitan Community, Québec, Canada. Following a few basic steps of data control, potential outliers were detected using two simple automated steps: (1) identifying water levels that are deeper than the 99th percentile of a high-reliability dataset compiled by groundwater professionals and assumed to adequately represent depths to static water level, and (2) using moving averages within a search radius of 250 m calculated around each well. All detected potential outliers were visually examined in a geographic information system and compared to neighbouring data before being kept or discarded. To evaluate the efficiency of the procedure, exploratory statistics, histograms and semi-variograms of the initial, intermediate and filtered datasets were compared to the high-reliability dataset. Objective interpolation was then performed using ordinary kriging. A cross-validation analysis showed a less biased and more accurate interpolation after applying the proposed outlier filtering procedure. Qualitative knowledge of the hydrogeological settings is an important component of this procedure which combines advantages of both manual and automated processing, making the procedure adaptive and easy to use. The final outcome of the proposed procedure is an improved interpolation map of depth to static water level along with minimised and low squared estimation errors.

  14. MRI scanner variability studies using a semi-automated analysis system.

    PubMed

    Hyde, R J; Ellis, J H; Gardner, E A; Zhang, Y; Carson, P L

    1994-01-01

    Due to the unique design of the Parallel Rod Test Object (PRoTO) and the associated semi-automated analysis program, it was necessary to test it extensively for precision and accuracy, and preliminarily for utility, before its distribution for wider use in MRI system quality control (QC). The test object and analysis program measured the desired quantities reproducibly and they accurately measured predicted changes from intentionally adjusted imaging system parameters, yielding sensitivity of the various test measures to deviation in the system operating parameters. From a single scan of the most recent revision of the test object, multiple quantitative quality control measures were obtained throughout the scanning volume on two MR imaging systems over periods of six and twelve months, respectively. From these and earlier trials, an initial indication was obtained of which performance measures are worth monitoring for QC. This experience suggests that signal-to-noise ratio (SNR) and distortion (including display scale) should be monitored but not necessarily the resolution. The latter was only found to alter at the same time or later than other parameters such as SNR had changed. Slice thickness was found to vary on some units and this measure was also used in normalizing the SNR by voxel volume. SNR, distortion, and resolution measurements using field-echo sequences were less stable than those using spin-echo sequences. Use of this QC program to test a wide variety of image quality measures allowed timely assessment of the long-term variability of the units tested. Long-term variability may become among the most important measures for comparison of system performance and maintenance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7997096

  15. A Neural-Network-Based Semi-Automated Geospatial Classification Tool

    NASA Astrophysics Data System (ADS)

    Hale, R. G.; Herzfeld, U. C.

    2014-12-01

    North America's largest glacier system, the Bering Bagley Glacier System (BBGS) in Alaska, surged in 2011-2013, as shown by rapid mass transfer, elevation change, and heavy crevassing. Little is known about the physics controlling surge glaciers' semi-cyclic patterns; therefore, it is crucial to collect and analyze as much data as possible so that predictive models can be made. In addition, physical signs frozen in ice in the form of crevasses may help serve as a warning for future surges. The BBGS surge provided an opportunity to develop an automated classification tool for crevasse classification based on imagery collected from small aircraft. The classification allows one to link image classification to geophysical processes associated with ice deformation. The tool uses an approach that employs geostatistical functions and a feed-forward perceptron with error back-propagation. The connectionist-geostatistical approach uses directional experimental (discrete) variograms to parameterize images into a form that the Neural Network (NN) can recognize. In an application to preform analysis on airborne video graphic data from the surge of the BBGS, an NN was able to distinguish 18 different crevasse classes with 95 percent or higher accuracy, for over 3,000 images. Recognizing that each surge wave results in different crevasse types and that environmental conditions affect the appearance in imagery, we designed the tool's semi-automated pre-training algorithm to be adaptable. The tool can be optimized to specific settings and variables of image analysis: (airborne and satellite imagery, different camera types, observation altitude, number and types of classes, and resolution). The generalization of the classification tool brings three important advantages: (1) multiple types of problems in geophysics can be studied, (2) the training process is sufficiently formalized to allow non-experts in neural nets to perform the training process, and (3) the time required to

  16. Feasibility study of semi-automated measurements of finger joint space widths.

    PubMed

    Pfeil, Alexander; Sommerfeld, Julia; Fröber, Rosemarie; Lehmann, Gabriele; Malich, Ansgar; Hansch, Andreas; Wolf, Gunter; Böttcher, Joachim

    2011-10-01

    The purpose of this study is to evaluate technical feasibility based on image capturing conditions (film-focus distance (FFD), film sensitivity, film brand, exposure level and tube voltage) that potentially alter radiographs and consequently may influence the semi-automated measurement of joint space distance (JSD) by computer-aided joint space analysis (CAJSA) in rheumatoid arthritis and osteoarthritis. The radiographs of a left hand (deceased man) were acquired under systematically changing image capturing conditions (exposure level: 4-8 mAs; FFD: 90-130 cm; film sensitivity: 200/400 and tube voltage: 40-52 kV with different image modalities: conventional radiographs, original digital radiographs, digital print-outs). All JSD-measurements were performed with the CAJSA-technology (Radiogrammetry Kit, Version 1.3.6; Sectra; Sweden) at the metacarpal-phalangeal articulation. JSD-analysis was not influenced by changes of FFD, exposure level, film sensitivity or film brand. JSD showed significant variation caused by tube voltage (conventional: CV = 1.913% for Agfa and CV = 2.448% for Kodak; digital: CV = 0.741% for Philips print-outs and CV = 0.620% with original digital images versus CV = 2.185% for Siemens print-outs and 0.951% with original digital images). Computer-aided joint space analysis for JSD-measurements is unaffected by the following image capturing parameters: film-focus distance, film sensitivity, film brand and exposure level. An influence of tube voltage was detected in a lesser extent for original digital images compared to the printed digital as well as conventional versions. Consequently, a standardized tube voltage is essential for accurate reproductions of CAJSA-measurements in rheumatoid arthritis and osteoarthritis. PMID:20401484

  17. Semi-automated scar detection in delayed enhanced cardiac magnetic resonance images

    NASA Astrophysics Data System (ADS)

    Morisi, Rita; Donini, Bruno; Lanconelli, Nico; Rosengarden, James; Morgan, John; Harden, Stephen; Curzen, Nick

    2015-06-01

    Late enhancement cardiac magnetic resonance images (MRI) has the ability to precisely delineate myocardial scars. We present a semi-automated method for detecting scars in cardiac MRI. This model has the potential to improve routine clinical practice since quantification is not currently offered due to time constraints. A first segmentation step was developed for extracting the target regions for potential scar and determining pre-candidate objects. Pattern recognition methods are then applied to the segmented images in order to detect the position of the myocardial scar. The database of late gadolinium enhancement (LE) cardiac MR images consists of 111 blocks of images acquired from 63 patients at the University Hospital Southampton NHS Foundation Trust (UK). At least one scar was present for each patient, and all the scars were manually annotated by an expert. A group of images (around one third of the entire set) was used for training the system which was subsequently tested on all the remaining images. Four different classifiers were trained (Support Vector Machine (SVM), k-nearest neighbor (KNN), Bayesian and feed-forward neural network) and their performance was evaluated by using Free response Receiver Operating Characteristic (FROC) analysis. Feature selection was implemented for analyzing the importance of the various features. The segmentation method proposed allowed the region affected by the scar to be extracted correctly in 96% of the blocks of images. The SVM was shown to be the best classifier for our task, and our system reached an overall sensitivity of 80% with less than 7 false positives per patient. The method we present provides an effective tool for detection of scars on cardiac MRI. This may be of value in clinical practice by permitting routine reporting of scar quantification.

  18. Semi-automated procedures for shoreline extraction using single RADARSAT-1 SAR image

    NASA Astrophysics Data System (ADS)

    Al Fugura, A.'kif; Billa, Lawal; Pradhan, Biswajeet

    2011-12-01

    Coastline identification is important for surveying and mapping reasons. Coastline serves as the basic point of reference and is used on nautical charts for navigation purposes. Its delineation has become crucial and more important in the wake of the many recent earthquakes and tsunamis resulting in complete change and redraw of some shorelines. In a tropical country like Malaysia, presence of cloud cover hinders the application of optical remote sensing data. In this study a semi-automated technique and procedures are presented for shoreline delineation from RADARSAT-1 image. A scene of RADARSAT-1 satellite image was processed using enhanced filtering technique to identify and extract the shoreline coast of Kuala Terengganu, Malaysia. RADSARSAT image has many advantages over the optical data because of its ability to penetrate cloud cover and its night sensing capabilities. At first, speckles were removed from the image by using Lee sigma filter which was used to reduce random noise and to enhance the image and discriminate the boundary between land and water. The results showed an accurate and improved extraction and delineation of the entire coastline of Kuala Terrenganu. The study demonstrated the reliability of the image averaging filter in reducing random noise over the sea surface especially near the shoreline. It enhanced land-water boundary differentiation, enabling better delineation of the shoreline. Overall, the developed techniques showed the potential of radar imagery for accurate shoreline mapping and will be useful for monitoring shoreline changes during high and low tides as well as shoreline erosion in a tropical country like Malaysia.

  19. TOBAGO — a semi-automated approach for the generation of 3-D building models

    NASA Astrophysics Data System (ADS)

    Gruen, Armin

    3-D city models are in increasing demand for a great number of applications. Photogrammetry is a relevant technology that can provide an abundance of geometric, topologic and semantic information concerning these models. The pressure to generate a large amount of data with high degree of accuracy and completeness poses a great challenge to phtogrammetry. The development of automated and semi-automated methods for the generation of those data sets is therefore a key issue in photogrammetric research. We present in this article a strategy and methodology for an efficient generation of even fairly complex building models. Within this concept we request the operator to measure the house roofs from a stereomodel in form of an unstructured point cloud. According to our experience this can be done very quickly. Even a non-experienced operator can measure several hundred roofs or roof units per day. In a second step we fit generic building models fully automatically to these point clouds. The structure information is inherently included in these building models. In such a way geometric, topologic and even semantic data can be handed over to a CAD-system, in our case AutoCad, for further visualization and manipulation. The structuring is achieved in three steps. In a first step a classifier is initiated which recognizes the class of houses a particular roof point cloud belongs to. This recognition step is primarily based on the analysis of the number of ridge points. In the second and third steps the concrete topological relations between roof points are investigated and generic building models are fitted to the point clouds. Based on the technique of constraint-based reasoning two geometrical parsers are solving this problem. We have tested the methodology under a variety of different conditions in several pilot projects. The results will indicate the good performance of our approach. In addition we will demonstrate how the results can be used for visualization (texture

  20. Semi-automated method to measure pneumonia severity in mice through computed tomography (CT) scan analysis

    NASA Astrophysics Data System (ADS)

    Johri, Ansh; Schimel, Daniel; Noguchi, Audrey; Hsu, Lewis L.

    2010-03-01

    Imaging is a crucial clinical tool for diagnosis and assessment of pneumonia, but quantitative methods are lacking. Micro-computed tomography (micro CT), designed for lab animals, provides opportunities for non-invasive radiographic endpoints for pneumonia studies. HYPOTHESIS: In vivo micro CT scans of mice with early bacterial pneumonia can be scored quantitatively by semiautomated imaging methods, with good reproducibility and correlation with bacterial dose inoculated, pneumonia survival outcome, and radiologists' scores. METHODS: Healthy mice had intratracheal inoculation of E. coli bacteria (n=24) or saline control (n=11). In vivo micro CT scans were performed 24 hours later with microCAT II (Siemens). Two independent radiologists scored the extent of airspace abnormality, on a scale of 0 (normal) to 24 (completely abnormal). Using the Amira 5.2 software (Mercury Computer Systems), a histogram distribution of voxel counts between the Hounsfield range of -510 to 0 was created and analyzed, and a segmentation procedure was devised. RESULTS: A t-test was performed to determine whether there was a significant difference in the mean voxel value of each mouse in the three experimental groups: Saline Survivors, Pneumonia Survivors, and Pneumonia Non-survivors. It was found that the voxel count method was able to statistically tell apart the Saline Survivors from the Pneumonia Survivors, the Saline Survivors from the Pneumonia Non-survivors, but not the Pneumonia Survivors vs. Pneumonia Non-survivors. The segmentation method, however, was successfully able to distinguish the two Pneumonia groups. CONCLUSION: We have pilot-tested an evaluation of early pneumonia in mice using micro CT and a semi-automated method for lung segmentation and scoring system. Statistical analysis indicates that the system is reliable and merits further evaluation.

  1. Methods for semi-automated indexing for high precision information retrieval

    NASA Technical Reports Server (NTRS)

    Berrios, Daniel C.; Cucina, Russell J.; Fagan, Lawrence M.

    2002-01-01

    OBJECTIVE: To evaluate a new system, ISAID (Internet-based Semi-automated Indexing of Documents), and to generate textbook indexes that are more detailed and more useful to readers. DESIGN: Pilot evaluation: simple, nonrandomized trial comparing ISAID with manual indexing methods. Methods evaluation: randomized, cross-over trial comparing three versions of ISAID and usability survey. PARTICIPANTS: Pilot evaluation: two physicians. Methods evaluation: twelve physicians, each of whom used three different versions of the system for a total of 36 indexing sessions. MEASUREMENTS: Total index term tuples generated per document per minute (TPM), with and without adjustment for concordance with other subjects; inter-indexer consistency; ratings of the usability of the ISAID indexing system. RESULTS: Compared with manual methods, ISAID decreased indexing times greatly. Using three versions of ISAID, inter-indexer consistency ranged from 15% to 65% with a mean of 41%, 31%, and 40% for each of three documents. Subjects using the full version of ISAID were faster (average TPM: 5.6) and had higher rates of concordant index generation. There were substantial learning effects, despite our use of a training/run-in phase. Subjects using the full version of ISAID were much faster by the third indexing session (average TPM: 9.1). There was a statistically significant increase in three-subject concordant indexing rate using the full version of ISAID during the second indexing session (p < 0.05). SUMMARY: Users of the ISAID indexing system create complex, precise, and accurate indexing for full-text documents much faster than users of manual methods. Furthermore, the natural language processing methods that ISAID uses to suggest indexes contributes substantially to increased indexing speed and accuracy.

  2. Semi-automated method for delineation of landmarks on models of the cerebral cortex

    PubMed Central

    Shattuck, David W.; Joshi, Anand A.; Pantazis, Dimitrios; Kan, Eric; Dutton, Rebecca A.; Sowell, Elizabeth R.; Thompson, Paul M.; Toga, Arthur W.; Leahy, Richard M.

    2009-01-01

    Sulcal and gyral landmarks on the human cerebral cortex are required for various studies of the human brain. Whether used directly to examine sulcal geometry, or indirectly to drive cortical surface registration methods, the accuracy of these landmarks is essential. While several methods have been developed to automatically identify sulci and gyri, their accuracy may be insufficient for certain neuroanatomical studies. We describe a semi-automated procedure that delineates a sulcus or gyrus given a limited number of user-selected points. The method uses a graph theory approach to identify the lowest-cost path between the points, where the cost is a combination of local curvature features and the distance between vertices on the surface representation. We implemented the algorithm in an interface that guides the user through a cortical surface delineation protocol, and we incorporated this tool into our BrainSuite software. We performed a study to compare the results produced using our method with results produced using Display, a popular tool that has been used extensively for manual delineation of sulcal landmarks. Six raters were trained on the delineation protocol. They performed delineations on 12 brains using both software packages. We performed a statistical analysis of 3 aspects of the delineation task: time required to delineate the surface, registration accuracy achieved compared to an expert-delineated gold-standard, and variation among raters. Our new method was shown to be faster to use, to provide reduced inter-rater variability, and to provide results that were at least as accurate as those produced using Display. PMID:19162074

  3. Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms.

    PubMed

    Khare, Siddharth M; Awasthi, Anjali; Venkataraman, V; Koushika, Sandhya P

    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 enhances contrast making it easier to detect and track pillar displacement driven by the organism. Second, we developed a semi-automated graphical user interface to analyze the images for pillar displacement, thus reducing the analysis time for each animal to minutes. The addition of color reduced the Young's modulus of PDMS. Therefore, the dye-PDMS composite was characterized using Yeoh's hyperelastic model and the pillars were calibrated using a silicon based force sensor. We used our device to measure forces exerted by wild type and mutant Caenorhabditis elegans moving on an agarose surface. Wild type C. elegans exert an average force of ∼1 μN on an individual pillar and a total average force of ∼7.68 μN. We show that the middle of C. elegans exerts more force than its extremities. We find that C. elegans mutants with defective body wall muscles apply significantly lower force on individual pillars, while mutants defective in sensing externally applied mechanical forces still apply the same average force per pillar compared to wild type animals. Average forces applied per pillar are independent of the length, diameter, or cuticle stiffness of the animal. We also used the device to measure, for the first time, forces applied by Drosophila melanogaster larvae. Peristaltic waves occurred at 0.4 Hz applying an average force of ∼1.58 μN on a single pillar. Our colored microfluidic device along with its displacement tracking software allows us to measure forces

  4. Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms

    PubMed Central

    Khare, Siddharth M.; Awasthi, Anjali; Venkataraman, V.; Koushika, Sandhya P.

    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 enhances contrast making it easier to detect and track pillar displacement driven by the organism. Second, we developed a semi-automated graphical user interface to analyze the images for pillar displacement, thus reducing the analysis time for each animal to minutes. The addition of color reduced the Young's modulus of PDMS. Therefore, the dye-PDMS composite was characterized using Yeoh's hyperelastic model and the pillars were calibrated using a silicon based force sensor. We used our device to measure forces exerted by wild type and mutant Caenorhabditis elegans moving on an agarose surface. Wild type C. elegans exert an average force of ∼1 μN on an individual pillar and a total average force of ∼7.68 μN. We show that the middle of C. elegans exerts more force than its extremities. We find that C. elegans mutants with defective body wall muscles apply significantly lower force on individual pillars, while mutants defective in sensing externally applied mechanical forces still apply the same average force per pillar compared to wild type animals. Average forces applied per pillar are independent of the length, diameter, or cuticle stiffness of the animal. We also used the device to measure, for the first time, forces applied by Drosophila melanogaster larvae. Peristaltic waves occurred at 0.4 Hz applying an average force of ∼1.58 μN on a single pillar. Our colored microfluidic device along with its displacement tracking software allows us to measure forces

  5. High-throughput SNP scoring with GAMMArrays: genomic analysis using multiplexed microsphere arrays

    NASA Astrophysics Data System (ADS)

    Green, Lance D.; Cai, Hong; Torney, David C.; Wood, Diane J.; Uribe-Romeo, Francisco J.; Kaderali, Lars; Nolan, John P.; White, P. S.

    2002-06-01

    We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.

  6. High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions

    PubMed Central

    2012-01-01

    Background Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. Methods A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. Results Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. Conclusions The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors. PMID:22929810

  7. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  8. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  9. Semi-automated extraction of landslides in Taiwan based on SPOT imagery and DEMs

    NASA Astrophysics Data System (ADS)

    Eisank, Clemens; Hölbling, Daniel; Friedl, Barbara; Chen, Yi-Chin; Chang, Kang-Tsung

    2014-05-01

    The vast availability and improved quality of optical satellite data and digital elevation models (DEMs), as well as the need for complete and up-to-date landslide inventories at various spatial scales have fostered the development of semi-automated landslide recognition systems. Among the tested approaches for designing such systems, object-based image analysis (OBIA) stepped out to be a highly promising methodology. OBIA offers a flexible, spatially enabled framework for effective landslide mapping. Most object-based landslide mapping systems, however, have been tailored to specific, mainly small-scale study areas or even to single landslides only. Even though reported mapping accuracies tend to be higher than for pixel-based approaches, accuracy values are still relatively low and depend on the particular study. There is still room to improve the applicability and objectivity of object-based landslide mapping systems. The presented study aims at developing a knowledge-based landslide mapping system implemented in an OBIA environment, i.e. Trimble eCognition. In comparison to previous knowledge-based approaches, the classification of segmentation-derived multi-scale image objects relies on digital landslide signatures. These signatures hold the common operational knowledge on digital landslide mapping, as reported by 25 Taiwanese landslide experts during personal semi-structured interviews. Specifically, the signatures include information on commonly used data layers, spectral and spatial features, and feature thresholds. The signatures guide the selection and implementation of mapping rules that were finally encoded in Cognition Network Language (CNL). Multi-scale image segmentation is optimized by using the improved Estimation of Scale Parameter (ESP) tool. The approach described above is developed and tested for mapping landslides in a sub-region of the Baichi catchment in Northern Taiwan based on SPOT imagery and a high-resolution DEM. An object

  10. Semi-automated Digital Imaging and Processing System for Measuring Lake Ice Thickness

    NASA Astrophysics Data System (ADS)

    Singh, Preetpal

    to detect equipment failure and identify defective products at the assembly line. The research work in this thesis combines machine vision and image processing technology to build a digital imaging and processing system for monitoring and measuring lake ice thickness in real time. An ultra-compact USB camera is programmed to acquire and transmit high resolution imagery for processing with MATLAB Image Processing toolbox. The image acquisition and transmission process is fully automated; image analysis is semi-automated and requires limited user input. Potential design changes to the prototype and ideas on fully automating the imaging and processing procedure are presented to conclude this research work.

  11. Semi-automated Data Set Submission Work Flow for Archival with the ORNL DAAC

    NASA Astrophysics Data System (ADS)

    Wright, D.; Beaty, T.; Cook, R. B.; Devarakonda, R.; Eby, P.; Heinz, S. L.; Hook, L. A.; McMurry, B. F.; Shanafield, H. A.; Sill, D.; Santhana Vannan, S.; Wei, Y.

    2013-12-01

    The ORNL DAAC archives and publishes, free of charge, data and information relevant to biogeochemical, ecological, and environmental processes. The ORNL DAAC primarily archives data produced by NASA's Terrestrial Ecology Program; however, any data that are pertinent to the biogeochemical and ecological community are of interest. The data set submission process to the ORNL DAAC has been recently updated and semi-automated to provide a consistent data provider experience and to create a uniform data product. The data archived at the ORNL DAAC must be well formatted, self-descriptive, and documented, as well as referenced in a peer-reviewed publication. If the ORNL DAAC is the appropriate archive for a data set, the data provider will be sent an email with several URL links to guide them through the submission process. The data provider will be asked to fill out a short online form to help the ORNL DAAC staff better understand the data set. These questions cover information about the data set, a description of the data set, temporal and spatial characteristics of the data set, and how the data were prepared and delivered. The questionnaire is generic and has been designed to gather input on the various diverse data sets the ORNL DAAC archives. A data upload module and metadata editor further guide the data provider through the submission process. For submission purposes, a complete data set includes data files, document(s) describing data, supplemental files, metadata record(s), and an online form. There are five major functions the ORNL DAAC performs during the process of archiving data: 1) Ingestion is the ORNL DAAC side of submission; data are checked, metadata records are compiled, and files are converted to archival formats. 2) Metadata records and data set documentation made searchable and the data set is given a permanent URL. 3) The data set is published, assigned a DOI, and advertised. 4) The data set is provided long-term post-project support. 5) Stewardship

  12. A Semi-Automated Image Analysis Procedure for In Situ Plankton Imaging Systems

    PubMed Central

    Bi, Hongsheng; Guo, Zhenhua; Benfield, Mark C.; Fan, Chunlei; Ford, Michael; Shahrestani, Suzan; Sieracki, Jeffery M.

    2015-01-01

    Plankton imaging systems are capable of providing fine-scale observations that enhance our understanding of key physical and biological processes. However, processing the large volumes of data collected by imaging systems remains a major obstacle for their employment, and existing approaches are designed either for images acquired under laboratory controlled conditions or within clear waters. In the present study, we developed a semi-automated approach to analyze plankton taxa from images acquired by the ZOOplankton VISualization (ZOOVIS) system within turbid estuarine waters, in Chesapeake Bay. When compared to images under laboratory controlled conditions or clear waters, images from highly turbid waters are often of relatively low quality and more variable, due to the large amount of objects and nonlinear illumination within each image. We first customized a segmentation procedure to locate objects within each image and extracted them for classification. A maximally stable extremal regions algorithm was applied to segment large gelatinous zooplankton and an adaptive threshold approach was developed to segment small organisms, such as copepods. Unlike the existing approaches for images acquired from laboratory, controlled conditions or clear waters, the target objects are often the majority class, and the classification can be treated as a multi-class classification problem. We customized a two-level hierarchical classification procedure using support vector machines to classify the target objects (< 5%), and remove the non-target objects (> 95%). First, histograms of oriented gradients feature descriptors were constructed for the segmented objects. In the first step all non-target and target objects were classified into different groups: arrow-like, copepod-like, and gelatinous zooplankton. Each object was passed to a group-specific classifier to remove most non-target objects. After the object was classified, an expert or non-expert then manually removed the

  13. Semi-automated calibration method for modelling of mountain permafrost evolution in Switzerland

    NASA Astrophysics Data System (ADS)

    Marmy, A.; Rajczak, J.; Delaloye, R.; Hilbich, C.; Hoelzle, M.; Kotlarski, S.; Lambiel, C.; Noetzli, J.; Phillips, M.; Salzmann, N.; Staub, B.; Hauck, C.

    2015-09-01

    Permafrost is a widespread phenomenon in the European Alps. Many important topics such as the future evolution of permafrost related to climate change and the detection of permafrost related to potential natural hazards sites are of major concern to our society. Numerical permafrost models are the only tools which facilitate the projection of the future evolution of permafrost. Due to the complexity of the processes involved and the heterogeneity of Alpine terrain, models must be carefully calibrated and results should be compared with observations at the site (borehole) scale. However, a large number of local point data are necessary to obtain a broad overview of the thermal evolution of mountain permafrost over a larger area, such as the Swiss Alps, and the site-specific model calibration of each point would be time-consuming. To face this issue, this paper presents a semi-automated calibration method using the Generalized Likelihood Uncertainty Estimation (GLUE) as implemented in a 1-D soil model (CoupModel) and applies it to six permafrost sites in the Swiss Alps prior to long-term permafrost evolution simulations. We show that this automated calibration method is able to accurately reproduce the main thermal condition characteristics with some limitations at sites with unique conditions such as 3-D air or water circulation, which have to be calibrated manually. The calibration obtained was used for RCM-based long-term simulations under the A1B climate scenario specifically downscaled at each borehole site. The projection shows general permafrost degradation with thawing at 10 m, even partially reaching 20 m depths until the end of the century, but with different timing among the sites. The degradation is more rapid at bedrock sites whereas ice-rich sites with a blocky surface cover showed a reduced sensitivity to climate change. The snow cover duration is expected to be reduced drastically (between -20 to -37 %) impacting the ground thermal regime. However

  14. A semi-automated image analysis procedure for in situ plankton imaging systems.

    PubMed

    Bi, Hongsheng; Guo, Zhenhua; Benfield, Mark C; Fan, Chunlei; Ford, Michael; Shahrestani, Suzan; Sieracki, Jeffery M

    2015-01-01

    Plankton imaging systems are capable of providing fine-scale observations that enhance our understanding of key physical and biological processes. However, processing the large volumes of data collected by imaging systems remains a major obstacle for their employment, and existing approaches are designed either for images acquired under laboratory controlled conditions or within clear waters. In the present study, we developed a semi-automated approach to analyze plankton taxa from images acquired by the ZOOplankton VISualization (ZOOVIS) system within turbid estuarine waters, in Chesapeake Bay. When compared to images under laboratory controlled conditions or clear waters, images from highly turbid waters are often of relatively low quality and more variable, due to the large amount of objects and nonlinear illumination within each image. We first customized a segmentation procedure to locate objects within each image and extracted them for classification. A maximally stable extremal regions algorithm was applied to segment large gelatinous zooplankton and an adaptive threshold approach was developed to segment small organisms, such as copepods. Unlike the existing approaches for images acquired from laboratory, controlled conditions or clear waters, the target objects are often the majority class, and the classification can be treated as a multi-class classification problem. We customized a two-level hierarchical classification procedure using support vector machines to classify the target objects (< 5%), and remove the non-target objects (> 95%). First, histograms of oriented gradients feature descriptors were constructed for the segmented objects. In the first step all non-target and target objects were classified into different groups: arrow-like, copepod-like, and gelatinous zooplankton. Each object was passed to a group-specific classifier to remove most non-target objects. After the object was classified, an expert or non-expert then manually removed the

  15. Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

    SciTech Connect

    Heusinkveld, Harm J.; Westerink, Remco H.S.

    2011-08-15

    Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca{sup 2+}]{sub i}, e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca{sup 2+}]{sub i} are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca{sup 2+}]{sub i} in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca{sup 2+}]{sub i} a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca{sup 2+}]{sub i}. Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with caveats and limitations that require further investigation. - Research Highlights: > The use of plate readers for high-throughput screening of intracellular

  16. Use of a Fluorometric Imaging Plate Reader in high-throughput screening

    NASA Astrophysics Data System (ADS)

    Groebe, Duncan R.; Gopalakrishnan, Sujatha; Hahn, Holly; Warrior, Usha; Traphagen, Linda; Burns, David J.

    1999-04-01

    High-throughput screening (HTS) efforts at Abbott Laboratories have been greatly facilitated by the use of a Fluorometric Imaging Plate Reader. The FLIPR consists of an incubated cabinet with integrated 96-channel pipettor and fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorometer. An argon laser is used to excite fluorophores in a 96-well microtiter plate and the emitted fluorescence is imaged by a cooled CCD camera. The image data is downloaded from the camera and processed to average the signal form each well of the microtiter pate for each time point. The data is presented in real time on the computer screen, facilitating interpretation and trouble-shooting. In addition to fluorescence, the camera can also detect luminescence form firefly luciferase.

  17. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

  18. A high-throughput label-free nanoparticle analyser

    NASA Astrophysics Data System (ADS)

    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.

  19. High-throughput theoretical design of lithium battery materials

    NASA Astrophysics Data System (ADS)

    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).

  20. Direct assembling methodologies for high-throughput bioscreening

    PubMed Central

    Rodríguez-Dévora, Jorge I.; Shi, Zhi-dong; Xu, Tao

    2012-01-01

    Over the last few decades, high-throughput (HT) bioscreening, a technique that allows rapid screening of biochemical compound libraries against biological targets, has been widely used in drug discovery, stem cell research, development of new biomaterials, and genomics research. To achieve these ambitions, scaffold-free (or direct) assembly of biological entities of interest has become critical. Appropriate assembling methodologies are required to build an efficient HT bioscreening platform. The development of contact and non-contact assembling systems as a practical solution has been driven by a variety of essential attributes of the bioscreening system, such as miniaturization, high throughput, and high precision. The present article reviews recent progress on these assembling technologies utilized for the construction of HT bioscreening platforms. PMID:22021162

  1. Fusion genes and their discovery using high throughput sequencing.

    PubMed

    Annala, M J; Parker, B C; Zhang, W; Nykter, M

    2013-11-01

    Fusion genes are hybrid genes that combine parts of two or more original genes. They can form as a result of chromosomal rearrangements or abnormal transcription, and have been shown to act as drivers of malignant transformation and progression in many human cancers. The biological significance of fusion genes together with their specificity to cancer cells has made them into excellent targets for molecular therapy. Fusion genes are also used as diagnostic and prognostic markers to confirm cancer diagnosis and monitor response to molecular therapies. High-throughput sequencing has enabled the systematic discovery of fusion genes in a wide variety of cancer types. In this review, we describe the history of fusion genes in cancer and the ways in which fusion genes form and affect cellular function. We also describe computational methodologies for detecting fusion genes from high-throughput sequencing experiments, and the most common sources of error that lead to false discovery of fusion genes. PMID:23376639

  2. Portable thermo-powered high-throughput visual electrochemiluminescence sensor.

    PubMed

    Hao, Nan; Xiong, Meng; Zhang, Jia-dong; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-12-17

    This paper describes a portable thermo-powered high-throughput visual electrochemiluminescence (ECL) sensor for the first time. This sensor is composed of a tiny power supply device based on thermal-electrical conversion and a facile prepared array electrode. The ECL detection could be conducted with thermo-power, which is easily accessible. For example, hot water, a bonfire, or a lighted candle enables the detection to be conducted. And the assay can be directly monitored by the naked eye semiquantitatively or smart phones quantitatively. Combined with transparent electrode and array microreactors, a portable high-throughput sensor was achieved. The portable device, avoiding the use of an electrochemical workstation to generate potential and a photomultiplier tube to receive the signal, is not only a valuable addition for traditional methods but also a suitable device for field operation or point-of-care testing. PMID:24215560

  3. High-throughput patterning of photonic structures with tunable periodicity

    PubMed Central

    Kempa, Thomas J.; Bediako, D. Kwabena; Kim, Sun-Kyung; Park, Hong-Gyu; Nocera, Daniel G.

    2015-01-01

    A patterning method termed “RIPPLE” (reactive interface patterning promoted by lithographic electrochemistry) is applied to the fabrication of arrays of dielectric and metallic optical elements. This method uses cyclic voltammetry to impart patterns onto the working electrode of a standard three-electrode electrochemical setup. Using this technique and a template stripping process, periodic arrays of Ag circular Bragg gratings are patterned in a high-throughput fashion over large substrate areas. By varying the scan rate of the cyclically applied voltage ramps, the periodicity of the gratings can be tuned in situ over micrometer and submicrometer length scales. Characterization of the periodic arrays of periodic gratings identified point-like and annular scattering modes at different planes above the structured surface. Facile, reliable, and rapid patterning techniques like RIPPLE may enable the high-throughput and low-cost fabrication of photonic elements and metasurfaces for energy conversion and sensing applications. PMID:25870280

  4. High throughput screening of starch structures using carbohydrate microarrays.

    PubMed

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  5. High-throughput screening to identify inhibitors of lysine demethylases

    PubMed Central

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several high-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the high-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors. PMID:25687466

  6. Perspectives on high-throughput technologies applied to protein crystallization.

    PubMed

    Saridakis, Emmanuel

    2012-07-01

    High-throughput crystallisation requires the rapid and accurate dispensing of protein and precipitating agent solutions at nanovolumes, but does not end there. The choice of the initial screens is very important, especially with respect to the availability of protein material. Data from previous crystallisation experiments that are scattered in the literature and only partially available in databases have to be analysed in efficient ways that will maximise their utility for designing new screens. A larger portion of crystallisation parameter space should be made accessible to screening, through the use of nucleants and seeding. Observation, assessment and scaling up of the crystallisation trials should be efficiently performed and, finally yet importantly, optimisation of conditions must also be adapted to the high-throughput environment. The above requirements are briefly addressed in the following paper. PMID:22489783

  7. High throughput screening of starch structures using carbohydrate microarrays

    PubMed Central

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  8. Characterization of biomimetic calcium phosphate labeled with fluorescent dextran for quantification of osteoclastic activity.

    PubMed

    Maria, Salwa M; Prukner, Christiane; Sheikh, Zeeshan; Müller, Frank A; Komarova, Svetlana V; Barralet, Jake E

    2015-07-01

    Bone resorbing osteoclasts represent an important therapeutic target for diseases associated with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis. The quantification of osteoclast resorptive activity in vitro is widely used for screening new anti-resorptive medications. The aim of this paper was to develop a simplified semi-automated method for the quantification of osteoclastic resorption using fluorescently labeled biomimetic mineral layers which can replace time intensive, often subjective and clearly non-sustainable use of translucent slices of tusks from vulnerable or endangered species such as the elephant. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We confirmed that fluorescent labeling did not interfere with the biomimetic features of hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption. Correlation between our assay and traditional manual measurement techniques was found to be very strong (R(2)=0.99). In addition, we modified the technique to provide depth and volume data of the resorption pits by confocal imaging at defined depths. Thus, our method allows automatic quantification of total osteoclastic resorption as well as additional data not obtainable by the current tusk slice technique offering a better alternative for high throughput screening of potential antiresorptives. PMID:25829107

  9. High-throughput evaluation of synthetic metabolic pathways

    PubMed Central

    Klesmith, Justin R.; Whitehead, Timothy A.

    2016-01-01

    A central challenge in the field of metabolic engineering is the efficient identification of a metabolic pathway genotype that maximizes specific productivity over a robust range of process conditions. Here we review current methods for optimizing specific productivity of metabolic pathways in living cells. New tools for library generation, computational analysis of pathway sequence-flux space, and high-throughput screening and selection techniques are discussed. PMID:27453919

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

    PubMed Central

    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

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

    PubMed

    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

  12. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

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

    PubMed Central

    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

  14. Condor-COPASI: high-throughput computing for biochemical networks

    PubMed Central

    2012-01-01

    Background Mathematical modelling has become a standard technique to improve our understanding of complex biological systems. As models become larger and more complex, simulations and analyses require increasing amounts of computational power. Clusters of computers in a high-throughput computing environment can help to provide the resources required for computationally expensive model analysis. However, exploiting such a system can be difficult for users without the necessary expertise. Results We present Condor-COPASI, a server-based software tool that integrates COPASI, a biological pathway simulation tool, with Condor, a high-throughput computing environment. Condor-COPASI provides a web-based interface, which makes it extremely easy for a user to run a number of model simulation and analysis tasks in parallel. Tasks are transparently split into smaller parts, and submitted for execution on a Condor pool. Result output is presented to the user in a number of formats, including tables and interactive graphical displays. Conclusions Condor-COPASI can effectively use a Condor high-throughput computing environment to provide significant gains in performance for a number of model simulation and analysis tasks. Condor-COPASI is free, open source software, released under the Artistic License 2.0, and is suitable for use by any institution with access to a Condor pool. Source code is freely available for download at http://code.google.com/p/condor-copasi/, along with full instructions on deployment and usage. PMID:22834945

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

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    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

  16. High-Throughput Computational and Experimental Techniques in Structural Genomics

    PubMed Central

    Chance, Mark R.; Fiser, Andras; Sali, Andrej; Pieper, Ursula; Eswar, Narayanan; Xu, Guiping; Fajardo, J. Eduardo; Radhakannan, Thirumuruhan; Marinkovic, Nebojsa

    2004-01-01

    Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell. PMID:15489337

  17. High throughput screening of ferroelectric thin film libraries

    NASA Astrophysics Data System (ADS)

    Schroeter, Christian; Wessler, Berit; Schoenecker, Andreas; Keitel, Uwe; Eng, Lukas M.

    2006-12-01

    High throughput methods can significantly speed up the search for advanced materials in a multidimensional configuration space, hence keeping innovation cycles short. In the search for improved materials, high throughput methods are wanted to optimize composition and processing of promising systems, and to find candidate compounds. Such a method is described here which is applicable to the development of ferroelectric thin films. Libraries with samples of varying chemical composition were produced via the sol-gel route on structured and metallized silicon wafers. To determine the permittivity of the films, automated measurements of film thickness and capacity were established. Furthermore, ferroelectric hysterisis measurements were performed on samples with a particularly high permittivity. This high throughput route, which allows for synthesis and characterization of over hundred samples per day, was proved and tested by means of lead zirconate titanate as a standard material. It was possible to obtain films with remarkable high permittivity and low coercive field at optimal lead zirconate/lead titanate ratio and by compensating for lead loss during processing by finding the optimal lead excess added to the precursor solutions.

  18. Promises and Pitfalls of High-Throughput Biological Assays.

    PubMed

    Finak, Greg; Gottardo, Raphael

    2016-01-01

    This chapter discusses some of the pitfalls encountered when performing biomedical research involving high-throughput "omics" data and presents some strategies and guidelines that researchers should follow when undertaking such studies. We discuss common errors in experimental design and data analysis that lead to irreproducible and non-replicable research and provide some guidelines to avoid these common mistakes so that researchers may have confidence in study outcomes, even if the results are negative. We discuss the importance of ranking and prespecifying hypotheses, performing power analysis, careful experimental design, and preplanning of statistical analyses in order to avoid the "fishing expedition" data analysis strategy, which is doomed to fail. The impact of multiple testing on false-positive rates is discussed, particularly in the context of the analysis of high-throughput data, and methods to correct for it are presented, as well as approaches to detect and correct for experimental biases and batch effects, which often plague high-throughput assays. We highlight the importance of sharing data and analysis code to facilitate reproducibility and present tools and software that are appropriate for this purpose. PMID:27115636

  19. High-Throughput Analysis of RNA Structure and Ribonucleoprotein Assembly

    PubMed Central

    McGinnis, Jennifer L.; Duncan, Caia D. S.; Weeks, Kevin M.

    2016-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes. PMID:20946765

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

    PubMed

    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

  1. High-Throughput Optical Sensing Immunoassays on Smartphone.

    PubMed

    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

  2. Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

    PubMed

    Jang, Mi Seon; Sahastrabuddhe, Sushant; Yun, Cheol-Heui; Han, Seung Hyun; Yang, Jae Seung

    2016-08-01

    Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines. PMID:27216239

  3. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9

    PubMed Central

    Varshney, Gaurav K.; Pei, Wuhong; LaFave, Matthew C.; Idol, Jennifer; Xu, Lisha; Gallardo, Viviana; Carrington, Blake; Bishop, Kevin; Jones, MaryPat; Li, Mingyu; Harper, Ursula; Huang, Sunny C.; Prakash, Anupam; Chen, Wenbiao; Sood, Raman; Ledin, Johan; Burgess, Shawn M.

    2015-01-01

    The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies. PMID:26048245

  4. High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

    PubMed Central

    Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

    2015-01-01

    The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

  5. High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

    PubMed Central

    MacGregor, Paula; Ivens, Alasdair; Shave, Steven; Collie, Iain; Gray, David; Auer, Manfred

    2014-01-01

    In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes. PMID:24442893

  6. A semi-automated system for analysis and storage of SNPs.

    PubMed

    Lehnert, V; Holzwarth, J; Ott, M; Thompson, A; Demmak, S; Foernzler, D

    2001-04-01

    The discovery of single nucleotide polymorphisms ( SNPs) is currently pursued with a tremendous effort. SNPs represent a rich source for molecular markers, since estimations predict six to seven million of these DNA variations in the human genome. A subset of these genetic variants is thought to have a pervasive impact on modern medicine, be it for the elucidation of differential pharmacological response or for the facilitated identification of genes involved in monogenetic and complex human diseases. Here we describe the overall process that leads to the set up of a SNP database. We describe a high-throughput sequencing assay for SNP discovery, automation of the dataflow from the DNA sequencer to the SNP analysis, and the tools to facilitate it. At the end of the process, a web-accessible interface collects the SNP information, which is processed in order to be written into the SNP database and to be available for end users who would like to select appropriate SNPs for their special screening needs. PMID:11295821

  7. Successful completion of a semi-automated enzyme-free cloning method.

    PubMed

    Bonacci, Stefano; Buccato, Scilla; Maione, Domenico; Petracca, Roberto

    2016-09-01

    Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99 %) were effectively amplified using the automated procedure and 286 (87 %) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5 %, respectively). PMID:27507291

  8. High-throughput enantiopurity analysis using enantiomeric DNA-based sensors.

    PubMed

    Feagin, Trevor A; Olsen, David P V; Headman, Zachary C; Heemstra, Jennifer M

    2015-04-01

    Distinguishing between the two enantiomers of a molecule is a challenging task due to their nearly identical physical properties. Time-consuming chromatography methods are typically required for this task, which greatly limits the throughput of analysis. Here we describe a fluorescence-based method for the rapid and high-throughput analysis of both small-molecule enantiopurity and concentration. Our approach relies on selective molecular recognition of one enantiomer of the target molecule using a DNA aptamer, and the ability of aptamer-based biosensors to transduce the presence of a target molecule into a dose-dependent fluorescence signal. The key novel aspect of our approach is the implementation of enantiomeric DNA biosensors, which are synthesized from D- and L-DNA, but labeled with orthogonal fluorophores. According to the principle of reciprocal chiral substrate specificity, these biosensors will bind to opposite enantiomers of the target with equal affinity and selectivity, enabling simultaneous quantification of both enantiomers of the target. Using the previously reported DNA biosensor for L-tyrosinamide (L-Tym), we demonstrate the ability to rapidly and accurately measure both enantiopurity and concentration for mixtures of L- and D-Tym. We also apply our enantiomeric biosensors to the optimization of reaction conditions for the synthesis of D-Tym and provide mathematical modeling to suggest that DNA biosensors having only modest binding selectivity can also be used for fluorescence-based enantiopurity measurement. This research provides a generalizable method for high-throughput analysis of reaction mixtures, which is anticipated to significantly accelerate reaction optimization for the synthesis of high-value chiral small molecules. PMID:25747268

  9. Current in vitro high throughput screening approaches to assess nuclear receptor activation.

    PubMed

    Raucy, Judy L; Lasker, Jerome M

    2010-11-01

    The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening. PMID:21189134

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

    PubMed

    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

  11. Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

    PubMed

    Jully, Vanessa; Mathot, Frédéric; Moniotte, Nicolas; Préat, Véronique; Lemoine, Dominique

    2016-06-01

    The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), β-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of β-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < β-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant. PMID:27238481

  12. A High-Throughput Biophotonics Instrument to Screen for Novel Ocular Photosensitizing Therapeutic Agents

    PubMed Central

    Butler, Mark C.; Itotia, Patrick N.

    2010-01-01

    Purpose. High-throughput techniques are needed to identify and optimize novel photodynamic therapy (PDT) agents with greater efficacy and to lower toxicity. Novel agents with the capacity to completely ablate pathologic angiogenesis could be of substantial utility in diseases such as wet age-related macular degeneration (AMD). Methods. An instrument and approach was developed based on light-emitting diode (LED) technology for high-throughput screening (HTS) of libraries of potential chemical and biological photosensitizing agents. Ninety-six-well LED arrays were generated at multiple wavelengths and under rigorous intensity control. Cell toxicity was measured in 96-well culture arrays with the nuclear dye SYTOX Green (Invitrogen-Molecular Probes, Eugene, OR). Results. Rapid screening of photoactivatable chemicals or biological molecules has been realized in 96-well arrays of cultured human cells. This instrument can be used to identify new PDT agents that exert cell toxicity on presentation of light of the appropriate energy. The system is further demonstrated through determination of the dose dependence of model compounds having or lacking cellular phototoxicity. Killer Red (KR), a genetically encoded red fluorescent protein expressed from transfected plasmids, is examined as a potential cellular photosensitizing agent and offers unique opportunities as a cell-type–specific phototoxic protein. Conclusions. This instrument has the capacity to screen large chemical or biological libraries for rapid identification and optimization of potential novel phototoxic lead candidates. KR and its derivatives have unique potential in ocular gene therapy for pathologic angiogenesis or tumors. PMID:19834043

  13. High-throughput transformation method for Yarrowia lipolytica mutant library screening.

    PubMed

    Leplat, Christophe; Nicaud, Jean-Marc; Rossignol, Tristan

    2015-09-01

    As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided. PMID:26100263

  14. A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus

    PubMed Central

    Cai, Wei; Tang, Zi-Min; Wen, Gui-Ping; Wang, Si-Ling; Ji, Wen-Fang; Yang, Min; Ying, Dong; Zheng, Zi-Zheng; Xia, Ning-Shao

    2016-01-01

    Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368–606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques’ sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses. PMID:27122081

  15. High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

    PubMed Central

    Faruqi, A Fawad; Hosono, Seiyu; Driscoll, Mark D; Dean, Frank B; Alsmadi, Osama; Bandaru, Rajanikanta; Kumar, Gyanendra; Grimwade, Brian; Zong, Qiuling; Sun, Zhenyu; Du, Yuefen; Kingsmore, Stephen; Knott, Tim; Lasken, Roger S

    2001-01-01

    Background Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. Results SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. Conclusions Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring. PMID:11511324

  16. A semi-automated “blanket” method for renal segmentation from non-contrast T1-weighted MR images

    PubMed Central

    Lim, Jeremy C.; Wake, Nicole; Seah, Jas-mine; Botterill, Elissa; Farquharson, Shawna; Mikheev, Artem; Lim, Ruth P.

    2016-01-01

    Objective To investigate the precision and accuracy of a new semi-automated method for kidney segmentation from single-breath-hold non-contrast MRI. Materials and methods The user draws approximate kidney contours on every tenth slice, focusing on separating adjacent organs from the kidney. The program then performs a sequence of fully automatic steps: contour filling, interpolation, non-uniformity correction, sampling of representative parenchyma signal, and 3D binary morphology. Three independent observers applied the method to images of 40 kidneys ranging in volume from 94.6 to 254.5 cm3. Manually constructed reference masks were used to assess accuracy. Results The volume errors for the three readers were: 4.4 % ± 3.0 %, 2.9 % ± 2.3 %, and 3.1 % ± 2.7 %. The relative discrepancy across readers was 2.5 % ± 2.1 %. The interactive processing time on average was 1.5 min per kidney. Conclusions Pending further validation, the semi-automated method could be applied for monitoring of renal status using non-contrast MRI. PMID:26516082

  17. Neurodegenerative changes in Alzheimer's disease: a comparative study of manual, semi-automated, and fully automated assessment using MRI

    NASA Astrophysics Data System (ADS)

    Fritzsche, Klaus H.; Giesel, Frederik L.; Heimann, Tobias; Thomann, Philipp A.; Hahn, Horst K.; Pantel, Johannes; Schröder, Johannes; Essig, Marco; Meinzer, Hans-Peter

    2008-03-01

    Objective quantification of disease specific neurodegenerative changes can facilitate diagnosis and therapeutic monitoring in several neuropsychiatric disorders. Reproducibility and easy-to-perform assessment are essential to ensure applicability in clinical environments. Aim of this comparative study is the evaluation of a fully automated approach that assesses atrophic changes in Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI). 21 healthy volunteers (mean age 66.2), 21 patients with MCI (66.6), and 10 patients with AD (65.1) were enrolled. Subjects underwent extensive neuropsychological testing and MRI was conducted on a 1.5 Tesla clinical scanner. Atrophic changes were measured automatically by a series of image processing steps including state of the art brain mapping techniques. Results were compared with two reference approaches: a manual segmentation of the hippocampal formation and a semi-automated estimation of temporal horn volume, which is based upon interactive selection of two to six landmarks in the ventricular system. All approaches separated controls and AD patients significantly (10 -5 < p < 10 -4) and showed a slight but not significant increase of neurodegeneration for subjects with MCI compared to volunteers. The automated approach correlated significantly with the manual (r = -0.65, p < 10 -6) and semi automated (r = -0.83, p < 10 -13) measurements. It proved high accuracy and at the same time maximized observer independency, time reduction and thus usefulness for clinical routine.

  18. Controlling high-throughput manufacturing at the nano-scale

    NASA Astrophysics Data System (ADS)

    Cooper, Khershed P.

    2013-09-01

    Interest in nano-scale manufacturing research and development is growing. The reason is to accelerate the translation of discoveries and inventions of nanoscience and nanotechnology into products that would benefit industry, economy and society. Ongoing research in nanomanufacturing is focused primarily on developing novel nanofabrication techniques for a variety of applications—materials, energy, electronics, photonics, biomedical, etc. Our goal is to foster the development of high-throughput methods of fabricating nano-enabled products. Large-area parallel processing and highspeed continuous processing are high-throughput means for mass production. An example of large-area processing is step-and-repeat nanoimprinting, by which nanostructures are reproduced again and again over a large area, such as a 12 in wafer. Roll-to-roll processing is an example of continuous processing, by which it is possible to print and imprint multi-level nanostructures and nanodevices on a moving flexible substrate. The big pay-off is high-volume production and low unit cost. However, the anticipated cost benefits can only be realized if the increased production rate is accompanied by high yields of high quality products. To ensure product quality, we need to design and construct manufacturing systems such that the processes can be closely monitored and controlled. One approach is to bring cyber-physical systems (CPS) concepts to nanomanufacturing. CPS involves the control of a physical system such as manufacturing through modeling, computation, communication and control. Such a closely coupled system will involve in-situ metrology and closed-loop control of the physical processes guided by physics-based models and driven by appropriate instrumentation, sensing and actuation. This paper will discuss these ideas in the context of controlling high-throughput manufacturing at the nano-scale.

  19. Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    PubMed Central

    Duffy, Sandra; Avery, Vicky M.

    2012-01-01

    With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4′,6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5–0.6, with signal-to-noise ratios of 12. PMID:22232455

  20. Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

    PubMed Central

    Park, Sung Yong; Goeken, Nolan; Lee, Hyo Jin; Bolan, Robert; Dubé, Michael P.

    2014-01-01

    ABSTRACT Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10−4 per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. IMPORTANCE Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic

  1. High-throughput sequencing: a roadmap toward community ecology.

    PubMed

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-04-01

    High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

  2. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  3. Adaptive Sampling for High Throughput Data Using Similarity Measures

    SciTech Connect

    Bulaevskaya, V.; Sales, A. P.

    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.

  4. Creation of a small high-throughput screening facility.

    PubMed

    Flak, Tod

    2009-01-01

    The creation of a high-throughput screening facility within an organization is a difficult task, requiring a substantial investment of time, money, and organizational effort. Major issues to consider include the selection of equipment, the establishment of data analysis methodologies, and the formation of a group having the necessary competencies. If done properly, it is possible to build a screening system in incremental steps, adding new pieces of equipment and data analysis modules as the need grows. Based upon our experience with the creation of a small screening service, we present some guidelines to consider in planning a screening facility. PMID:19551356

  5. Orchestrating high-throughput genomic analysis with Bioconductor.

    PubMed

    Huber, Wolfgang; Carey, Vincent J; Gentleman, Robert; Anders, Simon; Carlson, Marc; Carvalho, Benilton S; Bravo, Hector Corrada; Davis, Sean; Gatto, Laurent; Girke, Thomas; Gottardo, Raphael; Hahne, Florian; Hansen, Kasper D; Irizarry, Rafael A; Lawrence, Michael; Love, Michael I; MacDonald, James; Obenchain, Valerie; Oleś, Andrzej K; Pagès, Hervé; Reyes, Alejandro; Shannon, Paul; Smyth, Gordon K; Tenenbaum, Dan; Waldron, Levi; Morgan, Martin

    2015-02-01

    Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors. PMID:25633503

  6. High throughput computing: a solution for scientific analysis

    USGS Publications Warehouse

    O'Donnell, M.

    2011-01-01

    handle job failures due to hardware, software, or network interruptions (obviating the need to manually resubmit the job after each stoppage); be affordable; and most importantly, allow us to complete very large, complex analyses that otherwise would not even be possible. In short, we envisioned a job-management system that would take advantage of unused FORT CPUs within a local area network (LAN) to effectively distribute and run highly complex analytical processes. What we found was a solution that uses High Throughput Computing (HTC) and High Performance Computing (HPC) systems to do exactly that (Figure 1).

  7. A probabilistic approach to high throughput drug discovery.

    PubMed

    Labute, Paul; Nilar, Shahul; Williams, Christopher

    2002-03-01

    A methodology is presented in which high throughput screening experimental data are used to construct a probabilistic QSAR model which is subsequently used to select building blocks for a virtual combinatorial library. The methodology is based upon statistical probability estimation and not regression. The methodology is applied to the construction of two focused virtual combinatorial libraries: one for cyclic GMP phosphodiesterase type V inhibitors and one for acyl-CoA:cholesterol O-acyltransferase inhibitors. The results suggest that the methodology is capable of selecting combinatorial substituents that lead to active compounds starting with binary (pass/fail) activity measurements. PMID:11966422

  8. SSFinder: high throughput CRISPR-Cas target sites prediction tool.

    PubMed

    Upadhyay, Santosh Kumar; Sharma, Shailesh

    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 with Windows, Mac OS, and Linux operating systems, and freely available online. PMID:25089276

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

    SciTech Connect

    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.

  10. Towards A Fully Automated High-Throughput Phototransfection System

    PubMed Central

    Cappelleri, David J.; Halasz, Adam; Sul, Jai-Yoon; Kim, Tae Kyung; Eberwine, James; Kumar, Vijay

    2010-01-01

    We have designed and implemented a framework for creating a fully automated high-throughput phototransfection system. Integrated image processing, laser target position calculation, and stage movements show a throughput increase of > 23X over the current manual phototransfection method while the potential for even greater throughput improvements (> 110X) is described. A software tool for automated off-line single cell morphological measurements, as well as real-time image segmentation analysis, has also been constructed and shown to be able quantify changes in the cell before and after the process, successfully characterizing them, using metrics such as cell perimeter, area, major and minor axis length, and eccentricity values. PMID:20706617

  11. Extended length microchannels for high density high throughput electrophoresis systems

    DOEpatents

    Davidson, James C.; Balch, Joseph W.

    2000-01-01

    High throughput electrophoresis systems which provide extended well-to-read distances on smaller substrates, thus compacting the overall systems. The electrophoresis systems utilize a high density array of microchannels for electrophoresis analysis with extended read lengths. The microchannel geometry can be used individually or in conjunction to increase the effective length of a separation channel while minimally impacting the packing density of channels. One embodiment uses sinusoidal microchannels, while another embodiment uses plural microchannels interconnected by a via. The extended channel systems can be applied to virtually any type of channel confined chromatography.

  12. Orchestrating high-throughput genomic analysis with Bioconductor

    PubMed Central

    Huber, Wolfgang; Carey, Vincent J.; Gentleman, Robert; Anders, Simon; Carlson, Marc; Carvalho, Benilton S.; Bravo, Hector Corrada; Davis, Sean; Gatto, Laurent; Girke, Thomas; Gottardo, Raphael; Hahne, Florian; Hansen, Kasper D.; Irizarry, Rafael A.; Lawrence, Michael; Love, Michael I.; MacDonald, James; Obenchain, Valerie; Oleś, Andrzej K.; Pagès, Hervé; Reyes, Alejandro; Shannon, Paul; Smyth, Gordon K.; Tenenbaum, Dan; Waldron, Levi; Morgan, Martin

    2015-01-01

    Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors. PMID:25633503

  13. Live Cell Optical Sensing for High Throughput Applications

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    Live cell optical sensing employs label-free optical biosensors to non-invasively measure stimulus-induced dynamic mass redistribution (DMR) in live cells within the sensing volume of the biosensor. The resultant DMR signal is an integrated cellular response, and reflects cell signaling mediated through the cellular target(s) with which the stimulus intervenes. This article describes the uses of live cell optical sensing for probing cell biology and ligand pharmacology, with an emphasis of resonant waveguide grating biosensor cellular assays for high throughput applications.

  14. High-Throughput Sequencing: A Roadmap Toward Community Ecology

    PubMed Central

    Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

    2013-01-01

    High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

  15. A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

    PubMed Central

    Fan, Jun; de Jonge, Boudewijn L. M.; MacCormack, Kathy; Sriram, Shubha; McLaughlin, Robert E.; Plant, Helen; Preston, Marian; Fleming, Paul R.; Albert, Robert; Foulk, Melinda

    2014-01-01

    Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening. PMID:25246396

  16. Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis

    PubMed Central

    Yu, Ming; Wang, Qingsong; Patterson, James E.; Woolley, Adam T.

    2011-01-01

    It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to 8 samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to 8 samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid and high-throughput analysis. PMID:21449615

  17. Robo-Lector – a novel platform for automated high-throughput cultivations in microtiter plates with high information content

    PubMed Central

    Huber, Robert; Ritter, Daniel; Hering, Till; Hillmer, Anne-Kathrin; Kensy, Frank; Müller, Carsten; Wang, Le; Büchs, Jochen

    2009-01-01

    Background In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays. Results To meet this demand, a novel automated microfermentation platform consisting of a BioLector and a liquid-handling robot (Robo-Lector) was sucessfully built and tested. The BioLector provides a cultivation system that is able to permanently monitor microbial growth and the fluorescence of reporter proteins under defined conditions in microtiter plates. Three examplary methods were programed on the Robo-Lector platform to study in detail high-throughput cultivation processes and especially recombinant protein expression. The host/vector system E. coli BL21(DE3) pRhotHi-2-EcFbFP, expressing the fluorescence protein EcFbFP, was hereby investigated. With the method 'induction profiling' it was possible to conduct 96 different induction experiments (varying inducer concentrations from 0 to 1.5 mM IPTG at 8 different induction times) simultaneously in an automated way. The method 'biomass-specific induction' allowed to automatically induce cultures with different growth kinetics in a microtiter plate at the same biomass concentration, which resulted in a relative standard deviation of the EcFbFP production of only ± 7%. The third method 'biomass-specific replication' enabled to generate equal initial biomass concentrations in main cultures from precultures with different growth kinetics. This was realized by automatically transferring an appropiate inoculum volume from the different preculture microtiter wells to respective wells of the main culture plate, where

  18. An Improved Method for High-throughput Discrimination and Enumeration of Sedimentary Cells Using Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Morono, Y.; Kallmeyer, J.; Terada, T.; Inagaki, F.; IODP Expedition 329 Shipboard Science Party

    2011-12-01

    Detection and enumeration of microbial life in marine subsurface environments provides primary information on the extent and habitability of the Earth's biosphere. Flow cytometry (FCM) is a powerful tool for identifying and enumerating fluorescence-stained cells with high throughput, using fluorescent intensity, range of wavelength, and cell size. FCM is widely used in medical sciences and aquatic microbial ecology. However, mineral grains and difficulties in distinguishing between life cells and non-specific background fluorescence prevented FCM to be applied for counting microbial cells in sediment or rock samples. SYBR Green I-stained cells can be distinguished from non-biological background signals based on differences in their fluorescence spectra. Here we extended this technique to FCM analysis by modifying the cell detachment protocol using a density gradient method, and then standardized an FCM cell counting method for various types of marine subsurface sediments. Microbial cells in sediment samples could effectively be detached and analyzed discriminatively with FCM. The high capacity of FCM to count particles (up to 10,000 cells/sec) and its high sensitivity will provide information about microbial cell abundance at high spatial resolution and with unprecedented accuracy. This improved cell count method will be useful to evaluate samples with high depth resolution, including narrow geochemical and geological interfaces as potential specific microbial niches, and may even help to asses very low population densities at the fringe of the biosphere.

  19. High-Throughput Screening for Ligands of the HEPN Domain of Sacsin

    PubMed Central

    Li, Xinlu; Ménade, Marie; Kozlov, Guennadi; Hu, Zheping; Dai, Zheng; McPherson, Peter S.; Brais, Bernard; Gehring, Kalle

    2015-01-01

    Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons. PMID:26366743

  20. Robotic injection of zebrafish embryos for high-throughput screening in disease models.

    PubMed

    Spaink, Herman P; Cui, Chao; Wiweger, Malgorzata I; Jansen, Hans J; Veneman, Wouter J; Marín-Juez, Rubén; de Sonneville, Jan; Ordas, Anita; Torraca, Vincenzo; van der Ent, Wietske; Leenders, William P; Meijer, Annemarie H; Snaar-Jagalska, B Ewa; Dirks, Ron P

    2013-08-15

    The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines. PMID:23769806

  1. A Quantitative High-Throughput Screening Data Analysis Pipeline for Activity Profiling.

    PubMed

    Huang, Ruili

    2016-01-01

    The US Tox21 program has developed in vitro assays to test large collections of environmental chemicals in a quantitative high-throughput screening (qHTS) format, using triplicate 15-dose titrations to generate over 50 million data points to date. Counter screens are also employed to minimize interferences from non-target-specific assay artifacts, such as compound auto fluorescence and cytotoxicity. New data analysis approaches are needed to integrate these data and characterize the activities observed from these assays. Here, we describe a complete analysis pipeline that evaluates these qHTS data for technical quality in terms of signal reproducibility. We integrate signals from repeated assay runs, primary readouts, and counter screens to produce a final call on on-target compound activity. PMID:27518629

  2. Computational analysis of high-throughput flow cytometry data

    PubMed Central

    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

  3. High-throughput protein analysis integrating bioinformatics and experimental assays.

    PubMed

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

  4. High-throughput technology for novel SO2 oxidation catalysts

    NASA Astrophysics Data System (ADS)

    Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F.

    2011-10-01

    We review the state of the art and explain the need for better SO2 oxidation catalysts for the production of sulfuric acid. A high-throughput technology has been developed for the study of potential catalysts in the oxidation of SO2 to SO3. High-throughput methods are reviewed and the problems encountered with their adaptation to the corrosive conditions of SO2 oxidation are described. We show that while emissivity-corrected infrared thermography (ecIRT) can be used for primary screening, it is prone to errors because of the large variations in the emissivity of the catalyst surface. UV-visible (UV-Vis) spectrometry was selected instead as a reliable analysis method of monitoring the SO2 conversion. Installing plain sugar absorbents at reactor outlets proved valuable for the detection and quantitative removal of SO3 from the product gas before the UV-Vis analysis. We also overview some elements used for prescreening and those remaining after the screening of the first catalyst generations.

  5. Empirical assessment of sequencing errors for high throughput pyrosequencing data

    PubMed Central

    2013-01-01

    Background Sequencing-by-synthesis technologies significantly improve over the Sanger method in terms of speed and cost per base. However, they still usually fail to compete in terms of read length and quality. Current high-throughput implementations of the pyrosequencing technique yield reads whose length approach those of the capillary electrophoresis method. A less obvious question is whether their quality is affected by platform-specific sequencing errors. Results We present an empirical study aimed at assessing the quality and characterising sequencing errors for high throughput pyrosequencing data. We have developed a procedure for extracting sequencing error data from genome assemblies and study their characteristics, in particular the length distribution of indel gaps and their relation to the sequence contexts where they occur. We used this procedure to analyse data from three prokaryotic genomes sequenced with the GS FLX technology. We also compared two models previously employed with success for peptide sequence alignment. Conclusions We observed an overall very low error rate in the analysed data, with indel errors being much more abundant than substitutions. We also observed a dependence between the length of the gaps and that of the homopolymer context where they occur. As with protein alignments, a power-law model seems to approximate the indel errors more accurately, although the results are not so conclusive as to justify a depart from the commonly used affine gap penalty scheme. In whichever case, however, our procedure can be used to estimate more realistic error model parameters. PMID:23339526

  6. Human transcriptome array for high-throughput clinical studies

    PubMed Central

    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

  7. Advances, practice, and clinical perspectives in high-throughput sequencing.

    PubMed

    Park, S-J; Saito-Adachi, M; Komiyama, Y; Nakai, K

    2016-07-01

    Remarkable advances in high-throughput sequencing technologies have fundamentally changed our understanding of the genetic and epigenetic molecular bases underlying human health and diseases. As these technologies continue to revolutionize molecular biology leading to fresh perspectives, it is imperative to thoroughly consider the enormous excitement surrounding the technologies by highlighting the characteristics of platforms and their global trends as well as potential benefits and limitations. To date, with a variety of platforms, the technologies provide an impressive range of applications, including sequencing of whole genomes and transcriptomes, identifying of genome modifications, and profiling of protein interactions. Because these applications produce a flood of data, simultaneous development of bioinformatics tools is required to efficiently deal with the big data and to comprehensively analyze them. This review covers the major achievements and performances of the high-throughput sequencing and further summarizes the characteristics of their applications along with introducing applicable bioinformatics tools. Moreover, a step-by-step procedure for a practical transcriptome analysis is described employing an analytical pipeline. Clinical perspectives with special consideration to human oral health and diseases are also covered. PMID:26602181

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

    PubMed

    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

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

    SciTech Connect

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

    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.

  10. Polymer Microarrays for High Throughput Discovery of Biomaterials

    PubMed Central

    Hook, Andrew L.; Chang, Chien-Yi; Yang, Jing; Scurr, David J.; Langer, Robert; Anderson, Daniel G.; Atkinson, Steve; Williams, Paul; Davies, Martyn C.; Alexander, Morgan R.

    2012-01-01

    The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3. PMID:22314927

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

    NASA Astrophysics Data System (ADS)

    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.

  12. High-throughput screening to enhance oncolytic virus immunotherapy.

    PubMed

    Allan, K J; Stojdl, David F; Swift, S L

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms - including those based on herpes simplex virus, reovirus, and vaccinia virus - have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  13. High-throughput characterization for solar fuels materials discovery

    NASA Astrophysics Data System (ADS)

    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.

  14. Piezo-thermal Probe Array for High Throughput Applications

    PubMed Central

    Gaitas, Angelo; French, Paddy

    2012-01-01

    Microcantilevers are used in a number of applications including atomic-force microscopy (AFM). In this work, deflection-sensing elements along with heating elements are integrated onto micromachined cantilever arrays to increase sensitivity, and reduce complexity and cost. An array of probes with 5–10 nm gold ultrathin film sensors on silicon substrates for high throughput scanning probe microscopy is developed. The deflection sensitivity is 0.2 ppm/nm. Plots of the change in resistance of the sensing element with displacement are used to calibrate the probes and determine probe contact with the substrate. Topographical scans demonstrate high throughput and nanometer resolution. The heating elements are calibrated and the thermal coefficient of resistance (TCR) is 655 ppm/K. The melting temperature of a material is measured by locally heating the material with the heating element of the cantilever while monitoring the bending with the deflection sensing element. The melting point value measured with this method is in close agreement with the reported value in literature. PMID:23641125

  15. Versatile protein biotinylation strategies for potential high-throughput proteomics.

    PubMed

    Lue, Rina Y P; Chen, Grace Y J; Hu, Yi; Zhu, Qing; Yao, Shao Q

    2004-02-01

    We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo. PMID:14746473

  16. Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms

    PubMed Central

    Pacheco, Maria P.; Pfau, Thomas; Sauter, Thomas

    2016-01-01

    Recent progress in high-throughput data acquisition has shifted the focus from data generation to processing and understanding of how to integrate collected information. Context specific reconstruction based on generic genome scale models like ReconX or HMR has the potential to become a diagnostic and treatment tool tailored to the analysis of specific individuals. The respective computational algorithms require a high level of predictive power, robustness and sensitivity. Although multiple context specific reconstruction algorithms were published in the last 10 years, only a fraction of them is suitable for model building based on human high-throughput data. Beside other reasons, this might be due to problems arising from the limitation to only one metabolic target function or arbitrary thresholding. This review describes and analyses common validation methods used for testing model building algorithms. Two major methods can be distinguished: consistency testing and comparison based testing. The first is concerned with robustness against noise, e.g., missing data due to the impossibility to distinguish between the signal and the background of non-specific binding of probes in a microarray experiment, and whether distinct sets of input expressed genes corresponding to i.e., different tissues yield distinct models. The latter covers methods comparing sets of functionalities, comparison with existing networks or additional databases. We test those methods on several available algorithms and deduce properties of these algorithms that can be compared with future developments. The set of tests performed, can therefore serve as a benchmarking procedure for future algorithms. PMID:26834640

  17. High-throughput screening to enhance oncolytic virus immunotherapy

    PubMed Central

    Allan, KJ; Stojdl, David F; Swift, SL

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  18. High-throughput proteomics and the fight against pathogens.

    PubMed

    Horvatić, Anita; Kuleš, Josipa; Guillemin, Nicolas; Galan, Asier; Mrljak, Vladimir; Bhide, Mangesh

    2016-07-19

    Pathogens pose a major threat to human and animal welfare. Understanding the interspecies host-pathogen protein-protein interactions could lead to the development of novel strategies to combat infectious diseases through the rapid development of new therapeutics. The first step in understanding the host-pathogen crosstalk is to identify interacting proteins in order to define crucial hot-spots in the host-pathogen interactome, such as the proposed pharmaceutical targets by means of high-throughput proteomic methodologies. In order to obtain holistic insight into the inter- and intra-species bimolecular interactions, apart from the proteomic approach, sophisticated in silico modeling is used to correlate the obtained large data sets with other omics data and clinical outcomes. Since the main focus in this area has been directed towards human medicine, it is time to extrapolate the existing expertise to a new emerging field: the 'systems veterinary medicine'. Therefore, this review addresses high-throughput mass spectrometry-based technology for monitoring protein-protein interactions in vitro and in vivo and discusses pathogen cultivation, model host cells and available bioinformatic tools employed in vaccine development. PMID:27227577

  19. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  20. High-throughput search for improved transparent conducting oxides

    NASA Astrophysics Data System (ADS)

    Miglio, Anna

    High-throughput methodologies are a very useful computational tool to explore the space of binary and ternary oxides. We use these methods to search for new and improved transparent conducting oxides (TCOs). TCOs exhibit both visible transparency and good carrier mobility and underpin many energy and electronic applications (e.g. photovoltaics, transparent transistors). We find several potential new n-type and p-type TCOs with a low effective mass. Combining different ab initio approaches, we characterize candidate oxides by their effective mass (mobility), band gap (transparency) and dopability. We present several compounds, not considered previously as TCOs, and discuss the chemical rationale for their promising properties. This analysis is useful to formulate design strategies for future high mobility oxides and has led to follow-up studies including preliminary experimental characterization of a p-type TCO candidate with unexpected chemistry. G. Hautier, A. Miglio, D. Waroquiers, G.-M. Rignanese, and X. Gonze, ``How Does Chemistry Influence Electron Effective Mass in Oxides? A High-Throughput Computational Analysis'', Chem. Mater. 26, 5447 (2014). G. Hautier, A. Miglio, G. Ceder, G.-M. Rignanese, and X. Gonze, ``Identification and design principles of low hole effective mass p-type transparent conducting oxides'', Nature Commun. 4, 2292 (2013).

  1. A High Throughput Mechanical Screening Device for Cartilage Tissue Engineering

    PubMed Central

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

    2014-01-01

    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. PMID:24275442

  2. Structuring intuition with theory: The high-throughput way

    NASA Astrophysics Data System (ADS)

    Fornari, Marco

    2015-03-01

    First principles methodologies have grown in accuracy and applicability to the point where large databases can be built, shared, and analyzed with the goal of predicting novel compositions, optimizing functional properties, and discovering unexpected relationships between the data. In order to be useful to a large community of users, data should be standardized, validated, and distributed. In addition, tools to easily manage large datasets should be made available to effectively lead to materials development. Within the AFLOW consortium we have developed a simple frame to expand, validate, and mine data repositories: the MTFrame. Our minimalistic approach complement AFLOW and other existing high-throughput infrastructures and aims to integrate data generation with data analysis. We present few examples from our work on materials for energy conversion. Our intent s to pinpoint the usefulness of high-throughput methodologies to guide the discovery process by quantitatively structuring the scientific intuition. This work was supported by ONR-MURI under Contract N00014-13-1-0635 and the Duke University Center for Materials Genomics.

  3. High-throughput GPU-based LDPC decoding

    NASA Astrophysics Data System (ADS)

    Chang, Yang-Lang; Chang, Cheng-Chun; Huang, Min-Yu; Huang, Bormin

    2010-08-01

    Low-density parity-check (LDPC) code is a linear block code known to approach the Shannon limit via the iterative sum-product algorithm. LDPC codes have been adopted in most current communication systems such as DVB-S2, WiMAX, WI-FI and 10GBASE-T. LDPC for the needs of reliable and flexible communication links for a wide variety of communication standards and configurations have inspired the demand for high-performance and flexibility computing. Accordingly, finding a fast and reconfigurable developing platform for designing the high-throughput LDPC decoder has become important especially for rapidly changing communication standards and configurations. In this paper, a new graphic-processing-unit (GPU) LDPC decoding platform with the asynchronous data transfer is proposed to realize this practical implementation. Experimental results showed that the proposed GPU-based decoder achieved 271x speedup compared to its CPU-based counterpart. It can serve as a high-throughput LDPC decoder.

  4. A timetable organizer for the planning and implementation of screenings in manual or semi-automation mode.

    PubMed

    Goktug, Asli N; Chai, Sergio C; Chen, Taosheng

    2013-09-01

    We have designed an Excel spreadsheet to facilitate the planning and execution of screenings performed manually or in semi-automation mode, following a sequential set of events. Many assays involve multiple steps, often including time-sensitive stages, thus complicating the proper implementation to ensure that all plates are treated equally to achieve reliable outcomes. The spreadsheet macro presented in this study analyzes and breaks down the timings for all tasks, calculates the limitation in the number of plates that suit the desired parameters, and allows for optimization based on tolerance of time delay and equal treatment of plates when possible. The generated Gantt charts allow for visual inspection of the screening process and provide timings in a tabulated form to assist the user to conduct the experiments as projected by the software. The program can be downloaded from http://sourceforge.net/projects/sams-hts/. PMID:23653394

  5. Semi-Automated Detection Of Terrain Activity In The Swiss Alpine Periglacial Environment From DInSAR Scenes

    NASA Astrophysics Data System (ADS)

    Barboux, Chloe; Dalaloye, Reynald; Lambiel, Christophe; Strozzi, Tazio; Raetzo, Hugo; Collet, Claude

    2013-12-01

    This paper proposes a semi-automated method to update inventory of moving slopes. First a Map of Terrain Activity (MTA) is created by partitioning an interferogram, using segmentation and classification processes, into 3 regions: stable areas, coherently moving parts and decorrelated areas (due to motion or not). Then, a Combined-Map of Terrain Activity (C- MTA) is computed describing the general behavior of the terrain at a specific time-lapse. Finally, C-MTA is used to determine the potential change in the activity rate of moving slopes. Tests are performed in a small area using large set of TSX DInSAR scenes from summers 2008 to 2012 in order to update past moving slope inventories produced from ERS DInSAR data.

  6. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    PubMed Central

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  7. Semi-automated 2D Bruch's membrane shape analysis in papilledema using spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Wang, Jui-Kai; Sibony, Patrick A.; Kardon, Randy H.; Kupersmith, Mark J.; Garvin, Mona K.

    2015-03-01

    Recent studies have shown that the Bruch's membrane (BM) and retinal pigment epithelium (RPE), visualized on spectral-domain optical coherence tomography (SD-OCT), is deformed anteriorly towards the vitreous in patients with intracranial hypertension and papilledema. The BM/RPE shape has been quantified using a statistical-shape-model approach; however, to date, the approach has involved the tedious and time-consuming manual placement of landmarks and correspondingly, only the shape (and shape changes) of a limited number of patients has been studied. In this work, we first present a semi-automated approach for the extraction of 20 landmarks along the BM from an optic-nerve-head (ONH) centered OCT slice from each patient. In the approach, after the manual placement of the two Bruch's membrane opening (BMO) points, the remaining 18 landmarks are automatically determined using a graph-based segmentation approach. We apply the approach to the OCT scans of 116 patients (at baseline) enrolled in the Idiopathic Intracranial Hypertension Treatment Trial and generate a statistical shape model using principal components analysis. Using the resulting shape model, the coefficient (shape measure) corresponding to the second principal component (eigenvector) for each set of landmarks indicates the degree of the BM/RPE is oriented away from the vitreous. Using a subset of 20 patients, we compare the shape measure computed using this semi-automated approach with the resulting shape measure when (1) all landmarks are specified manually (Experiment I); and (2) a different expert specifies the two BMO points (Experiment II). In each case, a correlation coefficient >= 0.99 is obtained.

  8. RootScan: Software for high-throughput analysis of root anatomical traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RootScan is a program for semi-automated image analysis of anatomical phenes in root cross-sections. RootScan uses pixel value thresholds to separate the cross-section from its background and to visually dissect it into tissue regions. Area measurements and object counts are performed within various...

  9. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

    PubMed

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  10. Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

    NASA Astrophysics Data System (ADS)

    Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

    2016-03-01

    Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

  11. Intelligent, net or wireless enabled fluorosensors for high throughput monitoring of assorted crops

    NASA Astrophysics Data System (ADS)

    Barócsi, Attila

    2013-02-01

    Phenotypic characterization of assorted crops of different genotypes requires large data sets of diverse types for statistical reliability. Temporal monitoring of plant fluorescence is able to capture the dynamics of the photosynthesis process that is summarized in a number of parameters for which the genotypic heritability can be calculated. In this paper, an intelligent sensor system is presented that is capable of high-throughput production of baseline-corrected temporal fluorescence curves with many feature points. These are obtained by integrating several (direct and modulated) measurement methods applied at different wavelengths. Simultaneously, temporal change of the sample's emission and the ambient reference temperatures are recorded. Multiple sensors can be deployed easily in large span greenhouse environments with centralized data collection over wired or wireless infrastructure. The unique features of the sensors are a compact, embedded signal guiding fibre optic system, instrument-standard variable tubular detector and source modules, net or wireless enabling for remote control and fast, quasi real-time data collection. Along with the instrumentation, some representative phenotyping data are also presented that were taken on a subset of pepper recombinant inbred line population. It is also demonstrated that transient fluorescence feature points yield high heritability, offering a high confidence level for distinguishing the pepper genotypes.

  12. Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.

    PubMed

    Paintdakhi, Ahmad; Parry, Bradley; Campos, Manuel; Irnov, Irnov; Elf, Johan; Surovtsev, Ivan; Jacobs-Wagner, Christine

    2016-02-01

    With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills. PMID:26538279

  13. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    PubMed Central

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  14. Ultra-Sensitive, High Throughput and Quantitative Proteomics Measurements

    SciTech Connect

    Jacobs, Jon M.; Monroe, Matthew E.; Qian, Weijun; Shen, Yufeng; Anderson, Gordon A.; Smith, Richard D.

    2005-02-01

    We describe the broad basis and application of an approach for very high throughput, ultra-sensitive, and quantitative proteomic measurements based upon the use of ultra-high performance separations and mass spectrometry. An overview of the accurate mass and time (AMT) tag approach and a description of the incorporated data analysis pipeline necessary for efficient proteomic studies are presented. Adjunct technologies, including stable-isotope labeling methodologies and improvements in the utilization of LC-MS peak intensity information for quantitative purposes are discussed. Related areas include the use of automated sample handling for improving analysis reproducibility, methods for using information from the separation for more confident peptide peak identification, and the utilization of smaller diameter capillary columns having lower volumetric flow rates to increase electrospray ionization efficiency and allow for more predictable and quantitative results. The developments are illustrated in the context of studies of complex biological systems.

  15. High-throughput analysis of behavior for drug discovery.

    PubMed

    Alexandrov, Vadim; Brunner, Dani; Hanania, Taleen; Leahy, Emer

    2015-03-01

    Drug testing with traditional behavioral assays constitutes a major bottleneck in the development of novel therapies. PsychoGenics developed three comprehensive high-throughput systems, SmartCube(®), NeuroCube(®) and PhenoCube(®) systems, to increase the efficiency of the drug screening and phenotyping in rodents. These three systems capture different domains of behavior, namely, cognitive, motor, circadian, social, anxiety-like, gait and others, using custom-built computer vision software and machine learning algorithms for analysis. This review exemplifies the use of the three systems and explains how they can advance drug screening with their applications to phenotyping of disease models, drug screening, selection of lead candidates, behavior-driven lead optimization, and drug repurposing. PMID:25592319

  16. Proteomics equipped with multiplexing toward ultra high throughput.

    PubMed

    Kim, Min-Sik

    2015-01-01

    MS-based quantitative proteomics is a powerful technology to study virtually almost all biological and clinical samples. Although it has been known to be a high-throughput method, an MS analysis of a higher number of samples remains to be challenging practically and economically. In this issue, the use of multiplexing strategy for quantitative analysis of proteomes and phosphoproteomes has been demonstrated by Paulo et al. (Proteomics 2015, 15, 462-473) to better understand in vivo effects of two small molecule inhibitors on a mouse model. Within the short period of drug treatment, it has been found that the protein alteration is minimal in three tissues tested, whereas the phosphorylation level was widely altered. PMID:25522341

  17. Characterizing immune repertoires by high throughput sequencing: strategies and applications

    PubMed Central

    Calis, Jorg J.A.; Rosenberg, Brad R.

    2014-01-01

    As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system. PMID:25306219

  18. Towards high throughput screening of nanoparticle flotation collectors.

    PubMed

    Abarca, Carla; Yang, Songtao; Pelton, Robert H

    2015-12-15

    To function as flotation collectors for mineral processing, polymeric nanoparticles require a delicate balance of surface properties to give mineral-specific deposition and colloidal stability in high ionic strength alkaline media, while remaining sufficiently hydrophobic to promote flotation. Combinatorial nanoparticle surface modification, in conjunction with high throughput screening, is a promising approach for nanoparticle development. However, efficient automated screening assays are required to reject ineffective particles without having to undergo time consuming flotation testing. Herein we demonstrate that determining critical coagulation concentrations of sodium carbonate in combination with measuring the advancing water contact angle of nanoparticle-saturated glass surfaces can be used to screen ineffective nanoparticles. Finally, none of our first nanoparticle library based on poly(ethylene glycol) methyl ether methacrylate (PEG-methacrylate) were effective flotation collectors because the nanoparticles were too hydrophilic. PMID:26319325

  19. High-throughput screening of microbial adaptation to environmental stress.

    PubMed

    Bélanger, Pier-Anne; Beaudin, Julie; Roy, Sébastien

    2011-05-01

    We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance. PMID:21315114

  20. High Throughput In Situ XAFS Screening of Catalysts

    SciTech Connect

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Schroeder, Sven L. M.; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu

    2007-02-02

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on {gamma}-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

  1. Design and implementation of high throughput screening assays.

    PubMed

    Macarrón, Ricardo; Hertzberg, Robert P

    2011-03-01

    High throughput screening (HTS) is 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 analysis of many variables, starting with the choice of assay target 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 insure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:20865348

  2. Design of a High-Throughput Plasma-Processing System

    SciTech Connect

    Darkazalli, Ghazi; Matthei, Keith; Ruby, Douglas S.

    1999-07-20

    Sandia National Laboratories has demonstrated significant performance gains in crystalline silicon solar cell technology through the use of plasma-processing for the deposition of silicon nitride by Plasma Enhanced Chemical Vapor Deposition (PECVD), plasma-hydrogenation of the nitride layer, and reactive-ion etching of the silicon surface prior to the deposition to decrease the reflectivity of the surface. One of the major problems of implementing plasma processing into a cell production line is the batch configuration and/or low throughput of the systems currently available. This report describes the concept of a new in-line plasma processing system that could meet the industrial requirements for a high-throughput and cost effective solution for mass production of solar cells.

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

    PubMed Central

    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

  4. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

  7. High-throughput sequencing in veterinary infection biology and diagnostics.

    PubMed

    Belák, S; Karlsson, O E; Leijon, M; Granberg, F

    2013-12-01

    Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine. PMID:24761741

  8. Predicting Novel Bulk Metallic Glasses via High- Throughput Calculations

    NASA Astrophysics Data System (ADS)

    Perim, E.; Lee, D.; Liu, Y.; Toher, C.; Gong, P.; Li, Y.; Simmons, W. N.; Levy, O.; Vlassak, J.; Schroers, J.; Curtarolo, S.

    Bulk metallic glasses (BMGs) are materials which may combine key properties from crystalline metals, such as high hardness, with others typically presented by plastics, such as easy processability. However, the cost of the known BMGs poses a significant obstacle for the development of applications, which has lead to a long search for novel, economically viable, BMGs. The emergence of high-throughput DFT calculations, such as the library provided by the AFLOWLIB consortium, has provided new tools for materials discovery. We have used this data to develop a new glass forming descriptor combining structural factors with thermodynamics in order to quickly screen through a large number of alloy systems in the AFLOWLIB database, identifying the most promising systems and the optimal compositions for glass formation. National Science Foundation (DMR-1436151, DMR-1435820, DMR-1436268).

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

    PubMed Central

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

    2015-01-01

    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. (2005). Our learning algorithm has identified, with high confidence, several new crosstalk mechanisms in the T-cell signalling network. Many of them have already been confirmed experimentally in the recent literature and six new crosstalk mechanisms await experimental validation. PMID:19525198

  10. High-throughput sequencing of immune repertoires in multiple sclerosis.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Holmøy, Trygve

    2016-04-01

    T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T- and B-cell receptors. The antigen receptors are highly polymorphic due to recombination (T- and B-cell receptors) and mutation (B-cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T- and B-cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T- and B-cell receptor repertoires using earlier methodological approaches, and we focus on how high-throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth. PMID:27081660

  11. Interactive Visual Analysis of High Throughput Text Streams

    SciTech Connect

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

    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.

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

    SciTech Connect

    Gay, E.C.; Miller, W.E.; Laidler, J.J.

    1996-05-01

    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.

  13. High-throughput determination of RNA structure by proximity ligation.

    PubMed

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-09-01

    We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures. PMID:26237516

  14. Printing Proteins as Microarrays for High-Throughput Function Determination

    NASA Astrophysics Data System (ADS)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  15. High Throughput Substrate Phage Display for Protease Profiling

    PubMed Central

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2012-01-01

    Summary The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  16. High throughput substrate phage display for protease profiling.

    PubMed

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  17. High-throughput ballistic injection nanorheology to measure cell mechanics.

    PubMed

    Wu, Pei-Hsun; Hale, Christopher M; Chen, Wei-Chiang; Lee, Jerry S H; Tseng, Yiider; Wirtz, Denis

    2012-01-01

    High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of the cytoplasm-embedded particles are transformed into mean-squared displacements, which are subsequently transformed into frequency-dependent viscoelastic moduli and time-dependent creep compliance of the cytoplasm. This method allows for the study of a wide range of cellular conditions, including cells inside a 3D matrix, cell subjected to shear flows and biochemical stimuli, and cells in a live animal. Ballistic injection lasts <1 min and is followed by overnight incubation. Multiple particle tracking for one cell lasts <1 min. Forty cells can be examined in <1 h. PMID:22222790

  18. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  19. High-throughput characterization of protein–RNA interactions

    PubMed Central

    Cook, Kate B.; Hughes, Timothy R.

    2015-01-01

    RNA-binding proteins (RBPs) are important regulators of eukaryotic gene expression. Genomes typically encode dozens to hundreds of proteins containing RNA-binding domains, which collectively recognize diverse RNA sequences and structures. Recent advances in high-throughput methods for assaying the targets of RBPs in vitro and in vivo allow large-scale derivation of RNA-binding motifs as well as determination of RNA–protein interactions in living cells. In parallel, many computational methods have been developed to analyze and interpret these data. The interplay between RNA secondary structure and RBP binding has also been a growing theme. Integrating RNA–protein interaction data with observations of post-transcriptional regulation will enhance our understanding of the roles of these important proteins. PMID:25504152

  20. A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

    PubMed Central

    Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

    2015-01-01

    We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

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

    PubMed

    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

  2. Representation and classification for high-throughput data

    NASA Astrophysics Data System (ADS)

    Wessels, Lodewyk F. A.; Reinders, Marcel J. T.; van Welsem, Tibor; Nederlof, Petra M.

    2002-06-01

    Survival prediction and optimal treatment choice for cancer patients are dependent on correct disease classification. This classification can be improved significantly when high- throughput data such as microarray expression analysis is employed. These data sets usually suffer from the dimensionality problem: many features and few patients. Consequently, care must be taken when feature selection is performed and classifiers for disease classification are designed. In this paper we investigate several issues associated with this problem, including 1) data representation; 2) the type of classifier employed and 3) classifier construction, with specific emphasis on feature selection approaches. More specifically, 'filter' and 'wrapper' approaches for feature selection are studied. The different representations, selection criteria, classifiers and feature selection approaches are evaluated with regard to the effect on true classification performance. As test cases we employ a Comparative Genomic Hybridization breast cancer data sets and two publicly available gene expression data sets.

  3. Integrated control system environment for high-throughput tomography

    NASA Astrophysics Data System (ADS)

    Khokhriakov, Igor; Lottermoser, Lars; Gehrke, Rainer; Kracht, Thorsten; Wintersberger, Eugen; Kopmann, Andreas; Vogelgesang, Matthias; Beckmann, Felix

    2014-09-01

    A new control system for high-throughput experiments (X-Ray, Neutrons) is introduced in this article. The system consists of several software components which are required to make optimized use of the beamtime and to fulfill the demand to implement the new standardized data format established within the Helmholtz Association in Germany. The main components are: PreExperiment Data Collector; Status server; Data Format Server. Especially for tomography a concept for an online reconstruction based on GPU computing is presented. One of the main goals of the system is to collect data that extends standard experimental data, e.g. instrument's hardware state, preinvestigation data, experiment description data etc. The collected data is stored together with the experiment data in the permanent storage of the user. The stored data is then used for post processing and analysis of the experiment.

  4. High-throughput rheology in a microfluidic device

    NASA Astrophysics Data System (ADS)

    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).

  5. Biological Processes Discovered by High-Throughput Sequencing.

    PubMed

    Reon, Brian J; Dutta, Anindya

    2016-04-01

    Advances in DNA and RNA sequencing technologies have completely transformed the field of genomics. High-throughput sequencing (HTS) is now a widely used and accessible technology that allows scientists to sequence an entire transcriptome or genome in a timely and cost-effective manner. Application of HTS techniques has led to many key discoveries, including the identification of long noncoding RNAs, microDNAs, a family of small extrachromosomal circular DNA species, and tRNA-derived fragments, which are a group of small non-miRNAs that are derived from tRNAs. Furthermore, public sequencing repositories provide unique opportunities for laboratories to parse large sequencing databases to identify proteins and noncoding RNAs at a scale that was not possible a decade ago. Herein, we review how HTS has led to the discovery of novel nucleic acid species and uncovered new biological processes during the course. PMID:26828742

  6. High throughput parametric studies of the structure of complex nanomaterials

    NASA Astrophysics Data System (ADS)

    Tian, Peng

    The structure of nanoscale materials is difficult to study because crystallography, the gold-standard for structure studies, no longer works at the nanoscale. New tools are needed to study nanostructure. Furthermore, it is important to study the evolution of nanostructure of complex nanostructured materials as a function of various parameters such as temperature or other environmental variables. These are called parametric studies because an environmental parameter is being varied. This means that the new tools for studying nanostructure also need to be extended to work quickly and on large numbers of datasets. This thesis describes the development of new tools for high throughput studies of complex and nanostructured materials, and their application to study the structural evolution of bulk, and nanoparticles of, MnAs as a function of temperature. The tool for high throughput analysis of the bulk material was developed as part of this PhD thesis work and is called SrRietveld. A large part of making a new tool is to validate it and we did this for SrRietveld by carrying out a high-throughput study of uncertainties coming from the program using different ways of estimating the uncertainty. This tool was applied to study structural changes in MnAs as a function of temperature. We were also interested in studying different MnAs nanoparticles fabricated through different methods because of their applications in information storage. PDFgui, an existing tool for analyzing nanoparticles using Pair distribution function (PDF) refinement, was used in these cases. Comparing the results from the analysis by SrRietveld and PDFgui, we got more comprehensive structure information about MnAs. The layout of the thesis is as follows. First, the background knowledge about material structures is given. The conventional crystallographic analysis is introduced in both theoretical and practical ways. For high throughput study, the next-generation Rietveld analysis program: Sr

  7. EDITORIAL: Combinatorial and High-Throughput Materials Research

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Takeuchi, Ichiro

    2005-01-01

    The success of combinatorial and high-throughput methodologies relies greatly on the availability of various characterization tools with new and improved capabilities [1]. Indeed, how useful can a combinatorial library of 250, 400, 25 000 or 2 000 000 compounds be [2-5] if one is unable to characterize its properties of interest fairly quickly? How useful can a set of thousands of spectra or chromatograms be if one is unable to analyse them in a timely manner? For these reasons, the development of new approaches for materials characterization is one of the most active areas in combinatorial materials science. The importance of this aspect of research in the field has been discussed in numerous conferences including the Pittsburgh Conferences, the American Chemical Society Meetings, the American Physical Society Meetings, the Materials Research Society Symposia and various Gordon Research Conferences. Naturally, the development of new measurement instrumentation attracts the attention not only of practitioners of combinatorial materials science but also of those who design new software for data manipulation and mining. Experimental designs of combinatorial libraries are pursued with available and realistic synthetic and characterization capabilities in mind. It is becoming increasingly critical to link the design of new equipment for high-throughput parallel materials synthesis with integrated measurement tools in order to enhance the efficacy of the overall experimental strategy. We have received an overwhelming response to our proposal and call for papers for this Special Issue on Combinatorial Materials Science. The papers in this issue of Measurement Science and Technology are a very timely collection that captures the state of modern combinatorial materials science. They demonstrate the significant advances that are taking place in the field. In some cases, characterization tools are now being operated in the factory mode. At the same time, major challenges

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

    PubMed

    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 <150 nm), the centrifugation-decantation step was not required as the interference was less than with larger particles. Parallel measurements with a ChemiDoc MP plate scanner gave indications of aggregation; however, the results were less sensitive than the absorbance measurements. PMID:26857643

  9. High-Throughput Sequencing of Complete Mitochondrial Genomes.

    PubMed

    Briscoe, Andrew George; Hopkins, Kevin Peter; Waeschenbach, Andrea

    2016-01-01

    Next-generation sequencing has revolutionized mitogenomics, turning a cottage industry into a high throughput process. This chapter outlines methodologies used to sequence, assemble, and annotate mitogenomes of non-model organisms using Illumina sequencing technology, utilizing either long-range PCR amplicons or gDNA as starting template. Instructions are given on how to extract DNA, conduct long-range PCR amplifications, generate short Sanger barcode tag sequences, prepare equimolar sample pools, construct and assess quality library preparations, assemble Illumina reads using either seeded reference mapping or de novo assembly, and annotate mitogenomes in the absence of an automated pipeline. Notes and recommendations, derived from our own experience, are given throughout this chapter. PMID:27460369

  10. Numerical techniques for high-throughput reflectance interference biosensing

    NASA Astrophysics Data System (ADS)

    Sevenler, Derin; Ünlü, M. Selim

    2016-06-01

    We have developed a robust and rapid computational method for processing the raw spectral data collected from thin film optical interference biosensors. We have applied this method to Interference Reflectance Imaging Sensor (IRIS) measurements and observed a 10,000 fold improvement in processing time, unlocking a variety of clinical and scientific applications. Interference biosensors have advantages over similar technologies in certain applications, for example highly multiplexed measurements of molecular kinetics. However, processing raw IRIS data into useful measurements has been prohibitively time consuming for high-throughput studies. Here we describe the implementation of a lookup table (LUT) technique that provides accurate results in far less time than naive methods. We also discuss an additional benefit that the LUT method can be used with a wider range of interference layer thickness and experimental configurations that are incompatible with methods that require fitting the spectral response.

  11. High Throughput In Situ XAFS Screening of Catalysts

    NASA Astrophysics Data System (ADS)

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu; Schroeder, Sven L. M.

    2007-02-01

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on γ-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

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

    PubMed

    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

  13. High-throughput determination of RNA structure by proximity ligation

    PubMed Central

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-01-01

    We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA Proximity Ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle, and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures. PMID:26237516

  14. Microfluidic cell chips for high-throughput drug screening.

    PubMed

    Chi, Chun-Wei; Ahmed, Ah Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

    2016-05-01

    The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

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

    PubMed

    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

  16. Quantitative high throughput analytics to support polysaccharide production process development.

    PubMed

    Noyes, Aaron; Godavarti, Ranga; Titchener-Hooker, Nigel; Coffman, Jonathan; Mukhopadhyay, Tarit

    2014-05-19

    The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned to be central to the improvement of existing polysaccharide manufacturing processes through the identification of critical process parameters that potentially impact the quality attributes of the vaccine and to the development of de novo processes for clinical candidates, across the spectrum of downstream processing. The availability of a fast and automated analytics platform will expand the scope, robustness, and evolution of Design of Experiment (DOE) studies. This paper details recent advances in improving the speed, throughput, and success of in-process analytics at the micro-scale. Two methods, based on modifications of existing procedures, are described for the rapid measurement of polysaccharide titre in microplates without the need for heating steps. A simplification of a commercial endotoxin assay is also described that features a single measurement at room temperature. These assays, along with existing assays for protein and nucleic acids are qualified for deployment in the high throughput screening of polysaccharide feedstreams. Assay accuracy, precision, robustness, interference, and ease of use are assessed and described. In combination, these assays are capable of measuring the product concentration and impurity profile of a microplate of 96 samples in less than one day. This body of work relies on the evaluation of a combination of commercially available and clinically relevant polysaccharides to ensure maximum versatility and reactivity of the final assay suite. Together, these advancements reduce overall process time by up to 30-fold and significantly reduce sample volume over current practices. The

  17. High-Throughput Genomics Enhances Tomato Breeding Efficiency

    PubMed Central

    Barone, A; Di Matteo, A; Carputo, D; Frusciante, L

    2009-01-01

    Tomato (Solanum lycopersicum) is considered a model plant species for a group of economically important crops, such as potato, pepper, eggplant, since it exhibits a reduced genomic size (950 Mb), a short generation time, and routine transformation technologies. Moreover, it shares with the other Solanaceous plants the same haploid chromosome number and a high level of conserved genomic organization. Finally, many genomic and genetic resources are actually available for tomato, and the sequencing of its genome is in progress. These features make tomato an ideal species for theoretical studies and practical applications in the genomics field. The present review describes how structural genomics assist the selection of new varieties resistant to pathogens that cause damage to this crop. Many molecular markers highly linked to resistance genes and cloned resistance genes are available and could be used for a high-throughput screening of multiresistant varieties. Moreover, a new genomics-assisted breeding approach for improving fruit quality is presented and discussed. It relies on the identification of genetic mechanisms controlling the trait of interest through functional genomics tools. Following this approach, polymorphisms in major gene sequences responsible for variability in the expression of the trait under study are then exploited for tracking simultaneously favourable allele combinations in breeding programs using high-throughput genomic technologies. This aims at pyramiding in the genetic background of commercial cultivars alleles that increase their performances. In conclusion, tomato breeding strategies supported by advanced technologies are expected to target increased productivity and lower costs of improved genotypes even for complex traits. PMID:19721805

  18. A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics

    NASA Astrophysics Data System (ADS)

    Lagus, Todd P.; Edd, Jon F.

    2013-03-01

    Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.

  19. High-throughput detection and quantification of mitochondrial fusion through imaging flow cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2016-08-01

    Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to

  20. Semi-automated segmentation of neuroblastoma nuclei using the gradient energy tensor: a user driven approach

    NASA Astrophysics Data System (ADS)

    Kromp, Florian; Taschner-Mandl, Sabine; Schwarz, Magdalena; Blaha, Johanna; Weiss, Tamara; Ambros, Peter F.; Reiter, Michael

    2015-02-01

    We propose a user-driven method for the segmentation of neuroblastoma nuclei in microscopic fluorescence images involving the gradient energy tensor. Multispectral fluorescence images contain intensity and spatial information about antigene expression, fluorescence in situ hybridization (FISH) signals and nucleus morphology. The latter serves as basis for the detection of single cells and the calculation of shape features, which are used to validate the segmentation and to reject false detections. Accurate segmentation is difficult due to varying staining intensities and aggregated cells. It requires several (meta-) parameters, which have a strong influence on the segmentation results and have to be selected carefully for each sample (or group of similar samples) by user interactions. Because our method is designed for clinicians and biologists, who may have only limited image processing background, an interactive parameter selection step allows the implicit tuning of parameter values. With this simple but intuitive method, segmentation results with high precision for a large number of cells can be achieved by minimal user interaction. The strategy was validated on handsegmented datasets of three neuroblastoma cell lines.

  1. Accurate high-throughput identification of parallel G-quadruplex topology by a new tetraaryl-substituted imidazole.

    PubMed

    Hu, Ming-Hao; Chen, Shuo-Bin; Wang, Yu-Qing; Zeng, You-Mei; Ou, Tian-Miao; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-09-15

    G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures. PMID:27104587

  2. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

    PubMed

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R; Fry, Stephen C

    2015-09-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  3. Demonstration of submersible high-throughput microfluidic immunosensors for underwater explosives detection.

    PubMed

    Adams, André A; Charles, Paul T; Deschamps, Jeffrey R; Kusterbeck, Anne W

    2011-11-15

    Significant security threats posed by highly energetic nitroaromatic compounds in aquatic environments and the demilitarization and pending cleanup of areas previously used for munitions manufacture and storage represent a challenge for less expensive, faster, and more sensitive systems capable of analyzing groundwater and seawater samples for trace levels of explosive materials. Presented here is an inexpensive high throughput microfluidic immunosensor (HTMI) platform intended for the rapid, highly selective quantitation of nitroaromatic compounds in the field. Immunoaffinity and fluorescence detection schemes were implemented in tandem on a novel microfluidic device containing 39 parallel microchannels that were 500 μm tall, 250 μm wide, and 2.54 cm long with covalently tethered antibodies that was engineered for high-throughput high-volume sample processing. The devices were produced via a combination of high precision micromilling and hot embossing. Mass transfer limitations were found in conventional microsystems and were minimized due to higher surface area to volume ratios that exceeded those possessed by conventional microdevices and capillaries. Until now, these assays were limited to maximum total volume flow rates of ~1 mL/min due in part to kinetics and high head pressures of single microchannels. In the design demonstrated here, highly parallelized microchannels afforded up to a 100-fold increase in total volume flow rate while maintaining favorable kinetic constraints for efficient antigen-antibody interaction. The assay employed total volume throughput of up to 6 mL/min while yielding signal-to-noise ratios of >15 in all cases. In addition to samples being processed up to 60 times faster than in conventional displacement-based immunoassays, the current system was capable of quantitating 0.01 ng/mL TNT samples without implementing offline preconcentration, thereby, demonstrating the ability to improve sensitivity by as much as 2 orders of magnitude

  4. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

    PubMed Central

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R.; Fry, Stephen C.

    2015-01-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  5. Fabrication of a hybrid plastic-silicon microfluidic device for high-throughput genotyping

    NASA Astrophysics Data System (ADS)

    Chartier, Isabelle; Sudor, J.; Fouillet, Yves; Sarrut, N.; Bory, C.; Gruss, A.

    2003-01-01

    The lab-on-a-chip approach has been increasingly present in biological research over the last ten years, high-throughput analyses being one of the promising utilization. The work presented here has consisted in developing an automated genotyping system based on a continuous flow analysis which integrates all the steps of the genotyping process (PCR, purification and sequencing). The genotyping device consists of a disposable hybrid silicon-plastic microfluidic chip, equipped with a permanent external, heating/cooling system, syringe-pumps based injection systems and on-line fluorescence detection. High throughput is obtained by performing the reaction in a continuous flow (1 reaction every 6min per channel) and in parallel (48 channels). We are presenting here the technical solutions developed to fabricate the hybrid silicon-plastic microfluidic device. It includes a polycarbonate substrate having 48 parallel grooves sealed by film lamination techniques to create the channels. Two different solutions for the sealing of the channels are compared in relation to their biocompatibility, fluidic behavior and fabrication process yield. Surface roughness of the surface of the channels is the key point of this step. Silicon fluidic chips are used for thermo-cycled reactions. A specific bonding technique has been developed to bond silicon chips onto the plastic part which ensures alignment and hermetic fluidic connexion. Surface coatings are studied to enhance the PCR biocompatibility and fluidic behavior of the two-phase liquid flow. We then demonstrate continuous operation over more than 20 hours of the component and validate PCR protocol on microliter samples in a continuous flow reaction.

  6. Diagnostic markers of ovarian cancer by high-throughput antigen cloning and detection on arrays.

    PubMed

    Chatterjee, Madhumita; Mohapatra, Saroj; Ionan, Alexei; Bawa, Gagandeep; Ali-Fehmi, Rouba; Wang, Xiaoju; Nowak, James; Ye, Bin; Nahhas, Fatimah A; Lu, Karen; Witkin, Steven S; Fishman, David; Munkarah, Adnan; Morris, Robert; Levin, Nancy K; Shirley, Natalie N; Tromp, Gerard; Abrams, Judith; Draghici, Sorin; Tainsky, Michael A

    2006-01-15

    A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations. PMID:16424057

  7. High-throughput FCS using an LCOS spatial light modulator and an 8 × 1 SPAD array

    PubMed Central

    Colyer, Ryan A.; Scalia, Giuseppe; Rech, Ivan; Gulinatti, Angelo; Ghioni, Massimo; Cova, Sergio; Weiss, Shimon; Michalet, Xavier

    2010-01-01

    We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed. PMID:21258559

  8. Parallel tools in HEVC for high-throughput processing

    NASA Astrophysics Data System (ADS)

    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

  9. Silicon microphysiometer for high-throughput drug screening

    NASA Astrophysics Data System (ADS)

    Verhaegen, Katarina; Baert, Christiaan; Puers, Bob; Sansen, Willy; Simaels, Jeannine; Van Driessche, Veerle; Hermans, Lou; Mertens, Robert P.

    1999-06-01

    We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A prototype twin microcalorimeter based on the Seebeck effect has been fabricated by IC technology and micromachined postprocessing techniques. A biocompatible liquid rubber membrane supports two identical 0.5 X 2 cm2 measurement chambers, situated at the cold and hot junction of a 666-junction aluminum/p+-polysilicon thermopile. The chambers can house up to 106 eukaryotic cells cultured to confluence. The advantage of the device over microcalorimeters on the market, is the integration of the measurement channels on chip, rendering microvolume reaction vessels, ranging from 10 to 600 (mu) l, in the closest possible contact with the thermopile sensor (no springs are needed). Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The small thermal inertia of the microchannels results in the short response time of 70 s, when filled with 50 (mu) l of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 20%. Our results show that it is feasible to apply this large-area, small- volume whole-cell biosensor for drug discovery, where the binding assays that are commonly used to provide high- throughput need to be complemented with a functional assay. Solutions are brought onto the sensor by a simple pipette, making the use of an industrial microtiterplate dispenser feasible on a nx96-array of the microcalorimeter biosensor. Such an array of biosensors has been designed based on a new set of requirements as set forth by people in the field as this project moved on. The results obtained from the prototype large-area sensor were used to obtain an accurate model of the calorimeter, checked for by the simulation software ANSYS. At

  10. High throughput optoelectronic smart pixel systems using diffractive optics

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

    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

  12. Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

    PubMed Central

    Pino, Elizabeth C.; Webster, Christopher M.; Carr, Christopher E.; Soukas, Alexander A.

    2013-01-01

    The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research. PMID:23568026

  13. Biochemical and high throughput microscopic assessment of fat mass in Caenorhabditis elegans.

    PubMed

    Pino, Elizabeth C; Webster, Christopher M; Carr, Christopher E; Soukas, Alexander A

    2013-01-01

    The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research. PMID:23568026

  14. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain

    PubMed Central

    Madisen, Linda; Zwingman, Theresa A.; Sunkin, Susan M.; Oh, Seung Wook; Zariwala, Hatim A.; Gu, Hong; Ng, Lydia L.; Palmiter, Richard D.; Hawrylycz, Michael J.; Jones, Allan R.; Lein, Ed S.; Zeng, Hongkui

    2009-01-01

    The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universal responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in a number of Cre-driver lines, including novel Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation. PMID:20023653

  15. Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds

    PubMed Central

    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 developed a microtiter assay of FN deposition by human fibroblasts. The assay provides a robust read-out of FN assembly. Alexa 488-FN (A488-FN) was added to cell monolayers, and the total fluorescence intensity of deposited A488-FN was quantified. The fluorescence intensity of deposited A488-FN correlated with the presence of FN fibrils visualized by fluorescence microscopy. The assay Z’ values were 0.67 or 0.54, respectively, when using background values of fluorescence either with no added A488-FN or with A488-FN added together with a known inhibitor of FN deposition. The assay was used to screen libraries comprising 4160 known bioactive compounds. Nine compounds were identified as non- or low-cytotoxic inhibitors of FN assembly. Four (ML-9, HA-100, tyrphostin and imatinib mesylate) are kinase inhibitors, a category of compounds known to inhibit FN assembly; two (piperlongumine and cantharidin) are promoters of cancer cell apoptosis; and three (maprotiline, CGS12066B, and aposcopolamine) are modulators of biogenic amine signaling. The latter six compounds have not been recognized heretofore as affecting FN assembly. The assay is straight-forward, adapts to 96- and 384-well formats, and should be useful for routine measurement of FN deposition and HTS. Screening of more diverse chemical libraries and identification of specific and efficient modulators of FN fibrillogenesis may result in therapeutics to control excessive connective tissue deposition. PMID:22986508

  16. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    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 = 103 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. PMID:24278480

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

    PubMed

    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. PMID:26466349

  18. A high throughput screening for rarely transcribed differentially expressed genes.

    PubMed Central

    von Stein, O D; Thies, W G; Hofmann, M

    1997-01-01

    A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined. PMID:9185570

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

    SciTech Connect

    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.

  20. High-Throughput Screening Based Identification of Paramyxovirus Inhibitors

    PubMed Central

    Yoon, Jeong-Jeong; Chawla, Dhruv; Paal, Tanja; Ndungu, Maina; Du, Yuhong; Kurtkaya, Serdar; Sun, Aiming; Snyder, James P; Plemper, Richard K

    2008-01-01

    Paramyxoviruses are negative strand non-segmented RNA viruses. Several members of this family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to ultimately develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, we report a high-throughput screening protocol that allows hit identification using non-recombinant primary MV strains as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations ≤15.3 μM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with re-testing of identified candidates reveals a low false-negative rate and, thus, a high accuracy of primary hit identification. Eleven of the confirmed hits were found to interfere with the viral entry machinery, while the remaining 24 compounds target post-entry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals three patterns of activity: 1) exclusively MV-specific blockers; 2) inhibitors of MV and related viruses of the same genus; 3) broader-range inhibitors with activity against a different paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry. PMID:18626114

  1. Translational informatics: enabling high-throughput research paradigms

    PubMed Central

    Embi, Peter J.; Sen, Chandan K.

    2009-01-01

    A common thread throughout the clinical and translational research domains is the need to collect, manage, integrate, analyze, and disseminate large-scale, heterogeneous biomedical data sets. However, well-established and broadly adopted theoretical and practical frameworks and models intended to address such needs are conspicuously absent in the published literature or other reputable knowledge sources. Instead, the development and execution of multidisciplinary, clinical, or translational studies are significantly limited by the propagation of “silos” of both data and expertise. Motivated by this fundamental challenge, we report upon the current state and evolution of biomedical informatics as it pertains to the conduct of high-throughput clinical and translational research and will present both a conceptual and practical framework for the design and execution of informatics-enabled studies. The objective of presenting such findings and constructs is to provide the clinical and translational research community with a common frame of reference for discussing and expanding upon such models and methodologies. PMID:19737991

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

    PubMed

    Poddar, Rajesh; Kawai, Risa; Ölveczky, Bence P

    2013-01-01

    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. PMID:24349451

  3. Savant: genome browser for high-throughput sequencing data

    PubMed Central

    Fiume, Marc; Williams, Vanessa; Brook, Andrew; Brudno, Michael

    2010-01-01

    Motivation: The advent of high-throughput sequencing (HTS) technologies has made it affordable to sequence many individuals' genomes. Simultaneously the computational analysis of the large volumes of data generated by the new sequencing machines remains a challenge. While a plethora of tools are available to map the resulting reads to a reference genome, and to conduct primary analysis of the mappings, it is often necessary to visually examine the results and underlying data to confirm predictions and understand the functional effects, especially in the context of other datasets. Results: We introduce Savant, the Sequence Annotation, Visualization and ANalysis Tool, a desktop visualization and analysis browser for genomic data. Savant was developed for visualizing and analyzing HTS data, with special care taken to enable dynamic visualization in the presence of gigabases of genomic reads and references the size of the human genome. Savant supports the visualization of genome-based sequence, point, interval and continuous datasets, and multiple visualization modes that enable easy identification of genomic variants (including single nucleotide polymorphisms, structural and copy number variants), and functional genomic information (e.g. peaks in ChIP-seq data) in the context of genomic annotations. Availability: Savant is freely available at http://compbio.cs.toronto.edu/savant Contact: savant@cs.toronto.edu PMID:20562449

  4. Validation of high throughput sequencing and microbial forensics applications

    PubMed Central

    2014-01-01

    High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security. PMID:25101166

  5. BOOGIE: Predicting Blood Groups from High Throughput Sequencing Data

    PubMed Central

    Giollo, Manuel; Minervini, Giovanni; Scalzotto, Marta; Leonardi, Emanuela; Ferrari, Carlo; Tosatto, Silvio C. E.

    2015-01-01

    Over the last decade, we have witnessed an incredible growth in the amount of available genotype data due to high throughput sequencing (HTS) techniques. This information may be used to predict phenotypes of medical relevance, and pave the way towards personalized medicine. Blood phenotypes (e.g. ABO and Rh) are a purely genetic trait that has been extensively studied for decades, with currently over thirty known blood groups. Given the public availability of blood group data, it is of interest to predict these phenotypes from HTS data which may translate into more accurate blood typing in clinical practice. Here we propose BOOGIE, a fast predictor for the inference of blood groups from single nucleotide variant (SNV) databases. We focus on the prediction of thirty blood groups ranging from the well known ABO and Rh, to the less studied Junior or Diego. BOOGIE correctly predicted the blood group with 94% accuracy for the Personal Genome Project whole genome profiles where good quality SNV annotation was available. Additionally, our tool produces a high quality haplotype phase, which is of interest in the context of ethnicity-specific polymorphisms or traits. The versatility and simplicity of the analysis make it easily interpretable and allow easy extension of the protocol towards other phenotypes. BOOGIE can be downloaded from URL http://protein.bio.unipd.it/download/. PMID:25893845

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

    SciTech Connect

    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.

  7. High Throughput Profiling of Molecular Shapes in Crystals

    NASA Astrophysics Data System (ADS)

    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.

  8. High-throughput optical screening of cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    Compton, Jonathan L.; Luo, Justin C.; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated microcavitation bubbles without requiring flow chambers or microfluidics. These microcavitation bubbles expose adherent cells to a microtsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1 mm2. We demonstrate microtsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation, resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microtsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling and its modulation by exogenous molecules demonstrates the capacity to initiate and assess cellular mechanosignalling in real time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry.

  9. High-throughput screening of phosphodiesterase activity in living cells.

    PubMed

    Rich, Thomas C; Karpen, Jeffrey W

    2005-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP. PMID:15988054

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data

    PubMed Central

    Zhang, Shaokang; Yin, Yanlong; Jones, Marcus B.; Zhang, Zhenzhen; Deatherage Kaiser, Brooke L.; Dinsmore, Blake A.; Fitzgerald, Collette; Fields, Patricia I.

    2015-01-01

    Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1–3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization. PMID:25762776

  12. Automated Filtration-Based High-Throughput Plasmid Preparation System

    PubMed Central

    Itoh, Masayoshi; Kitsunai, Tokuji; Akiyama, Junichi; Shibata, Kazuhiro; Izawa, Masaki; Kawai, Jun; Tomaru, Yasuhiro; Carninci, Piero; Shibata, Yuko; Ozawa, Yasuhiro; Muramatsu, Masami; Okazaki, Yasushi; Hayashizaki, Yoshihide

    1999-01-01

    Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing. PMID:10330126

  13. High throughput determination of glucan and xylan fractions in lignocelluloses.

    PubMed

    Selig, Michael J; Tucker, Melvin P; Law, Cody; Doeppke, Crissa; Himmel, Michael E; Decker, Stephen R

    2011-05-01

    The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96×1.8 ml-well Hastelloy reactor plates (128×86×51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128×86×18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially. PMID:21287235

  14. High-throughput concept for tailoring switchable mirrors

    NASA Astrophysics Data System (ADS)

    Borgschulte, A.; Gremaud, R.; de Man, S.; Westerwaal, R. J.; Rector, J. H.; Dam, B.; Griessen, R.

    2006-11-01

    The optical properties, the switching kinetics and the lifetime of hydrogen switchable mirrors based on Mg-Ni alloys are determined with particular regard to the composition of the optically active metal-hydride layer in combination with the thickness of the catalytic capping layer. For this, a high-throughput experiment is introduced. The switching kinetics and the reversibility of switchable mirrors are strongly thickness dependent, though the details hinge on the fine structure of the clustered capping layer. Therefore, the kinetics is correlated with the surface structures of Pd on Mg yNi 1- y as investigated by scanning tunneling microscopy. The results are explained by the so-called strong metal-support interaction (SMSI) state, characterized by a complete encapsulation of the capping layer clusters by oxidized species originating from the support. The SMSI-effect is less important with increasing Pd-layer thickness, and is suppressed by a good wetting of the Pd-clusters on the optically active film. This explains the critical thickness for the catalyzed hydrogen uptake observed in many switchable mirror systems. Moreover, the degradation of the kinetics during cycling is found to depend on the Pd-layer thickness and on the gas environment. Only films, covered with at least 15 nm Pd, show small degradation caused by the SMSI-effect. The SMSI-effect is partly reversible: after changing the gas environment from hydrogen to oxygen, the oxide on the Pd-clusters can be partly removed.

  15. High-Throughput, Data-Rich Cellular RNA Device Engineering

    PubMed Central

    Townshend, Brent; Kennedy, Andrew B.; Xiang, Joy S.; Smolke, Christina D.

    2015-01-01

    Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing, and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary interaction RNA devices exhibit improved performance in terms of gene silencing, activation ratio, and ligand sensitivity as compared to optimized RNA devices that rely on secondary structure changes. We apply our method to building biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate understanding of the underlying sequence-structure-function relationships that empower rational design of complex biomolecules. PMID:26258292

  16. High Throughput Profiling of Molecular Shapes in Crystals.

    PubMed

    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

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

    PubMed

    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

  18. High throughput cherry-picking of solvated samples.

    PubMed

    Schmitt, Robert; Traphagen, Linda; Hajduk, Phillip

    2010-07-01

    Advances in the design of automated compound storage systems have made it possible to store large collections of research compounds in individual single-use aliquots dissolved in dimethyl sulfoxide and rapidly retrieve a specific group off them. This 'cherry-picking' approach offers researchers the opportunity to request large numbers of compounds desired for testing without having to also retrieve all the other compounds stored on the same rack or plate. This makes it possible to meet the increasing demand for samples from High Throughput Screening and Therapeutic Area teams without adding staff to dispense from powder each time, without the constraints imposed by storing in solvated compounds in fixed-well 96- or 384-way plates, and without sacrificing sample quality or shelf life by storing at room temperature. We describe how this approach has been implemented at Abbott Laboratories' central compound repository to provide smaller amounts of more compounds faster and with high quality. In doing so, we have been able to better support the innovation of our Drug Discovery colleagues. PMID:20426754

  19. Probabilistic Assessment of High-Throughput Wireless Sensor Networks

    PubMed Central

    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

  20. High-throughput optical screening of cellular mechanotransduction.

    PubMed

    Compton, Jonathan L; Luo, Justin C; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm(2). We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca(2+) release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca(2+) signalling by introducing a known inhibitor to this pathway. The imaging of Ca(2+) signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry. PMID:25309621