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Sample records for high-throughput microarray applications

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

    PubMed Central

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

    2011-01-01

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

  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. 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.

  4. 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.

  5. 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

  6. 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

  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. From High-Throughput Microarray-Based Screening to Clinical Application: The Development of a Second Generation Multigene Test for Breast Cancer Prognosis

    PubMed Central

    Brase, Jan C.; Kronenwett, Ralf; Petry, Christoph; Denkert, Carsten; Schmidt, Marcus

    2013-01-01

    Several multigene tests have been developed for breast cancer patients to predict the individual risk of recurrence. Most of the first generation tests rely on proliferation-associated genes and are commonly carried out in central reference laboratories. Here, we describe the development of a second generation multigene assay, the EndoPredict test, a prognostic multigene expression test for estrogen receptor (ER) positive, human epidermal growth factor receptor (HER2) negative (ER+/HER2−) breast cancer patients. The EndoPredict gene signature was initially established in a large high-throughput microarray-based screening study. The key steps for biomarker identification are discussed in detail, in comparison to the establishment of other multigene signatures. After biomarker selection, genes and algorithms were transferred to a diagnostic platform (reverse transcription quantitative PCR (RT-qPCR)) to allow for assaying formalin-fixed, paraffin-embedded (FFPE) samples. A comprehensive analytical validation was performed and a prospective proficiency testing study with seven pathological laboratories finally proved that EndoPredict can be reliably used in the decentralized setting. Three independent large clinical validation studies (n = 2,257) demonstrated that EndoPredict offers independent prognostic information beyond current clinicopathological parameters and clinical guidelines. The review article summarizes several important steps that should be considered for the development process of a second generation multigene test and offers a means for transferring a microarray signature from the research laboratory to clinical practice.

  9. High-Throughput Analysis of Serum Antigens Using Sandwich ELISAs on Microarrays

    SciTech Connect

    Servoss, Shannon; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2009-05-11

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection and validation of disease biomarkers. ELISA microarrays are capable of simultaneous detection of many proteins using a small sample volume. Although there are many potential pitfalls to the use of ELISA microarrays, these can be avoided by careful planning of experiments. In this chapter we describe a high-throughput protocol for processing ELISA microarrays that will result in reliable and reproducible data.

  10. High-throughput allogeneic antibody detection using protein microarrays.

    PubMed

    Paul, Jed; Sahaf, Bita; Perloff, Spenser; Schoenrock, Kelsi; Wu, Fang; Nakasone, Hideki; Coller, John; Miklos, David

    2016-05-01

    Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. PMID:26902899

  11. High-throughput variation detection and genotyping using microarrays.

    PubMed

    Cutler, D J; Zwick, M E; Carrasquillo, M M; Yohn, C T; Tobin, K P; Kashuk, C; Mathews, D J; Shah, N A; Eichler, E E; Warrington, J A; Chakravarti, A

    2001-11-01

    The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

  12. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies)...

  13. A New Versatile Microarray-based Method for High Throughput Screening of Carbohydrate-active Enzymes*

    PubMed Central

    Vidal-Melgosa, Silvia; Pedersen, Henriette L.; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B.; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G. T.

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths. PMID:25657012

  14. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    PubMed

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

  15. A high-throughput microparticle microarray platform for dendritic cell-targeting vaccines.

    PubMed

    Acharya, Abhinav P; Clare-Salzler, Michael J; Keselowsky, Benjamin G

    2009-09-01

    Immunogenomic approaches combined with advances in adjuvant immunology are guiding progress toward rational design of vaccines. Furthermore, drug delivery platforms (e.g., synthetic particles) are demonstrating promise for increasing vaccine efficacy. Currently there are scores of known antigenic epitopes and adjuvants, and numerous synthetic delivery systems accessible for formulation of vaccines for various applications. However, the lack of an efficient means to test immune cell responses to the abundant combinations available represents a significant blockade on the development of new vaccines. In order to overcome this barrier, we report fabrication of a new class of microarray consisting of antigen/adjuvant-loadable poly(D,L lactide-co-glycolide) microparticles (PLGA MPs), identified as a promising carrier for immunotherapeutics, which are co-localized with dendritic cells (DCs), key regulators of the immune system and prime targets for vaccines. The intention is to utilize this high-throughput platform to optimize particle-based vaccines designed to target DCs in vivo for immune system-related disorders, such as autoimmune diseases, cancer and infection. Fabrication of DC/MP arrays leverages the use of standard contact printing miniarraying equipment in conjunction with surface modification to achieve co-localization of particles/cells on isolated islands while providing background non-adhesive surfaces to prevent off-island cell migration. We optimized MP overspotting pin diameter, accounting for alignment error, to allow construction of large, high-fidelity arrays. Reproducible, quantitative delivery of as few as 16+/-2 MPs per spot was demonstrated and two-component MP dosing arrays were constructed, achieving MP delivery which was independent of formulation, with minimal cross-contamination. Furthermore, quantification of spotted, surface-adsorbed MP degradation was demonstrated, potentially useful for optimizing MP release properties. Finally, we

  16. Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    PubMed

    Onuki-Nagasaki, Reiko; Nagasaki, Akira; Hakamada, Kazumi; Uyeda, Taro Q P; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2010-01-01

    Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening. PMID:20387151

  17. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  18. Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

    SciTech Connect

    Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.; West, Jason A. A.; Hux, Gary A.

    2006-10-01

    The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. The development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.

  19. Use of Microarrays as a High-Throughput Platform for Label-Free Biosensing.

    PubMed

    Sun, Yung-Shin

    2015-08-01

    In recent years, various label-free biosensing technologies have been developed for studying the real-time kinetics of diverse biomolecular interactions. These biosensors partially take the place of fluorescence-based methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. However, to completely eliminate the need of fluorescence, throughput is the next big consideration. Microarrays provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, and many compatible fluorescent scanners have been commercially available. The combination of microarrays and label-free biosensors will be of great interest to researchers in related fields. Microarrays are fabricated by spotting, imprinting, or directly synthesizing biomolecules on solid supports such as glasses, silicon wafers, and other functionalized substrates, and they have been applied to detect DNAs, proteins, toxins, and so on in surface plasmon resonance (SPR) imaging systems and oblique-incidence reflectivity difference (OI-RD) microscopes. Current challenges include increasing sensitivity, reducing sampling time, improving surface chemistry, identifying captured molecules, and minimizing reagent consumption. Future research directions are to improve the instruments themselves, modify the microarray surface for more efficient analyte capture, and combine the systems with mass spectrometry and microfluidics. PMID:25812567

  20. Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

    PubMed

    Baek, I C; Jang, J-P; Choi, H-B; Choi, E-J; Ko, W-Y; Kim, T-G

    2013-10-01

    The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

  1. Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

    PubMed Central

    Baross, Ágnes; Delaney, Allen D; Li, H Irene; Nayar, Tarun; Flibotte, Stephane; Qian, Hong; Chan, Susanna Y; Asano, Jennifer; Ally, Adrian; Cao, Manqiu; Birch, Patricia; Brown-John, Mabel; Fernandes, Nicole; Go, Anne; Kennedy, Giulia; Langlois, Sylvie; Eydoux, Patrice; Friedman, JM; Marra, Marco A

    2007-01-01

    Background Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays. Results We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection. Conclusion We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity. PMID:17910767

  2. The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection.

    PubMed

    Maitra, Anirban; Cohen, Yoram; Gillespie, Susannah E D; Mambo, Elizabeth; Fukushima, Noriyoshi; Hoque, Mohammad O; Shah, Nila; Goggins, Michael; Califano, Joseph; Sidransky, David; Chakravarti, Aravinda

    2004-05-01

    Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.

  3. Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function

    PubMed Central

    Bochner, Barry R.; Gadzinski, Peter; Panomitros, Eugenia

    2001-01-01

    The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes. PMID:11435407

  4. A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.

    PubMed

    Gehring, Andrew; He, Xiaohua; Fratamico, Pina; Lee, Joseph; Bagi, Lori; Brewster, Jeffrey; Paoli, George; He, Yiping; Xie, Yanping; Skinner, Craig; Barnett, Charlie; Harris, Douglas

    2014-06-11

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1-2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

  5. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h. PMID:25881200

  6. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

  7. A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

    PubMed Central

    Gehring, Andrew; He, Xiaohua; Fratamico, Pina; Lee, Joseph; Bagi, Lori; Brewster, Jeffrey; Paoli, George; He, Yiping; Xie, Yanping; Skinner, Craig; Barnett, Charlie; Harris, Douglas

    2014-01-01

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef. PMID:24921195

  8. Label-Free and High-Throughput Detection of Protein Microarrays by Oblique-Incidence Reflectivity Difference Method

    NASA Astrophysics Data System (ADS)

    Wang, Xu; Lu, Heng; Wen, Juan; Yuan, Kun; LÜ, Hui-Bin; Jin, Kui-Juan; Zhou, Yue-Liang; Yang, Guo-Zhen

    2010-10-01

    We label-free detected the biological process of preparing a microarray that includes 400 spots of mouse immunoglobulin G (IgG) as well as the specific hybridization between mouse IgG and goat anti-mouse IgG by an oblique-incidence reflectivity difference (OI-RD) method. The detection results after each process including printing, washing, blocking, and hybridization, demonstrate that the OI-RD method can trace the preparation process of a microarray and detect the specific hybridization between antigens and antibodies. OI-RD is a promising method for label-free and high-throughput detection of biological microarrays.

  9. MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms.

    PubMed

    Shao, Ning; Jiang, Shi-Meng; Zhang, Miao; Wang, Jing; Guo, Shu-Juan; Li, Yang; Jiang, He-Wei; Liu, Cheng-Xi; Zhang, Da-Bing; Yang, Li-Tao; Tao, Sheng-Ce

    2014-01-21

    The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test. PMID:24359455

  10. MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms.

    PubMed

    Shao, Ning; Jiang, Shi-Meng; Zhang, Miao; Wang, Jing; Guo, Shu-Juan; Li, Yang; Jiang, He-Wei; Liu, Cheng-Xi; Zhang, Da-Bing; Yang, Li-Tao; Tao, Sheng-Ce

    2014-01-21

    The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test.

  11. A multivariate approach for high throughput pectin profiling by combining glycan microarrays with monoclonal antibodies.

    PubMed

    Sousa, António G; Ahl, Louise I; Pedersen, Henriette L; Fangel, Jonatan U; Sørensen, Susanne O; Willats, William G T

    2015-05-29

    Pectin-one of the most complex biomacromolecules in nature has been extensively studied using various techniques. This has been done so in an attempt to understand the chemical composition and conformation of pectin, whilst discovering and optimising new industrial applications of the polymer. For the last decade the emergence of glycan microarray technology has led to a growing capacity of acquiring simultaneous measurements related to various carbohydrate characteristics while generating large collections of data. Here we used a multivariate analysis approach in order to analyse a set of 359 pectin samples probed with 14 different monoclonal antibodies (mAbs). Principal component analysis (PCA) and partial least squares (PLS) regression were utilised to obtain the most optimal qualitative and quantitative information from the spotted microarrays. The potential use of microarray technology combined with chemometrics for the accurate determination of degree of methyl-esterification (DM) and degree of blockiness (DB) was assessed. PMID:25950120

  12. 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.

  13. A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

    PubMed Central

    Fei, Y.Y.; Landry, J.P.; Sun, Y.S.; Luo, J.T.; Wang, X.B.; Lam, K.S; Zhu, X.D.

    2009-01-01

    We describe a novel scanning optical microscope based on a polarization-modulated nulling ellipsometry. The new microscope employs a combination of scanning mirror and sample translation and thus enables high-throughput label-free detection of biomolecular microarrays with more than 10,000 protein or small molecule targets. For illustration, we show the image of a 2760-spot protein microarray on a functionalized glass slide obtained with such a microscope. The new scanning microscope is also capable of determining, in parallel, the real-time binding kinetics of multiple molecular species under aqueous conditions. PMID:18248040

  14. High-throughput insect cell protein expression applications.

    PubMed

    Buchs, Mirjam; Kim, Ernie; Pouliquen, Yann; Sachs, Michael; Geisse, Sabine; Mahnke, Marion; Hunt, Ian

    2009-01-01

    The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.

  15. Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

    PubMed Central

    Lu, Chen; Wonsidler, Joshua L.; Li, Jianwei; Du, Yanming; Block, Timothy; Haab, Brian; Chen, Songming

    2012-01-01

    In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed. Introduction Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies

  16. Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

    SciTech Connect

    Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2009-05-11

    The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

  17. Microarray Applications in Microbial Ecology Research.

    SciTech Connect

    Gentry, T.; Schadt, C.; Zhou, J.

    2006-04-06

    Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. This review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.

  18. 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

  19. 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.

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

    PubMed Central

    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

  1. 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

  2. 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 ...

  3. Development of an antigen microarray for high throughput monoclonal antibody selection

    PubMed Central

    Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J.

    2014-01-01

    Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host. PMID:24472540

  4. ELISA microarray technology as a high-throughput system for cancer biomarker validation

    SciTech Connect

    Zangar, Richard C.; Daly, Don S.; White, Amanda M.

    2006-01-01

    A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. Another major challenge with cancer screening is that most cancers are rare in the general population, meaning that the specificity of an assay must be very high if the number of false positive is not going to be much greater than the number of true positives. Because of these challenges with biomarker validation, it is necessary to analysis of thousands of samples before a clear idea of the utility of a screening assay can be determined. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and has the potential to accelerate biomarker validation. In this review, we discuss current ELISA microarray technology and the enabling advances needed to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.

  5. Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation

    PubMed Central

    Tourovskaia, Anna; Figueroa-Masot, Xavier; Folch, Albert

    2015-01-01

    We have developed a microfluidic cell culture method that allows for the formation of linear isolated myotubes organized in a parallel microarray. Attachment and spreading of cells are confined within microtracks of cell-adherent proteins separated by a protein-repellent coating. Signaling molecules or other molecules of interest can be focally delivered to the myotubes using heterogeneous microfluidic streams. We have used the method to focally deliver agrin (a molecule implicated as a postsynaptic organizer), which leads to localized acetylcholine receptor clustering. These techniques can be modified to accommodate other cell types and can be adapted to virtually any bioactive molecule such as signaling factors or drugs. This protocol features two major techniques that can be utilized simultaneously or independently to (i) micropattern cells using surface chemical modification and (ii) use a microfluidic platform for culturing and focal stimulation of cells with molecules of interest. Device design, fabrication and assembly can be completed in 3 days. PMID:17406389

  6. High-throughput investigation of endothelial-to-mesenchymal transformation (EndMT) with combinatorial cellular microarrays.

    PubMed

    Wang, Zongjie; Calpe, Blaise; Zerdani, Jalil; Lee, Youngsang; Oh, Jonghyun; Bae, Hojae; Khademhosseini, Ali; Kim, Keekyoung

    2016-07-01

    In the developing heart, a specific subset of endocardium undergoes an endothelial-to-mesenchymal transformation (EndMT) thus forming nascent valve leaflets. Extracellular matrix (ECM) proteins and growth factors (GFs) play important roles in regulating EndMT but the combinatorial effect of GFs with ECM proteins is less well understood. Here we use microscale engineering techniques to create single, binary, and tertiary component microenvironments to investigate the combinatorial effects of ECM proteins and GFs on the attachment and transformation of adult ovine mitral valve endothelial cells to a mesenchymal phenotype. With the combinatorial microenvironment microarrays, we utilized 60 different combinations of ECM proteins (Fibronectin, Collagen I, II, IV, Laminin) and GFs (TGF-β1, bFGF, VEGF) and were able to identify new microenvironmental conditions capable of modulating EndMT in MVECs. Experimental results indicated that TGF-β1 significantly upregulated the EndMT while either bFGF or VEGF downregulated EndMT process markedly. Also, ECM proteins could influence both the attachment of MVECs and the response of MVECs to GFs. In terms of attachment, fibronectin is significantly better for the adhesion of MVECs among the five tested proteins. Overall collagen IV and fibronectin appeared to play important roles in promoting EndMT process. Great consistency between macroscale and microarrayed experiments and present studies demonstrates that high-throughput cellular microarrays are a promising approach to study the regulation of EndMT in valvular endothelium. Biotechnol. Bioeng. 2016;113: 1403-1412. © 2015 Wiley Periodicals, Inc.

  7. High-throughput carbohydrate microarray profiling of 27 antibodies demonstrates widespread specificity problems.

    PubMed

    Manimala, Joseph C; Roach, Timothy A; Li, Zhitao; Gildersleeve, Jeffrey C

    2007-08-01

    Progress toward understanding the biological roles of carbohydrates has been remarkably slow, and efforts to exploit this class of biopolymers as diagnostic and therapeutic targets have proven extremely challenging. Both basic and clinical research rely heavily on identifying and monitoring expression levels of carbohydrates. Over the last 30 years, the majority of expression information has been derived from antibody- and lectin-binding studies. Using a carbohydrate microarray containing 80 different glycans and glycoproteins, the specificities of 27 antiglycan antibodies were evaluated, including antibodies to histo-blood group A, B, and H antigens (81FR2.2, CLCP-19B, B389, 92FR-A2, B480, B460, B376, and B393), Lewis antigens (7LE, 15C02, 28, ZC-18C, 121SLE, CA199.02, PR.5C5, 2-25LE, BR55, T174, T218, F3, A70-C/C8, FR4A5, and K21), and other tumor-associated antigens (B389, 1A4, B1.1, and 5B5). In total, evaluation of over 2000 individual carbohydrate-protein interactions was carried out. More than half of the antibodies considered to be specific for their designated antigen were found to cross-react with other glycans. The cross-reactive glycans could be mistaken for the designated antigen in biopsy samples or other biological samples, leading to inaccurate conclusions. PMID:17483136

  8. Applications of Biophysics in High-Throughput Screening Hit Validation.

    PubMed

    Genick, Christine Clougherty; Barlier, Danielle; Monna, Dominique; Brunner, Reto; Bé, Céline; Scheufler, Clemens; Ottl, Johannes

    2014-06-01

    For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein's amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of ~2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article.

  9. Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

    PubMed

    Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

    2015-07-01

    Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

  10. Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format

    NASA Astrophysics Data System (ADS)

    Zhu, Xiangdong; Landry, James P.; Sun, Yung-Shin; Gregg, Jeff P.; Lam, Kit S.; Guo, Xiaowen

    2007-04-01

    We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in.×3 in. (2.54 cm×7.62 cm) microscope slide.

  11. Silicon photonic crystal microarrays for high throughput label-free detection of lung cancer cell line lysates with sensitivity and specificity

    NASA Astrophysics Data System (ADS)

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Gemmill, Robert M.; Chen, Ray T.

    2013-03-01

    Detection of biomolecules on microarrays based on label-free on-chip optical biosensors is very attractive since this format avoids complex chemistries caused by steric hindrance of labels. Application areas include the detection of cancers and allergens, and food-borne pathogens to name a few. We have demonstrated photonic crystal microcavity biosensors with high sensitivity down to 1pM concentrations (67pg/ml). High sensitivities were achieved by slow light engineering which reduced the radiation loss and increased the stored energy in the photonic crystal microcavity resonance mode. Resonances with high quality factor Q~26,760 in liquid ambient, coupled with larger optical mode volumes allowed enhanced interaction with the analyte biomolecules which resulted in sensitivities down to 10 cells per micro-liter to lung cancer cell lysates. The specificity of detection was ensured by multiplexed detections from multiple photonic crystal microcavities arrayed on the arms of a multimode interference power splitter. Specific binding interactions and control experiments were performed simultaneously at the same instant of time with the same 60 microliter sample volume. Specificity is further ensured by sandwich assay methods in the multiplexed experiment. Sandwich assay based amplification increased the sensitivity further resulting in the detection of lung cancer cell lysates down to concentrations of 2 cells per micro-liter. The miniaturization enabled by photonic crystal biosensors coupled with waveguide interconnected layout thus offers the potential of high throughput proteomics with high sensitivity and specificity.

  12. Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

    NASA Astrophysics Data System (ADS)

    Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

    2013-06-01

    High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome

  13. Acoustic Droplet Ejection Applications for High-Throughput Screening of Infectious Agents.

    PubMed

    Rasmussen, Lynn; White, E Lucile; Bostwick, James R

    2016-02-01

    When acoustic droplet ejection technology was first introduced for high-throughput applications, it was used primarily for dispensing compounds dissolved in DMSO. The high precision and accuracy achieved for low-volume transfers in this application were noted by those working outside of the compound management area, and interest was generated in expanding the scope of the technology to include other liquid types. Later-generation instruments included calibrations for several aqueous buffers that were applicable to the life sciences. The High Throughput Screening Center at Southern Research has made use of this range of liquid calibrations for the Infectious Disease Program. The original calibration for DMSO has allowed the preparation of assay-ready plates that can be sent to remote locations. This process was used as part of the collaboration between Southern Research and Galveston National Laboratory, University of Texas Medical Branch, to develop high-throughput screening for biological safety level 4 containment and to provide compounds for two pilot screens that were run there with BSL-4-level pathogens. The aqueous calibrations have been instrumental in miniaturizing assays used for infectious disease, such as qPCR, tissue culture infectious dose 50, and bacterial motility, to make them compatible with HTS operations. PMID:26663786

  14. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  15. Application of high-throughput sequencing in understanding human oral microbiome related with health and disease

    PubMed Central

    Chen, Hui; Jiang, Wen

    2014-01-01

    The oral microbiome is one of most diversity habitat in the human body and they are closely related with oral health and disease. As the technique developing, high-throughput sequencing has become a popular approach applied for oral microbial analysis. Oral bacterial profiles have been studied to explore the relationship between microbial diversity and oral diseases such as caries and periodontal disease. This review describes the application of high-throughput sequencing for characterization of oral microbiota and analyzing the changes of the microbiome in the states of health or disease. Deep understanding the knowledge of microbiota will pave the way for more effective prevent dentistry and contribute to the development of personalized dental medicine. PMID:25352835

  16. High-Throughput Quantification of Bioactive Lipids by MALDI Mass Spectrometry: Application to Prostaglandins

    PubMed Central

    Manna, Joseph D.; Reyzer, Michelle L.; Latham, Joey C.; Weaver, C. David; Marnett, Lawrence J.; Caprioli, Richard M.

    2011-01-01

    Analysis and quantification of analytes in biological systems is a critical component of metabolomic investigations of cell function. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We introduce a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted metabolomes. Proof-of-concept is demonstrated by analysis of prostaglandins and glyceryl prostaglandins. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increases the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15 fmol – 2000 fmol on plate) and improved linearity (r2= 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated. PMID:21770391

  17. A synchronous Gigabit Ethernet protocol stack for high-throughput UDP/IP applications

    NASA Astrophysics Data System (ADS)

    Födisch, P.; Lange, B.; Sandmann, J.; Büchner, A.; Enghardt, W.; Kaever, P.

    2016-01-01

    State of the art detector readout electronics require high-throughput data acquisition (DAQ) systems. In many applications, e. g. for medical imaging, the front-end electronics are set up as separate modules in a distributed DAQ. A standardized interface between the modules and a central data unit is essential. The requirements on such an interface are varied, but demand almost always a high throughput of data. Beyond this challenge, a Gigabit Ethernet interface is predestined for the broad requirements of Systems-on-a-Chip (SoC) up to large-scale DAQ systems. We have implemented an embedded protocol stack for a Field Programmable Gate Array (FPGA) capable of high-throughput data transmission and clock synchronization. A versatile stack architecture for the User Datagram Protocol (UDP) and Internet Control Message Protocol (ICMP) over Internet Protocol (IP) such as Address Resolution Protocol (ARP) as well as Precision Time Protocol (PTP) is presented. With a point-to-point connection to a host in a MicroTCA system we achieved the theoretical maximum data throughput limited by UDP both for 1000BASE-T and 1000BASE-KX links. Furthermore, we show that the random jitter of a synchronous clock over a 1000BASE-T link for a PTP application is below 60 ps.

  18. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  19. Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

    PubMed Central

    Weingeist, David M.; Ge, Jing; Wood, David K.; Mutamba, James T.; Huang, Qiuying; Rowland, Elizabeth A.; Yaffe, Michael B.; Floyd, Scott; Engelward, Bevin P.

    2013-01-01

    A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects. PMID:23422001

  20. 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.

  1. Examination of a genetic algorithm for the application in high-throughput downstream process development.

    PubMed

    Treier, Katrin; Berg, Annette; Diederich, Patrick; Lang, Katharina; Osberghaus, Anna; Dismer, Florian; Hubbuch, Jürgen

    2012-10-01

    Compared to traditional strategies, application of high-throughput experiments combined with optimization methods can potentially speed up downstream process development and increase our understanding of processes. In contrast to the method of Design of Experiments in combination with response surface analysis (RSA), optimization approaches like genetic algorithms (GAs) can be applied to identify optimal parameter settings in multidimensional optimizations tasks. In this article the performance of a GA was investigated applying parameters applicable in high-throughput downstream process development. The influence of population size, the design of the initial generation and selection pressure on the optimization results was studied. To mimic typical experimental data, four mathematical functions were used for an in silico evaluation. The influence of GA parameters was minor on landscapes with only one optimum. On landscapes with several optima, parameters had a significant impact on GA performance and success in finding the global optimum. Premature convergence increased as the number of parameters and noise increased. RSA was shown to be comparable or superior for simple systems and low to moderate noise. For complex systems or high noise levels, RSA failed, while GA optimization represented a robust tool for process optimization. Finally, the effect of different objective functions is shown exemplarily for a refolding optimization of lysozyme.

  2. High-Throughput Heterogeneous Integration of Diverse Nanomaterials on a Single Chip for Sensing Applications

    PubMed Central

    MacNaughton, Samuel; Ammu, Srikanth; Manohar, Sanjeev K.; Sonkusale, Sameer

    2014-01-01

    There is a large variety of nanomaterials each with unique electronic, optical and sensing properties. However, there is currently no paradigm for integration of different nanomaterials on a single chip in a low-cost high-throughput manner. We present a high throughput integration approach based on spatially controlled dielectrophoresis executed sequentially for each nanomaterial type to realize a scalable array of individually addressable assemblies of graphene, carbon nanotubes, metal oxide nanowires and conductive polymers on a single chip. This is a first time where such a diversity of nanomaterials has been assembled on the same layer in a single chip. The resolution of assembly can range from mesoscale to microscale and is limited only by the size and spacing of the underlying electrodes on chip used for assembly. While many applications are possible, the utility of such an array is demonstrated with an example application of a chemical sensor array for detection of volatile organic compounds below parts-per-million sensitivity. PMID:25350279

  3. Differentiation of whole bacterial cells based on high-throughput microarray chip printing and infrared microspectroscopic readout.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Burke, Tara L; Fry, Frederick S

    2009-10-01

    Using robotic automation, a microarray printing protocol for whole bacterial cells was developed for subsequent label-free and nondestructive infrared microspectroscopic detection. Using this contact microspotting system, 24 microorganisms were printed on zinc selenide slides; these were 6 species of Listeria, 10 species of Vibrio, 2 strains of Photobacterium damselae, Yersinia enterocolitica 289, Bacillus cereus ATCC 14529, Staphylococcus aureus, ATCC 19075 (serotype 104 B), Shigella sonnei 20143, Klebsiella pneumoniae KP73, Enterobacter cloacae, Citrobacter freundii 200, and Escherichia coli. Microarrays consisting of separate spots of bacterial deposits gave consistent and reproducible infrared spectra, which were differentiated by unsupervised pattern recognition algorithms. Two multivariate analysis algorithms, principal component analysis and hierarchical cluster analysis, successfully separated most, but not all, the bacteria investigated down to the species level.

  4. Differentiation of whole bacterial cells based on high-throughput microarray chip printing and infrared microspectroscopic readout.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Burke, Tara L; Fry, Frederick S

    2009-10-01

    Using robotic automation, a microarray printing protocol for whole bacterial cells was developed for subsequent label-free and nondestructive infrared microspectroscopic detection. Using this contact microspotting system, 24 microorganisms were printed on zinc selenide slides; these were 6 species of Listeria, 10 species of Vibrio, 2 strains of Photobacterium damselae, Yersinia enterocolitica 289, Bacillus cereus ATCC 14529, Staphylococcus aureus, ATCC 19075 (serotype 104 B), Shigella sonnei 20143, Klebsiella pneumoniae KP73, Enterobacter cloacae, Citrobacter freundii 200, and Escherichia coli. Microarrays consisting of separate spots of bacterial deposits gave consistent and reproducible infrared spectra, which were differentiated by unsupervised pattern recognition algorithms. Two multivariate analysis algorithms, principal component analysis and hierarchical cluster analysis, successfully separated most, but not all, the bacteria investigated down to the species level. PMID:19630511

  5. High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

    PubMed

    Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

    2015-01-01

    Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

  6. BR 07-1 DEVELOPMENT OF THE CELL MICROARRAY FOR HIGH-THROUGHPUT ANALYSIS OF GUT MICROBIOTA.

    PubMed

    Hong, Seong-Tshool

    2016-09-01

    The human intestine contains a massive and complex microbial community called gut microbiota. A typical human carries 100 trillion microbes in his/her body which is 10 times greater than the number of their host cells, i.e. whole number of human cells. A combined microbial genome constituting gut microbiota is well excess our own human genome. The microbial composition of gut microbiotata and its role on diseases became a booming area of research, presenting a new paradigm of opportunities for modern medicines. Recent evidences showed that gut microbiota acts as a very important determining factor for the development of almost all complex diseases such as primary hypertension, obesity, depression, diabetes, autism, asthma, bowl diseases, rheumatic arthritis, systemic lupus erythematosus, Crohn's disease, Parkinson's disease, Alzheimer's disease, epilepsy, schizophrenia, etc. In spite of the significant role of gut microbiota in the development of complex diseases, the elucidation of the mechanistic pathway on the development of complex diseases by gut microbiota is not moving forward as expected. Current methods to identify alteration of gut microbiota in patients and healthy controls are basically based on the metagenomic sequencings of DNA samples extracted from feces by using next-generation sequencing machines. Although the metagenomic sequencing approaches proved association of gut microbiota with various complex diseases, those methods failed to accurately pinpoint the etiological agents in gut microbiota for complex diseases. The metagenomic sequencing approaches are not only difficult to identify the etiological agent of complex diseases at species level but also difficult to use, requiring complex bioinformatic analyses, and expensive. To overcome the current challenges in analysis of gut microbiota, we developed a novel cell microarray to analyze the constituent microbial organisms of gut microbiota very accurately and fast by using a drop of blood. The

  7. BR 07-1 DEVELOPMENT OF THE CELL MICROARRAY FOR HIGH-THROUGHPUT ANALYSIS OF GUT MICROBIOTA.

    PubMed

    Hong, Seong-Tshool

    2016-09-01

    The human intestine contains a massive and complex microbial community called gut microbiota. A typical human carries 100 trillion microbes in his/her body which is 10 times greater than the number of their host cells, i.e. whole number of human cells. A combined microbial genome constituting gut microbiota is well excess our own human genome. The microbial composition of gut microbiotata and its role on diseases became a booming area of research, presenting a new paradigm of opportunities for modern medicines. Recent evidences showed that gut microbiota acts as a very important determining factor for the development of almost all complex diseases such as primary hypertension, obesity, depression, diabetes, autism, asthma, bowl diseases, rheumatic arthritis, systemic lupus erythematosus, Crohn's disease, Parkinson's disease, Alzheimer's disease, epilepsy, schizophrenia, etc. In spite of the significant role of gut microbiota in the development of complex diseases, the elucidation of the mechanistic pathway on the development of complex diseases by gut microbiota is not moving forward as expected. Current methods to identify alteration of gut microbiota in patients and healthy controls are basically based on the metagenomic sequencings of DNA samples extracted from feces by using next-generation sequencing machines. Although the metagenomic sequencing approaches proved association of gut microbiota with various complex diseases, those methods failed to accurately pinpoint the etiological agents in gut microbiota for complex diseases. The metagenomic sequencing approaches are not only difficult to identify the etiological agent of complex diseases at species level but also difficult to use, requiring complex bioinformatic analyses, and expensive. To overcome the current challenges in analysis of gut microbiota, we developed a novel cell microarray to analyze the constituent microbial organisms of gut microbiota very accurately and fast by using a drop of blood. The

  8. HTSstation: A Web Application and Open-Access Libraries for High-Throughput Sequencing Data Analysis

    PubMed Central

    David, Fabrice P. A.; Delafontaine, Julien; Carat, Solenne; Ross, Frederick J.; Lefebvre, Gregory; Jarosz, Yohan; Sinclair, Lucas; Noordermeer, Daan; Rougemont, Jacques; Leleu, Marion

    2014-01-01

    The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch. PMID:24475057

  9. High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis

    PubMed Central

    Ferraro, Daniel J; Bhave, Sandeep R; Kotipatruni, Rama P; Hunn, Jeremy C; Wildman, Scott A; Hong, Charles; Dadey, David Y. A.; Muhoro, Lincoln K.; Jaboin, Jerry J; Thotala, Dinesh; Hallahan, Dennis E

    2013-01-01

    Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in the cell surface in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal/mol, −487.239 kcal/mol, and −595.328 kcal/mol for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide in vitro via electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor. PMID:23147990

  10. Solid electrodes in electroanalytical chemistry: present applications and prospects for high throughput screening of drug compounds.

    PubMed

    Uslu, Bengi; Ozkan, Sibel A

    2007-08-01

    This review summarizes recent progress in the development and application of solid electrodes to the screening of pharmaceutical dosage forms and biological fluids. Recent trends and advances in the electroanalytical chemistry of solid electrodes, microelectrodes and electrochemical sensors are reviewed. The varieties of solid electrodes and their basic physico-chemical properties and some specific characteristics including some supramolecular phenomena at their surface are surveyed. This review also includes some selected designs and their applications. Despite many reviews about individual solid electrodes in the literature, this review offers the first comprehensive report on all forms of solid electrodes. Special attention is paid to the possibilities of solid electrodes in high throughput electroanalytical investigation of drug dosage forms and biological samples using modern electroanalytical techniques. Various selected studies on these subjects since 1996 are reviewed in this paper.

  11. Development and Applications of the Lectin Microarray.

    PubMed

    Hirabayashi, Jun; Kuno, Atsushi; Tateno, Hiroaki

    2015-01-01

    The lectin microarray is an emerging technology for glycomics. It has already found maximum use in diverse fields of glycobiology by providing simple procedures for differential glycan profiling in a rapid and high-throughput manner. Since its first appearance in the literature in 2005, many application methods have been developed essentially on the same platform, comprising a series of glycan-binding proteins immobilized on an appropriate substrate such as a glass slide. Because the lectin microarray strategy does not require prior liberation of glycans from the core protein in glycoprotein analysis, it should encourage researchers not familiar with glycotechnology to use glycan analysis in future work. This feasibility should provide a broader range of experimental scientists with good opportunities to investigate novel aspects of glycoscience. Applications of the technology include not only basic sciences but also the growing fields of bio-industry. This chapter describes first the essence of glycan profiling and the basic fabrication of the lectin microarray for this purpose. In the latter part the focus is on diverse applications to both structural and functional glycomics, with emphasis on the wide applicability now available with this new technology. Finally, the importance of developing advanced lectin engineering is discussed.

  12. High-Throughput Profiling of the Humoral Immune Responses Against Thirteen Human Papillomavirus Types by Proteome Microarrays

    PubMed Central

    Luevano, Martha; Bernard, Hans-Ulrich; Barrera-Saldaña, Hugo A.; Trevino, Victor; Garcia-Carranca, Alejandro; Villa, Luisa L.; Monk, Bradley J.; Tan, Xiaolin; Davies, D. Huw; Felgner, Phil L.; Kalantari, Mina

    2010-01-01

    We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, 18, 31, 33, 35, 45, 53), genital warts (HPV-6, 11), or skin lesions (HPV-1, 2, 4, 5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence, but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins. PMID:20554302

  13. Applications in high-content functional protein microarrays.

    PubMed

    Moore, Cedric D; Ajala, Olutobi Z; Zhu, Heng

    2016-02-01

    Protein microarray technology provides a versatile platform for characterization of hundreds to thousands of proteins in a parallel and high-throughput manner. Over the last decade, applications of functional protein microarrays in particular have flourished in studying protein function at a systems level and have led to the construction of networks and pathways describing these functions. Relevant areas of research include the detection of various binding properties of proteins, the study of enzyme-substrate relationships, the analysis of host-microbe interactions, and profiling antibody specificity. In addition, discovery of novel biomarkers in autoimmune diseases and cancers is emerging as a major clinical application of functional protein microarrays. In this review, we will summarize the recent advances of functional protein microarrays in both basic and clinical applications. PMID:26599287

  14. CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells.

    PubMed

    Ge, Jing; Prasongtanakij, Somsak; Wood, David K; Weingeist, David M; Fessler, Jessica; Navasummrit, Panida; Ruchirawat, Mathuros; Engelward, Bevin P

    2014-10-18

    DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

  15. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray

    PubMed Central

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J.

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  16. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams

    PubMed Central

    Georgantzoglou, Antonios; Merchant, Michael J.; Jeynes, Jonathan C. G.; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E.; Jena, Rajesh

    2016-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells’ clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x–y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells’ response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell’s capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  17. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams.

    PubMed

    Georgantzoglou, Antonios; Merchant, Michael J; Jeynes, Jonathan C G; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E; Jena, Rajesh

    2015-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells' clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells' response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell's capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  18. Applications of Functional Protein Microarrays in Basic and Clinical Research

    PubMed Central

    Zhu, Heng; Qian, Jiang

    2013-01-01

    The protein microarray technology provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput manner. It is viewed as a new tool that overcomes the limitation of DNA microarrays. On the basis of its application, protein microarrays fall into two major classes: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be directly spotted on a slide to form the so-called “reverse-phase” protein microarray. In the last decade, applications of functional protein microarrays in particular have flourished in studying protein function and construction of networks and pathways. In this chapter, we will review the recent advancements in the protein microarray technology, followed by presenting a series of examples to illustrate the power and versatility of protein microarrays in both basic and clinical research. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:22989767

  19. A low-cost, portable, high-throughput wireless sensor system for phonocardiography applications.

    PubMed

    Sa-Ngasoongsong, Akkarapol; Kunthong, Jakkrit; Sarangan, Venkatesh; Cai, Xinwei; Bukkapatnam, Satish T S

    2012-01-01

    This paper presents the design and testing of a wireless sensor system developed using a Microchip PICDEM developer kit to acquire and monitor human heart sounds for phonocardiography applications. This system can serve as a cost-effective option to the recent developments in wireless phonocardiography sensors that have primarily focused on Bluetooth technology. This wireless sensor system has been designed and developed in-house using off-the-shelf components and open source software for remote and mobile applications. The small form factor (3.75 cm × 5 cm × 1 cm), high throughput (6,000 Hz data streaming rate), and low cost ($13 per unit for a 1,000 unit batch) of this wireless sensor system make it particularly attractive for phonocardiography and other sensing applications. The experimental results of sensor signal analysis using several signal characterization techniques suggest that this wireless sensor system can capture both fundamental heart sounds (S1 and S2), and is also capable of capturing abnormal heart sounds (S3 and S4) and heart murmurs without aliasing. The results of a denoising application using Wavelet Transform show that the undesirable noises of sensor signals in the surrounding environment can be reduced dramatically. The exercising experiment results also show that this proposed wireless PCG system can capture heart sounds over different heart conditions simulated by varying heart rates of six subjects over a range of 60-180 Hz through exercise testing.

  20. A Low-Cost, Portable, High-Throughput Wireless Sensor System for Phonocardiography Applications

    PubMed Central

    Sa-ngasoongsong, Akkarapol; Kunthong, Jakkrit; Sarangan, Venkatesh; Cai, Xinwei; Bukkapatnam, Satish T. S.

    2012-01-01

    This paper presents the design and testing of a wireless sensor system developed using a Microchip PICDEM developer kit to acquire and monitor human heart sounds for phonocardiography applications. This system can serve as a cost-effective option to the recent developments in wireless phonocardiography sensors that have primarily focused on Bluetooth technology. This wireless sensor system has been designed and developed in-house using off-the-shelf components and open source software for remote and mobile applications. The small form factor (3.75 cm × 5 cm × 1 cm), high throughput (6,000 Hz data streaming rate), and low cost ($13 per unit for a 1,000 unit batch) of this wireless sensor system make it particularly attractive for phonocardiography and other sensing applications. The experimental results of sensor signal analysis using several signal characterization techniques suggest that this wireless sensor system can capture both fundamental heart sounds (S1 and S2), and is also capable of capturing abnormal heart sounds (S3 and S4) and heart murmurs without aliasing. The results of a denoising application using Wavelet Transform show that the undesirable noises of sensor signals in the surrounding environment can be reduced dramatically. The exercising experiment results also show that this proposed wireless PCG system can capture heart sounds over different heart conditions simulated by varying heart rates of six subjects over a range of 60–180 Hz through exercise testing. PMID:23112633

  1. A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening.

    PubMed

    Kim, Eunha; Lee, Sanghee; Park, Seung Bum

    2012-02-25

    We developed a novel fluorescent bioprobe (SF44) that can specifically visualize the cellular lipid droplets in in vitro and in vivo systems and illustrated the mechanistic rationale of its fluorogenic property. Its application to image-based high throughput screening led us to the identification of a new small-molecule modulator of lipid droplet formation.

  2. Computer controlled cryo-electron microscopy--TOM² a software package for high-throughput applications.

    PubMed

    Korinek, Andreas; Beck, Florian; Baumeister, Wolfgang; Nickell, Stephan; Plitzko, Jürgen M

    2011-09-01

    Automated data acquisition expedites structural studies by electron microscopy and it allows to collect data sets of unprecedented size and consistent quality. In electron tomography it greatly facilitates the systematic exploration of large cellular landscapes and in single particle analysis it allows to generate data sets for an exhaustive classification of coexisting molecular states. Here we describe a novel software philosophy and architecture that can be used for a great variety of automated data acquisition scenarios. Based on our original software package TOM, the new TOM(2) package has been designed in an object-oriented way. The whole program can be seen as a collection of self-sufficient modules with defined relationships acting in a concerted manner. It subdivides data acquisition into a set of hierarchical tasks, bonding data structure and the operations to be performed tightly together. To demonstrate its capacity for high-throughput data acquisition it has been used in conjunction with instrumentation combining the latest technological achievements in electron optics, cryogenics and robotics. Its performance is demonstrated with a single particle analysis case study and with a batch tomography application.

  3. Development of a Framework for High-Throughput Calculations and its Application to Energy Storage Challenges

    NASA Astrophysics Data System (ADS)

    Kirklin, Scott

    From a historical perspective, the progress of humanity has been measured by the materials that mankind has been able to use. Looking forward, future technological developments will continue to hinge on the development of materials with precisely tailored properties and performance. In pursuit of this goal, this thesis presents a framework for the high-throughput handling of first principles materials modeling. This framework takes the form of the Open Quantum Materials Database (OQMD - www.oqmd.org), a repository of crystal structures, computed materials properties, and a host of tools for data storage, retrieval, and analysis. At present, the OQMD contains over 300,000 materials, and over 1.3 million completed density functional theory calculations. We set forth to demonstrate the usefulness of the OQMD for materials discovery by using it to search for materials for three applications: 1) conversion reaction anode materials for Li-ion batteries, 2) electrode materials for a novel hybrid Li-ion/Li-O2 battery chemistry, and 3) precipitation strengtheners for a suite of structural metals. In each of these materials discovery projects, we first determine the scope of relevant materials to consider, then develop a set of screens based on DFT calculable bulk materials properties, implement the specified filters, and finally consider the apparent advantages and disadvantages of the predicted materials.

  4. Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

    PubMed Central

    Nebane, N. Miranda; Coric, Tatjana; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

    2016-01-01

    The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens. PMID:26663785

  5. High throughput fabrication of plasmonic nanostructures in nanofluidic pores for biosensing applications

    NASA Astrophysics Data System (ADS)

    Mazzotta, Francesco; Höök, Fredrik; Jonsson, Magnus P.

    2012-10-01

    One of the primary advantages of nanoscale sensors is that they often can provide conceptually new ways of performing sensing that are not feasible with their large-scale analogs. For example, the small size of nanoscale sensor elements, such as plasmonic metal nanoparticles, allows them to be combined with nanofluidic systems. Among the potential applications of such a combination is the efficient delivery of analyte to the sensor surface. With this in mind, in this work we look to address the challenge of creating and positioning nanoplasmonic sensor elements within nanofluidic pores. A scheme is presented that allows for the production of arrays of pores in a thin (220 nm) silicon nitride membrane with one plasmonic nanoparticle sensor element in each pore. The high throughput fabrication protocol is parallel and enables multiple sensor chips to be produced simultaneously, yet with accurate tuning of the dimension and shape of the nanoparticles. The presented system is shown to possess polarization-sensitive plasmonic resonances that can be tuned significantly in the visible wavelength range by just varying one process parameter. The thickness of the membrane could be optimized to minimize the influence of the optical membrane interference on the plasmonic readout. The sensitivity of the plasmon resonances to changes in refractive index, which forms the basis for using the system for biosensing, was found to be competitive with other nanoplasmonic sensors.

  6. Development of high-throughput glass inkjet devices for pharmaceutical applications.

    PubMed

    Ehtezazi, Touraj; Dempster, Nicola M; Martin, Graham D; Hoath, Stephen D; Hutchings, Ian M

    2014-11-01

    The application of the inkjet method to pharmaceutical products is promising. To make this realistic, not only does the throughput of this method need to be increased, but also the components should be inert to pharmaceutical preparations. We present designs of glass-based inkjet devices that are capable of producing droplets at high rates. To achieve this, inkjet devices from glass capillary tubes were manufactured with orifice diameters of 5, 10 and 20 μm and were actuated with diaphragm piezoelectric disks. Also, a pressure capsule was formed by creating a manifold at a distance from the orifice tip. Placing the piezoelectric disk at 0.5 mm distance from the tip allowed the formation of a jet at 3.2 MHz in certain designs, but for a short period of time because of overheating. The length of the pressure capsule, its inlet diameter, and the nozzle tip geometry were crucial to lower the required power. Actuating an inkjet device with 10 μm orifice diameter comfortably at 900 kHz and drying the droplets from 1% salbutamol sulphate solution allowed the formation of particles with diameters of 1.76 ± 0.15 μm and the geometric standard deviation of 1.08. In conclusion, optimising internal design of glass inkjet devices allowed the production of high-throughput droplet ejectors.

  7. Light extinction and scattering by agarose based resin beads and applications in high-throughput screening.

    PubMed

    Kittelmann, Jörg; Hämmerling, Frank; Ebeler, Moritz; Hubbuch, Jürgen

    2015-06-01

    Optimization of chromatographic processes by high-throughput screening (HTS) methodologies have become a critical part of downstream process development. Nevertheless there are still no non-invasive optical methods to characterize resin as well as protein-resin interaction on liquid-handling platforms available. Several approaches to automated resin screening in microplates are described in literature, yet all those methods involve indirect measurements by removal of, and sample quantification within, supernatant volumes. In this work, we introduce light extinction by light scattering to directly assess resin volume and bead density within microplates. Methods for this novel resin characterization are described for 96 and 384-well microplates. An example application demonstrates ligand concentration measurement in microplates with four commercial SP Sepharose™ Fast Flow batches. Further, direct quantification of adsorbent bound biomolecules is shown in an example with kinetic protein-resin interaction measurement in a batch screening process. This new approach is expected to promote batch-based resin characterization and monitoring on HTS platforms and further miniaturization and increase in throughput of chromatographic HTS processes.

  8. Development and application of a high-throughput platform for perfusion-based cell culture processes.

    PubMed

    Villiger-Oberbek, Agata; Yang, Yang; Zhou, Weichang; Yang, Jianguo

    2015-10-20

    A high-throughput (HT) cell culture model has been established for the support of perfusion-based cell culture processes operating at high cell densities. To mimic perfusion, the developed platform takes advantage of shake tubes and operates them in a batch-refeed mode with daily medium exchange to supply the cultures with nutrients and remove toxic byproducts. By adjusting the shaking parameters, such as the speed and setting angle, we have adapted the shake tubes to a semi-continuous production of a recombinant enzyme in a perfusion-like mode. We have demonstrated that the developed model can be used to select clones and cell culture media ahead of process optimization studies in bioreactors and confirmed the applicability of shake tubes to a perfusion-like cell culture reaching ∼50E6 viable cells/mL. Furthermore, through regular cell mass removal and periodic medium exchange we have successfully maintained satellite cultures of bench-top perfusion bioreactors, achieving a sustainable cell culture performance at ≥30E6 viable cells/mL and viabilities >80% for over 58 days. The established HT model is a unique and powerful tool that can be used for the development and screening of media formulations, or for testing selected process parameters during both process optimization and manufacturing support campaigns.

  9. Discovery of Novel NOx Catalysts for CIDI Applications by High-throughput Methods

    SciTech Connect

    Blint, Richard J.

    2007-12-31

    DOE project DE-PS26-00NT40758 has developed very active, lean exhaust, NOx reduction catalysts that have been tested on the discovery system, laboratory reactors and engine dynamometer systems. The goal of this project is the development of effective, affordable NOx reduction catalysts for lean combustion engines in the US light duty vehicle market which can meet Tier II emission standards with hydrocarbons based reductants for reducing NOx. General Motors (prime contractor) along with subcontractors BASF (Engelhard) (a catalytic converter developer) and ACCELRYS (an informatics supplier) carried out this project which began in August of 2002. BASF (Engelhard) has run over 16,000 tests of 6100 possible catalytic materials on a high throughput discovery system suitable for automotive catalytic materials. Accelrys developed a new database informatics system which allowed material tracking and data mining. A program catalyst was identified and evaluated at all levels of the program. Dynamometer evaluations of the program catalyst both with and without additives show 92% NOx conversions on the HWFET, 76% on the US06, 60% on the cold FTP and 65% on the Set 13 heavy duty test using diesel fuel. Conversions of over 92% on the heavy duty FTP using ethanol as a second fluid reductant have been measured. These can be competitive with both of the alternative lean NOx reduction technologies presently in the market. Conversions of about 80% were measured on the EUDC for lean gasoline applications without using active dosing to adjust the C:N ratio for optimum NOx reduction at all points in the certification cycle. A feasibility analysis has been completed and demonstrates the advantages and disadvantages of the technology using these materials compared with other potential technologies. The teaming agreements among the partners contain no obstacles to commercialization of new technologies to any potential catalyst customers.

  10. Comparison of mapping algorithms used in high-throughput sequencing: application to Ion Torrent data

    PubMed Central

    2014-01-01

    Background The rapid evolution in high-throughput sequencing (HTS) technologies has opened up new perspectives in several research fields and led to the production of large volumes of sequence data. A fundamental step in HTS data analysis is the mapping of reads onto reference sequences. Choosing a suitable mapper for a given technology and a given application is a subtle task because of the difficulty of evaluating mapping algorithms. Results In this paper, we present a benchmark procedure to compare mapping algorithms used in HTS using both real and simulated datasets and considering four evaluation criteria: computational resource and time requirements, robustness of mapping, ability to report positions for reads in repetitive regions, and ability to retrieve true genetic variation positions. To measure robustness, we introduced a new definition for a correctly mapped read taking into account not only the expected start position of the read but also the end position and the number of indels and substitutions. We developed CuReSim, a new read simulator, that is able to generate customized benchmark data for any kind of HTS technology by adjusting parameters to the error types. CuReSim and CuReSimEval, a tool to evaluate the mapping quality of the CuReSim simulated reads, are freely available. We applied our benchmark procedure to evaluate 14 mappers in the context of whole genome sequencing of small genomes with Ion Torrent data for which such a comparison has not yet been established. Conclusions A benchmark procedure to compare HTS data mappers is introduced with a new definition for the mapping correctness as well as tools to generate simulated reads and evaluate mapping quality. The application of this procedure to Ion Torrent data from the whole genome sequencing of small genomes has allowed us to validate our benchmark procedure and demonstrate that it is helpful for selecting a mapper based on the intended application, questions to be addressed, and the

  11. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications

    NASA Astrophysics Data System (ADS)

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, Sanghyeon; Choi, Won Jun

    2016-02-01

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates.

  12. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications

    PubMed Central

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun

    2016-01-01

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates. PMID:26864968

  13. Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications.

    PubMed

    Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun

    2016-02-11

    Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called "Si photonics"). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates.

  14. Quantifying the Onset and Progression of Plant Senescence by Color Image Analysis for High Throughput Applications

    PubMed Central

    Cai, Jinhai; Okamoto, Mamoru; Atieno, Judith; Sutton, Tim; Li, Yongle; Miklavcic, Stanley J.

    2016-01-01

    Leaf senescence, an indicator of plant age and ill health, is an important phenotypic trait for the assessment of a plant’s response to stress. Manual inspection of senescence, however, is time consuming, inaccurate and subjective. In this paper we propose an objective evaluation of plant senescence by color image analysis for use in a high throughput plant phenotyping pipeline. As high throughput phenotyping platforms are designed to capture whole-of-plant features, camera lenses and camera settings are inappropriate for the capture of fine detail. Specifically, plant colors in images may not represent true plant colors, leading to errors in senescence estimation. Our algorithm features a color distortion correction and image restoration step prior to a senescence analysis. We apply our algorithm to two time series of images of wheat and chickpea plants to quantify the onset and progression of senescence. We compare our results with senescence scores resulting from manual inspection. We demonstrate that our procedure is able to process images in an automated way for an accurate estimation of plant senescence even from color distorted and blurred images obtained under high throughput conditions. PMID:27348807

  15. Quantifying the Onset and Progression of Plant Senescence by Color Image Analysis for High Throughput Applications.

    PubMed

    Cai, Jinhai; Okamoto, Mamoru; Atieno, Judith; Sutton, Tim; Li, Yongle; Miklavcic, Stanley J

    2016-01-01

    Leaf senescence, an indicator of plant age and ill health, is an important phenotypic trait for the assessment of a plant's response to stress. Manual inspection of senescence, however, is time consuming, inaccurate and subjective. In this paper we propose an objective evaluation of plant senescence by color image analysis for use in a high throughput plant phenotyping pipeline. As high throughput phenotyping platforms are designed to capture whole-of-plant features, camera lenses and camera settings are inappropriate for the capture of fine detail. Specifically, plant colors in images may not represent true plant colors, leading to errors in senescence estimation. Our algorithm features a color distortion correction and image restoration step prior to a senescence analysis. We apply our algorithm to two time series of images of wheat and chickpea plants to quantify the onset and progression of senescence. We compare our results with senescence scores resulting from manual inspection. We demonstrate that our procedure is able to process images in an automated way for an accurate estimation of plant senescence even from color distorted and blurred images obtained under high throughput conditions.

  16. Quantifying the Onset and Progression of Plant Senescence by Color Image Analysis for High Throughput Applications.

    PubMed

    Cai, Jinhai; Okamoto, Mamoru; Atieno, Judith; Sutton, Tim; Li, Yongle; Miklavcic, Stanley J

    2016-01-01

    Leaf senescence, an indicator of plant age and ill health, is an important phenotypic trait for the assessment of a plant's response to stress. Manual inspection of senescence, however, is time consuming, inaccurate and subjective. In this paper we propose an objective evaluation of plant senescence by color image analysis for use in a high throughput plant phenotyping pipeline. As high throughput phenotyping platforms are designed to capture whole-of-plant features, camera lenses and camera settings are inappropriate for the capture of fine detail. Specifically, plant colors in images may not represent true plant colors, leading to errors in senescence estimation. Our algorithm features a color distortion correction and image restoration step prior to a senescence analysis. We apply our algorithm to two time series of images of wheat and chickpea plants to quantify the onset and progression of senescence. We compare our results with senescence scores resulting from manual inspection. We demonstrate that our procedure is able to process images in an automated way for an accurate estimation of plant senescence even from color distorted and blurred images obtained under high throughput conditions. PMID:27348807

  17. Development and application of high-throughput techniques for evaluation of photopolymerizable monomer formulations

    NASA Astrophysics Data System (ADS)

    Johnson, Peter Michael

    Photopolymerization of multicomponent mixtures is widely used in industry to create a wide range of material properties for a variety of applications including microelectronics, contact lenses, dental restorations, adhesives and coatings. Under typical photopolymerization conditions, crosslinking polymerization exhibit non-classical phenomena such as autoacceleration, autodeceleration, incomplete conversion, and reaction diffusion controlled termination. These non-classical behaviors present difficult challenges to predicting network properties over various polymerization conditions. However, the complex photopolymerization reaction that generates these properties is modulated by the variety of conditions that these materials are subjected to both during and after the photopolymerization. A design of experiments approach limits the number of systems that are required for analysis, but a significant fraction of photopolymer analysis techniques are time consuming and the conclusions are limited to the region analyzed. This process creates a large bottleneck in the ability to optimize formulations and understand the effects that develop such advantageous properties. The wide spectrum of monomer chemistries add to this complexity and hinder the efficient optimization of various control parameters to produce a resulting polymer with the suitable material properties. In this work, high-throughput techniques for analyzing photopolymer conversion and modeling copolymerizations were developed. Controllable gradients of significant properties in photopolymerization were employed to produce a two factor system with all available analysis conditions on a single substrate. Gradients of composition, light intensity, exposure time, and temperature were all developed to produce combinatorial samples that were subsequently analyzed for their conversion. This technique allows for the rapid assessment of conversion over the entire substrate, generating a large set of data in a

  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. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  20. Automated high-throughput fatty acid analysis of umbilical cord serum and application to an epidemiological study.

    PubMed

    Lin, Yu Hong; Salem, Norman; Wells, Ellen M; Zhou, Weiyin; Loewke, James D; Brown, James A; Lands, William E M; Goldman, Lynn R; Hibbeln, Joseph R

    2012-05-01

    Large population studies show that polyunsaturated fatty acids are important for human health, but determining relationships between the health benefits and the fatty acid content has been hampered by the unavailability of labor-effective high-throughput technologies. An automated high throughput fatty acid analysis was developed from a previous procedure based on direct transesterification including the automation of chemical procedures, data acquisition and automatic data processing. The method was validated and applied to umbilical cord serum samples in an epidemiological study. The method was linear in the range of 1-600 μg/mL serum with r² ≥ 0.99. The within-run CV was <5.4% for 23 fatty acids and a range of recoveries over three concentrations were 76-119% in a low-lipid matrix with the exception of 14:0. The fatty acid concentration as measured by the robotic method for human plasma was in good agreement with the Lepage & Roy method. The fatty acid profile in umbilical cord serum from American subjects (n = 287) showed an average of 38.0, 24.9, 32.0 and 4.6% of total fatty acids for saturates, monounsaturates, n-6 and n-3 polyunsaturates, respectively. This is the first report of a complete, validated, cost-effective, automated, high throughput fatty acid measurement method along with application to a population-based study. Automated fatty acid analysis coupled with automated data processing greatly facilitates the high throughput, 72 samples transesterified in 6 h, required for large population-based studies. PMID:22430941

  1. Development of Control Applications for High-Throughput Protein Crystallography Experiments

    SciTech Connect

    Gaponov, Yurii A.; Matsugaki, Naohiro; Honda, Nobuo; Sasajima, Kumiko; Igarashi, Noriyuki; Hiraki, Masahiko; Yamada, Yusuke; Wakatsuki, Soichi

    2007-01-19

    An integrated client-server control system (PCCS) with a unified relational database (PCDB) has been developed for high-throughput protein crystallography experiments on synchrotron beamlines. The major steps in protein crystallographic experiments (purification, crystallization, crystal harvesting, data collection, and data processing) are integrated into the software. All information necessary for performing protein crystallography experiments is stored in the PCDB database (except raw X-ray diffraction data, which is stored in the Network File Server). To allow all members of a protein crystallography group to participate in experiments, the system was developed as a multi-user system with secure network access based on TCP/IP secure UNIX sockets. Secure remote access to the system is possible from any operating system with X-terminal and SSH/X11 (Secure Shell with graphical user interface) support. Currently, the system covers the high-throughput X-ray data collection stages and is being commissioned at BL5A and NW12A (PF, PF-AR, KEK, Tsukuba, Japan)

  2. High throughput sequencing methods for microbiome profiling: application to food animal systems.

    PubMed

    Highlander, Sarah K

    2012-06-01

    Analysis of microbial communities using high throughput sequencing methods began in the mid 2000s permitting the production of 1000s to 10,000s of sequence reads per sample and megabases of data per sequence run. This then unprecedented depth of sequencing allowed, for the first time, the discovery of the 'rare biosphere' in environmental samples. The technology was quickly applied to studies in several human subjects. Perhaps these early studies served as a reminder that though the microbes that inhabit mammals are known to outnumber host cells by an order of magnitude or more, most of these are unknown members of our second genome, or microbiome (as coined by Joshua Lederberg), because of our inability to culture them. High throughput methods for microbial 16S ribosomal RNA gene and whole genome shotgun (WGS) sequencing have now begun to reveal the composition and identity of archaeal, bacterial and viral communities at many sites, in and on the human body. Surveys of the microbiota of food production animals have been published in the past few years and future studies should benefit from protocols and tools developed from large-scale human microbiome studies. Nevertheless, production animal-related resources, such as improved host genome assemblies and increased numbers and diversity of host-specific microbial reference genome sequences, will be needed to permit meaningful and robust analysis of 16S rDNA and WGS sequence data.

  3. BatchPrimer3: A high throughput web application for PCR and sequencing primer design

    PubMed Central

    You, Frank M; Huo, Naxin; Gu, Yong Qiang; Luo, Ming-cheng; Ma, Yaqin; Hane, Dave; Lazo, Gerard R; Dvorak, Jan; Anderson, Olin D

    2008-01-01

    Background Microsatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Results A new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories

  4. An Automated Application Framework to Model Disordered Materials Based on a High Throughput First Principles Approach

    NASA Astrophysics Data System (ADS)

    Oses, Corey; Yang, Kesong; Curtarolo, Stefano; Duke Univ Collaboration; UC San Diego Collaboration

    Predicting material properties of disordered systems remains a long-standing and formidable challenge in rational materials design. To address this issue, we introduce an automated software framework capable of modeling partial occupation within disordered materials using a high-throughput (HT) first principles approach. At the heart of the approach is the construction of supercells containing a virtually equivalent stoichiometry to the disordered material. All unique supercell permutations are enumerated and material properties of each are determined via HT electronic structure calculations. In accordance with a canonical ensemble of supercell states, the framework evaluates ensemble average properties of the system as a function of temperature. As proof of concept, we examine the framework's final calculated properties of a zinc chalcogenide (ZnS1-xSex), a wide-gap oxide semiconductor (MgxZn1-xO), and an iron alloy (Fe1-xCux) at various stoichiometries.

  5. Experiment and Modeling of Spatially Indexed Microbead Arrays for High-Throughput Screening Applications

    NASA Astrophysics Data System (ADS)

    Leary, Thomas; Maldarelli, Charles; Couzis, Alexander

    2011-11-01

    The development of platforms for multiplexed, high throughput screening of the binding interactions of target biomolecules against a library of potential binding probes enables progress in many areas in medicine and biology. Formats in which probes are linked to microbeads arrayed in a microfluidic channel offer high sensitivity, reduced reagent consumption and are easily parallelized for multiplexed detection. This presentation describes a microfluidically assembled microbead array in which beads are streamed through a channel with an array of wells inscribed in the floor of the channel. The beads are captured in the wells via gravity. We demonstrate that an array of beads displaying different receptors can be assembled in this format, indexed by sequential depostion and used for a prototype assay. Solutions for a two dimensional mass transfer model of the conjugation of the probe to the receptor on the bead surface identify kinetically limited regimes which are used to measure the binding kinetics of the prototype assay.

  6. High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation.

    PubMed

    Allmendinger, Andrea; Dieu, Le-Ha; Fischer, Stefan; Mueller, Robert; Mahler, Hanns-Christian; Huwyler, Jörg

    2014-10-01

    Viscosity characterization of protein formulations is of utmost importance for the development of subcutaneously administered formulations. However, viscosity determinations are time-consuming and require large sample volumes in the range of hundreds of microliters to a few milliliters, depending on the method used. In this article, an automated, high-throughput method is described to determine dynamic viscosity of Newtonian fluids using standard capillary electrophoresis (CE) equipment. CE is an analytical method routinely used for the separation and characterization of proteins. In our set-up, the capillary is filled with the test sample, and a constant pressure is applied. A small aliquot of riboflavin is subsequently loaded into the capillary and used as a dye to monitor movement of protein samples. Migration time of the riboflavin peak moving through the filled capillary is converted to the viscosity by applying the Hagen-Poiseuille's law. The instrument is operated without using an electrical field. Repeatability, robustness, linearity, and reproducibility were demonstrated for different capillary lots and instruments, as well as for different capillary lengths and diameters. Accuracy was verified by comparing the viscosity data obtained by CE instrumentation with those obtained by plate/cone rheometry. The suitability of the method for protein formulations was demonstrated, and limitations were discussed. Typical viscosities in the range of 5-40mPas were reliably measured with this method. Advantages of the CE instrumentation-based method included short measurement times (1-15min), small sample volumes (few microliters) for a capillary with a diameter of 50μm and a length of 20.5cm as well as potential to be suitable for high-throughput measurements.

  7. High-throughput genotyping: practical considerations concerning the day-to-day application

    NASA Astrophysics Data System (ADS)

    McIndoe, Richard A.; Bumgarner, R. E.; Welti, Russ; Hood, Leroy

    1996-04-01

    Advances in high throughput genotyping protocols over the past few years have been remarkable. Most protocols developed to increase the throughput of genotyping rely on fluorescent based technologies for data acquisition and capture. In general, the number of genotypes per day quoted for these protocols are the result of extrapolations based on ideal situations. Here we present our experience with respect to the day to day problems of high throughput genotyping. Our laboratory is currently working on several genetic mapping projects in both mouse and man. For example, we are looking at the genetic basis for susceptibility to rheumatoid arthritis in a local native American tribe as well as a mouse animal model for the same disease. The machines used to collect gel image data are two Li- Cor infrared DNA sequencers adapted for genotyping. During the evolution of these projects, we have addressed issues concerning the tracking and flow of information from the initial extraction of DNA to the calling of the genotypes. In particular, we have focused on designing methods that are efficient, cost effective and can be easily taught to the technical staff. Computer programs have been written that record gel specific information (e.g. ID information), archive data and capture genotypes in a simple point and click environment. Instrumentation was purchased to ease the repetitive nature of sample allocation, reagent disbursement and gel loading. Using this system, we can produce genotype data on 96 individuals for 20 loci (1920 genotypes) in one day. Solutions to the overall flow of information at each of these junctions are discussed.

  8. High-throughput protein crystallization.

    PubMed

    Stevens, R C

    2000-10-01

    The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.

  9. Strategies and applications of combinatorial methods and high throughput screening to the discovery of non-noble metal catalyst

    NASA Astrophysics Data System (ADS)

    Bricker, Maureen L.; Sachtler, J. W. Adriaan; Gillespie, Ralph D.; McGonegal, Charles P.; Vega, Honorio; Bem, Dave S.; Holmgren, Jennifer S.

    2004-02-01

    The integrated End-to-End™ combinatorial process for catalyst preparation and screening, with emphasis on its capability to vary both process and compositional parameters will be demonstrated. Additionally, each step of the combinatorial screening process has been validated against results from traditional screening methods. The greatest challenge of all has been the adherence to the core concepts of the combinatorial approach. Catalyst libraries have been made and tested for naphthalene dehydrogenation chemistry. The preparation of these libraries has included the application of high throughput techniques for: metal impregnation; catalyst finishing; catalyst screening. The catalyst screening system has been used to find a non-noble metal catalyst system that can replace Pt in dehydrogenation applications in the petroleum industry. A proprietary catalytic composition was developed for the dehydrogenation of methylcyclohexane (MCH) to toluene starting with four non-noble metals of different proportions and four different supports (alumina, titania, zirconia and silica) prepared in different ways and applying a statistical design of experiments. These data demonstrate that all steps of catalyst preparation and screening are performed in a rapid, useful, high throughput manner. Data will be presented from the catalyst screening efforts will demonstrate that optimized metal composition is dependent on the support type.

  10. Facile and rapid generation of 3D chemical gradients within hydrogels for high-throughput drug screening applications.

    PubMed

    Ahadian, Samad; Ramón-Azcón, Javier; Estili, Mehdi; Obregón, Raquel; Shiku, Hitoshi; Matsue, Tomokazu

    2014-09-15

    We propose a novel application of dielectrophoresis (DEP) to make three-dimensional (3D) methacrylated gelatin (GelMA) hydrogels with gradients of micro- and nanoparticles. DEP forces were able to manipulate micro- and nanoparticles of different sizes and materials (i.e., C2C12 myoblasts, polystyrene beads, gold microparticles, and carbon nanotubes) within GelMA hydrogels in a rapid and facile way and create 3D gradients of these particles in a microchamber. Immobilization of drugs, such as fluorescein isothiocyanate-dextran (FITC-dextran) and 6-hydroxydopamine (6-OHDA), on gold microparticles allowed us to investigate the high-throughput release of these drugs from GelMA-gold microparticle gradient systems. The latter gradient constructs were incubated with C2C12 myoblasts for 24h to examine the cell viability through the release of 6-OHDA. The drug was released from the microparticles in a gradient manner, inducing a cell viability gradient. This novel approach to create 3D chemical gradients within hydrogels is scalable to any arbitrary length scale. It is useful for making anisotropic biomimetic materials and high-throughput platforms to investigate cell-microenvironment interactions in a rapid, simple, cost-effective, and reproducible manner.

  11. Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and...

  12. Application of phage display to high throughput antibody generation and characterization

    PubMed Central

    Schofield, Darren J; Pope, Anthony R; Clementel, Veronica; Buckell, Jenny; Chapple, Susan DJ; Clarke, Kay F; Conquer, Jennie S; Crofts, Anna M; Crowther, Sandra RE; Dyson, Michael R; Flack, Gillian; Griffin, Gareth J; Hooks, Yvette; Howat, William J; Kolb-Kokocinski, Anja; Kunze, Susan; Martin, Cecile D; Maslen, Gareth L; Mitchell, Joanne N; O'Sullivan, Maureen; Perera, Rajika L; Roake, Wendy; Shadbolt, S Paul; Vincent, Karen J; Warford, Anthony; Wilson, Wendy E; Xie, Jane; Young, Joyce L; McCafferty, John

    2007-01-01

    We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation. PMID:18047641

  13. The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

    PubMed

    Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

    2014-05-01

    The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

  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. High-Throughput Sequencing, a Versatile Weapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology

    PubMed Central

    Caboche, Ségolène; Audebert, Christophe; Hot, David

    2014-01-01

    The recent progresses of high-throughput sequencing (HTS) technologies enable easy and cost-reduced access to whole genome sequencing (WGS) or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose. PMID:25437800

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

    PubMed

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

    2016-02-01

    Inspired by the paper platforms for 3-D cell culture, a paper-based microfluidic device containing drug concentration gradient was designed and constructed for investigating cell response to drugs based on high throughput analysis. This drug gradient generator was applied to generate concentration gradients of doxorubicin (DOX) as the model drug. HeLa cells encapsulated in collagen hydrogel were incubated in the device reservoirs to evaluate the cell viability based on the controlled release of DOX spatially. It was demonstrated that drug diffusion through the paper fibers created a gradient of drug concentration, which influenced cell viability. This drug screening platform has a great opportunity to be applied for drug discovery and diagnostic studies with simultaneous and parallel tests of drugs under various gradient concentrations. PMID:26864970

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

    PubMed

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

    2016-02-01

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

  18. 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.

  19. Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications.

    PubMed

    Fuchs, David; Pedersen-Bjergaard, Stig; Jensen, Henrik; Rand, Kasper D; Honoré Hansen, Steen; Petersen, Nickolaj Jacob

    2016-07-01

    The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC-MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; R(2), 0.994) and finally, the EME-autosampler was used to analyze in vitro conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis. PMID:27237618

  20. [Low field nuclear magnetic resonance for rapid quantitation of microalgae lipid and its application in high throughput screening].

    PubMed

    Liu, Tingting; Yang, Yi; Wang, Zejian; Zhuang, Yingping; Chu, Ju; Guoi, Meijin

    2016-03-01

    A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2 > 0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency. PMID:27349121

  1. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Lu, Guoxin

    2007-01-01

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

  2. [Double-stranded DNA microarray: principal, techniques and applications].

    PubMed

    Pan, Yan; Wang, Jin-Ke

    2013-03-01

    Double-stranded DNA (dsDNA) microarray, also known as protein binding microarray (PBM), is an important technique that can be used to assay the interaction of DNA-binding protein (such as transcription factor, TF) with vast amount of DNA molecules in high-throughput format. This technique immobilizes large amount of various dsDNA molecules on the surface of a solid support (such as glass slide) for detecting the binding interaction of a DNA-binding protein with all of the immobilized dsDNA molecules, and thus determining the DNA-binding affinity, specificity and preference of TFs. In recent years, this technique has demonstrated its valuable applications in several aspects, including rapidly characterizing DNA-binding specificity of large number of TFs, building DNA-binding profiles of TFs, identifying DNA-binding sites and target genes of TFs, discriminating the subtle DNA-binding preferences of members and their dimmers of a TF family, and examining the effects of a cofactor on the DNA-binding specificity of TFs. This paper reviews the principal, techniques, and applications of dsDNA microarray.

  3. High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

    PubMed Central

    Moller, Isabel; Marcus, Susan E.; Haeger, Ash; Verhertbruggen, Yves; Verhoef, Rene; Schols, Henk; Ulvskov, Peter; Mikkelsen, Jørn Dalgaard; Knox, J. Paul

    2007-01-01

    Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. PMID:17629746

  4. High-throughput spectrometer designs in a compact form-factor: principles and applications

    NASA Astrophysics Data System (ADS)

    Norton, S. M.

    2013-05-01

    Many compact, portable Raman spectrometers have entered the market in the past few years with applications in narcotics and hazardous material identification, as well as verification applications in pharmaceuticals and security screening. Often, the required compact form-factor has forced designers to sacrifice throughput and sensitivity for portability and low-cost. We will show that a volume phase holographic (VPH)-based spectrometer design can achieve superior throughput and thus sensitivity over conventional Czerny-Turner reflective designs. We will look in depth at the factors influencing throughput and sensitivity and illustrate specific VPH-based spectrometer examples that highlight these design principles.

  5. Laser Desorption Mass Spectrometry for High Throughput DNA Analysis and Its Applications

    SciTech Connect

    Allman, S.L.; Chen, C.H.; Golovlev, V.V.; Isola, N.R.; Matteson, K.J.; Potter, N.T.; Taranenko, N.I.

    1999-01-23

    Laser desorption mass spectrometry (LDMS) has been developed for DNA sequencing, disease diagnosis, and DNA Fingerprinting for forensic applications. With LDMS, the speed of DNA analysis can be much faster than conventional gel electrophoresis. No dye or radioactive tagging to DNA segments for detection is needed. LDMS is emerging as a new alternative technology for DNA analysis.

  6. Laser desorption mass spectrometry for high-throughput DNA analysis and its applications

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Golovlev, Valeri V.; Taranenko, N. I.; Allman, S. L.; Isola, Narayana R.; Potter, N. T.; Matteson, K. J.; Chang, Linus Y.

    1999-05-01

    Laser desorption mass spectrometry (LDMS) has been developed for DNA sequencing, disease diagnosis, and DNA fingerprinting for forensic applications. With LDMS, the speed of DNA analysis can be much faster than conventional gel electrophoresis. No dye or radioactive tagging to DNA segments for detection is needed. LDMS is emerging as a new alternative technology for DNA analysis.

  7. STITCHER: A web resource for high-throughput design of primers for overlapping PCR applications.

    PubMed

    O'Halloran, Damien M

    2015-06-01

    Overlapping PCR is routinely used in a wide number of molecular applications. These include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping by traditional PCR techniques and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online (http://ohalloranlab.net/STITCHER.html). STITCHER can handle both single sequence and multi-sequence input, and specific features facilitate numerous other PCR applications, including assembly PCR, adapter PCR, and primer walking. Field PCR, and in particular, LAMP, offers promise as an on site tool for pathogen detection in underdeveloped areas, and STITCHER includes off-target detection features for pathogens commonly targeted using LAMP technology.

  8. Self-sealed vertical polymeric nanoporous-junctions for high-throughput nanofluidic applications.

    PubMed

    Kim, Sung Jae; Han, Jongyoon

    2008-05-01

    We developed a reliable but simple integration method of polymeric nanostructure in a poly(dimethylsiloxane) (PDMS)-based microfluidic channel, for nanofluidic applications. The Nafion polymer junction was creased by infiltrating polymer solution between the gaps created by mechanical cutting, without any photolithography or etching processes. The PDMS can seal itself with the heterogeneous polymeric nanoporous material between the PDMS/PDMS gap due to its flexibility without any (covalent) bonding between PDMS and the polymer materials. Thus, one can easily integrate the nanoporous-junction into a PDMS microchip in a leak-free manner with excellent repeatability. We demonstrated nanofluidic preconcentration of proteins (beta-phycoerythrin) using the device. Because the polymeric junction spans across the entire microchannel height, the preconcentration was achieved with high-pressure field or even in large channels, with the dimensions of 1000 microm width x 100 microm depth.

  9. Application of microarray technology in pulmonary diseases

    PubMed Central

    Tzouvelekis, Argyris; Patlakas, George; Bouros, Demosthenes

    2004-01-01

    Microarrays are a powerful tool that have multiple applications both in clinical and cell biology arenas of common lung diseases. To exemplify how this tool can be useful, in this review, we will provide an overview of the application of microarray technology in research relevant to common lung diseases and present some of the future perspectives. PMID:15585067

  10. High-throughput proteomics

    NASA Astrophysics Data System (ADS)

    Lesley, Scott A.; Nasoff, Marc; Kreusch, Andreas; Spraggon, Glen

    2001-04-01

    Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. We are addressing this challenge through instrumentation and approaches to rapidly express, purify, crystallize, and mutate large numbers of human gene products. Our approach applies the principles of HTS technologies commonly used in pharmaceutical development. Genes are cloned, expressed, and purified in parallel to achieve a throughput potential of hundreds per day. Our instrumentation allows us to produce tens of milligrams of protein from 96 separate clones simultaneously. Purified protein is used for several applications including a high-throughput crystallographic screening approach for structure determination using automated image analysis. To further understand protein function, we are integrating a mutagenesis and screening approach. By combining these key technologies, we hope to provide a fundamental basis for understanding gene function at the protein level.

  11. High-throughput design of low-activation, high-strength creep-resistant steels for nuclear-reactor applications

    NASA Astrophysics Data System (ADS)

    Lu, Qi; van der Zwaag, Sybrand; Xu, Wei

    2016-02-01

    Reduced-activation ferritic/martensitic steels are prime candidate materials for structural applications in nuclear power reactors. However, their creep strength is much lower than that of creep-resistant steel developed for conventional fossil-fired power plants as alloying elements with a high neutron activation cannot be used. To improve the creep strength and to maintain a low activation, a high-throughput computational alloy design model coupling thermodynamics, precipitate-coarsening kinetics and an optimization genetic algorithm, is developed. Twelve relevant alloying elements with either low or high activation are considered simultaneously. The activity levels at 0-10 year after the end of irradiation are taken as optimization parameter. The creep-strength values (after exposure for 10 years at 650 °C) are estimated on the basis of the solid-solution strengthening and the precipitation hardening (taking into account precipitate coarsening). Potential alloy compositions leading to a high austenite fraction or a high percentage of undesirable second phase particles are rejected automatically in the optimization cycle. The newly identified alloys have a much higher precipitation hardening and solid-solution strengthening at the same activity level as existing reduced-activation ferritic/martensitic steels.

  12. 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...

  13. Identifying Toxicity Pathways with ToxCast High-Throughput Screening and Applications to Predicting Developmental Toxicity

    EPA Science Inventory

    Results from rodent and non-rodent prenatal developmental toxicity tests for over 300 chemicals have been curated into the relational database ToxRefDB. These same chemicals have been run in concentration-response format through over 500 high-throughput screening assays assessin...

  14. A low-cost, high-throughput, automated single nucleotide polymorphism assay for forensic human DNA applications.

    PubMed

    Pomeroy, Robert; Duncan, George; Sunar-Reeder, Bulbin; Ortenberg, Elen; Ketchum, Melba; Wasiluk, Hannah; Reeder, Dennis

    2009-12-01

    Single nucleotide polymorphism (SNP) analysis of human DNA for the purpose of identification has some promising attributes. The question of approach is critical to the eventual adoption of this technology. The use of a low-volume open array platform was tested with a small selected set of eight SNP primers that have a low F(ST) (the proportion of the total genetic variance contained in a subpopulation [S subscript] relative to the total genetic variance [T subscript]) in human populations. Because multiple SNPs must be interrogated, issues concerning DNA concentration, total DNA, and whole genome amplification were investigated. Excellent correlations were obtained for seven of the eight SNP assays on a set of DNA samples of known configuration over a broad concentration range spanning 25-150ng/microl in blind studies. These seven SNP assays were then applied to 39 DNA samples in a population from southern India. These SNPs were sufficient to individualize each member of this sample population. In a paternity study, these same SNPs showed clear parental relationships. For low amounts of genomic DNA, the use of a commercially available whole genome amplification kit showed promise for genotyping sub-nanogram samples. Discrimination against nonhuman DNA was also demonstrated successfully. Because of the very low quantities of reagents used in the assay, the cost per test becomes reasonably inexpensive. Overall, using commercially available SNP assays, the OpenArray platform showed excellent promise as a highly automated, low-volume, high-throughput system for SNP analysis with potential applications to relevant forensic analyses such as identification and paternity.

  15. Plant seed species identification from chemical fingerprints: a high-throughput application of direct analysis in real time mass spectrometry.

    PubMed

    Lesiak, Ashton D; Cody, Robert B; Dane, A John; Musah, Rabi A

    2015-09-01

    Plant species identification based on the morphological features of plant parts is a well-established science in botany. However, species identification from seeds has largely been unexplored, despite the fact that the seeds contain all of the genetic information that distinguishes one plant from another. Using seeds of genus Datura plants, we show here that the mass spectrum-derived chemical fingerprints for seeds of the same species are similar. On the other hand, seeds from different species within the same genus display distinct chemical signatures, even though they may contain similar characteristic biomarkers. The intraspecies chemical signature similarities on the one hand, and interspecies fingerprint differences on the other, can be processed by multivariate statistical analysis methods to enable rapid species-level identification and differentiation. The chemical fingerprints can be acquired rapidly and in a high-throughput manner by direct analysis in real time mass spectrometry (DART-MS) analysis of the seeds in their native form, without use of a solvent extract. Importantly, knowledge of the identity of the detected molecules is not required for species level identification. However, confirmation of the presence within the seeds of various characteristic tropane and other alkaloids, including atropine, scopolamine, scopoline, tropine, tropinone, and tyramine, was accomplished by comparison of the in-source collision-induced dissociation (CID) fragmentation patterns of authentic standards, to the fragmentation patterns observed in the seeds when analyzed under similar in-source CID conditions. The advantages, applications, and implications of the chemometric processing of DART-MS derived seed chemical signatures for species level identification and differentiation are discussed.

  16. 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.

  17. Progress in the application of DNA microarrays.

    PubMed Central

    Lobenhofer, E K; Bushel, P R; Afshari, C A; Hamadeh, H K

    2001-01-01

    Microarray technology has been applied to a variety of different fields to address fundamental research questions. The use of microarrays, or DNA chips, to study the gene expression profiles of biologic samples began in 1995. Since that time, the fundamental concepts behind the chip, the technology required for making and using these chips, and the multitude of statistical tools for analyzing the data have been extensively reviewed. For this reason, the focus of this review will be not on the technology itself but on the application of microarrays as a research tool and the future challenges of the field. PMID:11673116

  18. Protein Microarrays: Novel Developments and Applications

    PubMed Central

    Berrade, Luis; Garcia, Angie E.

    2011-01-01

    Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays. PMID:21116694

  19. 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

  20. Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

    PubMed

    Holt, Tom G; Choi, Bernard K; Geoghagen, Neil S; Jensen, Kristian K; Luo, Qi; LaMarr, William A; Makara, Gergely M; Malkowitz, Lorraine; Ozbal, Can C; Xiong, Yusheng; Dufresne, Claude; Luo, Ming-Juan

    2009-10-01

    Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

  1. Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications.

    PubMed

    Hughes, Stephen R; Butt, Tauseef R; Bartolett, Scott; Riedmuller, Steven B; Farrelly, Philip

    2011-08-01

    The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries.

  2. Microfluidics for cell-based high throughput screening platforms - A review.

    PubMed

    Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

    2016-01-15

    In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.

  3. High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses.

    PubMed

    Abd El-Rehim, Dalia M; Ball, Graham; Pinder, Sarah E; Rakha, Emad; Paish, Claire; Robertson, John F R; Macmillan, Douglas; Blamey, Roger W; Ellis, Ian O

    2005-09-01

    Recent studies on gene molecular profiling using cDNA microarray in a relatively small series of breast cancer have identified biologically distinct groups with apparent clinical and prognostic relevance. The validation of such new taxonomies should be confirmed on larger series of cases prior to acceptance in clinical practice. The development of tissue microarray (TMA) technology provides methodology for high-throughput concomitant analyses of multiple proteins on large numbers of archival tumour samples. In our study, we have used immunohistochemistry techniques applied to TMA preparations of 1,076 cases of invasive breast cancer to study the combined protein expression profiles of a large panel of well-characterized commercially available biomarkers related to epithelial cell lineage, differentiation, hormone and growth factor receptors and gene products known to be altered in some forms of breast cancer. Using hierarchical clustering methodology, 5 groups with distinct patterns of protein expression were identified. A sixth group of only 4 cases was also identified but deemed too small for further detailed assessment. Further analysis of these clusters was performed using multiple layer perceptron (MLP)-artificial neural network (ANN) with a back propagation algorithm to identify key biomarkers driving the membership of each group. We have identified 2 large groups by their expression of luminal epithelial cell phenotypic characteristics, hormone receptors positivity, absence of basal epithelial phenotype characteristics and lack of c-erbB-2 protein overexpression. Two additional groups were characterized by high c-erbB-2 positivity and negative or weak hormone receptors expression but showed differences in MUC1 and E-cadherin expression. The final group was characterized by strong basal epithelial characteristics, p53 positivity, absent hormone receptors and weak to low luminal epithelial cytokeratin expression. In addition, we have identified significant

  4. 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.

  5. Diffractive micro-arrays for active spectroscopy and interconnect applications

    NASA Astrophysics Data System (ADS)

    Castracane, James; Xu, Bai; Gutin, Olga N.; Lavrijsen, Rein; Stollenwerk, Andrew

    2002-06-01

    The use of Micro-Electro-Mechanical Systems (MEMS) technology has opened the door for many applications. In particular, by exploiting the reconfigurability of optical surfaces fabricated with this technology, many sensor, communication and spectroscopic systems can benefit. The controlled re-direction of single or multiple optical input sources can lend itself to high throughput sample analysis or massively parallel optical connectivity. In addition, the change in a MEMS-based optical surface can result in a flexible spectral analysis of incoming radiation. We report on the recent advances in our projects which are focused on the design/simulation, materials processing and integration issues involved with the creation and optimized operation of such diffractive micro-arrays. In this presentation, the state of the art in such devices will be presented which will include the process flow associated with production, structural metrology, optical performance, and parallel switching capabilities of the systems. The use of numerous materials including polysilicon, silicon dioxide and selected polymers as structural layers has enabled the production of devices which can be tailored for specific, performance related applications. Examples to be presented include diffractive surfaces with substantial (1 cm x 1 cm) active areas as well as large arrays with sub-micron feature sizes. Functional integration of the prototype devices include optical interconnects, active spectroscopy and bio/chem diagnostic systems.

  6. Geochip: A high throughput genomic tool for linking community structure to functions

    SciTech Connect

    Van Nostrand, Joy D.; Liang, Yuting; He, Zhili; Li, Guanghe; Zhou, Jizhong

    2009-01-30

    GeoChip is a comprehensive functional gene array that targets key functional genes involved in the geochemical cycling of N, C, and P, sulfate reduction, metal resistance and reduction, and contaminant degradation. Studies have shown the GeoChip to be a sensitive, specific, and high-throughput tool for microbial community analysis that has the power to link geochemical processes with microbial community structure. However, several challenges remain regarding the development and applications of microarrays for microbial community analysis.

  7. Simultaneous determination of multiple angiotensin type 1 receptor antagonists and its application to high-throughput pharmacokinetic study

    NASA Astrophysics Data System (ADS)

    Zhu, Xiaoyan; Sun, Jianguo; Hao, Haiping; Wang, Guangji; Hu, Xiaoling; Lv, Hua; Gu, Shenghua; Wu, Xiaoming; Xu, Jinyi

    2008-05-01

    A rapid and sensitive high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) detection was developed for the simultaneous determination of multiple angiotensin type 1 receptor antagonists (AT1RAs) WX472, WX581, 1b and telmisartan in rat plasma for the purpose of high-throughout pharmacokinetic screening. The method was operated under selected reaction monitoring (SRM) mode in the positive ion mode. The analytes and the internal standard (pitavastatin) were extracted from 100 [mu]L rat plasma under acidic conditions by liquid-liquid extraction with ethyl acetate. The analytes and internal standard were baseline separated on a Gemini analytical column (3 [mu]m, 150 mm × 2.0 mm) with the adoption of a gradient elution using acetonitrile and 0.05% aqueous formic acid. The standard curves were linear in the concentration ranges of 4.5-900 ng/mL for WX472, 5-1000 ng/mL for WX581 and 0.5-100 ng/mL for 1b and telmisartan. Intra- and inter-batch precisions (R.S.D.%) were all within 15% and the method assessed a quite good accuracy (R.E.%). Recoveries were found to be >65% for all the compounds and no obvious matrix effects were found. This method has been successfully applied to the high-throughput pharmacokinetic screening study for both cassette dosing and cassette analysis of four compounds to rats. Significant drug-drug interactions were observed after cassette dosing. The study suggested that cassette analysis of pooled samples would be a better choice for the high-throughput pharmacokinetic screening of angiotensin type 1 receptor antagonists.

  8. Turbulence in microfluidics: Cleanroom-free, fast, solventless, and bondless fabrication and application in high throughput liquid-liquid extraction.

    PubMed

    de Camargo, Camila L; Shiroma, Letícia S; Giordano, Gabriela F; Gobbi, Angelo L; Vieira, Luis C S; Lima, Renato S

    2016-10-12

    This paper addresses an important breakthrough in the deployment of ultra-high adhesion strength microfluidic technologies to provide turbulence at harsh flow rate conditions. This paper is only, to our knowledge, the second reporting on the generation of high flow rate-assisted turbulence in microchannels. This flow solves a crucial bottleneck in microfluidics: the generation of high throughput homogeneous mixings. We focused on the fabrication of bulky polydimethylsiloxane (PDMS) microchips (without any interfaces) rather than the laborious surface modifications that were employed in the first reporting about turbulence-assisted microfluidics. The fabrication is cleanroom-free, simple, low-cost, fast, solventless, and bondless requiring only a laboratory oven. More specifically, our method relies on the shaping of a nylon scaffold, cure of PDMS with embedded nylon, and removal of this scaffold. The scaffold was obtained by manually wrapping nylon threads. The withdrawing out of the scaffold was completed in few seconds using only a plier. Such microchannels endured flow rates of up to 60.0 mL min(-1) with a strikingly low elastic deformation. The importance in producing turbulence into microscale channels was successfully shown in liquid-liquid extractions. The great energy dissipation rate relative to the turbulence created high throughput and efficient extractions in microfluidics for the first time. The residence time was only 0.01 s at 25.0 mL min(-1) (total flow rate of the immiscible phases). In addition, the partition coefficient determined in a single run was similar to that obtained by the conventional batch shake-flask method that was realized in triplicate. PMID:27662761

  9. Turbulence in microfluidics: Cleanroom-free, fast, solventless, and bondless fabrication and application in high throughput liquid-liquid extraction.

    PubMed

    de Camargo, Camila L; Shiroma, Letícia S; Giordano, Gabriela F; Gobbi, Angelo L; Vieira, Luis C S; Lima, Renato S

    2016-10-12

    This paper addresses an important breakthrough in the deployment of ultra-high adhesion strength microfluidic technologies to provide turbulence at harsh flow rate conditions. This paper is only, to our knowledge, the second reporting on the generation of high flow rate-assisted turbulence in microchannels. This flow solves a crucial bottleneck in microfluidics: the generation of high throughput homogeneous mixings. We focused on the fabrication of bulky polydimethylsiloxane (PDMS) microchips (without any interfaces) rather than the laborious surface modifications that were employed in the first reporting about turbulence-assisted microfluidics. The fabrication is cleanroom-free, simple, low-cost, fast, solventless, and bondless requiring only a laboratory oven. More specifically, our method relies on the shaping of a nylon scaffold, cure of PDMS with embedded nylon, and removal of this scaffold. The scaffold was obtained by manually wrapping nylon threads. The withdrawing out of the scaffold was completed in few seconds using only a plier. Such microchannels endured flow rates of up to 60.0 mL min(-1) with a strikingly low elastic deformation. The importance in producing turbulence into microscale channels was successfully shown in liquid-liquid extractions. The great energy dissipation rate relative to the turbulence created high throughput and efficient extractions in microfluidics for the first time. The residence time was only 0.01 s at 25.0 mL min(-1) (total flow rate of the immiscible phases). In addition, the partition coefficient determined in a single run was similar to that obtained by the conventional batch shake-flask method that was realized in triplicate.

  10. Application of Phenotype Microarray technology to soil microbiology

    NASA Astrophysics Data System (ADS)

    Mocali, Stefano

    2016-04-01

    It is well established that soil microorganisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Furthermore, addressing the functionality of genomes is one of the most important and challenging tasks of today's biology. In particular the ability to link genotypes to corresponding phenotypes is of interest in the reconstruction and biotechnological manipulation of metabolic pathways. High-throughput culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. Thus, over the last years, Phenotype Microarray (PM) technology has been used to address many specific issues related to the metabolic functionality of microorganisms. However, computational tools that could directly link PM data with the gene(s) of interest followed by the extraction of information on gene-phenotype correlation are still missing. Here potential applications of phenotype arrays to soil microorganisms, use of the plates in stress response studies and for assessment of phenotype of environmental communities are described. Considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting are also discussed. In particular, here we present DuctApe, a suite that allows the analysis of both genomic sequences and PM data, to find metabolic differences among PM experiments and to correlate them with KEGG pathways and gene presence/absence patterns.

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

    PubMed

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

    2014-07-08

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

  12. High throughput screening informatics.

    PubMed

    Ling, Xuefeng Bruce

    2008-03-01

    High throughput screening (HTS), an industrial effort to leverage developments in the areas of modern robotics, data analysis and control software, liquid handling devices, and sensitive detectors, has played a pivotal role in the drug discovery process, allowing researchers to efficiently screen millions of compounds to identify tractable small molecule modulators of a given biological process or disease state and advance them into high quality leads. As HTS throughput has significantly increased the volume, complexity, and information content of datasets, lead discovery research demands a clear corporate strategy for scientific computing and subsequent establishment of robust enterprise-wide (usually global) informatics platforms, which enable complicated HTS work flows, facilitate HTS data mining, and drive effective decision-making. The purpose of this review is, from the data analysis and handling perspective, to examine key elements in HTS operations and some essential data-related activities supporting or interfacing the screening process, and outline properties that various enabling software should have. Additionally, some general advice for corporate managers with system procurement responsibilities is offered.

  13. 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.

  14. Application of Cassette Ultracentrifugation Using Non-labeled Compounds and Liquid Chromatography-Tandem Mass Spectrometry Analysis for High-Throughput Protein Binding Determination.

    PubMed

    Kieltyka, Kasia; McAuliffe, Brian; Cianci, Christopher; Drexler, Dieter M; Shou, Wilson; Zhang, Jun

    2016-03-01

    Membrane-based devices typically used for serum protein binding determination are not fully applicable to highly lipophilic compounds because of nonspecific binding to the device membrane. Ultracentrifugation, however, completely eliminates the issue by using a membrane-free approach, although its wide application has been limited. This lack of utilization is mainly attributed to 2 factors: the high cost in acquiring and handling of radiolabeled compounds and low assay throughput owing to the difficulties in process automation. To overcome these challenges, we report a high-throughput workflow by cassette ultracentrifugation of nonradiolabeled compounds followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Twenty compounds with diverse physicochemical and protein binding properties were selected for the evaluation of the workflow. To streamline the working process, approaches of matrix balancing for all the samples for LC-MS/MS analysis and determining free fraction without analytical calibration curves were adopted. Both the discrete ultracentrifugation of individual compounds and cassette ultracentrifugation of all the test compounds followed by simultaneous LC-MS/MS analysis exhibited a linear correlation with literature values, demonstrating respectively the validity of the ultracentrifugation process and the cassette approach. The cassette ultracentrifugation using nonradiolabeled compounds followed by LC-MS/MS analysis has greatly facilitated its application for high-throughput protein binding screening in drug discovery.

  15. Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles

    PubMed Central

    Alsenaidy, Mohammad A.; Jain, Nishant K.; Kim, Jae H.; Middaugh, C. Russell; Volkin, David B.

    2014-01-01

    In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies. PMID:24659968

  16. Determination of ligand-MurB interactions by isothermal denaturation: application as a secondary assay to complement high throughput screening.

    PubMed

    Sarver, Ronald W; Rogers, Joseph M; Epps, Dennis E

    2002-02-01

    We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism-as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target. PMID:11897052

  17. A novel method for the identification of conserved structural patterns in RNA: From small scale to high-throughput applications

    PubMed Central

    Pietrosanto, Marco; Mattei, Eugenio; Helmer-Citterich, Manuela; Ferrè, Fabrizio

    2016-01-01

    Functional RNA regions are often related to recurrent secondary structure patterns (or motifs), which can exert their role in several different ways, particularly in dictating the interaction with RNA-binding proteins, and acting in the regulation of a large number of cellular processes. Among the available motif-finding tools, the majority focuses on sequence patterns, sometimes including secondary structure as additional constraints to improve their performance. Nonetheless, secondary structures motifs may be concurrent to their sequence counterparts or even encode a stronger functional signal. Current methods for searching structural motifs generally require long pipelines and/or high computational efforts or previously aligned sequences. Here, we present BEAM (BEAr Motif finder), a novel method for structural motif discovery from a set of unaligned RNAs, taking advantage of a recently developed encoding for RNA secondary structure named BEAR (Brand nEw Alphabet for RNAs) and of evolutionary substitution rates of secondary structure elements. Tested in a varied set of scenarios, from small- to large-scale, BEAM is successful in retrieving structural motifs even in highly noisy data sets, such as those that can arise in CLIP-Seq or other high-throughput experiments. PMID:27580722

  18. A High-Throughput Study of PtNiZr Catalysts for Application in PEM Fuel Cells

    SciTech Connect

    Whitacre, J.F.; Valdez, T.I.; Narayanan, S.R.

    2009-05-26

    The effects of adding Zr to PtNi oxygen reduction reaction (ORR) electrocatalyst alloys were examined in a study aimed at probing the possibility of creating catalysts with enhanced resistance to corrosion in a PEM fuel cell environment. Samples consisting of pure Pt or PtNiZr alloys with a range of compositions (not exceeding 11 at.% Zr) were fabricated using co-sputter deposition. A high-throughput fabrication approach was used wherein 18 distinct thin film catalyst alloy samples with varying compositions were deposited onto a large-area substrate with individual Au current collector structures. A multi-channel pseudo-potentiostat allowed for the simultaneous quantitative study of catalytic activity for all 18 electrodes in a single test bath, a first for the study of ORR electrocatalysts. A properly stirred oxygenated 1 M H{sub 2}SO{sub 4} electrolyte solution was used to provide each electrode with a steady-state flow of reactants during electrochemical evaluation. The onset potentials, absolute current density values, and Tafel analysis data obtained using this technique were compared with literature reports. The analyses showed that most PtNiZr alloys tested offered improvements over pure Pt, however those surfaces with a high mole fraction (>4 at.%) of Zr exhibited reduced activity that was roughly inversely correlated to the amount of Zr present. Film composition, morphology, and crystallographic properties were examined using X-ray energy dispersive spectroscopy (XEDS), X-ray photoelectron spectroscopy (XPS), SEM, and synchrotron X-ray diffraction. These data were then correlated with electrochemical data to elucidate the relationships between composition, structure, and relative performance for this ternary system.

  19. The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

    PubMed

    Davies, John K; Harrison, Paul F; Lin, Ya-Hsun; Bartley, Stephanie; Khoo, Chen Ai; Seemann, Torsten; Ryan, Catherine S; Kahler, Charlene M; Hill, Stuart A

    2014-01-01

    Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

  20. Marine Invertebrate Xenobiotic-Activated Nuclear Receptors: Their Application as Sensor Elements in High-Throughput Bioassays for Marine Bioactive Compounds

    PubMed Central

    Richter, Ingrid; Fidler, Andrew E.

    2014-01-01

    Developing high-throughput assays to screen marine extracts for bioactive compounds presents both conceptual and technical challenges. One major challenge is to develop assays that have well-grounded ecological and evolutionary rationales. In this review we propose that a specific group of ligand-activated transcription factors are particularly well-suited to act as sensors in such bioassays. More specifically, xenobiotic-activated nuclear receptors (XANRs) regulate transcription of genes involved in xenobiotic detoxification. XANR ligand-binding domains (LBDs) may adaptively evolve to bind those bioactive, and potentially toxic, compounds to which organisms are normally exposed to through their specific diets. A brief overview of the function and taxonomic distribution of both vertebrate and invertebrate XANRs is first provided. Proof-of-concept experiments are then described which confirm that a filter-feeding marine invertebrate XANR LBD is activated by marine bioactive compounds. We speculate that increasing access to marine invertebrate genome sequence data, in combination with the expression of functional recombinant marine invertebrate XANR LBDs, will facilitate the generation of high-throughput bioassays/biosensors of widely differing specificities, but all based on activation of XANR LBDs. Such assays may find application in screening marine extracts for bioactive compounds that could act as drug lead compounds. PMID:25421319

  1. High-throughput phenotyping of plant shoots.

    PubMed

    Berger, Bettina; de Regt, Bas; Tester, Mark

    2012-01-01

    Advances in automated plant handling and image acquisition now make it possible to use digital imaging for the high-throughput phenotyping of plants. Various traits can be extracted from individual images. However, the potential of this technology lies in the acquisition of time series. Since whole shoot imaging is nondestructive, plants can now be monitored throughout their lifecycle, and dynamic traits such as plant growth and development can be captured and quantified. The technique is applicable to a wide range of plants and research areas and makes high-throughput screens possible, reducing the time and labor needed for the phenotypic characterization of plants.

  2. Novel heparan sulfate assay by using automated high-throughput mass spectrometry: Application to monitoring and screening for mucopolysaccharidoses.

    PubMed

    Shimada, Tsutomu; Kelly, Joan; LaMarr, William A; van Vlies, Naomi; Yasuda, Eriko; Mason, Robert W; Mackenzie, William; Kubaski, Francyne; Giugliani, Roberto; Chinen, Yasutsugu; Yamaguchi, Seiji; Suzuki, Yasuyuki; Orii, Kenji E; Fukao, Toshiyuki; Orii, Tadao; Tomatsu, Shunji

    2014-01-01

    Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity

  3. Technological advances in high-throughput screening.

    PubMed

    Liu, Bailing; Li, Songjun; Hu, Jie

    2004-01-01

    High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the

  4. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    PubMed

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  5. Application of DNA microarray for screening metagenome library clones.

    PubMed

    Park, Soo-Je; Chae, Jong-Chan; Rhee, Sung-Keun

    2010-01-01

    Sequence-based screening tools of a metagenome library can expedite metagenome researches considering tremendous metagenome diversities. Several critical disadvantages of activity-based screening of metagenome libraries could be overcome by sequence-based screening approaches. DNA microarray technology widely used for monitoring environmental genes can be employed for screening environmental fosmid and BAC clones harboring target genes due to its high throughput nature. DNAs of fosmid clones are extracted and spotted on a glass slide and fluorescence-labeled probes are hybridized to the microarray. Specific hybridization signals can be obtained only for the fosmid clones that contain the target gene with high sensitivity (10 ng/μL of fosmid clone DNA) and quantitativeness. PMID:20830574

  6. Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.

    PubMed

    Janakiraman, Vijay; Kwiatkowski, Chris; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

    2015-01-01

    High-throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high-throughput mini-bioreactor system viz. the Advanced Microscale Bioreactor (ambr15(TM) ), to perform process characterization in less than a month and develop an input control strategy. As a pre-requisite to process characterization, a scale-down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO2 profiles as well as several other process and product quality parameters. The scale-down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15(TM) system is capable of replacing the bench scale bioreactor system for routine process development and process characterization.

  7. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

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

    PubMed

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

    2007-07-01

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

  9. Bulk Combinatorial Synthesis and High Throughput Characterization for Rapid Assessment of Magnetic Materials: Application of Laser Engineered Net Shaping (LENS™)

    NASA Astrophysics Data System (ADS)

    Geng, J.; Nlebedim, I. C.; Besser, M. F.; Simsek, E.; Ott, R. T.

    2016-07-01

    A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS™; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS™ system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. The Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

  10. 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.

  11. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  12. Application of a kosmotrope-based solubility assay to multiple protein therapeutic classes indicates broad use as a high-throughput screen for protein therapeutic aggregation propensity.

    PubMed

    Yamniuk, Aaron P; Ditto, Noah; Patel, Mehul; Dai, Jun; Sejwal, Preeti; Stetsko, Paul; Doyle, Michael L

    2013-08-01

    Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.

  13. Functional Protein Microarray Technology

    PubMed Central

    Hu, Shaohui; Xie, Zhi; Qian, Jiang; Blackshaw, Seth; Zhu, Heng

    2010-01-01

    Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we will review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We will also report recent progress of using functional protein microarrays in profiling protein posttranslational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we will discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. PMID:20872749

  14. Fabrication of continuous flow microfluidics device with 3D electrode structures for high throughput DEP applications using mechanical machining.

    PubMed

    Zeinali, Soheila; Çetin, Barbaros; Oliaei, Samad Nadimi Bavil; Karpat, Yiğit

    2015-07-01

    Microfluidics is the combination of micro/nano fabrication techniques with fluid flow at microscale to pursue powerful techniques in controlling and manipulating chemical and biological processes. Sorting and separation of bio-particles are highly considered in diagnostics and biological analyses. Dielectrophoresis (DEP) has offered unique advantages for microfluidic devices. In DEP devices, asymmetric pair of planar electrodes could be employed to generate non-uniform electric fields. In DEP applications, facing 3D sidewall electrodes is considered to be one of the key solutions to increase device throughput due to the generated homogeneous electric fields along the height of microchannels. Despite the advantages, fabrication of 3D vertical electrodes requires a considerable challenge. In this study, two alternative fabrication techniques have been proposed for the fabrication of a microfluidic device with 3D sidewall electrodes. In the first method, both the mold and the electrodes are fabricated using high precision machining. In the second method, the mold with tilted sidewalls is fabricated using high precision machining and the electrodes are deposited on the sidewall using sputtering together with a shadow mask fabricated by electric discharge machining. Both fabrication processes are assessed as highly repeatable and robust. Moreover, the two methods are found to be complementary with respect to the channel height. Only the manipulation of particles with negative-DEP is demonstrated in the experiments, and the throughput values up to 105 particles / min is reached in a continuous flow. The experimental results are compared with the simulation results and the limitations on the fabrication techniques are also discussed.

  15. Microfluidic Cell Culture and Its Application in High Throughput Drug Screening: Cardiotoxicity Assay for hERG Channels

    PubMed Central

    Su, Xiaojing; Young, Edmond W.K.; Underkofler, Heather A. S.; Kamp, Timothy J.; January, Craig T.; Beebe, David J.

    2011-01-01

    Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval, and hence risk for life threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, we demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. We validate our microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP) and poly(dimethylsiloxane) (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG), and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Our results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. We demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. We further showed that experimental procedures can be streamlined by using direct in-channel immunofluorescent staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with five different drugs suggested that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic

  16. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    PubMed

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. PMID:27590051

  17. High-Throughput UPLC-MS Method for the Determination of N-Acetyl-l-Cysteine: Application in Tissue Distribution Study in Wistar Rats.

    PubMed

    Siddiqui, Masoom Raza; Wabaidur, Saikh Mohammad; Ola, Mohammad Shamsul; AlOthman, Zeid A; Rafiquee, Mohammad Zulfiqar Ali; Khan, Moonis Ali

    2016-08-01

    N-Acetylcysteine (NAC) is the N-acetyl derivative of the amino acid l-cysteine and is extensively used as a medicine to treat a variety of diseases. High-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method has been developed for the quantitative assessment of N-acetyl-l-cysteine. The method was further applied to study the distribution of the intraperitoneal injected drug into different tissues and plasma of Wistar rats, including liver, kidney, heart, lungs and spleen. The drug was having highest concentration in plasma and liver followed by kidney, lungs, heart and spleen. Method validation studies suggested being linear in the range of 1-15 µg mL(-1) for liver, kidney, heart, lungs and spleen and 1-120 µg mL(-1) for the plasma. The limit of detection and limit of quantitation were found to be 0.20 and 0.66 µg mL(-1), respectively. The recovery studies suggested that in all the cases, the obtained recovery was in the range of 98.51-101.88%. Our analyses provide a validated UPLC-MS method for the determination of NAC and its successful application for the analysis in plasma and tissues obtained from Wistar rats. PMID:27102930

  18. 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.

  19. High-throughput neuro-imaging informatics

    PubMed Central

    Miller, Michael I.; Faria, Andreia V.; Oishi, Kenichi; Mori, Susumu

    2013-01-01

    This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high-throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high-dimensional neuroinformatic representation index containing O(1000–10,000) discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high-throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high-throughput machine learning methods for supporting (i) cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii) integration of image and personal medical record non-image information for diagnosis and prognosis. PMID:24381556

  20. DNA Microarrays in Herbal Drug Research

    PubMed Central

    Chavan, Preeti; Joshi, Kalpana; Patwardhan, Bhushan

    2006-01-01

    Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts. PMID:17173108

  1. Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

    EPA Science Inventory

    Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

  2. 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.

  3. 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

  4. Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings.

    PubMed

    Britton, Sumudu; Cheng, Qin; McCarthy, James S

    2016-01-01

    As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination. PMID:26879936

  5. 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.

  6. Robin: An Intuitive Wizard Application for R-Based Expression Microarray Quality Assessment and Analysis1[W][OA

    PubMed Central

    Lohse, Marc; Nunes-Nesi, Adriano; Krüger, Peter; Nagel, Axel; Hannemann, Jan; Giorgi, Federico M.; Childs, Liam; Osorio, Sonia; Walther, Dirk; Selbig, Joachim; Sreenivasulu, Nese; Stitt, Mark; Fernie, Alisdair R.; Usadel, Björn

    2010-01-01

    The wide application of high-throughput transcriptomics using microarrays has generated a plethora of technical platforms, data repositories, and sophisticated statistical analysis methods, leaving the individual scientist with the problem of choosing the appropriate approach to address a biological question. Several software applications that provide a rich environment for microarray analysis and data storage are available (e.g. GeneSpring, EMMA2), but these are mostly commercial or require an advanced informatics infrastructure. There is a need for a noncommercial, easy-to-use graphical application that aids the lab researcher to find the proper method to analyze microarray data, without this requiring expert understanding of the complex underlying statistics, or programming skills. We have developed Robin, a Java-based graphical wizard application that harnesses the advanced statistical analysis functions of the R/BioConductor project. Robin implements streamlined workflows that guide the user through all steps of two-color, single-color, or Affymetrix microarray analysis. It provides functions for thorough quality assessment of the data and automatically generates warnings to notify the user of potential outliers, low-quality chips, or low statistical power. The results are generated in a standard format that allows ready use with both specialized analysis tools like MapMan and PageMan and generic spreadsheet applications. To further improve user friendliness, Robin includes both integrated help and comprehensive external documentation. To demonstrate the statistical power and ease of use of the workflows in Robin, we present a case study in which we apply Robin to analyze a two-color microarray experiment comparing gene expression in tomato (Solanum lycopersicum) leaves, flowers, and roots. PMID:20388663

  7. Application of DNA Microarray to Clinical Diagnostics.

    PubMed

    Patel, Ankita; Cheung, Sau W

    2016-01-01

    Microarray-based technology to conduct array comparative genomic hybridization (aCGH) has made a significant impact on the diagnosis of human genetic diseases. Such diagnoses, previously undetectable by traditional G-banding chromosome analysis, are now achieved by identifying genomic copy number variants (CNVs) using the microarray. Not only can hundreds of well-characterized genetic syndromes be detected in a single assay, but new genomic disorders and disease-causing genes can also be discovered through the utilization of aCGH technology. Although other platforms such as single nucleotide polymorphism (SNP) arrays can be used for detecting CNVs, in this chapter we focus on describing the methods for performing aCGH using Agilent oligonucleotide arrays for both prenatal (e.g., amniotic fluid and chorionic villus sample) and postnatal samples (e.g., blood).

  8. [Genomic medicine. Polymorphisms and microarray applications].

    PubMed

    Spalvieri, Mónica P; Rotenberg, Rosa G

    2004-01-01

    This update shows new concepts related to the significance of DNA variations among individuals, as well as to their detection by using a new technology. The sequencing of the human genome is only the beginning of what will enable us to understand genetic diversity. The unit of DNA variability is the polymorphism of a single nucleotide (SNP). At present, studies on SNPs are restricted to basic research but the large number of papers on this subject makes feasible their entrance into clinical practice. We illustrate here the use of SNPs as molecular markers in ethnical genotyping, gene expression in some diseases and as potential targets in pharmacological response, and also introduce the technology of arrays. Microarrays experiments allow the quantification and comparison of gene expression on a large scale, at the same time, by using special chips and array designs. Conventional methods provide data from up to 20 genes, while a single microarray may provide information about thousands of them simultaneously, leading to a more rapid and accurate genotyping. Biotechnology improvements will facilitate our knowledge of each gene sequence, the frequency and exact location of SNPs and their influence on cellular behavior. Although experimental efficiency and validity of results from microarrays are still controversial, the knowledge and characterization of a patient's genetic profile will lead, undoubtedly, to advances in prevention, diagnosis, prognosis and treatment of human diseases. PMID:15637833

  9. NCBI GEO: archive for high-throughput functional genomic data.

    PubMed

    Barrett, Tanya; Troup, Dennis B; Wilhite, Stephen E; Ledoux, Pierre; Rudnev, Dmitry; Evangelista, Carlos; Kim, Irene F; Soboleva, Alexandra; Tomashevsky, Maxim; Marshall, Kimberly A; Phillippy, Katherine H; Sherman, Patti M; Muertter, Rolf N; Edgar, Ron

    2009-01-01

    The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) is the largest public repository for high-throughput gene expression data. Additionally, GEO hosts other categories of high-throughput functional genomic data, including those that examine genome copy number variations, chromatin structure, methylation status and transcription factor binding. These data are generated by the research community using high-throughput technologies like microarrays and, more recently, next-generation sequencing. The database has a flexible infrastructure that can capture fully annotated raw and processed data, enabling compliance with major community-derived scientific reporting standards such as 'Minimum Information About a Microarray Experiment' (MIAME). In addition to serving as a centralized data storage hub, GEO offers many tools and features that allow users to effectively explore, analyze and download expression data from both gene-centric and experiment-centric perspectives. This article summarizes the GEO repository structure, content and operating procedures, as well as recently introduced data mining features. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/.

  10. SaDA: From Sampling to Data Analysis-An Extensible Open Source Infrastructure for Rapid, Robust and Automated Management and Analysis of Modern Ecological High-Throughput Microarray Data.

    PubMed

    Singh, Kumar Saurabh; Thual, Dominique; Spurio, Roberto; Cannata, Nicola

    2015-06-01

    One of the most crucial characteristics of day-to-day laboratory information management is the collection, storage and retrieval of information about research subjects and environmental or biomedical samples. An efficient link between sample data and experimental results is absolutely important for the successful outcome of a collaborative project. Currently available software solutions are largely limited to large scale, expensive commercial Laboratory Information Management Systems (LIMS). Acquiring such LIMS indeed can bring laboratory information management to a higher level, but most of the times this requires a sufficient investment of money, time and technical efforts. There is a clear need for a light weighted open source system which can easily be managed on local servers and handled by individual researchers. Here we present a software named SaDA for storing, retrieving and analyzing data originated from microorganism monitoring experiments. SaDA is fully integrated in the management of environmental samples, oligonucleotide sequences, microarray data and the subsequent downstream analysis procedures. It is simple and generic software, and can be extended and customized for various environmental and biomedical studies.

  11. SaDA: From Sampling to Data Analysis—An Extensible Open Source Infrastructure for Rapid, Robust and Automated Management and Analysis of Modern Ecological High-Throughput Microarray Data

    PubMed Central

    Singh, Kumar Saurabh; Thual, Dominique; Spurio, Roberto; Cannata, Nicola

    2015-01-01

    One of the most crucial characteristics of day-to-day laboratory information management is the collection, storage and retrieval of information about research subjects and environmental or biomedical samples. An efficient link between sample data and experimental results is absolutely important for the successful outcome of a collaborative project. Currently available software solutions are largely limited to large scale, expensive commercial Laboratory Information Management Systems (LIMS). Acquiring such LIMS indeed can bring laboratory information management to a higher level, but most of the times this requires a sufficient investment of money, time and technical efforts. There is a clear need for a light weighted open source system which can easily be managed on local servers and handled by individual researchers. Here we present a software named SaDA for storing, retrieving and analyzing data originated from microorganism monitoring experiments. SaDA is fully integrated in the management of environmental samples, oligonucleotide sequences, microarray data and the subsequent downstream analysis procedures. It is simple and generic software, and can be extended and customized for various environmental and biomedical studies. PMID:26047146

  12. A brief introduction to tiling microarrays: principles, concepts, and applications.

    PubMed

    Lemetre, Christophe; Zhang, Zhengdong D

    2013-01-01

    Technological achievements have always contributed to the advancement of biomedical research. It has never been more so than in recent times, when the development and application of innovative cutting-edge technologies have transformed biology into a data-rich quantitative science. This stunning revolution in biology primarily ensued from the emergence of microarrays over two decades ago. The completion of whole-genome sequencing projects and the advance in microarray manufacturing technologies enabled the development of tiling microarrays, which gave unprecedented genomic coverage. Since their first description, several types of application of tiling arrays have emerged, each aiming to tackle a different biological problem. Although numerous algorithms have already been developed to analyze microarray data, new method development is still needed not only for better performance but also for integration of available microarray data sets, which without doubt constitute one of the largest collections of biological data ever generated. In this chapter we first introduce the principles behind the emergence and the development of tiling microarrays, and then discuss with some examples how they are used to investigate different biological problems.

  13. An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.

    PubMed

    Morono, Yuki; Terada, Takeshi; Kallmeyer, Jens; Inagaki, Fumio

    2013-10-01

    Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ~50% of cells were recovered from deep samples (100-365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10(4)-10(8) cells cm(-3). We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere. PMID:23731283

  14. Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

    PubMed

    Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

    2015-01-01

    Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species.

  15. An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting

    PubMed Central

    Morono, Yuki; Terada, Takeshi; Kallmeyer, Jens; Inagaki, Fumio

    2013-01-01

    Summary Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104–108 cells cm−3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere. PMID:23731283

  16. Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

    PubMed

    Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

    2015-01-01

    Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species. PMID:24842551

  17. Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) with silver colloids in 96-well plates: Application to ultra fast and sensitive immunoassays, High Throughput Screening and drug discovery.

    PubMed

    Aslan, Kadir; Holley, Patrick; Geddes, Chris D

    2006-05-30

    Fluorescence detection is the basis of most assays used in drug discovery and High Throughput Screening (HTS) today. In all of these assays, assay rapidity and sensitivity is a primary concern, the sensitivity determined by both the quantum yield of the fluorophores and efficiency of the detection system, while rapidity is determined by the physical and biophysical parameters of temperature, concentration, assay bioaffinity, etc. In this paper we describe a platform technology that promises to fundamentally address these two physical constraints of sensitivity and rapidity. By combining the use of Metal-Enhanced Fluorescence (MEF), a near-field effect that can significantly enhance fluorescence signatures, with low power microwave heating, we can significantly increase the sensitivity of surface assays as well as >95% kinetically complete the assay within a few seconds. In addition, the metallic nanostructures used to facilitate MEF appear to be preferentially heated as compared to the surface assay fluid, advantageously localizing the MEF and heating around the nanostructures. To demonstrate proof of principle, a 96-well plate has been functionalized with silver nanostructures, and a model protein avidin-biotin assay studied. In our findings, a greater than 5-fold fluorescence enhancement coupled with a approximately 90-fold increase in assay kinetics was observed, but with no assay washing steps needed due to the silver-enhanced evanescent field mode of excitation. These findings promise to strongly facilitate high throughput fluorescence-based processes, such as in biology, drug discovery and general compound screening.

  18. High-Throughput Nonlinear Optical Microscopy

    PubMed Central

    So, Peter T.C.; Yew, Elijah Y.S.; Rowlands, Christopher

    2013-01-01

    High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field. PMID:24359736

  19. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

    PubMed Central

    Miller, Melissa B.; Tang, Yi-Wei

    2009-01-01

    Summary: The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles. PMID:19822891

  20. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications. PMID:27477388

  1. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications.

  2. Large-scale application of high-throughput molecular mechanics with Poisson-Boltzmann surface area for routine physics-based scoring of protein-ligand complexes.

    PubMed

    Brown, Scott P; Muchmore, Steven W

    2009-05-28

    We apply a high-throughput formulation of the molecular mechanics with Poisson-Boltzmann surface area (htMM-PBSA) to estimate relative binding potencies on a set of 308 small-molecule ligands in complex with the proteins urokinase, PTP-1B, and Chk-1. We observe statistically significant correlation to experimentally measured potencies and report correlation coefficients for the three proteins in the range 0.72-0.83. The htMM-PBSA calculations illustrate the feasibility of procedural automation of physics-based scoring calculations to produce rank-ordered binding-potency estimates for protein-ligand complexes, with sufficient throughput for realization of practical implementation into scientist workflows in an industrial drug discovery research setting.

  3. Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening.

    PubMed

    Tavares, Clint D J; Devkota, Ashwini K; Dalby, Kevin N; Cho, Eun Jeong

    2016-01-01

    Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.

  4. Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening.

    PubMed

    Tavares, Clint D J; Devkota, Ashwini K; Dalby, Kevin N; Cho, Eun Jeong

    2016-01-01

    Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction. PMID:26501899

  5. High Throughput Screening Tools for Thermoelectric Materials

    NASA Astrophysics Data System (ADS)

    Wong-Ng, W.; Yan, Y.; Otani, M.; Martin, J.; Talley, K. R.; Barron, S.; Carroll, D. L.; Hewitt, C.; Joress, H.; Thomas, E. L.; Green, M. L.; Tang, X. F.

    2015-06-01

    A suite of complementary high-throughput screening systems for combinatorial films was developed at National Institute of Standards and Technology to facilitate the search for efficient thermoelectric materials. These custom-designed capabilities include a facility for combinatorial thin film synthesis and a suite of tools for screening the Seebeck coefficient, electrical resistance (electrical resistivity), and thermal effusivity (thermal conductivity) of these films. The Seebeck coefficient and resistance are measured via custom-built automated apparatus at both ambient and high temperatures. Thermal effusivity is measured using a frequency domain thermoreflectance technique. This paper will discuss applications using these tools on representative thermoelectric materials, including combinatorial composition-spread films, conventional films, single crystals, and ribbons.

  6. 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

  7. Nanoliter high throughput quantitative PCR

    PubMed Central

    Morrison, Tom; Hurley, James; Garcia, Javier; Yoder, Karl; Katz, Arrin; Roberts, Douglas; Cho, Jamie; Kanigan, Tanya; Ilyin, Sergey E.; Horowitz, Daniel; Dixon, James M.; Brenan, Colin J.H.

    2006-01-01

    Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols. PMID:17000636

  8. High-throughput DNA methylation datasets for evaluating false discovery rate methodologies.

    PubMed

    Asomaning, N; Archer, K J

    2012-06-01

    When analyzing high-throughput genomic data, the multiple comparison problem is most often addressed through estimation of the false discovery rate (FDR), using methods such as the Benjamini & Hochberg, Benjamini & Yekutieli, the q-value method, or in controlling the family-wise error rate (FWER) using Holm's step down method. To date, research studies that have compared various FDR/FWER methodologies have made use of limited simulation studies and/or have applied the methods to one or more microarray gene expression dataset(s). However, for microarray datasets the veracity of each null hypothesis tested is unknown so that an objective evaluation of performance cannot be rendered for application data. Due to the role of methylation in X-chromosome inactivation, we postulate that high-throughput methylation datasets may provide an appropriate forum for assessing the performance of commonly used FDR methodologies. These datasets preserve the complex correlation structure between probes, offering an advantage over simulated datasets. Using several methylation datasets, commonly used FDR methods including the q-value, Benjamini & Hochberg, and Benjamini & Yekutieli procedures as well as Holm's step down method were applied to identify CpG sites that are differentially methylated when comparing healthy males to healthy females. The methods were compared with respect to their ability to identify CpG sites located on sex chromosomes as significant, by reporting the sensitivity, specificity, and observed FDR. These datasets are useful for characterizing the performance of multiple comparison procedures, and may find further utility in other tasks such as comparing variable selection capabilities of classification methods and evaluating the performance of meta-analytic methods for microarray data.

  9. Microsphere-enhanced label-free high-throughput molecular detection

    NASA Astrophysics Data System (ADS)

    Deng, Cheng; Huang, Guoliang; Xu, Shukuan; Zhu, Jing; Ma, Rui; Han, Chao; Chen, Shengyi; Ma, Chen

    2008-12-01

    In this paper, we developed a microsphere enhanced and label free high throughput molecular detection based on SPRI and microarray chips, and a self-built surface plasmon resonance imaging instrument was set. One label-free protein chip forming a 7× probe array was designed and fabricated using a commercial microarray robot spotter on chemical modified gold-coated glass slide. Antibody molecules were successfully detected in label-free and high-throughput method by using this chip. The detection signals on the chip was successfully enhanced by using microspheres with a diameter of 1 μm.

  10. Functional protein microarray as molecular decathlete: a versatile player in clinical proteomics.

    PubMed

    Zhu, Heng; Cox, Eric; Qian, Jiang

    2012-12-01

    Functional protein microarrays were developed as a high-throughput tool to overcome the limitations of DNA microarrays and to provide a versatile platform for protein functional analyses. Recent years have witnessed tremendous growth in the use of protein microarrays, particularly functional protein microarrays, to address important questions in the field of clinical proteomics. In this review, we will summarize some of the most innovative and exciting recent applications of protein microarrays in clinical proteomics, including biomarker identification, pathogen-host interactions, and cancer biology. PMID:23027439

  11. Inkjet printing for high-throughput cell patterning.

    PubMed

    Roth, E A; Xu, T; Das, M; Gregory, C; Hickman, J J; Boland, T

    2004-08-01

    The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.

  12. 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

  13. Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.

    PubMed

    Palma, Bernardo Brito; Moutinho, Daniela; Urban, Philippe; Rueff, José; Kranendonk, Michel

    2016-08-01

    Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level. PMID:27476332

  14. Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays

    PubMed Central

    Rantala, Juha K.; Kwon, Sunjong; Korkola, James; Gray, Joe W.

    2013-01-01

    Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 μm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

  15. Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers.

    PubMed

    Stralis-Pavese, Nancy; Sessitsch, Angela; Weilharter, Alexandra; Reichenauer, Thomas; Riesing, Johann; Csontos, József; Murrell, J Colin; Bodrossy, Levente

    2004-04-01

    Landfill sites are responsible for 6-12% of global methane emission. Methanotrophs play a very important role in decreasing landfill site methane emissions. We investigated the methane oxidation capacity and methanotroph diversity in lysimeters simulating landfill sites with different plant vegetations. Methane oxidation rates were 35 g methane m-2 day-1 or higher for planted lysimeters and 18 g methane m-2 day-1 or less for bare soil controls. Best methane oxidation, as displayed by gas depth profiles, was found under a vegetation of grass and alfalfa. Methanotroph communities were analysed at high throughput and resolution using a microbial diagnostic microarray targeting the particulate methane monooxygenase (pmoA) gene of methanotrophs and functionally related bacteria. Members of the genera Methylocystis and Methylocaldum were found to be the dominant members in landfill site simulating lysimeters. Soil bacterial communities in biogas free control lysimeters, which were less abundant in methanotrophs, were dominated by Methylocaldum. Type Ia methanotrophs were found only in the top layers of bare soil lysimeters with relatively high oxygen and low methane concentrations. A competetive advantage of type II methanotrophs over type Ia methanotrophs was indicated under all plant covers investigated. Analysis of average and individual results from parallel samples was used to identify general trends and variations in methanotroph community structures in relation to depth, methane supply and plant cover. The applicability of the technology for the detection of environmental perturbations was proven by an erroneous result, where an unexpected community composition detected with the microarray indicated a potential gas leakage in the lysimeter being investigated.

  16. THINK Back: KNowledge-based Interpretation of High Throughput data.

    PubMed

    Farfán, Fernando; Ma, Jun; Sartor, Maureen A; Michailidis, George; Jagadish, Hosagrahar V

    2012-01-01

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways. PMID:22536867

  17. THINK Back: KNowledge-based Interpretation of High Throughput data

    PubMed Central

    2012-01-01

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways. PMID:22536867

  18. THINK Back: KNowledge-based Interpretation of High Throughput data.

    PubMed

    Farfán, Fernando; Ma, Jun; Sartor, Maureen A; Michailidis, George; Jagadish, Hosagrahar V

    2012-03-13

    Results of high throughput experiments can be challenging to interpret. Current approaches have relied on bulk processing the set of expression levels, in conjunction with easily obtained external evidence, such as co-occurrence. While such techniques can be used to reason probabilistically, they are not designed to shed light on what any individual gene, or a network of genes acting together, may be doing. Our belief is that today we have the information extraction ability and the computational power to perform more sophisticated analyses that consider the individual situation of each gene. The use of such techniques should lead to qualitatively superior results. The specific aim of this project is to develop computational techniques to generate a small number of biologically meaningful hypotheses based on observed results from high throughput microarray experiments, gene sequences, and next-generation sequences. Through the use of relevant known biomedical knowledge, as represented in published literature and public databases, we can generate meaningful hypotheses that will aide biologists to interpret their experimental data. We are currently developing novel approaches that exploit the rich information encapsulated in biological pathway graphs. Our methods perform a thorough and rigorous analysis of biological pathways, using complex factors such as the topology of the pathway graph and the frequency in which genes appear on different pathways, to provide more meaningful hypotheses to describe the biological phenomena captured by high throughput experiments, when compared to other existing methods that only consider partial information captured by biological pathways.

  19. High-throughput in vitro drug release and pharmacokinetic simulation as a tool for drug delivery system development: application to intravitreal ocular administration.

    PubMed

    Sarkhel, Sanjay; Ramsay, Eva; Kontturi, Leena-Stiina; Peltoniemi, Jonne; Urtti, Arto

    2014-12-30

    In vitro estimation of release kinetics from drug delivery systems is needed in formulation development. Cost-effective methods of assessment for delivery systems are needed particularly in the case of biologicals and drug administration routes that are difficult to screen in vivo (e.g. intraocular drug delivery). As a proof-of-concept, we demonstrate here a practical high-throughput methodology to investigate in vitro drug release and predict resulting drug concentrations in the eye after intravitreal administration. 96-well plate based assay aided with robotic sampling was used to study release of eight model drugs of varying physicochemical properties (dexamethasone, vancomycin, alpha-lactalbumin, lysozyme, myoglobin, albumin, lactoferrin, human IgG) from twelve alginate microsphere formulations. The amount of drug released over a period of time was assessed by photometric and fluorescence methods. In vitro drug release rates obtained were used in pharmacokinetic simulations using one-compartment model of the vitreal cavity with anatomical volume of distribution and clearance estimates based on the literature precedence. An integrated approach of drug release screening and pharmacokinetic simulations can prove to be a useful methodology in guiding formulation development for ocular delivery in animal models. In general, the methodology has the potential to be a cost-effective tool for early stage drug delivery system discovery and development.

  20. High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

    PubMed Central

    Chen, Li; Wang, Yemin; Guo, Hang; Xu, Min; Deng, Zixin

    2012-01-01

    A genomic cosmid library of Streptomyces clavuligerus was constructed and transferred efficiently by conjugation to Streptomyces lividans, and 12 distinct groups of overlapping cosmid clones that activated the silent actinorhodin biosynthesis gene cluster were identified. This generally applicable high-throughput screening procedure greatly facilitates the identification of antibiotic biosynthesis activators. PMID:22504805

  1. Disc-based microarrays: principles and analytical applications.

    PubMed

    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays. PMID:26922341

  2. A high-throughput antibody-based microarray typing platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing microbial contaminants thus facilitating...

  3. Advancing a distributed multi-scale computing framework for large-scale high-throughput discovery in materials science.

    PubMed

    Knap, J; Spear, C E; Borodin, O; Leiter, K W

    2015-10-30

    We describe the development of a large-scale high-throughput application for discovery in materials science. Our point of departure is a computational framework for distributed multi-scale computation. We augment the original framework with a specialized module whose role is to route evaluation requests needed by the high-throughput application to a collection of available computational resources. We evaluate the feasibility and performance of the resulting high-throughput computational framework by carrying out a high-throughput study of battery solvents. Our results indicate that distributed multi-scale computing, by virtue of its adaptive nature, is particularly well-suited for building high-throughput applications.

  4. Application of high-throughput sequencing to measure the performance of commonly used selective cultivation methods for the foodborne pathogen Campylobacter.

    PubMed

    Oakley, Brian B; Morales, Cesar A; Line, J Eric; Seal, Bruce S; Hiett, Kelli L

    2012-02-01

    Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (< 0.04%) of sequences present in the feces, but 88-97% of sequences from each media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes.

  5. Analysis of a high-throughput cone-and-plate apparatus for the application of defined spatiotemporal flow to cultured cells.

    PubMed

    Spruell, Christopher; Baker, Aaron B

    2013-06-01

    The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress-mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone-and-plate viscometer in which a rotating, low-angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high-throughput studies on shear-mediated mechanobiology through the rotation of cone-tipped shafts in a standard 96-well culture plate. Here, we present a model of the three-dimensional flow within the culture wells with a rotating, cone-tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady-state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non-linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and "parallel shaft" regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor-Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. PMID:23280552

  6. High-throughput and rapid quantification of lipids by nanoflow UPLC-ESI-MS/MS: application to the hepatic lipids of rabbits with nonalcoholic fatty liver disease.

    PubMed

    Byeon, Seul Kee; Lee, Jong Cheol; Chung, Bong Chul; Seo, Hong Seog; Moon, Myeong Hee

    2016-07-01

    A rapid and high-throughput quantification method (approximately 300 lipids within 20 min) was established using nanoflow ultrahigh-pressure liquid chromatography-tandem mass spectrometry (nUPLC-ESI-MS/MS) with selective reaction monitoring (SRM) and applied to the quantitative profiling of the hepatic lipids of rabbits with different metabolic conditions that stimulate the development of nonalcoholic fatty liver disease (NAFLD). Among the metabolic conditions of rabbits in this study [inflammation (I), high-cholesterol diet (HC), and high-cholesterol diet combined with inflammation (HCI)], significant perturbation in hepatic lipidome (>3-fold and p < 0.01) was observed in the HC and HCI groups, while no single lipid showed a significant change in group I. In addition, this study revealed a dramatic increase (>2-fold) in relatively high-abundant monohexosylceramides (MHCs), sphingomyelins (SMs), and triacylglycerols (TGs) in both the HC and HCI groups, especially in MHCs as all 11 MHCs increased by larger than 3- to 12-fold. As the levels of the relatively high-abundant lipids in the above classes increased, the total lipidome level of each class increased significantly by approximately 2-fold to 5-fold. Other classes of lipids also generally increased, which was likely induced by the increase in mitogenic and nonapoptotic MHCs and SMs, as they promote cell proliferation. On the other hand, a slight decrease in the level of apoptotic ceramides (Cers) was observed, which agreed with the general increase in total lipid level. As distinct changes in hepatic lipidome were observed from HC groups, this suggests that HC or HCI is highly associated with NAFLD but not inflammation alone itself. Graphical Abstract Schematic of lipidomic analysis from hepatic tissue using nanoflow LC-ESI-MS/MS and nUPLC-ESI-MS/MS.

  7. Microarrays in hematology.

    PubMed

    Walker, Josef; Flower, Darren; Rigley, Kevin

    2002-01-01

    Microarrays are fast becoming routine tools for the high-throughput analysis of gene expression in a wide range of biologic systems, including hematology. Although a number of approaches can be taken when implementing microarray-based studies, all are capable of providing important insights into biologic function. Although some technical issues have not been resolved, microarrays will continue to make a significant impact on hematologically important research. PMID:11753074

  8. Application of phenotypic microarrays to environmental microbiology

    SciTech Connect

    Borglin, sharon; Joyner, Dominique; DeAngelis, Kristen; Khudyakov, Jane; D'haeseleer, Patrik; Joachimiak, Marcin; Hazen, Terry C; Fagan, Lisa Anne

    2012-01-01

    Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting.

  9. Application of Tissue Microarray Technology to Stem Cell Research

    PubMed Central

    Spada, Alberto La; Rainoldi, Barnaba; Blasio, Andrea De; Biunno, Ida

    2014-01-01

    There is virtually an unlimited number of possible Tissue Microarray (TMA) applications in basic and clinical research and ultimately in diagnostics. However, to assess the functional importance of novel markers, researchers very often turn to cell line model systems. The appropriate choice of a cell line is often a difficult task, but the use of cell microarray (CMA) technology enables a quick screening of several markers in cells of different origins, mimicking a genomic-scale analysis. In order to improve the morphological evaluations of the CMA slides we harvested the cells by conventional trypsinization, mechanical scraping and cells grown on coverslips. We show that mechanical scraping is a good evaluation method since keeps the real morphology very similar to those grown on coverslips. Immunofluorescence images are of higher quality, facilitating the reading of the biomarker cellular and subcellular localization. Here, we describe CMA technology in stem cell research.

  10. DNA microarray application in ecotoxicology: experimental design, microarray scanning, and factors affecting transcriptional profiles in a small fish species.

    PubMed

    Wang, Rong-Lin; Biales, Adam; Bencic, David; Lattier, David; Kostich, Mitch; Villeneuve, Dan; Ankley, Gerald T; Lazorchak, Jim; Toth, Greg

    2008-03-01

    The research presented here is part of a larger study of the molecular mode of action of endocrine-disrupting chemicals targeting the hypothalamic-pituitary-gonadal axis in zebrafish (Danio rerio). It addresses several issues critical to microarray application in aquatic ecotoxicology: experimental design, microarray scanning, gene expression intensity distribution, and the effect of experimental parameters on the zebrafish transcriptome. Expression profiles from various tissues of individual zebrafish exposed to 17alpha-ethinylestradiol (30 ng/L), fadrozole (25 micro.g/L), or 17beta-trenbolone (3.0 microg/L) for 48 or 96 h were examined with the Agilent Oligo Microarray (G2518A). As a flexible and efficient alternative to the designs commonly used in microarray studies, an unbalanced incomplete block design was found to be well suited for this work, as evidenced by high data reproducibility, low microarray-to-microarray variability, and little gene-specific dye bias. Random scanner noise had little effect on data reproducibility. A low-level, slightly variable Cyanine 3 (Cy3) contaminant was revealed by hyperspectral imaging, suggesting fluorescence contamination as a potential contributor to the large variance associated with weakly expressed genes. Expression intensities of zebrafish genes were skewed toward the lower end of their distribution range, and more weakly expressed genes tended to have larger variances. Tissue type, followed in descending order by gender, chemical treatment, and exposure duration, had the greatest effect on the overall gene expression profiles, a finding potentially critical to experimental design optimization. Overall, congruence was excellent between quantitative polymerase chain reaction results and microarray profiles of 13 genes examined across a subset of 20 pairs of ovarian samples. These findings will help to improve applications of microarrays in future ecotoxicological studies.

  11. Detection of protein microarrays by oblique-incidence reflectivity difference technique

    NASA Astrophysics Data System (ADS)

    Wen, Juan; Lu, Heng; Wang, Xu; Yuan, Kun; Lü, Huibin; Zhou, Yueliang; Yin, Kuijuan; Yang, Guozhen; Li, Wei; Ruan, Kangcheng

    2010-02-01

    Biological microarrays with different proteins and different protein concentrations are detected without external labeling by an oblique-incidence reflectivity difference (OIRD) technique. The initial experiment results reveal that the intensities of OIRD signals can distinguish the different proteins and concentrations of protein. The OIRD technique promises feasible applications to life sciences for label-free and high-throughput detection.

  12. 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.

  13. A fully automated robotic system for high throughput fermentation.

    PubMed

    Zimmermann, Hartmut F; Rieth, Jochen

    2007-03-01

    High throughput robotic systems have been used since the 1990s to carry out biochemical assays in microtiter plates. However, before the application of such systems in industrial fermentation process development, some important specific demands should be taken into account. These are sufficient oxygen supply, optimal growth temperature, minimized sample evaporation, avoidance of contaminations, and simple but reliable process monitoring. A fully automated solution where all these aspects have been taken into account is presented.

  14. 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

  15. Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms.

    PubMed

    Pacheco, Maria P; Pfau, Thomas; Sauter, Thomas

    2015-01-01

    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.

  16. 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

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

    PubMed

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

    2016-06-09

    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.

  18. Sensing immune responses with customized peptide microarrays.

    PubMed

    Schirwitz, Christopher; Loeffler, Felix F; Felgenhauer, Thomas; Stadler, Volker; Breitling, Frank; Bischoff, F Ralf

    2012-12-01

    The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

  19. 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

  20. ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they ar...

  1. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

    PubMed

    Li, Yongjin

    2016-01-01

    The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use.

  2. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

    PubMed

    Li, Yongjin

    2016-01-01

    The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use. PMID:26860568

  3. Living-Cell Microarrays

    PubMed Central

    Yarmush, Martin L.; King, Kevin R.

    2011-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

  4. Up-to-Date Applications of Microarrays and Their Way to Commercialization.

    PubMed

    Schumacher, Sarah; Muekusch, Sandra; Seitz, Harald

    2015-04-23

    This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

  5. Up-to-Date Applications of Microarrays and Their Way to Commercialization

    PubMed Central

    Schumacher, Sarah; Muekusch, Sandra; Seitz, Harald

    2015-01-01

    This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made. PMID:27600220

  6. Up-to-Date Applications of Microarrays and Their Way to Commercialization

    PubMed Central

    Schumacher, Sarah; Muekusch, Sandra; Seitz, Harald

    2015-01-01

    This review addresses up-to-date applications of Protein Microarrays. Protein Microarrays play a significant role in basic research as well as in clinical applications and are applicable in a lot of fields, e.g., DNA, proteins and small molecules. Additionally they are on the way to enter clinics in routine diagnostics. Protein Microarrays can be powerful tools to improve healthcare. An overview of basic characteristics to mediate essential knowledge of this technique is given. To reach this goal, some challenges still have to be addressed. A few applications of Protein Microarrays in a medical context are shown. Finally, an outlook, where the potential of Protein Microarrays is depicted and speculations how the future of Protein Microarrays will look like are made.

  7. 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

  8. Viral detection by high-throughput sequencing.

    PubMed

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

    2015-01-01

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

  9. Microfabricated high-throughput electronic particle detector.

    PubMed

    Wood, D K; Requa, M V; Cleland, A N

    2007-10-01

    We describe the design, fabrication, and use of a radio frequency reflectometer integrated with a microfluidic system, applied to the very high-throughput measurement of micron-scale particles, passing in a microfluidic channel through the sensor region. The device operates as a microfabricated Coulter counter [U.S. Patent No. 2656508 (1953)], similar to a design we have described previously, but here with significantly improved electrode geometry as well as including electronic tuning of the reflectometer; the two improvements yielding an improvement by more than a factor of 10 in the signal to noise and in the diametric discrimination of single particles. We demonstrate the high-throughput discrimination of polystyrene beads with diameters in the 4-10 microm range, achieving diametric resolutions comparable to the intrinsic spread of diameters in the bead distribution, at rates in excess of 15 x 10(6) beads/h.

  10. Viral detection by high-throughput sequencing.

    PubMed

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

    2015-01-01

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

  11. PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

    PubMed Central

    Kojima, Takaaki; Takei, Yoshiaki; Ohtsuka, Miharu; Kawarasaki, Yasuaki; Yamane, Tsuneo; Nakano, Hideo

    2005-01-01

    We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale. PMID:16214800

  12. Network medicine and high throughput screening.

    PubMed

    Smith, Robert E; Tran, Kevin; Vocque, Ralph H

    2013-09-01

    A new paradigm is emerging in modern drug discovery. It is a fusion of traditional and modern medicine, phenotypic and targeted drug discovery, or systems and reductionist thinking. This is exemplified by using a combination of network medicine and high throughput screening. It blends the use of physiologically relevant biological systems with the high throughput and statistical robustness of modern assay technologies. The basic principles of network theory and tools of network medicine are described. Scale-free networks and their organizing principles are discussed. They are emergent properties of living, autopoietic systems. This includes networks of people who do high throughput screening (HTS), and microscopic networks of ions, metabolites, DNA, RNA, proteins, lipids, carbohydrates, viruses, bacteria, fungi, human cells and tissues. Databases have been constructed based on the metabolome, genome, transcriptome, proteome, lipidome, glycocode, virome, bacteriome and many others. Modern HTS can be used to examine the interactions of many parts of the complex human network. High content screening (HCS) can look at perturbations that occur when test compounds are added to single cells. Allo-network drugs can have effects far beyond a single protein and can be transmitted to other cells. Interactions and hidden connections can be revealed, with the goal of developing new drugs that have few, if any harmful side effects and are effective against multi-drug resistant cancer cells or bacteria.

  13. Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa.

    PubMed

    Kojima, Mikiko; Kamada-Nobusada, Tomoe; Komatsu, Hirokazu; Takei, Kentaro; Kuroha, Takeshi; Mizutani, Masaharu; Ashikari, Motoyuki; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto; Suzuki, Koji; Sakakibara, Hitoshi

    2009-07-01

    We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA and gibberellins. This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI; UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as gibberellins, we chemically derivatized fractions containing auxin, ABA and gibberellins with bromocholine that has a quaternary ammonium functional group. This modification, that we call 'MS-probe', makes these hormone derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of gibberellins increased up to 50-fold. Our current method needs <100 mg (FW) of plant tissues to determine phytohormone profiles and enables us to analyze >180 plant samples simultaneously. Application of this method to plant hormone profiling enabled us to draw organ distribution maps of hormone species in rice and also to identify interactions among the four major hormones in the rice gibberellin signaling mutants, gid1-3, gid2-1 and slr1. Combining the results of hormone profiling data with transcriptome data in the gibberellin signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.

  14. Application of 2,4-Dinitrophenylhydrazine (DNPH) in High-Throughput Screening for Microorganism Mutants Accumulating 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD)

    PubMed Central

    Liu, Yang; Cao, Fei; Xiong, Hui; Shen, Yanbing; Wang, Min

    2016-01-01

    To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9α-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application. PMID:27706217

  15. A rapid automatic processing platform for bead label-assisted microarray analysis: application for genetic hearing-loss mutation detection.

    PubMed

    Zhu, Jiang; Song, Xiumei; Xiang, Guangxin; Feng, Zhengde; Guo, Hongju; Mei, Danyang; Zhang, Guohao; Wang, Dong; Mitchelson, Keith; Xing, Wanli; Cheng, Jing

    2014-04-01

    Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.

  16. Protein Microarrays--Without a Trace

    SciTech Connect

    Camarero, J A

    2007-04-05

    Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.

  17. 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...

  18. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

  19. The Development and Application of a Quantitative Peptide Microarray Based Approach to Protein Interaction Domain Specificity Space*

    PubMed Central

    Engelmann, Brett W.; Kim, Yohan; Wang, Miaoyan; Peters, Bjoern; Rock, Ronald S.; Nash, Piers D.

    2014-01-01

    Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology. PMID:25135669

  20. 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.

  1. 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.

  2. 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

  3. Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening.

    PubMed

    Mere; Bennett; Coassin; England; Hamman; Rink; Zimmerman; Negulescu

    1999-08-01

    Assay miniaturization applicable across a wide range of target classes, along with automation and process integration, are well-recognized goals for ultra-high-throughput screening on an industrial scale. This report summarizes the implementation of fluorescence resonance energy transfer (FRET)-based biochemical and cell-based assays in 3456-well NanoWelltrade mark assay plates using key components of Aurora's ultra-high-throughput screening system.

  4. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    PubMed Central

    Christiansen, Anders; Kringelum, Jens V.; Hansen, Christian S.; Bøgh, Katrine L.; Sullivan, Eric; Patel, Jigar; Rigby, Neil M.; Eiwegger, Thomas; Szépfalusi, Zsolt; Masi, Federico de; Nielsen, Morten; Lund, Ole; Dufva, Martin

    2015-01-01

    Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds. PMID:26246327

  5. Next Generation Gene Synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device

    PubMed Central

    Matzas, Mark; Stähler, Peer F.; Kefer, Nathalie; Siebelt, Nicole; Boisguérin, Valesca; Leonard, Jack T.; Keller, Andreas; Stähler, Cord F.; Häberle, Pamela; Gharizadeh, Baback; Babrzadeh, Farbod; Church, George

    2012-01-01

    The setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications. PMID:21113166

  6. 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

  7. 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

  8. 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

  9. 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?

  10. High Throughput Optimization of Stem Cell Microenvironments

    PubMed Central

    Yang, Fan; Mei, Ying; Langer, Robert; Anderson, Daniel G.

    2009-01-01

    Stem cells have great potential as cell sources for regenerative medicine due to both their self-renewal and multi-lineage differentiation capacity. Despite advances in the field of stem cell biology, major challenges remain before stem cells can be widely used for therapeutic purposes. One challenge is to develop reproducible methods to control stem cell growth and differentiation. The niche in which stem cells reside is a complex, multi-factorial environment. In contrast to using cells alone, biomaterials can provide initial structural support, and allow cells to adhere, proliferate and differentiate in a three-dimensional environment. Researchers have incorporated signals into the biomaterials that can promote desired cell functions in a spatially and temporally controlled manner. Despite progress in biomaterial design and methods to modulate cellular behavior, many of the complex signal networks that regulate cell-material interactions remain unclear. Due to the vast numbers of material properties to be explored and the complexity of cell-surface interactions, it is often difficult to optimize stem cell microenvironments using conventional, iterative approaches. To address these challenges, high throughput screening of combinatorial libraries has emerged as a novel approach to achieve rapid screening with reduced materials and costs. In this review, we discuss recent research in the area of high throughput approaches for characterization and optimization of cellular interactions with their microenvironments. In contrast to conventional approaches, screening combinatorial libraries can result in the discovery of unexpected material solutions to these complex problems. PMID:19601753

  11. 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).

  12. Establishment of local searching methods for orbitrap-based high throughput metabolomics analysis.

    PubMed

    Tang, Haiping; Wang, Xueying; Xu, Lina; Ran, Xiaorong; Li, Xiangjun; Chen, Ligong; Zhao, Xinbin; Deng, Haiteng; Liu, Xiaohui

    2016-08-15

    Our method aims to establish local endogenous metabolite databases economically without purchasing chemical standards, giving strong bases for following orbitrap based high throughput untargeted metabolomics analysis. A new approach here is introduced to construct metabolite databases on the base of biological sample analysis and mathematic extrapolation. Building local metabolite databases traditionally requires expensive chemical standards, which is barely affordable for most research labs. As a result, most labs working on metabolomics analysis have to refer public libraries, which is time consuming and limited for high throughput analysis. Using this strategy, a high throughput orbitrap based metabolomics platform can be established at almost no cost within a couple of months. It enables to facilitate the application of high throughput metabolomics analysis to identify disease-related biomarkers or investigate biological functions using orbitrap. PMID:27260449

  13. Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    PubMed Central

    Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

    2010-01-01

    Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

  14. Fluidic and air-stable supported lipid bilayer and cell-mimicking microarrays.

    PubMed

    Deng, Yang; Wang, Yini; Holtz, Bryan; Li, Jingyi; Traaseth, Nathan; Veglia, Gianluigi; Stottrup, Benjamin J; Elde, Robert; Pei, Duanqing; Guo, Athena; Zhu, X-Y

    2008-05-14

    As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Cell membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), are currently not sufficiently robust to meet this need. Here we describe a method of forming fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet. Achieving air stability allows us to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. We demonstrate their application as cell membrane-mimicking microarrays by reconstituting peripheral as well as integral membrane components that can be recognized by their respective targets. These demonstrations establish the viability of the fluidic and air-stable SLB platform for generating content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors.

  15. Contributions to Statistical Problems Related to Microarray Data

    ERIC Educational Resources Information Center

    Hong, Feng

    2009-01-01

    Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

  16. Design, construction, characterization, and application of a hyperspectral microarray scanner.

    PubMed

    Sinclair, Michael B; Timlin, Jerilyn A; Haaland, David M; Werner-Washburne, Margaret

    2004-04-01

    We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research. The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 microm and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray. This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments. Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate. We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media.

  17. The Impact of Photobleaching on Microarray Analysis.

    PubMed

    von der Haar, Marcel; Preuß, John-Alexander; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2015-01-01

    DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner's laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube's voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results. PMID:26378589

  18. A high-throughput neutron spectrometer

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  19. 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

  20. 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

  1. High-throughput methods for electron crystallography.

    PubMed

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

    2013-01-01

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

  2. Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes.

    PubMed

    Tiersch, Terrence R; Yang, Huiping; Hu, E

    2011-08-01

    With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.

  3. New Lung Cancer Panel for High-Throughput Targeted Resequencing

    PubMed Central

    Kim, Eun-Hye; Lee, Sunghoon; Park, Jongsun; Lee, Kyusang; Bhak, Jong

    2014-01-01

    We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at 30× sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than 1,000× coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening. PMID:25031567

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

    PubMed

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

    2011-03-01

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

  5. 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

  6. 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

  7. 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.

  8. A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis.

    PubMed

    Chen, Qinghai; Wu, Nan; Xie, Meng; Zhang, Bo; Chen, Ming; Li, Jianjun; Zhuo, Lisha; Kuang, Hong; Fu, Weiling

    2012-04-01

    The accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations.

  9. A protein chip approach for high-throughput antigen identification and characterization.

    PubMed

    Hu, Shaohui; Li, Yu; Liu, Guozhen; Song, Qifeng; Wang, Li; Han, Yuning; Zhang, Yang; Song, Yali; Yao, Xiying; Tao, Yong; Zeng, Haipan; Yang, Huanming; Wang, Jian; Zhu, Heng; Chen, Zhi-Nan; Wu, Lin

    2007-06-01

    Proteomics research in humans and other eukaryotes demands a large number of high-quality mAbs. Here, we report a new approach to produce high-quality mAbs against human liver proteins using a combined force of high-throughput mAb production and protein microarrays. After immunizing mice with live cells from human livers, we isolated 54 hybridomas with binding activities to human cells and identified the corresponding antigens for five mAbs via screening on a protein microarray of 1058 unique human liver proteins. Finally, we demonstrated that using the five mAbs we could characterize the expression profiles of their corresponding antigens by using tissue microarrays. Among them, we discovered that eIF1A expressed only in normal liver tissues, not in hepatocellular carcinoma in humans.

  10. Oligonucleotide microarrays in constitutional genetic diagnosis.

    PubMed

    Keren, Boris; Le Caignec, Cedric

    2011-06-01

    Oligonucleotide microarrays such as comparative genomic hybridization arrays and SNP microarrays enable the identification of genomic imbalances - also termed copy-number variants - with increasing resolution. This article will focus on the most significant applications of high-throughput oligonucleotide microarrays, both in genetic diagnosis and research. In genetic diagnosis, the method is becoming a standard tool for investigating patients with unexplained developmental delay/intellectual disability, autism spectrum disorders and/or with multiple congenital anomalies. Oligonucleotide microarray have also been recently applied to the detection of genomic imbalances in prenatal diagnosis either to characterize a chromosomal rearrangement that has previously been identified by standard prenatal karyotyping or to detect a cryptic genomic imbalance in a fetus with ultrasound abnormalities and a normal standard prenatal karyotype. In research, oligonucleotide microarrays have been used for a wide range of applications, such as the identification of new genes responsible for monogenic disorders and the association of a copy-number variant as a predisposing factor to a common disease. Despite its widespread use, the interpretation of results is not always straightforward. We will discuss several unexpected results and ethical issues raised by these new methods.

  11. 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

  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. 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

  14. 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.

  15. 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

  16. 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

  17. Orthogonal NGS for High Throughput Clinical Diagnostics.

    PubMed

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

    2016-04-19

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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Recent advances and future applications of microfluidic live-cell microarrays.

    PubMed

    Rothbauer, Mario; Wartmann, David; Charwat, Verena; Ertl, Peter

    2015-11-01

    Microfluidic live-cell microarrays show much promise as screening tools for biomedical research because they could shed light on key biological processes such as cell signaling and cell-to-cell and cell-to-substrate dynamic responses. While miniaturization reduces the need for expensive clinical grade reagents, the integration of functional components including micropumps, biosensors, actuators, mixers and gradient generators results in improved assay reliability, reproducibility and well-defined cell culture conditions. The present review addresses recent technological advances in microfluidic live-cell microarray technology with a special focus on the applications of microfluidic single-cell, multi-cell and 3D cell microarrays.

  3. [Research progress of probe design software of oligonucleotide microarrays].

    PubMed

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software.

  4. Tissue Microarrays in Clinical Oncology

    PubMed Central

    Voduc, David; Kenney, Challayne; Nielsen, Torsten O.

    2008-01-01

    The tissue microarray is a recently-implemented, high-throughput technology for the analysis of molecular markers in oncology. This research tool permits the rapid assessment of a biomarker in thousands of tumor samples, using commonly available laboratory assays such as immunohistochemistry and in-situ hybridization. Although introduced less than a decade ago, the TMA has proven to be invaluable in the study of tumor biology, the development of diagnostic tests, and the investigation of oncological biomarkers. This review describes the impact of TMA-based research in clinical oncology and its potential future applications. Technical aspects of TMA construction, and the advantages and disadvantages inherent to this technology are also discussed. PMID:18314063

  5. 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).

  6. High-throughput flow cytometry for drug discovery.

    PubMed

    Edwards, Bruce S; Young, Susan M; Saunders, Matthew J; Bologa, Cristian; Oprea, Tudor I; Ye, Richard D; Prossnitz, Eric R; Graves, Steven W; Sklar, Larry A

    2007-05-01

    High-throughput flow cytometry exploits a novel many-samples/one-file approach to dramatically speed data acquisition, limit aspirated sample volume to as little as 2 μl/well and produce multisample data sets that facilitate automated analysis of samples in groups as well as individually. It has been successfully applied to both cell- and microsphere-based bioassays in 96- and 384-well formats, to screen tens-of-thousands of compounds and identify novel bioactive structures. High-content multiparametric analysis capabilities have been exploited for assay multiplexing, allowing the assessment of biologic selectivity and specificity to be an integral component of primary screens. These and other advances in the last decade have contributed to the application of flow cytometry as a uniquely powerful tool for probing biologic and chemical diversity and complex systems biology.

  7. 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

  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. High Throughput Gene Expression Measurement with Real Time PCR in a Microfluidic Dynamic Array

    PubMed Central

    Spurgeon, Sandra L.; Jones, Robert C.; Ramakrishnan, Ramesh

    2008-01-01

    We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays. PMID:18301740

  10. 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

  11. 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

  12. Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays

    PubMed Central

    Lim, Jiseok; Caen, Ouriel; Vrignon, Jérémy; Konrad, Manfred; Baret, Jean-Christophe

    2015-01-01

    Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of β-galactosidase in droplets using nine different concentrations of a fluorogenic substrate. PMID:26015838

  13. High-throughput parallel SPM for metrology, defect, and mask inspection

    NASA Astrophysics Data System (ADS)

    Sadeghian, H.; Herfst, R. W.; van den Dool, T. C.; Crowcombe, W. E.; Winters, J.; Kramer, G. F. I. J.

    2014-10-01

    Scanning probe microscopy (SPM) is a promising candidate for accurate assessment of metrology and defects on wafers and masks, however it has traditionally been too slow for high-throughput applications, although recent developments have significantly pushed the speed of SPM [1,2]. In this paper we present new results obtained with our previously presented high-throughput parallel SPM system [3,4] that showcase two key advances that are required for a successful deployment of SPM in high-throughput metrology, defect and mask inspection. The first is a very fast (up to 40 lines/s) image acquisition and a comparison of the image quality as function of speed. Secondly, a fast approach method: measurements of the scan-head approaching the sample from 0.2 and 1.0 mm distance in under 1.4 and 6 seconds respectively.

  14. Graphene oxide-based micropatterns via high-throughput multiphoton-induced reduction and ablation.

    PubMed

    Li, Yi-Cheng; Yeh, Te-Fu; Huang, Hsin-Chieh; Chang, Hsin-Yu; Lin, Chun-Yu; Cheng, Li-Chung; Chang, Chia-Yuan; Teng, Hsisheng; Chen, Shean-Jen

    2014-08-11

    In this study, a developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device to locally control the laser pulse numbers, GO-based micropatterns can be quickly achieved instantly. Furthermore, the degree of reduction and ablation can be precisely adjusted via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this approach offers a high-throughput and multiple-function approach to accomplish a large area of micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system fulfills the requirement of mass production for GO-based applications in microelectronic devices. PMID:25321055

  15. Review article: high-throughput affinity-based technologies for small-molecule drug discovery.

    PubMed

    Zhu, Zhengrong; Cuozzo, John

    2009-12-01

    High-throughput affinity-based technologies are rapidly growing in use as primary screening methods in drug discovery. In this review, their principles and applications are described and their impact on small-molecule drug discovery is evaluated. In general, these technologies can be divided into 2 groups: those that detect binding interactions by measuring changes to the protein target and those that detect bound compounds. Technologies detecting binding interactions by focusing on the protein have limited throughput but can reveal mechanistic information about the binding interaction; technologies detecting bound compounds have very high throughput, some even significantly higher than current high-throughput screening technologies, but offer limited information about the binding interaction. In addition, the appropriate use of affinity-based technologies is discussed. Finally, nanotechnology is predicted to generate a significant impact on the future of affinity-based technologies. PMID:19822881

  16. Medium to high throughput screening: microfabrication and chip-based technology.

    PubMed

    Wen, Yuan; Zhang, Xudong; Yang, Shang-Tian

    2012-01-01

    Medium to high throughput screening for toxicity testing can provide a wealth of information with significant time and cost savings. New technologies, such as microfabrication, microfluidics and chip-based technology, combined with advanced cell culture and detection techniques, open up new opportunities in toxicity testing. In this chapter, fundamentals of microfabrication and microfluidics are discussed with a focus on the broad and novel applications on toxicity studies enabled by these technologies. Emphasis is placed on microscale cell and tissue culture models for medium and high throughput systemic toxicity studies in vitro.

  17. High Throughput Proteomics Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    SciTech Connect

    Qian, Weijun; Camp, David G.; Smith, Richard D.

    2004-06-01

    The advent of high throughput proteomics technology for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of cellular machinery. Here, we review recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance (FTICR) mass spectrometry along with its potential application to high throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system-level.

  18. High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Qian, Wei-Jun; Camp, David G; Smith, Richard D

    2004-06-01

    The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.

  19. Microarrays for identifying binding sites and probing structure of RNAs

    PubMed Central

    Kierzek, Ryszard; Turner, Douglas H.; Kierzek, Elzbieta

    2015-01-01

    Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs. PMID:25505162

  20. A general approach for discriminative de novo motif discovery from high-throughput data

    PubMed Central

    Grau, Jan; Posch, Stefan; Grosse, Ivo; Keilwagen, Jens

    2013-01-01

    De novo motif discovery has been an important challenge of bioinformatics for the past two decades. Since the emergence of high-throughput techniques like ChIP-seq, ChIP-exo and protein-binding microarrays (PBMs), the focus of de novo motif discovery has shifted to runtime and accuracy on large data sets. For this purpose, specialized algorithms have been designed for discovering motifs in ChIP-seq or PBM data. However, none of the existing approaches work perfectly for all three high-throughput techniques. In this article, we propose Dimont, a general approach for fast and accurate de novo motif discovery from high-throughput data. We demonstrate that Dimont yields a higher number of correct motifs from ChIP-seq data than any of the specialized approaches and achieves a higher accuracy for predicting PBM intensities from probe sequence than any of the approaches specifically designed for that purpose. Dimont also reports the expected motifs for several ChIP-exo data sets. Investigating differences between in vitro and in vivo binding, we find that for most transcription factors, the motifs discovered by Dimont are in good accordance between techniques, but we also find notable exceptions. We also observe that modeling intra-motif dependencies may increase accuracy, which indicates that more complex motif models are a worthwhile field of research. PMID:24057214

  1. Reducing false positive findings in statistical analysis of pharmacogenomic biomarker studies using high-throughput technologies.

    PubMed

    Matsui, Shigeyuki

    2006-05-01

    The promise of pharmacogenomics lies in the potential to establish a personalized drug therapy with the intent of maximizing effectiveness and minimizing risk, through development of pharmacogenomics biomarkers. However, currently, most pharmacogenomic measurements are not considered valid biomarkers with clear clinical significance, thus this field is in early developmental stages. Recently, the development of comprehensive, high-throughput technologies such as gene expression microarrays has provided powerful new tools for these stages. This technological transformation is, at the same time, generating an increasing demand for statistical analysis of large and complex multivariate datasets from high-throughput assays. This article provides a review of the key features to be observed in statistical analyses of large amounts of data from pharmacogenomic biomarker studies with high-throughput assays. The problem of false positive can be very serious in such studies. The evaluation of stability and reproducibility of the results of statistical analysis are claimed to reduce chance that false positive findings are subject to further investigation in subsequent studies.

  2. Chemically diverse polymer microarrays and high throughput surface characterisation: a method for discovery of materials for stem cell culture† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4bm00054d Click here for additional data file.

    PubMed Central

    Celiz, A. D.; Smith, J. G. W.; Patel, A. K.; Langer, R.; Anderson, D. G.; Barrett, D. A.; Young, L. E.; Davies, M. C.; Denning, C.

    2014-01-01

    Materials discovery provides the opportunity to identify novel materials that are tailored to complex biological environments by using combinatorial mixing of monomers to form large libraries of polymers as micro arrays. The materials discovery approach is predicated on the use of the largest chemical diversity possible, yet previous studies into human pluripotent stem cell (hPSC) response to polymer microarrays have been limited to 20 or so different monomer identities in each study. Here we show that it is possible to print and assess cell adhesion of 141 different monomers in a microarray format. This provides access to the largest chemical space to date, allowing us to meet the regenerative medicine challenge to provide scalable synthetic culture ware. This study identifies new materials suitable for hPSC expansion that could not have been predicted from previous knowledge of cell-material interactions. PMID:25328672

  3. Chemically diverse polymer microarrays and high throughput surface characterisation: a method for discovery of materials for stem cell culture†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4bm00054dClick here for additional data file.

    PubMed

    Celiz, A D; Smith, J G W; Patel, A K; Langer, R; Anderson, D G; Barrett, D A; Young, L E; Davies, M C; Denning, C; Alexander, M R

    2014-11-30

    Materials discovery provides the opportunity to identify novel materials that are tailored to complex biological environments by using combinatorial mixing of monomers to form large libraries of polymers as micro arrays. The materials discovery approach is predicated on the use of the largest chemical diversity possible, yet previous studies into human pluripotent stem cell (hPSC) response to polymer microarrays have been limited to 20 or so different monomer identities in each study. Here we show that it is possible to print and assess cell adhesion of 141 different monomers in a microarray format. This provides access to the largest chemical space to date, allowing us to meet the regenerative medicine challenge to provide scalable synthetic culture ware. This study identifies new materials suitable for hPSC expansion that could not have been predicted from previous knowledge of cell-material interactions.

  4. DNA microarrays: experimental issues, data analysis, and application to bacterial systems.

    PubMed

    Dharmadi, Yandi; Gonzalez, Ramon

    2004-01-01

    DNA microarrays are currently used to study the transcriptional response of many organisms to genetic and environmental perturbations. Although there is much room for improvement of this technology, its potential has been clearly demonstrated in the past 5 years. The general consensus is that the bottleneck is now located in the processing and analysis of transcriptome data and its use for purposes other than the quantification of changes in gene expression levels. In this article we discuss technological aspects of DNA microarrays, statistical and biological issues pertinent to the design of microarray experiments, and statistical tools for microarray data analysis. A review on applications of DNA microarrays in the study of bacterial systems is presented. Special attention is given to studies in the following areas: (1) bacterial response to environmental changes; (2) gene identification, genome organization, and transcriptional regulation; and (3) genetic and metabolic engineering. Soon, the use of DNA microarray technologies in conjunction with other genome/system-wide analyses (e.g., proteomics, metabolomics, fluxomics, phenomics, etc.) will provide a better assessment of genotype-phenotype relationships in bacteria, which serve as a basis for understanding similar processes in more complex organisms.

  5. Incorporating Population Variability and Susceptible Subpopulations into Dosimetry for High-Throughput Toxicity Testing

    EPA Science Inventory

    Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assess...

  6. High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this work we report a high throughput mass spectrometry-based technique for rapid high resolution strain identification of Campylobacter jejuni. This method readily distinguishes C. jejuni from C. coli, has comparable resolving power to multi-locus sequence typing (MLST), is applicable to mixtur...

  7. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    PubMed

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale. PMID:27247981

  8. High-throughput synthesis and characterization of nanocrystalline porphyrinic zirconium metal-organic frameworks.

    PubMed

    Kelty, M L; Morris, W; Gallagher, A T; Anderson, J S; Brown, K A; Mirkin, C A; Harris, T D

    2016-06-14

    We describe and employ a high-throughput screening method to accelerate the synthesis and identification of pure-phase, nanocrystalline metal-organic frameworks (MOFs). We demonstrate the efficacy of this method through its application to a series of porphyrinic zirconium MOFs, resulting in the isolation of MOF-525, MOF-545, and PCN-223 on the nanoscale.

  9. Nanoelectrospray ion generation for high-throughput mass spectrometry using a micromachined ultrasonic ejector array

    SciTech Connect

    Aderogba, S.; Meacham, J.M.; Degertekin, F.L.; Fedorov, A.G.; Fernandez, F.M.

    2005-05-16

    Ultrasonic electrospray ionization (ESI) for high-throughput mass spectrometry is demonstrated using a silicon micromachined microarray. The device uses a micromachined ultrasonic atomizer operating in the 900 kHz-2.5 MHz range for droplet generation and a metal electrode in the fluid cavity for ionization. Since the atomization and ionization processes are separated, the ultrasonic ESI source shows the potential for operation at low voltages with a wide range of solvents in contrast with conventional capillary ESI technology. This is demonstrated using the ultrasonic ESI microarray to obtain the mass spectrum of a 10 {mu}M reserpine sample on a time of flight mass spectrometer with 197:1 signal-to-noise ratio at an ionization potential of 200 V.

  10. Development of high-throughput perfusion-based microbioreactor platform capable of providing tunable dynamic tensile loading to cells and its application for the study of bovine articular chondrocytes.

    PubMed

    Wu, Min-Hsien; Wang, Hsin-Yao; Liu, Heng-Liang; Wang, Shih-Siou; Liu, Yen-Ting; Chen, Yan-Ming; Tsai, Shiao-Wen; Lin, Chun-Li

    2011-08-01

    Mammalian cells are sensitive to extracellular microenvironments. In order to precisely explore the physiological responses of cells to tensile loading, a stable and well-defined culture condition is required. In this study, a high-throughput perfusion-based microbioreactor platform capable of providing dynamic equibiaxial tensile loading to the cultured cells under a steady culture condition was proposed. The mechanism of generating tensile stimulation to cells is based on the pneumatically-driven deformation of an elastic polydimethylsiloxan (PDMS) membrane which exerts tensile loading to the attached cells. By modulating the magnitude and frequency of the applied pneumatic pressure, various tensile loading can be generated in a controllable manner. In this study, the microbioreactor platform was designed with the aid of the experimentally-validated finite element (FE) analysis to ensure the loading of tensile strain to cells is uniform and definable. Based on this design, the quantitative relationship between the applied pneumatic pressure and the generated tensile strain on the PDMS membrane was established via FE analysis. Results demonstrated that the proposed device was able to generate the tensile strain range (0~0.12), which covers the physiological condition that articular chondrocytes experience tensile strain under human walking condition. In this study, moreover, the effect of tensile loading on the metabolic, biosynthetic and proliferation activities of articular chondrocytes was investigated. Results disclosed that the dynamic tensile loading of 0.12 strain at 1 Hz might significantly up-regulate the synthesis of glycosaminoglycans while such stimulation was found no significant influence on the metabolic activity, the synthesis of collagen, and the proliferation of chondrocytes. Overall, this study has presented a high throughput perfusion micro cell culture device that is suitable for precisely exploring the effect of tensile loading on cell

  11. ILOOP – a web application for two-channel microarray interwoven loop design

    PubMed Central

    Pirooznia, Mehdi; Gong, Ping; Yang, Jack Y; Yang, Mary Qu; Perkins, Edward J; Deng, Youping

    2008-01-01

    Microarray technology is widely applied to address complex scientific questions. However, there remain fundamental issues on how to design experiments to ensure that the resulting data enables robust statistical analysis. Interwoven loop design has several advantages over other designs. However it suffers in the complexity of design. We have implemented an online web application which allows users to find optimal loop designs for two-color microarray experiments. Given a number of conditions (such as treatments or time points) and replicates, the application will find the best possible design of the experiment and output experimental parameters. It is freely available from . PMID:18831776

  12. High-Throughput Screening to Identify Plant Derived Human LDH-A Inhibitors

    PubMed Central

    Deiab, S.; Mazzio, E.; Messeha, S.; Mack, N.; Soliman, K. F. A.

    2014-01-01

    Aims Lactate dehydrogenase (LDH)-A is highly expressed in diverse human malignant tumors, parallel to aggressive metastatic disease, resistance to radiation/chemotherapy and clinically poor outcome. Although this enzyme constitutes a plausible target in treatment of advanced cancer, there are few known LDH-A inhibitors. Study Design In this work, we utilized a high-throughput enzyme micro-array format to screen and evaluate > 900 commonly used medicinal plant extracts (0.00001-.5 mg/ml) for capacity to inhibit activity of recombinant full length human LDHA; EC .1.1.1.27. Methodology The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis- MALDI-TOF-MS/MS, enzyme activity was validated by oxidation of NADH (500μM) and kinetic inhibition established in the presence of a known inhibitor (Oxalic Acid). Results Of the natural extracts tested, the lowest IC50s [<0.001 mg/ml] were obtained by: Chinese Gallnut (Melaphis chinensis gallnut), Bladderwrack (Fucus vesiculosus), Kelp (Laminaria Japonica) and Babul (Acacia Arabica). Forty-six additional herbs contained significant LDH-A inhibitory properties with IC50s [<0.07 mg/ml], some of which have common names of Arjun, Pipsissewa, Cinnamon, Pink Rose Buds/Petals, Wintergreen, Cat’s Claw, Witch Hazel Root and Rhodiola Root. Conclusion These findings reflect relative potency by rank of commonly used herbs and plants that contain human LDH-A inhibitory properties. Future research will be required to isolate chemical constituents within these plants responsible for LDH-A inhibition and investigate potential therapeutic application. PMID:24478981

  13. Label-free high-throughput microRNA expression profiling from total RNA

    PubMed Central

    Duan, Demin; Zheng, Ke-xiao; Shen, Ye; Cao, Rong; Jiang, Li; Lu, Zhuoxuan; Yan, Xiyun; Li, Jiong

    2011-01-01

    MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays. Although effective, these labeling-based approaches are usually labor-intensive, time-consuming and liable to bias. Besides, the cross-hybridization of co-existing miRNA precursors (pre-miRNAs) is not adequately addressed in most assays that use total RNA as input. Here, we present a hybridization-triggered fluorescence strategy for label-free, microarray-based high-throughput miRNA expression profiling. The total RNA is directly applied to the microarray with a short fluorophore-linked oligonucleotide Universal Tag which can be selectively captured by the target-bound probes via base-stacking effects. This Stacking-Hybridized Universal Tag (SHUT) assay has been successfully used to analyze as little as 100 ng total RNA from human tissues, and found to be highly specific to homogenous miRNAs. Superb discrimination toward single-base mismatch at the 5′ or 3′ end has been demonstrated. Importantly, the pre-miRNAs generated negligible signals, validating the direct use of total RNA. PMID:21976734

  14. High-Throughput Sequencing in Mitochondrial DNA Research

    PubMed Central

    Ye, Fei; Samuels, David C.; Clark, Travis; Guo, Yan

    2014-01-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers’ ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control. PMID:24859348

  15. 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...

  16. High-throughput sequencing in mitochondrial DNA research.

    PubMed

    Ye, Fei; Samuels, David C; Clark, Travis; Guo, Yan

    2014-07-01

    Next-generation sequencing, also known as high-throughput sequencing, has greatly enhanced researchers' ability to conduct biomedical research on all levels. Mitochondrial research has also benefitted greatly from high-throughput sequencing; sequencing technology now allows for screening of all 16,569 base pairs of the mitochondrial genome simultaneously for SNPs and low level heteroplasmy and, in some cases, the estimation of mitochondrial DNA copy number. It is important to realize the full potential of high-throughput sequencing for the advancement of mitochondrial research. To this end, we review how high-throughput sequencing has impacted mitochondrial research in the categories of SNPs, low level heteroplasmy, copy number, and structural variants. We also discuss the different types of mitochondrial DNA sequencing and their pros and cons. Based on previous studies conducted by various groups, we provide strategies for processing mitochondrial DNA sequencing data, including assembly, variant calling, and quality control.

  17. AOPs & Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making.

    EPA Science Inventory

    As high throughput screening (HTS) approaches play a larger role in toxicity testing, computational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models for this purpose are becoming increasingly more sophisticated...

  18. Development of A High Throughput Method Incorporating Traditional Analytical Devices

    PubMed Central

    White, C. C.; Embree, E.; Byrd, W. E; Patel, A. R.

    2004-01-01

    A high-throughput (high throughput is the ability to process large numbers of samples) and companion informatics system has been developed and implemented. High throughput is defined as the ability to autonomously evaluate large numbers of samples, while an informatics system provides the software control of the physical devices, in addition to the organization and storage of the generated electronic data. This high throughput system includes both an ultra-violet and visible light spectrometer (UV-Vis) and a Fourier transform infrared spectrometer (FTIR) integrated with a multi sample positioning table. This method is designed to quantify changes in polymeric materials occurring from controlled temperature, humidity and high flux UV exposures. The integration of the software control of these analytical instruments within a single computer system is presented. Challenges in enhancing the system to include additional analytical devices are discussed. PMID:27366626

  19. 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, ...

  20. 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 ...

  1. Environmental surveillance and monitoring--The next frontiers for high-throughput toxicology.

    PubMed

    Schroeder, Anthony L; Ankley, Gerald T; Houck, Keith A; Villeneuve, Daniel L

    2016-03-01

    High-throughput toxicity testing technologies along with the World Wide Web are revolutionizing both generation of and access to data regarding the biological activities that chemicals can elicit when they interact with specific proteins, genes, or other targets in the body of an organism. To date, however, most of the focus has been on the application of such data to assessment of individual chemicals. The authors suggest that environmental surveillance and monitoring represent the next frontiers for high-throughput toxicity testing. Resources already exist in curated databases of chemical-biological interactions, including highly standardized quantitative dose-response data generated from nascent high-throughput toxicity testing programs such as ToxCast and Tox21, to link chemicals detected through environmental analytical chemistry to known biological activities. The emergence of the adverse outcome pathway framework and the associated knowledge base for linking molecular-level or pathway-level perturbations of biological systems to adverse outcomes traditionally considered in risk assessment and regulatory decision-making through a series of measurable biological changes provides a critical link between activity and hazard. Furthermore, environmental samples can be directly analyzed via high-throughput toxicity testing platforms to provide an unprecedented breadth of biological activity characterization that integrates the effects of all compounds present in a mixture, whether known or not. Novel application of these chemical-biological interaction data provides an opportunity to transform scientific characterization of potential hazards associated with exposure to complex mixtures of environmental contaminants. PMID:26923854

  2. Unconventional Architectures for High-Throughput Sciences

    SciTech Connect

    Nieplocha, Jarek; Marquez, Andres; Petrini, Fabrizio; Chavarría-Miranda, Daniel

    2007-06-15

    Science laboratories and sophisticated simulations are producing data of increasing volumes and complexities, and that’s posing significant challenges to current data infrastructures as terabytes to petabytes of data must be processed and analyzed. Traditional computing platforms, originally designed to support model-driven applications, are unable to meet the demands of the data-intensive scientific applications. Pacific Northwest National Laboratory (PNNL) research goes beyond “traditional supercomputing” applications to address emerging problems that need scalable, real-time solutions. The outcome is new unconventional architectures for data-intensive applications specifically designed to process the deluge of scientific data, including FPGAs, multithreaded architectures and IBM's Cell.

  3. Virtual high throughput screening in new lead identification.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2011-12-01

    Drug discovery continues to be one of the greatest contemporary challenges and rational application of modelling approaches is the first important step to obtain lead compounds, which can be optimised further. Virtual high throughput screening (VHTS) is one of the efficient approaches to obtain lead structures for a given target. Strategic application of different screening filters like pharmacophore mapping, shape-based, ligand-based, molecular similarity etc., in combination with other drug design protocols provide invaluable insights in lead identification and optimization. Screening of large databases using these computational methods provides potential lead compounds, thus triggering a meaningful interplay between computations and experiments. In this review, we present a critical account on the relevance of molecular modelling approaches in general, lead optimization and virtual screening methods in particular for new lead identification. The importance of developing reliable scoring functions for non-bonded interactions has been highlighted, as it is an extremely important measure for the reliability of scoring function. The lead optimization and new lead design has also been illustrated with examples. The importance of employing a combination of general and target specific screening protocols has also been highlighted. PMID:21843146

  4. 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.

  5. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening

    PubMed Central

    Friedmann, Michael P.; Torbeev, Vladimir; Zelenay, Viviane; Sobol, Alexander; Greenwald, Jason; Riek, Roland

    2015-01-01

    Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications. PMID:26650386

  6. High Throughput Profiling of Molecular Shapes in Crystals

    PubMed Central

    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

  7. 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

  8. Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses▿

    PubMed Central

    Dacheux, Laurent; Berthet, Nicolas; Dissard, Gabriel; Holmes, Edward C.; Delmas, Olivier; Larrous, Florence; Guigon, Ghislaine; Dickinson, Philip; Faye, Ousmane; Sall, Amadou A.; Old, Iain G.; Kong, Katherine; Kennedy, Giulia C.; Manuguerra, Jean-Claude; Cole, Stewart T.; Caro, Valérie; Gessain, Antoine; Bourhy, Hervé

    2010-01-01

    The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world. PMID:20610710

  9. Microarray-Based Phospho-Proteomic Profiling of Complex Biological Systems12

    PubMed Central

    Goodwin, C. Rory; Woodard, Crystal L.; Zhou, Xin; Pan, Jianbo; Olivi, Alessandro; Xia, Shuli; Bettegowda, Chetan; Sciubba, Daniel M.; Pevsner, Jonathan; Zhu, Heng; Laterra, John

    2016-01-01

    Protein microarray technology has been successfully used for identifying substrates of purified activated kinases. We used protein microarrays to globally interrogate the effects of PTEN and Akt activity on the phospho-kinome of in vitro and in vivo glioma models and validated results in clinical pathological specimens. Whole cell lysates extracted from tumor samples can be applied to human kinome chip microarrays to profile the global kinase phosphorylation patterns in a high-throughput manner and identify novel substrates inherent to the tumor cell and the interactions with tumor microenvironment. Our findings identify a novel microarray-based method for assessing intracellular signaling events applicable to human oncogenesis and other pathophysiologic states. PMID:27084428

  10. Microarray-Based Phospho-Proteomic Profiling of Complex Biological Systems.

    PubMed

    Goodwin, C Rory; Woodard, Crystal L; Zhou, Xin; Pan, Jianbo; Olivi, Alessandro; Xia, Shuli; Bettegowda, Chetan; Sciubba, Daniel M; Pevsner, Jonathan; Zhu, Heng; Laterra, John

    2016-04-01

    Protein microarray technology has been successfully used for identifying substrates of purified activated kinases. We used protein microarrays to globally interrogate the effects of PTEN and Akt activity on the phospho-kinome of in vitro and in vivo glioma models and validated results in clinical pathological specimens. Whole cell lysates extracted from tumor samples can be applied to human kinome chip microarrays to profile the global kinase phosphorylation patterns in a high-throughput manner and identify novel substrates inherent to the tumor cell and the interactions with tumor microenvironment. Our findings identify a novel microarray-based method for assessing intracellular signaling events applicable to human oncogenesis and other pathophysiologic states. PMID:27084428

  11. Application of a high-throughput analyzer in evaluating solid adsorbents for post-combustion carbon capture via multicomponent adsorption of CO2, N2, and H2O.

    PubMed

    Mason, Jarad A; McDonald, Thomas M; Bae, Tae-Hyun; Bachman, Jonathan E; Sumida, Kenji; Dutton, Justin J; Kaye, Steven S; Long, Jeffrey R

    2015-04-15

    Despite the large number of metal-organic frameworks that have been studied in the context of post-combustion carbon capture, adsorption equilibria of gas mixtures including CO2, N2, and H2O, which are the three biggest components of the flue gas emanating from a coal- or natural gas-fired power plant, have never been reported. Here, we disclose the design and validation of a high-throughput multicomponent adsorption instrument that can measure equilibrium adsorption isotherms for mixtures of gases at conditions that are representative of an actual flue gas from a power plant. This instrument is used to study 15 different metal-organic frameworks, zeolites, mesoporous silicas, and activated carbons representative of the broad range of solid adsorbents that have received attention for CO2 capture. While the multicomponent results presented in this work provide many interesting fundamental insights, only adsorbents functionalized with alkylamines are shown to have any significant CO2 capacity in the presence of N2 and H2O at equilibrium partial pressures similar to those expected in a carbon capture process. Most significantly, the amine-appended metal organic framework mmen-Mg2(dobpdc) (mmen = N,N'-dimethylethylenediamine, dobpdc (4-) = 4,4'-dioxido-3,3'-biphenyldicarboxylate) exhibits a record CO2 capacity of 4.2 ± 0.2 mmol/g (16 wt %) at 0.1 bar and 40 °C in the presence of a high partial pressure of H2O.

  12. Application of a High-Throughput Analyzer in Evaluating Solid Adsorbents for Post-Combustion Carbon Capture via Multicomponent Adsorption of CO2, N-2, and H2O

    SciTech Connect

    Mason, JA; McDonald, TM; Bae, TH; Bachman, JE; Sumida, K; Dutton, JJ; Kaye, SS; Long, JR

    2015-04-15

    Despite the large number of metal-organic frameworks that have been studied in the context of post-combustion carbon capture, adsorption equilibria of gas mixtures including CO2, N-2, and H2O, which are the three biggest components of the flue gas emanating from a coal- or natural gas-fired power plant, have never been reported. Here, we disclose the design and validation of a high-throughput multicomponent adsorption instrument that can measure equilibrium adsorption isotherms for mixtures of gases at conditions that are representative of an actual flue gas from a power plant. This instrument is used to study 15 different metal-organic frameworks, zeolites, mesoporous silicas, and activated carbons representative of the broad range of solid adsorbents that have received attention for CO2 capture. While the multicomponent results presented in this work provide many interesting fundamental insights, only adsorbents functionalized with alkylamines are shown to have any significant CO2 capacity in the presence of N-2 and H2O at equilibrium partial pressures similar to those expected in a carbon capture process. Most significantly, the amine-appended metal organic framework mmen-Mg-2(dobpdc) (mmen = N,N'-dimethylethylenediamine, dobpdc (4-) = 4,4'-dioxido-3,3'-biphenyldicarboxylate) exhibits a record CO2 capacity of 4.2 +/- 0.2 mmol/g (16 wt %) at 0.1 bar and 40 degrees C in the presence of a high partial pressure of H2O.

  13. Application of a high-throughput analyzer in evaluating solid adsorbents for post-combustion carbon capture via multicomponent adsorption of CO2, N2, and H2O.

    PubMed

    Mason, Jarad A; McDonald, Thomas M; Bae, Tae-Hyun; Bachman, Jonathan E; Sumida, Kenji; Dutton, Justin J; Kaye, Steven S; Long, Jeffrey R

    2015-04-15

    Despite the large number of metal-organic frameworks that have been studied in the context of post-combustion carbon capture, adsorption equilibria of gas mixtures including CO2, N2, and H2O, which are the three biggest components of the flue gas emanating from a coal- or natural gas-fired power plant, have never been reported. Here, we disclose the design and validation of a high-throughput multicomponent adsorption instrument that can measure equilibrium adsorption isotherms for mixtures of gases at conditions that are representative of an actual flue gas from a power plant. This instrument is used to study 15 different metal-organic frameworks, zeolites, mesoporous silicas, and activated carbons representative of the broad range of solid adsorbents that have received attention for CO2 capture. While the multicomponent results presented in this work provide many interesting fundamental insights, only adsorbents functionalized with alkylamines are shown to have any significant CO2 capacity in the presence of N2 and H2O at equilibrium partial pressures similar to those expected in a carbon capture process. Most significantly, the amine-appended metal organic framework mmen-Mg2(dobpdc) (mmen = N,N'-dimethylethylenediamine, dobpdc (4-) = 4,4'-dioxido-3,3'-biphenyldicarboxylate) exhibits a record CO2 capacity of 4.2 ± 0.2 mmol/g (16 wt %) at 0.1 bar and 40 °C in the presence of a high partial pressure of H2O. PMID:25844924

  14. A novel high-throughput irradiator for in vitro radiation sensitivity bioassays

    NASA Astrophysics Data System (ADS)

    Fowler, Tyler L.

    Given the emphasis on more personalized radiation therapy there is an ongoing and compelling need to develop high-throughput screening tools to further examine the biological effects of ionizing radiation on cells, tissues and organ systems in either the research or clinical setting. Conventional x-ray irradiators are designed to provide maximum versatility to radiobiology researchers, typically accommodating small animals, tissue or blood samples, and cellular applications. This added versatility often impedes the overall sensitivity and specificity of an experiment resulting in a trade-off between the number of absorbed doses (or dose rates) and biological endpoints that can be investigated in vitro in a reasonable amount of time. Therefore, modern irradiator designs are incompatible with current high-throughput bioassay technologies. Furthermore, important dosimetry and calibration characteristics (i.e. dose build-up region, beam attenuation, and beam scatter) of these irradiators are typically unknown to the end user, which can lead to significant deviation between delivered dose and intended dose to cells that adversely impact experimental results. Therefore, the overarching goal of this research is to design and develop a robust and fully automated high-throughput irradiator for in vitro radiation sensitivity investigations. Additionally, in vitro biological validation of this system was performed by assessing intracellular reactive oxygen species production, physical DNA double strand breaks, and activation of cellular DNA repair mechanisms. Finally, the high-throughput irradiator was used to investigate autophagic flux, a cellular adaptive response, as a potential biomarker of radiation sensitivity.

  15. A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridium acetobutylicum.

    PubMed

    Paredes, Carlos J; Senger, Ryan S; Spath, Iwona S; Borden, Jacob R; Sillers, Ryan; Papoutsakis, Eleftherios T

    2007-07-01

    While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism.

  16. 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

  17. Multiplexing a high-throughput liability assay to leverage efficiencies.

    PubMed

    Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

    2009-06-01

    In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

  18. Achieving High Throughput for Data Transfer over ATM Networks

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory J.; Townsend, Jeffrey N.

    1996-01-01

    File-transfer rates for ftp are often reported to be relatively slow, compared to the raw bandwidth available in emerging gigabit networks. While a major bottleneck is disk I/O, protocol issues impact performance as well. Ftp was developed and optimized for use over the TCP/IP protocol stack of the Internet. However, TCP has been shown to run inefficiently over ATM. In an effort to maximize network throughput, data-transfer protocols can be developed to run over UDP or directly over IP, rather than over TCP. If error-free transmission is required, techniques for achieving reliable transmission can be included as part of the transfer protocol. However, selected image-processing applications can tolerate a low level of errors in images that are transmitted over a network. In this paper we report on experimental work to develop a high-throughput protocol for unreliable data transfer over ATM networks. We attempt to maximize throughput by keeping the communications pipe full, but still keep packet loss under five percent. We use the Bay Area Gigabit Network Testbed as our experimental platform.

  19. High-Throughput Preparation of New Photoactive Nanocomposites.

    PubMed

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

    2016-06-01

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

  20. High-throughput measurements of hydrogel tissue construct mechanics.

    PubMed

    Marquez, Juan Pablo; Legant, Wesley; Lam, Vy; Cayemberg, Amy; Elson, Elliot; Wakatsuki, Tetsuro

    2009-06-01

    Engineered tissues represent a natural environment for studying cell physiology, mechanics, and function. Cellular interactions with the extracellular matrix proteins are important determinants of cell physiology and tissue mechanics. Dysregulation of these parameters can result in diseases such as cardiac fibrosis and atherosclerosis. In this report we present a novel system to produce hydrogel tissue constructs (HTCs) and to characterize their mechanical properties. HTCs are grown in custom chambers and a robotic system is used to indent them and measure the resulting forces. Force measurements are then used to estimate HTC pretension (cellular contractility). Pretension was reduced in a dose-dependent manner by cytochalasin D (CD) treatment; the highest concentration (2microM) resulted in 10-fold decrease. On the other hand, treatment with fetal bovine serum (20%) resulted in approximately threefold increase in pretension. Excellent repeatability and precision were observed in measurements from replicate HTCs. The coefficient of statistical variance of quantified pretension ranged from 7% to 15% (n=4). Due to the small size (4x4x0.8mm) of the HTCs, this system of profiling HTC mechanics can readily be used in high-throughput applications. In particular, it can be used for screening chemical libraries in search of drugs that can alter tissue mechanics.

  1. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles. PMID:25184988

  2. High-throughput protein crystallography and drug discovery.

    PubMed

    Tickle, Ian; Sharff, Andrew; Vinkovic, Mladen; Yon, Jeff; Jhoti, Harren

    2004-10-20

    Single crystal X-ray diffraction is the technique of choice for studying the interactions of small organic molecules with proteins by determining their three-dimensional structures; however the requirement for highly purified protein and lack of process automation have traditionally limited its use in this field. Despite these shortcomings, the use of crystal structures of therapeutically relevant drug targets in pharmaceutical research has increased significantly over the last decade. The application of structure-based drug design has resulted in several marketed drugs and is now an established discipline in most pharmaceutical companies. Furthermore, the recently published full genome sequences of Homo sapiens and a number of micro-organisms have provided a plethora of new potential drug targets that could be utilised in structure-based drug design programs. In order to take maximum advantage of this explosion of information, techniques have been developed to automate and speed up the various procedures required to obtain protein crystals of suitable quality, to collect and process the raw X-ray diffraction data into usable structural information, and to use three-dimensional protein structure as a basis for drug discovery and lead optimisation. This tutorial review covers the various technologies involved in the process pipeline for high-throughput protein crystallography as it is currently being applied to drug discovery. It is aimed at synthetic and computational chemists, as well as structural biologists, in both academia and industry, who are interested in structure-based drug design.

  3. Measuring intracellular calcium fluxes in high throughput mode.

    PubMed

    Chambers, Chris; Smith, Fiona; Williams, Christine; Marcos, Sandra; Liu, Zhen Han; Hayter, Paul; Ciaramella, Giuseppe; Keighley, Wilma; Gribbon, Phil; Sewing, Andreas

    2003-06-01

    The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.

  4. High-throughput screening in the diagnostics industry.

    PubMed

    Wilson, S; Howell, S

    2002-08-01

    The diagnostics industry is constantly under pressure to bring innovation quicker to market and so the impetus to speed up product-development cycle times becomes greater. There are a number of steps in the product-development cycle where the application of high-throughput screening can help. In the case of lateral-flow immuno-diagnostics the selection of antibody reagents is paramount. In particular, rapid identification of antibody pairs that are able to 'sandwich' around the target antigen is required. One screen that has been applied successfully is the use of surface plasmon resonance biosensors like Biacore. Using such a system one can evaluate over 400 antibody pairings in under 5 days. Conventional approaches to screen this number of antibody pairs would take many months. Other automated screening systems like DELFIA can be used in processing the vast amount of tests required for clinical trials. In addition, the use of robotics to automate routine product testing can be used to shorten the product-development cycle.

  5. High-Throughput Preparation of New Photoactive Nanocomposites.

    PubMed

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

    2016-06-01

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

  6. A High-Throughput Screen for Alpha Particle Radiation Protectants

    PubMed Central

    Seideman, Jonathan H.; Shum, David; Djaballah, Hakim

    2010-01-01

    Abstract Alpha-particle-emitting elements are of increasing importance as environmental and occupational carcinogens, toxic components of radiation dispersal devices and accidents, and potent therapeutics in oncology. Alpha particle radiation differs from radiations of lower linear energy transfer in that it predominantly damages DNA via direct action. Because of this, radical scavengers effective for other radiations have had only limited effect in mitigating alpha particle toxicity. We describe here a simple assay and a pilot screen of 3,119 compounds in a high-throughput screen (HTS), using the alpha-particle-emitting isotope, 225Ac, for the discovery of compounds that might protect mammalian cells from alpha particles through novel mechanisms. The assay, which monitored the viability of a myeloid leukemic cell line upon alpha particle exposure, was robust and reproducible, yielding a Z' factor of 0.66 and a signal-to-noise ratio of nearly 10 to 1. Surprisingly, 1 compound emerged from this screen, epoxy-4,5-α-dihydroxysantonin (EDHS), that showed considerable protective activity. While the value of EDHS remains to be determined, its discovery is a proof of concept and validation of the utility of this HTS methodology. Further application of the described assay could yield compounds useful in minimizing the toxicity and carcinogenesis associated with alpha particle exposure. PMID:20658946

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

    PubMed

    Trubetskoy, Olga; Finel, Moshe; Trubetskoy, Vladimir

    2008-08-01

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

  8. Analysis of DNA Sequence Variants Detected by High Throughput Sequencing

    PubMed Central

    Adams, David R; Sincan, Murat; Fajardo, Karin Fuentes; Mullikin, James C; Pierson, Tyler M; Toro, Camilo; Boerkoel, Cornelius F; Tifft, Cynthia J; Gahl, William A; Markello, Tom C

    2014-01-01

    The Undiagnosed Diseases Program at the National Institutes of Health uses High Throughput Sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The paper is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects. PMID:22290882

  9. The JCSG high-throughput structural biology pipeline.

    PubMed

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

    2010-10-01

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

  10. Multivariate curve resolution for hyperspectral image analysis :applications to microarray technology.

    SciTech Connect

    Van Benthem, Mark Hilary; Sinclair, Michael B.; Haaland, David Michael; Martinez, M. Juanita (University of New Mexico, Albuquerque, NM); Timlin, Jerilyn Ann; Werner-Washburne, Margaret C. (University of New Mexico, Albuquerque, NM); Aragon, Anthony D. (University of New Mexico, Albuquerque, NM)

    2003-01-01

    Multivariate curve resolution (MCR) using constrained alternating least squares algorithms represents a powerful analysis capability for a quantitative analysis of hyperspectral image data. We will demonstrate the application of MCR using data from a new hyperspectral fluorescence imaging microarray scanner for monitoring gene expression in cells from thousands of genes on the array. The new scanner collects the entire fluorescence spectrum from each pixel of the scanned microarray. Application of MCR with nonnegativity and equality constraints reveals several sources of undesired fluorescence that emit in the same wavelength range as the reporter fluorphores. MCR analysis of the hyperspectral images confirms that one of the sources of fluorescence is due to contaminant fluorescence under the printed DNA spots that is spot localized. Thus, traditional background subtraction methods used with data collected from the current commercial microarray scanners will lead to errors in determining the relative expression of low-expressed genes. With the new scanner and MCR analysis, we generate relative concentration maps of the background, impurity, and fluorescent labels over the entire image. Since the concentration maps of the fluorescent labels are relatively unaffected by the presence of background and impurity emissions, the accuracy and useful dynamic range of the gene expression data are both greatly improved over those obtained by commercial microarray scanners.

  11. Combinatorial and high-throughput screening approaches for strain engineering.

    PubMed

    Liu, Wenshan; Jiang, Rongrong

    2015-03-01

    Microbes have long been used in the industry to produce valuable biochemicals. Combinatorial engineering approaches, new strain engineering tools derived from inverse metabolic engineering, have started to attract attention in recent years, including genome shuffling, error-prone DNA polymerase, global transcription machinery engineering (gTME), random knockout/overexpression libraries, ribosome engineering, multiplex automated genome engineering (MAGE), customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER), and library construction of "tunable intergenic regions" (TIGR). Since combinatorial approaches and high-throughput screening methods are fundamentally interconnected, color/fluorescence-based, growth-based, and biosensor-based high-throughput screening methods have been reviewed. We believe that with the help of metabolic engineering tools and new combinatorial approaches, plus effective high-throughput screening methods, researchers will be able to achieve better results on improving microorganism performance under stress or enhancing biochemical yield.

  12. High throughput combinatorial screening of semiconductor materials

    NASA Astrophysics Data System (ADS)

    Mao, Samuel S.

    2011-11-01

    This article provides an overview of an advanced combinatorial material discovery platform developed recently for screening semiconductor materials with properties that may have applications ranging from radiation detectors to solar cells. Semiconductor thin-film libraries, each consisting of 256 materials of different composition arranged into a 16×16 matrix, were fabricated using laser-assisted evaporation process along with a combinatorial mechanism to achieve variations. The composition and microstructure of individual materials on each thin-film library were characterized with an integrated scanning micro-beam x-ray fluorescence and diffraction system, while the band gaps were determined by scanning optical reflection and transmission of the libraries. An ultrafast ultraviolet photon-induced charge probe was devised to measure the mobility and lifetime of individual thin-film materials on semiconductor libraries. Selected results on the discovery of semiconductors with desired band gaps and transport properties are illustrated.

  13. High-throughput sequencing and vaccine design.

    PubMed

    Luciani, F

    2016-04-01

    Next-generation sequencing (NGS) technologies have reshaped genome research. The resulting increase in sequencing depth and resolution has led to an unprecedented level of genomic detail and thus an increasing awareness of the complexity of animal, human and pathogen genomes. This has resulted in new approaches to vaccine research. On the one hand, the increase in genome complexity challenges our ability to study and understand pathogen biology and pathogen-host interactions. On the other hand, the increase in genomic data also provides key information for developing and designing improved vaccines against pathogens that were previously extremely difficult to deal with, such as rapidly mutating RNA viruses or bacteria that have complex interactions with the host immune system. This review describes how the broad application of NGS technologies to genome research is affecting vaccine research. It focuses on implications for the field of viral genomics, and includes recent animal and human studies.

  14. Qualifying high-throughput immune repertoire sequencing.

    PubMed

    Niklas, Norbert; Pröll, Johannes; Weinberger, Johannes; Zopf, Agnes; Wiesinger, Karin; Krismer, Konstantin; Bettelheim, Peter; Gabriel, Christian

    2014-01-01

    Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell's gene composition and variation makes deep analysis of one individual's immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform. PMID:24607567

  15. Insights to transcriptional networks by using high throughput RNAi strategies

    PubMed Central

    Mattila, Jaakko

    2010-01-01

    RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity. PMID:19952073

  16. 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

  17. CHALLENGES IN SECONDARY ANALYSIS OF HIGH THROUGHPUT SCREENING DATA

    PubMed Central

    BLUCHER, AURORA S.; MCWEENEY, SHANNON K.

    2014-01-01

    Repurposing an existing drug for an alternative use is not only a cost effective method of development, but also a faster process due to the drug's previous clinical testing and established pharmokinetic profiles. A potentially rich resource for computational drug repositioning approaches is publically available high throughput screening data, available in databases such as PubChem Bioassay and ChemBank. We examine statistical and computational considerations for secondary analysis of publicly available high throughput screening (HTS) data with respect to metadata, data quality, and completeness. We discuss developing methods and best practices that can help to ameliorate these issues. PMID:24297539

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

    PubMed

    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.

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

    PubMed

    Morgan, R E; Westwood, N J

    2004-01-01

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

  20. The synergy between combinatorial chemistry and high-throughput screening.

    PubMed

    Diller, David J

    2008-05-01

    Despite the initial promise of combinatorial chemistry, particularly large library combinatorial chemistry, to greatly accelerate drug discovery, this approach has not been fully utilized as a means to build the compound collections of pharmaceutical and biotechnology companies. This review highlights some of the strengths of large library combinatorial chemistry as a means of generating molecules for lead discovery, such as providing rich and robust structure-activity relationships around each hit series. The challenges and concepts emerging from traditional high-throughput screening and fragment-based drug design, how these methods influence the design of large combinatorial libraries and the interpretation of the ensuing high-throughput screening data are also highlighted.

  1. 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

  2. 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.

  3. 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

  4. High-throughput proteomics : optical approaches.

    SciTech Connect

    Davidson, George S.

    2008-09-01

    Realistic cell models could greatly accelerate our ability to engineer biochemical pathways and the production of valuable organic products, which would be of great use in the development of biofuels, pharmaceuticals, and the crops for the next green revolution. However, this level of engineering will require a great deal more knowledge about the mechanisms of life than is currently available. In particular, we need to understand the interactome (which proteins interact) as it is situated in the three dimensional geometry of the cell (i.e., a situated interactome), and the regulation/dynamics of these interactions. Methods for optical proteomics have become available that allow the monitoring and even disruption/control of interacting proteins in living cells. Here, a range of these methods is reviewed with respect to their role in elucidating the interactome and the relevant spatial localizations. Development of these technologies and their integration into the core competencies of research organizations can position whole institutions and teams of researchers to lead in both the fundamental science and the engineering applications of cellular biology. That leadership could be particularly important with respect to problems of national urgency centered around security, biofuels, and healthcare.

  5. Importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Szaniszlo, Peter; Prow, Tarl W.; Reece, Lisa M.; Wang, Nan; Asmuth, David M.

    2002-06-01

    Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. High-throughput cell separation technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using microamplification PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to

  6. Application of DNA microarray technology in determining breast cancer prognosis and therapeutic response.

    PubMed

    Brennan, Donal J; O'Brien, Sallyann L; Fagan, Ailís; Culhane, Aedín C; Higgins, Desmond G; Duffy, Michael J; Gallagher, William M

    2005-08-01

    There are > 1.15 million cases of breast cancer diagnosed worldwide annually, and it is the second leading cause of cancer death in the European Union. The optimum management of patients with breast cancer requires accurate prognostic and predictive factors. At present, only a small number of such factors are used clinically. DNA microarrays have the potential to measure the expression of tens of thousands of genes simultaneously. Recent preliminary findings suggest that DNA microarray-based gene expression profiling can provide powerful and independent prognostic information in patients with newly diagnosed breast cancer. As well as providing prognostic information, emerging results suggest that DNA microarrays can also be used for predicting response or resistance to treatment, especially to neoadjuvant chemotherapy. Prior to clinical application, these preliminary findings must be validated using large-scale prospective studies. This article reviews these advances and also examines the role of DNA microarrays in reducing the number of patients who receive inappropriate chemotherapy. The most recent data supporting the integration of various publicly available data sets is also reviewed in detail.

  7. High-throughput engineering and analysis of peptide binding to class II MHC.

    PubMed

    Jiang, Wei; Boder, Eric T

    2010-07-27

    Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.

  8. High-Throughput and Combinatorial Gene Expression on a Chip for Metabolism-Induced Toxicology Screening

    PubMed Central

    Kwon, Seok Joon; Lee, Dong Woo; Shah, Dhiral A.; Ku, Bosung; Jeon, Sang Youl; Solanki, Kusum; Ryan, Jessica D.; Clark, Douglas S.; Dordick, Jonathan S.; Lee, Moo-Yeal

    2014-01-01

    Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner. PMID:24799042

  9. A High-throughput O-Glycopeptide Discovery Platform for Seromic Profiling

    PubMed Central

    Blixt, Ola; Cló, Emiliano; Nudelman, Aaron S.; Sørensen, Kasper Kildegaard; Clausen, Thomas; Wandall, Hans H.; Livingston, Philip O.; Clausen, Henrik; Jensen, Knud J.

    2010-01-01

    Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introducing a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products were achieved on an amine-reactive hydrogel coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in-situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine induced disease free breast cancer patients and in patients, with confirmed disease at time of diagnosis. PMID:20726594

  10. glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data.

    PubMed

    Hutchins, Andrew Paul; Jauch, Ralf; Dyla, Mateusz; Miranda-Saavedra, Diego

    2014-01-01

    Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data), and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.

  11. Perspectives of DNA microarray and next-generation DNA sequencing technologies.

    PubMed

    Teng, XiaoKun; Xiao, HuaSheng

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  12. Bioinformatics and Microarray Data Analysis on the Cloud.

    PubMed

    Calabrese, Barbara; Cannataro, Mario

    2016-01-01

    High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data. PMID:25863787

  13. Bioinformatics and Microarray Data Analysis on the Cloud.

    PubMed

    Calabrese, Barbara; Cannataro, Mario

    2016-01-01

    High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data.

  14. 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...

  15. 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...

  16. 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...

  17. High-throughput glycoanalytical technology for systems glycobiology.

    PubMed

    Liu, Li; Telford, Jayne E; Knezevic, Ana; Rudd, Pauline M

    2010-10-01

    The development of glycoanalytical HPLC-based high-throughput technology has greatly enhanced the study of glycobiology, facilitating the discovery of disease-related solutions and providing an informative view of glycosylation and its relationship with other biological disciplines in a systems biology approach.

  18. 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...

  19. 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...

  20. High-throughput production of two disulphide-bridge toxins.

    PubMed

    Upert, Grégory; Mourier, Gilles; Pastor, Alexandra; Verdenaud, Marion; Alili, Doria; Servent, Denis; Gilles, Nicolas

    2014-08-01

    A quick and efficient production method compatible with high-throughput screening was developed using 36 toxins belonging to four different families of two disulphide-bridge toxins. Final toxins were characterized using HPLC co-elution, CD and pharmacological studies.

  1. High-Throughput Sequencing and Rare Genetic Diseases

    PubMed Central

    Makrythanasis, P.; Antonarakis, S.E.

    2012-01-01

    High-throughput sequencing has drastically changed the research of genes responsible for genetic disorders and is now gradually introduced as an additional genetic diagnostic testing in clinical practice. The current debates on the emerging technical, medical and ethical issues as well as the potential optimum use of the available technology are discussed. PMID:23293577

  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. 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...

  4. Towards high throughput screening of electrochemical stability of battery electrolytes.

    PubMed

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen. PMID:26266636

  5. High-throughput DNA extraction of forensic adhesive tapes.

    PubMed

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. PMID:27448236

  6. Towards high throughput screening of electrochemical stability of battery electrolytes

    NASA Astrophysics Data System (ADS)

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E.; Leiter, Kenneth W.; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen.

  7. High-throughput DNA extraction of forensic adhesive tapes.

    PubMed

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples.

  8. Commercially available high-throughput Dip Pen Nanolithography

    NASA Astrophysics Data System (ADS)

    Haaheim, J. R.; Tevaarwerk, E. R.; Fragala, J.; Shile, R.

    2008-04-01

    Dip Pen Nanolithography ® (DPN ®) is an inherently additive SPM-based technique which operates under ambient conditions, making it suitable to deposit a wide range of biological and inorganic materials. Massively parallel two-dimensional nanopatterning with DPN is now commercially available via NanoInk's 2D nano PrintArray TM, making DPN a high-throughput, flexible and versatile method for precision nanoscale pattern formation. By fabricating 55,000 tip-cantilevers across a 1 cm2 chip, we leverage the inherent versatility of DPN and demonstrate large area surface coverage, routinely achieving throughputs of 3x10 7 μm2 per hour. Further, we have engineered the device to be easy to use, wire-free, and fully integrated with the NSCRIPTOR's scanner, stage, and sophisticated lithography routines. In this talk we discuss the methods of operating this commercially available device, subsequent results showing sub-100 nm feature sizes and excellent uniformity (standard deviation < 16%), and our continuing development work. Simultaneous multiplexed deposition of a variety of molecules is a fundamental goal of massively parallel 2D nanopatterning, and we will discuss our progress on this front, including ink delivery methods, tip coating, and patterning techniques to generate combinatorial libraries of nanoscale patterns. Another fundamental challenge includes planar leveling of the 2D nano PrintArray, and herein we describe our successful implementation of device viewports and integrated software leveling routines that monitor cantilever deflection to achieve planarity and uniform surface contact. Finally, we will discuss the results of 2D nanopatterning applications such as: 1) rapidly and flexibly generating nanostructures; 2) chemically directed assembly and 3) directly writing biological materials.

  9. Towards high throughput screening of electrochemical stability of battery electrolytes.

    PubMed

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen.

  10. High-throughput neuroimaging-genetics computational infrastructure

    PubMed Central

    Dinov, Ivo D.; Petrosyan, Petros; Liu, Zhizhong; Eggert, Paul; Hobel, Sam; Vespa, Paul; Woo Moon, Seok; Van Horn, John D.; Franco, Joseph; Toga, Arthur W.

    2014-01-01

    Many contemporary neuroscientific investigations face significant challenges in terms of data management, computational processing, data mining, and results interpretation. These four pillars define the core infrastructure necessary to plan, organize, orchestrate, validate, and disseminate novel scientific methods, computational resources, and translational healthcare findings. Data management includes protocols for data acquisition, archival, query, transfer, retrieval, and aggregation. Computational processing involves the necessary software, hardware, and networking infrastructure required to handle large amounts of heterogeneous neuroimaging, genetics, clinical, and phenotypic data and meta-data. Data mining refers to the process of automatically extracting data features, characteristics and associations, which are not readily visible by human exploration of the raw dataset. Result interpretation includes scientific visualization, community validation of findings and reproducible findings. In this manuscript we describe the novel high-throughput neuroimaging-genetics computational infrastructure available at the Institute for Neuroimaging and Informatics (INI) and the Laboratory of Neuro Imaging (LONI) at University of Southern California (USC). INI and LONI include ultra-high-field and standard-field MRI brain scanners along with an imaging-genetics database for storing the complete provenance of the raw and derived data and meta-data. In addition, the institute provides a large number of software tools for image and shape analysis, mathematical modeling, genomic sequence processing, and scientific visualization. A unique feature of this architecture is the Pipeline environment, which integrates the data management, processing, transfer, and visualization. Through its client-server architecture, the Pipeline environment provides a graphical user interface for designing, executing, monitoring validating, and disseminating of complex protocols that utilize

  11. Potentiometric sensor for the high throughput determination of tetramisole hydrochloride.

    PubMed

    Gupta, Vinod Kumar; Singh, Ashok Kumar; Gupta, Barkha

    2007-08-01

    The electrochemical response characteristics of poly(vinyl)chloride (PVC) based membrane sensors for determination of tetramisole hydrochloride (TmCl) is described. The membranes of these electrodes consist of tetramisole-tetraphenyl borate (Tm-TPB), chlorophenyl borate (Tm-ClPB), and phosphotungstate (Tm(3)-PT) ion associations dispersed in a PVC matrix with dibutylpthalate as a plasticizer. The electrodes were fully characterized in terms of composition, life span, usable pH range, and working concentration range and ionic strength. The electrodes showed Nernstian response over the concentration ranges of 7.4 x 10(-7) to 1.0 x 10(-2) M, 1.7 x 10(-6) to 1.0 x 10(-2) M, and 5.6 x 10(-6) to 1.0 x 10(-2) M TmCl, respectively, and were applied to the potentiometric determination of tetramisole ion in pure solutions and pharmaceutical preparations. The potentiometric determination was also used in the determination of tetramisole in pharmaceutical preparations in four batches of different expiration dates. The electrodes exhibited good selectivity for TmCl with respect to a large number of excipients such as inorganic cations, organic cations, amino acids, and sugars. The solubility product of the ion-pair and the formation constant of the precipitation reaction leading to the ion-pair formation were determined conductometrically. The new potentiometric method offers the advantages of high-throughput determination, simplicity, accuracy, automation feasibility, and applicability to turbid and colored sample solutions. PMID:17979641

  12. A bead-based western for high-throughput cellular signal transduction analyses

    PubMed Central

    Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

    2016-01-01

    Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

  13. Identifying Fishes through DNA Barcodes and Microarrays

    PubMed Central

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

    Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. PMID

  14. Cell-based microarrays: current progress, future prospects.

    PubMed

    Palmer, Ella; Freeman, Tom

    2005-07-01

    Cell-based microarrays were first described by Ziauddin and Sabatini in 2001 as a novel method for performing high-throughput screens of gene function. In this study, expression vectors containing the open reading frame of human genes were printed onto glass microscope slides to form a microarray. Transfection reagents were added pre- or post-spotting, and cells grown over the surface of the array. They demonstrated that cells growing in the immediate vicinity of the expression vectors underwent 'reverse transfection', and that subsequent alterations in cell function could then be detected by secondary assays performed on the array. Subsequent publications have adapted the technique to a variety of applications, and have also shown that the approach works when arrays are fabricated using short interfering RNAs and compounds. The potential of this method for performing analyses of gene function and for identifying novel therapeutic agents has been clearly demonstrated, and current efforts are focused on improving and harnessing this technology for high-throughput screening applications. PMID:16014002

  15. Microarrays, Integrated Analytical Systems

    NASA Astrophysics Data System (ADS)

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  16. High-Throughput Metagenomic Technologies for Complex Microbial Community Analysis: Open and Closed Formats

    PubMed Central

    He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-01

    ABSTRACT   Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. PMID:25626903

  17. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

    PubMed Central

    Colot, Hildur V.; Park, Gyungsoon; Turner, Gloria E.; Ringelberg, Carol; Crew, Christopher M.; Litvinkova, Liubov; Weiss, Richard L.; Borkovich, Katherine A.; Dunlap, Jay C.

    2006-01-01

    The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination. PMID:16801547

  18. High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

    SciTech Connect

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G.; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied “open-format” and “closed-format” detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  19. High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry.

    PubMed

    Jagannadh, Veerendra Kalyan; Bhat, Bindu Prabhath; Nirupa Julius, Lourdes Albina; Gorthi, Sai Siva

    2016-03-01

    In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per μl) obtained from our instrument, with that of a commercially available hematology analyzer. PMID:26781098

  20. High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

    PubMed

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions.

  1. High-throughput methods for miniaturization and automation of monoclonal antibody purification processes.

    PubMed

    Treier, Katrin; Hansen, Sigrid; Richter, Carolin; Diederich, Patrick; Hubbuch, Jürgen; Lester, Philip

    2012-01-01

    In the last decade, high-throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high-throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab-scale and miniaturized afterwards. The scale-down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96-well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.

  2. High-throughput investigation of catalysts for JP-8 fuel cracking to liquefied petroleum gas.

    PubMed

    Bedenbaugh, John E; Kim, Sungtak; Sasmaz, Erdem; Lauterbach, Jochen

    2013-09-01

    Portable power technologies for military applications necessitate the production of fuels similar to LPG from existing feedstocks. Catalytic cracking of military jet fuel to form a mixture of C₂-C₄ hydrocarbons was investigated using high-throughput experimentation. Cracking experiments were performed in a gas-phase, 16-sample high-throughput reactor. Zeolite ZSM-5 catalysts with low Si/Al ratios (≤25) demonstrated the highest production of C₂-C₄ hydrocarbons at moderate reaction temperatures (623-823 K). ZSM-5 catalysts were optimized for JP-8 cracking activity to LPG through varying reaction temperature and framework Si/Al ratio. The reducing atmosphere required during catalytic cracking resulted in coking of the catalyst and a commensurate decrease in conversion rate. Rare earth metal promoters for ZSM-5 catalysts were screened to reduce coking deactivation rates, while noble metal promoters reduced onset temperatures for coke burnoff regeneration.

  3. An integrated probabilistic approach for gene function prediction using multiple sources of high-throughput data.

    PubMed

    Zhang, Chao; Joshi, Trupti; Lin, Guan Ning; Xu, Dong

    2008-01-01

    Characterising gene function is one of the major challenging tasks in the post-genomic era. Various approaches have been developed to integrate multiple sources of high-throughput data to predict gene function. Most of those approaches are just used for research purpose and have not been implemented as publicly available tools. Even for those implemented applications, almost all of them are still web-based 'prediction servers' that have to be managed by specialists. This paper introduces a systematic method for integrating various sources of high-throughput data to predict gene function and analyse our prediction results and evaluates its performances based on the competition for mouse gene function prediction (MouseFunc). A stand-alone Java-based software package 'GeneFAS' is freely available at http://digbio. missouri.eduigenefas.

  4. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  5. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  6. Development of a Chemoenzymatic-like and Photoswitchable Method for the High-Throughput creation of Protein Microarrays. Application to the Analysis of the Protein/Protein Interactions Involved in the YOP Virulon from Yersinia pestis.

    SciTech Connect

    Camarero, J A

    2006-12-07

    Protein arrays are ideal tools for the rapid analysis of whole proteomes as well as for the development of reliable and cheap biosensors. The objective of this proposal is to develop a new ligand assisted ligation method based in the naturally occurring protein trans-splicing process. This method has been used for the generation of spatially addressable arrays of multiple protein components by standard micro-lithographic techniques. Key to our approach is the use of the protein trans-splicing process. This naturally occurring process allows the development of a truly generic and highly efficient method for the covalent attachment of proteins through its C-terminus to any solid support. This technology has been used for the creation of protein chips containing several virulence factors from the human pathogen Y. pestis.

  7. High throughput automated colorimetric method for the screening of l-lactic acid producing microorganisms.

    PubMed

    Liaud, Nadège; Navarro, David; Vidal, Nicolas; Sigoillot, Jean-Claude; Raouche, Sana

    2014-01-01

    Lactic acid is a valuable and fully degradable organic acid with promising applications in poly-lactic acid production (Taskila S and Ojamo, 2013 [1]). Despite their efficiency, the cost of the current lactic acid bio-processes is still an obstacle to this application (Miller et al., 2011 [2]). To ameliorate lactic acid producing strains, researchers are using mutations and metabolic engineering techniques, as well as medium optimization. All these studies necessitate a good and high throughput screening method. Currently, researchers mostly use HPLC methods which often necessitate sample preparation, are not stereospecific and do not allow high throughput. To help optimizing l-lactic acid production, we developed a high throughput colorimetric method inspired by the blood l-lactic acid detection method used for diagnosis (Lin et al., 1999 [3]).•Two sequential enzymatic reactions using l-lactate oxidase, peroxidase and ABTS (2,2'-azino-di-[3-ethylbenzthiazoine-sulfonate]), a chromogenic peroxidase substrate, are used to quantify l-lactate between 13.8 and 90 mg/l.•The accuracy of the method was ascertained before automation.•The method was successfully applied for the direct determination of l-lactate content in fungal culture supernatants.

  8. Combinatorial/high throughput methods for the determination of polyanhydride phase behavior.

    PubMed

    Thorstenson, Jon B; Petersen, Latrisha K; Narasimhan, Balaji

    2009-01-01

    Combinatorial methods have been developed to study the phase behavior of biodegradable polyanhydrides for drug delivery applications. The polyanhydrides of interest are poly[1,6-bis(p-carboxyphenoxy) hexane] (CPH) and poly[sebacic anhydride] (SA). Both continuous and discrete polymer blend libraries were fabricated by using a combination of solution-based gradient deposition and rapid prototyping. Blend compositions were characterized via a high throughput transmission Fourier transform infrared (FTIR) sampling technique and compared against theoretical mass balance predictions. To obtain phase diagrams of CPH/SA, the effect of blend composition and annealing temperature on the miscibility of the blend was studied. This gradient library was observed with optical microscopy in order to determine cloud points. These results were compared with a theoretical phase diagram obtained from Flory-Huggins theory and with atomic force microscopy (AFM) experiments on blend libraries and the agreement between the methods was very good. The high throughput method demonstrates that the CPH/SA system exhibits upper critical solution temperature behavior. These libraries are amenable to other high throughput applications in biomaterials science including cell viability, cell activation, and protein/biomaterial interactions.

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

    PubMed

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

    2015-10-20

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

  10. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow

    PubMed Central

    Spencer, Adrianne; Baker, Aaron B.

    2016-01-01

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis. PMID:26816215

  11. 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

  12. The use of microarrays in microbial ecology

    SciTech Connect

    Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

    2009-09-15

    Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer

  13. Quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging method optimized for analysis of multicolor microarrays.

    PubMed

    Liu, Zhiyi; Ma, Suihua; Ji, Yanhong; Liu, Le; Hu, Zhaoxu; Guo, Jihua; Ma, Hui; He, Yonghong

    2010-09-15

    The microarray technique, which can provide parallel detection with high throughput in biomedical research, has generated considerable interest since the end of the 20th century. A number of instruments have been reported for microarray detection. In this paper, we have developed a quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging system for multicolor microarray research. Hyperspectral imaging records the entire emission spectrum for every voxel within the imaged area in contrast to recording only fluorescence intensities of filter-based scanners. When coupled with data analysis, the recorded spectral information allows for quantitative identification of the contributions of multiple, spectrally overlapping fluorescent dyes and elimination of unwanted artifacts. This system is improved with a specifically designed, high performance spectrometer which can offer a spectral resolution of 0.2 nm and operates with spatial resolutions ranging from 2 to 30 μm. We demonstrate the application of the system by reading out arrays for identification of bacteria. PMID:20718427

  14. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Li, Wei; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Ruan, Kangcheng; Yang, Guozhen

    2010-09-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml - 1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody-antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays.

  15. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-06

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

  16. An advanced application of protein microarrays: cell-based assays for functional genomics.

    PubMed

    Carbone, Roberta

    2009-01-01

    Microarrays have become common tools for approaching different experimental questions: DNA, protein and peptide arrays offer the power of multiplexing the assay and by means of miniaturization technology, the possibility to reduce cost and amount of samples and reagents. Recently, a novel technology for functional assays has been proposed. Sabatini and co-workers have shown a cell-based microarrays method (1) that relies on the deposition and immobilization of an array of cDNA plasmids on a slide where cells are subsequently plated; the cDNA is then internalized by "reverse transfection" and cells overexpress or downregulate in each single spot the genes of interest. This approach allows the screening of different phenotypes in living cells of many genes in parallel on a single slide. To overcome some relevant limitations of this approach, we have implemented the technology by means of viral immobilization (2) on a novel surface of cluster-assembled nanostructured TiO2 (3) previously functionalized with an array of a docking protein. In this work, we present the detailed development of the "reverse infection cell-microarray based technology" in U2OS cells on a novel coated slide that represents an advanced application of protein arrays.

  17. High-throughput fabrication and screening improves gold nanoparticle chemiresistor sensor performance.

    PubMed

    Hubble, Lee J; Cooper, James S; Sosa-Pintos, Andrea; Kiiveri, Harri; Chow, Edith; Webster, Melissa S; Wieczorek, Lech; Raguse, Burkhard

    2015-02-01

    Chemiresistor sensor arrays are a promising technology to replace current laboratory-based analysis instrumentation, with the advantage of facile integration into portable, low-cost devices for in-field use. To increase the performance of chemiresistor sensor arrays a high-throughput fabrication and screening methodology was developed to assess different organothiol-functionalized gold nanoparticle chemiresistors. This high-throughput fabrication and testing methodology was implemented to screen a library consisting of 132 different organothiol compounds as capping agents for functionalized gold nanoparticle chemiresistor sensors. The methodology utilized an automated liquid handling workstation for the in situ functionalization of gold nanoparticle films and subsequent automated analyte testing of sensor arrays using a flow-injection analysis system. To test the methodology we focused on the discrimination and quantitation of benzene, toluene, ethylbenzene, p-xylene, and naphthalene (BTEXN) mixtures in water at low microgram per liter concentration levels. The high-throughput methodology identified a sensor array configuration consisting of a subset of organothiol-functionalized chemiresistors which in combination with random forests analysis was able to predict individual analyte concentrations with overall root-mean-square errors ranging between 8-17 μg/L for mixtures of BTEXN in water at the 100 μg/L concentration. The ability to use a simple sensor array system to quantitate BTEXN mixtures in water at the low μg/L concentration range has direct and significant implications to future environmental monitoring and reporting strategies. In addition, these results demonstrate the advantages of high-throughput screening to improve the performance of gold nanoparticle based chemiresistors for both new and existing applications. PMID:25562398

  18. High throughput screening of particle conditioning operations: I. System design and method development.

    PubMed

    Noyes, Aaron; Huffman, Ben; Godavarti, Ranga; Titchener-Hooker, Nigel; Coffman, Jonathan; Sunasara, Khurram; Mukhopadhyay, Tarit

    2015-08-01

    The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale-down system (USD) for high throughput particle conditioning (HTPC) composed of off-the-shelf components. The apparatus is comprised of a temperature-controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot-scale performance of a non-geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale-up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale-up heuristics.

  19. Image Harvest: an open-source platform for high-throughput plant image processing and analysis

    PubMed Central

    Knecht, Avi C.; Campbell, Malachy T.; Caprez, Adam; Swanson, David R.; Walia, Harkamal

    2016-01-01

    High-throughput plant phenotyping is an effective approach to bridge the genotype-to-phenotype gap in crops. Phenomics experiments typically result in large-scale image datasets, which are not amenable for processing on desktop computers, thus creating a bottleneck in the image-analysis pipeline. Here, we present an open-source, flexible image-analysis framework, called Image Harvest (IH), for processing images originating from high-throughput plant phenotyping platforms. Image Harvest is developed to perform parallel processing on computing grids and provides an integrated feature for metadata extraction from large-scale file organization. Moreover, the integration of IH with the Open Science Grid provides academic researchers with the computational resources required for processing large image datasets at no cost. Image Harvest also offers functionalities to extract digital traits from images to interpret plant architecture-related characteristics. To demonstrate the applications of these digital traits, a rice (Oryza sativa) diversity panel was phenotyped and genome-wide association mapping was performed using digital traits that are used to describe different plant ideotypes. Three major quantitative trait loci were identified on rice chromosomes 4 and 6, which co-localize with quantitative trait loci known to regulate agronomically important traits in rice. Image Harvest is an open-source software for high-throughput image processing that requires a minimal learning curve for plant biologists to analyzephenomics datasets. PMID:27141917

  20. [Advances in high-throughput transcriptome research of traditional Chinese medicines].

    PubMed

    Zhang, Zhao-Bao; Hou, Lin; Pan, Qing; Wang, Xu-Min; Cui, Qing-Hua; Tian, Jing-Zhen; Ma, Lu-Yu

    2014-05-01

    Traditional Chinese medicine is a treasure of Chinese culture, absorbing the wisdom of the Chinese people. Continuous application of new technologies makes traditional Chinese medicine research advance with the times. After several years of development, high-throughput transcriptome study has become a mature research tool in biology. This paper reviewed the advances in medicine transcriptome study, and compared two sequencing platforms, Roche's GS FLX platform and Illumina's HiSeq 2000 platform. Moreover, this paper introduced medicine transcriptome analysis process, with Panax quinquefolius and Lonicera japonica for examples, showing the characteristics of traditional Chinese medicine transcriptome studies. High-throughput transcriptome studies facilitate traditional Chinese medicine research with overall understand of functional genes, give clear elucidation of metabolic pathways, lay molecular foundation for the traditional Chinese medicine research and offer modern interpretation for traditional Chinese medicine theory. However, the current study faces several difficulties, including weak molecular basis, high sequencing cost and staff shortages in data anaysis. In the future, with the development in sequencing technology, the combination of transcriptome and other genomics, such as proteome and metabolome, will lay a solid foundation for the new high-throughput screening and developing model for the traditional Chinese medicine industry.

  1. Improved workflows for high throughput library preparation using the transposome-based nextera system

    PubMed Central

    2013-01-01

    Background The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs. Results We show that an initial bead-based normalisation step can remove the need for quantification and improves sample purity. A 75% cost reduction was achieved with a low-volume modified protocol which was tested over genomes with different GC content to demonstrate its robustness. Finally we developed a custom set of index tags and primers which increase the number of samples that can simultaneously be sequenced on a single lane of an Illumina instrument. Conclusions We addressed the bottlenecks of Nextera library construction to produce a modified protocol which harnesses the full power of the Nextera kit and allows the reproducible construction of libraries on a high-throughput scale reducing the associated cost of the kit. PMID:24256843

  2. 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

  3. Image Harvest: an open-source platform for high-throughput plant image processing and analysis.

    PubMed

    Knecht, Avi C; Campbell, Malachy T; Caprez, Adam; Swanson, David R; Walia, Harkamal

    2016-05-01

    High-throughput plant phenotyping is an effective approach to bridge the genotype-to-phenotype gap in crops. Phenomics experiments typically result in large-scale image datasets, which are not amenable for processing on desktop computers, thus creating a bottleneck in the image-analysis pipeline. Here, we present an open-source, flexible image-analysis framework, called Image Harvest (IH), for processing images originating from high-throughput plant phenotyping platforms. Image Harvest is developed to perform parallel processing on computing grids and provides an integrated feature for metadata extraction from large-scale file organization. Moreover, the integration of IH with the Open Science Grid provides academic researchers with the computational resources required for processing large image datasets at no cost. Image Harvest also offers functionalities to extract digital traits from images to interpret plant architecture-related characteristics. To demonstrate the applications of these digital traits, a rice (Oryza sativa) diversity panel was phenotyped and genome-wide association mapping was performed using digital traits that are used to describe different plant ideotypes. Three major quantitative trait loci were identified on rice chromosomes 4 and 6, which co-localize with quantitative trait loci known to regulate agronomically important traits in rice. Image Harvest is an open-source software for high-throughput image processing that requires a minimal learning curve for plant biologists to analyzephenomics datasets.

  4. Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

    PubMed

    Huang, Haiming; Economopoulos, Nicolas O; Liu, Bernard A; Uetrecht, Andrea; Gu, Jun; Jarvik, Nick; Nadeem, Vincent; Pawson, Tony; Moffat, Jason; Miersch, Shane; Sidhu, Sachdev S

    2015-11-01

    Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.

  5. Image Harvest: an open-source platform for high-throughput plant image processing and analysis.

    PubMed

    Knecht, Avi C; Campbell, Malachy T; Caprez, Adam; Swanson, David R; Walia, Harkamal

    2016-05-01

    High-throughput plant phenotyping is an effective approach to bridge the genotype-to-phenotype gap in crops. Phenomics experiments typically result in large-scale image datasets, which are not amenable for processing on desktop computers, thus creating a bottleneck in the image-analysis pipeline. Here, we present an open-source, flexible image-analysis framework, called Image Harvest (IH), for processing images originating from high-throughput plant phenotyping platforms. Image Harvest is developed to perform parallel processing on computing grids and provides an integrated feature for metadata extraction from large-scale file organization. Moreover, the integration of IH with the Open Science Grid provides academic researchers with the computational resources required for processing large image datasets at no cost. Image Harvest also offers functionalities to extract digital traits from images to interpret plant architecture-related characteristics. To demonstrate the applications of these digital traits, a rice (Oryza sativa) diversity panel was phenotyped and genome-wide association mapping was performed using digital traits that are used to describe different plant ideotypes. Three major quantitative trait loci were identified on rice chromosomes 4 and 6, which co-localize with quantitative trait loci known to regulate agronomically important traits in rice. Image Harvest is an open-source software for high-throughput image processing that requires a minimal learning curve for plant biologists to analyzephenomics datasets. PMID:27141917

  6. Droplet microfluidic technology for single-cell high-throughput screening.

    PubMed

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  7. Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs.

    PubMed

    Chaudhary, Amit Kumar; Na, Dokyun; Lee, Eun Yeol

    2015-11-01

    Due to global crises such as pollution and depletion of fossil fuels, sustainable technologies based on microbial cell-factories have been garnering great interest as an alternative to chemical factories. The development of microbial cell-factories is imperative in cutting down the overall manufacturing cost. Thus, diverse metabolic engineering strategies and engineering tools have been established to obtain a preferred genotype and phenotype displaying superior productivity. However, these tools are limited to only a handful of genes with permanent modification of a genome and significant labor costs, and this is one of the bottlenecks associated with biofactory construction. Therefore, a groundbreaking rapid and high-throughput engineering tool is needed for efficient construction of microbial cell-factories. During the last decade, copious small noncoding RNAs (ncRNAs) have been discovered in bacteria. These are involved in substantial regulatory roles like transcriptional and post-transcriptional gene regulation by modulating mRNA elongation, stability, or translational efficiency. Because of their vulnerability, ncRNAs can be used as another layer of conditional control over gene expression without modifying chromosomal sequences, and hence would be a promising high-throughput tool for metabolic engineering. Here, we review successful design principles and applications of ncRNAs for high-throughput metabolic engineering or physiological studies of diverse industrially important microorganisms. PMID:26027891

  8. A versatile toolkit for high throughput functional genomics with Trichoderma reesei

    SciTech Connect

    Schuster, Andre; Bruno, Kenneth S.; Collett, James R.; Baker, Scott E.; Seiboth, Bernhard; Kubicek, Christian P.; Schmoll, Monika

    2012-01-02

    The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. RESULTS: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.CONCLUSIONS:Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.

  9. Liquid gradient in two-dimensional matrix for high throughput screening.

    PubMed

    Hu, Shan-Wen; Xu, Bi-Yi; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-01-01

    Based on the ingenious combination of two different gradient generation mechanisms, this work reports a novel approach for a high throughput linear liquid gradient in a two-dimensional (2D) matrix. Specifically, a typical Christmas Tree structure with two inlets was designed as the first mixture gradient generator, upon which the second diffusion gradient generator was coupled to produce the desired concentration series on the basis of the distance difference. Rather than a simple 1D line, the integration of the two generators would result in an innovative 2D matrix of reservoirs, which was then characterized both theoretically and experimentally. Theoretically, calculation of fluid field demonstrated the formation of a concentration gradient, which was then confirmed by the dye solution visualization analysis. For high throughput screening application, doxorubicin (Dox) was then selected as model medicine to treat the acute myeloblastic leukemia (HL-60) cells. Cell viability displayed that cell death rate enhanced with the increase of drug concentration, and this result was higher than that on a 96-well plate, and the corresponding mechanism was properly discussed. Subsequently, Dox and quercetin were employed simultaneously to generate an overlapping gradient and its effect on HL-60 cells was investigated. Due to the automatic formation of concentration gradient that could improve the work efficiency, this work provides a promising tool for future high throughput drug screening.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  11. 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

  12. A high-throughput microRNA expression profiling system.

    PubMed

    Guo, Yanwen; Mastriano, Stephen; Lu, Jun

    2014-01-01

    As small noncoding RNAs, microRNAs (miRNAs) regulate diverse biological functions, including physiological and pathological processes. The expression and deregulation of miRNA levels contain rich information with diagnostic and prognostic relevance and can reflect pharmacological responses. The increasing interest in miRNA-related research demands global miRNA expression profiling on large numbers of samples. We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings. PMID:25030917

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

    PubMed

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

    2014-01-01

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

  14. 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

  15. 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

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

    PubMed

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

    2008-12-24

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

  17. 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).

  18. Microarrays for the evaluation of cell-biomaterial surface interactions

    NASA Astrophysics Data System (ADS)

    Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

    2007-01-01

    The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

  19. Microarray technology: an increasing variety of screening tools for proteomic research.

    PubMed

    Stoll, Dieter; Bachmann, Jutta; Templin, Markus F; Joos, Thomas O

    2004-12-15

    Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently used for the identification, quantitation and functional analysis of proteins that are of interest for proteomic research in basic and applied biology and for diagnostic applications. Such novel assays are also of major interest for the pharmaceutical industry, focusing on the identification of biomarkers and the validation of potential target molecules. Sensitivity, reproducibility, robustness and automation have to be demonstrated before this technology will be suitable for high-throughput applications.

  20. 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

  1. 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

  2. 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

  3. 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

  4. High throughput and miniaturised systems for biodegradability assessments.

    PubMed

    Cregut, Mickael; Jouanneau, Sulivan; Brillet, François; Durand, Marie-José; Sweetlove, Cyril; Chenèble, Jean-Charles; L'Haridon, Jacques; Thouand, Gérald

    2014-01-01

    The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

  5. 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

  6. Computational approaches to phenotyping: high-throughput phenomics.

    PubMed

    Lussier, Yves A; Liu, Yang

    2007-01-01

    The recent completion of the Human Genome Project has made possible a high-throughput "systems approach" for accelerating the elucidation of molecular underpinnings of human diseases, and subsequent derivation of molecular-based strategies to more effectively prevent, diagnose, and treat these diseases. Although altered phenotypes are among the most reliable manifestations of altered gene functions, research using systematic analysis of phenotype relationships to study human biology is still in its infancy. This article focuses on the emerging field of high-throughput phenotyping (HTP) phenomics research, which aims to capitalize on novel high-throughput computation and informatics technology developments to derive genomewide molecular networks of genotype-phenotype associations, or "phenomic associations." The HTP phenomics research field faces the challenge of technological research and development to generate novel tools in computation and informatics that will allow researchers to amass, access, integrate, organize, and manage phenotypic databases across species and enable genomewide analysis to associate phenotypic information with genomic data at different scales of biology. Key state-of-the-art technological advancements critical for HTP phenomics research are covered in this review. In particular, we highlight the power of computational approaches to conduct large-scale phenomics studies.

  7. 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

  8. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA.

    PubMed

    Alquezar-Planas, David E; Fordyce, Sarah L

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred to as "ancient DNA," using the Roche GS FLX 454 platform.

  9. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA.

    PubMed

    Alquezar-Planas, David E; Fordyce, Sarah L

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred to as "ancient DNA," using the Roche GS FLX 454 platform. PMID:22665278

  10. Analysis of antimicrobial resistance mechanisms in MDR bacteria by microarray and high-throughput sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antimicrobial resistance in pathogenic bacteria is a major concern in human and animal health. The National Antimicrobial Resistance Monitoring System (NARMS) was designed by the CDC, FDA, and USDA to monitor antimicrobial resistance in the U.S. The Bacterial Epidemiology and Antimicrobial Resistanc...

  11. High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates.

    PubMed

    Hasegawa, Toshimi; Hayashi, Manabu; Nomura, Kazuki; Hayashi, Mayumi; Kido, Miyuki; Ohmori, Tsuneo; Fukuda, Masao; Iguchi, Akinori; Ueno, Shigeaki; Shigematsu, Toru; Hirayama, Masao; Fujii, Tomoyuki

    2012-06-01

    Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the high-pressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis.

  12. 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

  13. Advancing translational research with next-generation protein microarrays.

    PubMed

    Yu, Xiaobo; Petritis, Brianne; LaBaer, Joshua

    2016-04-01

    Protein microarrays are a high-throughput technology used increasingly in translational research, seeking to apply basic science findings to enhance human health. In addition to assessing protein levels, posttranslational modifications, and signaling pathways in patient samples, protein microarrays have aided in the identification of potential protein biomarkers of disease and infection. In this perspective, the different types of full-length protein microarrays that are used in translational research are reviewed. Specific studies employing these microarrays are presented to highlight their potential in finding solutions to real clinical problems. Finally, the criteria that should be considered when developing next-generation protein microarrays are provided. PMID:26749402

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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).

  2. High-throughput crystallography for lead discovery in drug design.

    PubMed

    Blundell, Tom L; Jhoti, Harren; Abell, Chris

    2002-01-01

    Knowledge of the three-dimensional structures of protein targets now emerging from genomic data has the potential to accelerate drug discovery greatly. X-ray crystallography is the most widely used technique for protein structure determination, but technical challenges and time constraints have traditionally limited its use primarily to lead optimization. Here, we describe how significant advances in process automation and informatics have aided the development of high-throughput X-ray crystallography, and discuss the use of this technique for structure-based lead discovery.

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

    PubMed

    Huang, G M

    1999-01-01

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

  4. 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.

  5. 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

  6. GNARE: an environment for Grid-based high-throughput genome analysis.

    SciTech Connect

    Sulakhe, D.; Rodriguez, A.; D'Souza, M.; Wilde, M.; Nefedova, V.; Foster, I.; Maltsev, N.; Mathematics and Computer Science; Univ. of Chicago

    2005-01-01

    Recent progress in genomics and experimental biology has brought exponential growth of the biological information available for computational analysis in public genomics databases. However, applying the potentially enormous scientific value of this information to the understanding of biological systems requires computing and data storage technology of an unprecedented scale. The grid, with its aggregated and distributed computational and storage infrastructure, offers an ideal platform for high-throughput bioinformatics analysis. To leverage this we have developed the Genome Analysis Research Environment (GNARE) - a scalable computational system for the high-throughput analysis of genomes, which provides an integrated database and computational backend for data-driven bioinformatics applications. GNARE efficiently automates the major steps of genome analysis including acquisition of data from multiple genomic databases; data analysis by a diverse set of bioinformatics tools; and storage of results and annotations. High-throughput computations in GNARE are performed using distributed heterogeneous grid computing resources such as Grid2003, TeraGrid, and the DOE science grid. Multi-step genome analysis workflows involving massive data processing, the use of application-specific toots and algorithms and updating of an integrated database to provide interactive Web access to results are all expressed and controlled by a 'virtual data' model which transparently maps computational workflows to distributed grid resources. This paper describes how Grid technologies such as Globus, Condor, and the Gryphyn virtual data system were applied in the development of GNARE. It focuses on our approach to Grid resource allocation and to the use of GNARE as a computational framework for the development of bioinformatics applications.

  7. Curating and Preparing High-Throughput Screening Data for Quantitative Structure-Activity Relationship Modeling.

    PubMed

    Kim, Marlene T; Wang, Wenyi; Sedykh, Alexander; Zhu, Hao

    2016-01-01

    Publicly available bioassay data often contains errors. Curating massive bioassay data, especially high-throughput screening (HTS) data, for Quantitative Structure-Activity Relationship (QSAR) modeling requires the assistance of automated data curation tools. Using automated data curation tools are beneficial to users, especially ones without prior computer skills, because many platforms have been developed and optimized based on standardized requirements. As a result, the users do not need to extensively configure the curation tool prior to the application procedure. In this chapter, a freely available automatic tool to curate and prepare HTS data for QSAR modeling purposes will be described.

  8. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    PubMed Central

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  9. Curating and Preparing High-Throughput Screening Data for Quantitative Structure-Activity Relationship Modeling.

    PubMed

    Kim, Marlene T; Wang, Wenyi; Sedykh, Alexander; Zhu, Hao

    2016-01-01

    Publicly available bioassay data often contains errors. Curating massive bioassay data, especially high-throughput screening (HTS) data, for Quantitative Structure-Activity Relationship (QSAR) modeling requires the assistance of automated data curation tools. Using automated data curation tools are beneficial to users, especially ones without prior computer skills, because many platforms have been developed and optimized based on standardized requirements. As a result, the users do not need to extensively configure the curation tool prior to the application procedure. In this chapter, a freely available automatic tool to curate and prepare HTS data for QSAR modeling purposes will be described. PMID:27518634

  10. High-throughput gated photon counter with two detection windows programmable down to 70 ps width

    SciTech Connect

    Boso, Gianluca; Tosi, Alberto Zappa, Franco; Mora, Alberto Dalla

    2014-01-15

    We present the design and characterization of a high-throughput gated photon counter able to count electrical pulses occurring within two well-defined and programmable detection windows. We extensively characterized and validated this instrument up to 100 Mcounts/s and with detection window width down to 70 ps. This instrument is suitable for many applications and proves to be a cost-effective and compact alternative to time-correlated single-photon counting equipment, thanks to its easy configurability, user-friendly interface, and fully adjustable settings via a Universal Serial Bus (USB) link to a remote computer.

  11. High-throughput gated photon counter with two detection windows programmable down to 70 ps width

    NASA Astrophysics Data System (ADS)

    Boso, Gianluca; Tosi, Alberto; Mora, Alberto Dalla; Zappa, Franco

    2014-01-01

    We present the design and characterization of a high-throughput gated photon counter able to count electrical pulses occurring within two well-defined and programmable detection windows. We extensively characterized and validated this instrument up to 100 Mcounts/s and with detection window width down to 70 ps. This instrument is suitable for many applications and proves to be a cost-effective and compact alternative to time-correlated single-photon counting equipment, thanks to its easy configurability, user-friendly interface, and fully adjustable settings via a Universal Serial Bus (USB) link to a remote computer.

  12. High-throughput gated photon counter with two detection windows programmable down to 70 ps width.

    PubMed

    Boso, Gianluca; Tosi, Alberto; Dalla Mora, Alberto; Zappa, Franco

    2014-01-01

    We present the design and characterization of a high-throughput gated photon counter able to count electrical pulses occurring within two well-defined and programmable detection windows. We extensively characterized and validated this instrument up to 100 Mcounts/s and with detection window width down to 70 ps. This instrument is suitable for many applications and proves to be a cost-effective and compact alternative to time-correlated single-photon counting equipment, thanks to its easy configurability, user-friendly interface, and fully adjustable settings via a Universal Serial Bus (USB) link to a remote computer.

  13. Automated sample preparation and LC-MS for high-throughput ADME quantification.

    PubMed

    O'Connor, Desmond

    2002-01-01

    Bioanalytical groups in the pharmaceutical industry provide quantitative data to support all stages of drug discovery. The increased use of 96-well plates and robotic liquid handling systems, the availability of robust triple quadruple mass spectrometers, and developments in chromatographic and samples preparation techniques, have all increased the rate at which this data can be generated. This review describes currently used methods and emerging technologies for automation of high-throughput quantitative bioanalysis. The focus is on recent applications of sample preparation and chromatography techniques compatible with detection by triple quadruple mass spectrometers.

  14. High-throughput realization of an infrared selective absorber/emitter by DUV microsphere projection lithography.

    PubMed

    Bonakdar, Alireza; Rezaei, Mohsen; Dexheimer, Eric; Mohseni, Hooman

    2016-01-22

    In this paper, we present a low-cost and high-throughput nanofabrication method to realize metasurfaces that have selective absorption/emission in the mid-infrared region of the electromagnetic spectrum. We have developed DUV projection lithography to produce arbitrary patterns with sub-80 nm feature sizes. As examples of practical applications, we experimentally demonstrate structures with single and double spectral absorption/emission features, and in close agreement with numerical simulation. The fundamental mechanism of perfect absorption is discussed as well. Selective infrared absorbers/emitters are critical elements in realizing efficient thermophotovoltaic cells and high-performance biosensors. PMID:26650855

  15. Microarray analysis reveals the actual specificity of enrichment media used for food safety assessment.

    PubMed

    Kostić, Tanja; Stessl, Beatrix; Wagner, Martin; Sessitsch, Angela

    2011-06-01

    Microbial diagnostic microarrays are tools for simultaneous detection and identification of microorganisms in food, clinical, and environmental samples. In comparison to classic methods, microarray-based systems have the potential for high throughput, parallelism, and miniaturization. High specificity and high sensitivity of detection have been demonstrated. A microbial diagnostic microarray for the detection of the most relevant bacterial food- and waterborne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labeling of oligonucleotides and the phylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus and/or species level) and sensitive (0.1% relative and 10(4) CFU absolute sensitivity) detection of the target pathogens. In initial challenge studies of the applicability of microarray-based food analysis, we obtained results demonstrating the questionable specificity of standardized culture-dependent microbiological detection methods. Taking into consideration the importance of reliable food safety assessment methods, comprehensive performance assessment is essential. Results demonstrate the potential of this new pathogen diagnostic microarray to evaluate culture-based standard methods in microbiological food analysis.

  16. Microarray applications to understand the impact of exposure to environmental contaminants in wild dolphins (Tursiops truncatus).

    PubMed

    Mancia, Annalaura; Abelli, Luigi; Kucklick, John R; Rowles, Teresa K; Wells, Randall S; Balmer, Brian C; Hohn, Aleta A; Baatz, John E; Ryan, James C

    2015-02-01

    It is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. The health of an ecosystem is largely reflected in the health of its inhabitants. As an apex predator, the common bottlenose dolphin (Tursiops truncatus) can reflect the health of near shore marine ecosystems, and reflect coastal threats that pose risk to human health, such as legacy contaminants or marine toxins, e.g. polychlorinated biphenyls (PCBs) and brevetoxins. Major advances in the understanding of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of the development of cell-lines for use in in vitro experiments, the production of monoclonal antibodies to recognize dolphin proteins, the development of dolphin DNA microarrays to measure global gene expression and the sequencing of the dolphin genome. These advances may play a central role in understanding the complex and specialized biology of the dolphin with regard to how this species responds to an array of environmental insults. This work presents the creation, characterization and application of a new molecular tool to better understand the complex and unique biology of the common bottlenose dolphin and its response to environmental stress and infection. A dolphin oligo microarray representing 24,418 unigene sequences was developed and used to analyze blood samples collected from 69 dolphins during capture-release health assessments at five geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). The microarray was validated and tested for its ability to: 1) distinguish male from female dolphins; 2) differentiate dolphins inhabiting different geographic locations (Atlantic coasts vs the Gulf of Mexico); and 3) study in detail dolphins resident in one site, the Georgia coast, known to

  17. Microarray applications to understand the impact of exposure to environmental contaminants in wild dolphins (Tursiops truncatus).

    PubMed

    Mancia, Annalaura; Abelli, Luigi; Kucklick, John R; Rowles, Teresa K; Wells, Randall S; Balmer, Brian C; Hohn, Aleta A; Baatz, John E; Ryan, James C

    2015-02-01

    It is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. The health of an ecosystem is largely reflected in the health of its inhabitants. As an apex predator, the common bottlenose dolphin (Tursiops truncatus) can reflect the health of near shore marine ecosystems, and reflect coastal threats that pose risk to human health, such as legacy contaminants or marine toxins, e.g. polychlorinated biphenyls (PCBs) and brevetoxins. Major advances in the understanding of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of the development of cell-lines for use in in vitro experiments, the production of monoclonal antibodies to recognize dolphin proteins, the development of dolphin DNA microarrays to measure global gene expression and the sequencing of the dolphin genome. These advances may play a central role in understanding the complex and specialized biology of the dolphin with regard to how this species responds to an array of environmental insults. This work presents the creation, characterization and application of a new molecular tool to better understand the complex and unique biology of the common bottlenose dolphin and its response to environmental stress and infection. A dolphin oligo microarray representing 24,418 unigene sequences was developed and used to analyze blood samples collected from 69 dolphins during capture-release health assessments at five geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). The microarray was validated and tested for its ability to: 1) distinguish male from female dolphins; 2) differentiate dolphins inhabiting different geographic locations (Atlantic coasts vs the Gulf of Mexico); and 3) study in detail dolphins resident in one site, the Georgia coast, known to

  18. A Bayesian approach to analyzing phenotype microarray data enables estimation of microbial growth parameters.

    PubMed

    Gerstgrasser, Matthias; Nicholls, Sarah; Stout, Michael; Smart, Katherine; Powell, Chris; Kypraios, Theodore; Stekel, Dov

    2016-06-01

    Biolog phenotype microarrays (PMs) enable simultaneous, high throughput analysis of cell cultures in different environments. The output is high-density time-course data showing redox curves (approximating growth) for each experimental condition. The software provided with the Omnilog incubator/reader summarizes each time-course as a single datum, so most of the information is not used. However, the time courses can be extremely varied and often contain detailed qualitative (shape of curve) and quantitative (values of parameters) information. We present a novel, Bayesian approach to estimating parameters from Phenotype Microarray data, fitting growth models using Markov Chain Monte Carlo (MCMC) methods to enable high throughput estimation of important information, including length of lag phase, maximal "growth" rate and maximum output. We find that the Baranyi model for microbial growth is useful for fitting Biolog data. Moreover, we introduce a new growth model that allows for diauxic growth with a lag phase, which is particularly useful where Phenotype Microarrays have been applied to cells grown in complex mixtures of substrates, for example in industrial or biotechnological applications, such as worts in brewing. Our approach provides more useful information from Biolog data than existing, competing methods, and allows for valuable comparisons between data series and across different models. PMID:26762475

  19. 3-D microarray and its microfabrication-free fluidic immunoassay device.

    PubMed

    Liu, Yingshuai; Zhang, Yuanyuan; Lu, Zhisong; Li, Chang Ming

    2015-08-19

    Conventional 2-D microarray is known to have high-throughput detection capability; however, the sensing spots density is significantly hindered by the spot-to-spot distance (gap) requirement for eliminating cross-talks between adjacent spots. Herein a new conceptual 3-D microarray device is proposed to significantly improve the spots density. To demonstrate advantages of the 3-D array, a microfabrication-free fluidic immunoassay device is further made by simply coupling an antibodies-arrayed glass cuboid into a circular glass tube. Rapid, sensitive and high-throughput flow-through immunoassays were accomplished with the 3-D array-based device for detection limits of 10-100 pg mL(-1) and wide dynamic range over 4-5 orders of magnitude in human serum with cancer biomarkers α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model targets, holding great promise for practical clinical applications. The 3-D microarray device not only significantly increases the density of sensing spots, but also greatly enhances the mass transport for rapid immunoassay when using in a flow-through device. PMID:26343442

  20. Morphology control in polymer blend fibers—a high throughput computing approach

    NASA Astrophysics Data System (ADS)

    Sesha Sarath Pokuri, Balaji; Ganapathysubramanian, Baskar

    2016-08-01

    Fibers made from polymer blends have conventionally enjoyed wide use, particularly in textiles. This wide applicability is primarily aided by the ease of manufacturing such fibers. More recently, the ability to tailor the internal morphology of polymer blend fibers by carefully designing processing conditions has enabled such fibers to be used in technologically relevant applications. Some examples include anisotropic insulating properties for heat and anisotropic wicking of moisture, coaxial morphologies for optical applications as well as fibers with high internal surface area for filtration and catalysis applications. However, identifying the appropriate processing conditions from the large space of possibilities using conventional trial-and-error approaches is a tedious and resource-intensive process. Here, we illustrate a high throughput computational approach to rapidly explore and characterize how processing conditions (specifically blend ratio and evaporation rates) affect the internal morphology of polymer blends during solvent based fabrication. We focus on a PS: PMMA system and identify two distinct classes of morphologies formed due to variations in the processing conditions. We subsequently map the processing conditions to the morphology class, thus constructing a ‘phase diagram’ that enables rapid identification of processing parameters for specific morphology class. We finally demonstrate the potential for time dependent processing conditions to get desired features of the morphology. This opens up the possibility of rational stage-wise design of processing pathways for tailored fiber morphology using high throughput computing.