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/.

Life in the fast lane for protein crystallization and X-ray crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2005-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2004-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

SMARTIV: combined sequence and structure de-novo motif discovery for in-vivo RNA binding data.

PubMed

Polishchuk, Maya; Paz, Inbal; Yakhini, Zohar; Mandel-Gutfreund, Yael

2018-05-25

Gene expression regulation is highly dependent on binding of RNA-binding proteins (RBPs) to their RNA targets. Growing evidence supports the notion that both RNA primary sequence and its local secondary structure play a role in specific Protein-RNA recognition and binding. Despite the great advance in high-throughput experimental methods for identifying sequence targets of RBPs, predicting the specific sequence and structure binding preferences of RBPs remains a major challenge. We present a novel webserver, SMARTIV, designed for discovering and visualizing combined RNA sequence and structure motifs from high-throughput RNA-binding data, generated from in-vivo experiments. The uniqueness of SMARTIV is that it predicts motifs from enriched k-mers that combine information from ranked RNA sequences and their predicted secondary structure, obtained using various folding methods. Consequently, SMARTIV generates Position Weight Matrices (PWMs) in a combined sequence and structure alphabet with assigned P-values. SMARTIV concisely represents the sequence and structure motif content as a single graphical logo, which is informative and easy for visual perception. SMARTIV was examined extensively on a variety of high-throughput binding experiments for RBPs from different families, generated from different technologies, showing consistent and accurate results. Finally, SMARTIV is a user-friendly webserver, highly efficient in run-time and freely accessible via http://smartiv.technion.ac.il/.

High-Throughput Synthesis and Structure of Zeolite ZSM-43 with Two-Directional 8-Ring Channels.

PubMed

Willhammar, Tom; Su, Jie; Yun, Yifeng; Zou, Xiaodong; Afeworki, Mobae; Weston, Simon C; Vroman, Hilda B; Lonergan, William W; Strohmaier, Karl G

2017-08-07

The aluminosilicate zeolite ZSM-43 (where ZSM = Zeolite Socony Mobil) was first synthesized more than 3 decades ago, but its chemical structure remained unsolved because of its poor crystallinity and small crystal size. Here we present optimization of the ZSM-43 synthesis using a high-throughput approach and subsequent structure determination by the combination of electron crystallographic methods and powder X-ray diffraction. The synthesis required the use of a combination of both inorganic (Cs + and K + ) and organic (choline) structure-directing agents. High-throughput synthesis enabled a screening of the synthesis conditions, which made it possible to optimize the synthesis, despite its complexity, in order to obtain a material with significantly improved crystallinity. When both rotation electron diffraction and high-resolution transmission electron microscopy imaging techniques are applied, the structure of ZSM-43 could be determined. The structure of ZSM-43 is a new zeolite framework type and possesses a unique two-dimensional channel system limited by 8-ring channels. ZSM-43 is stable upon calcination, and sorption measurements show that the material is suitable for adsorption of carbon dioxide as well as methane.

Data-driven high-throughput prediction of the 3-D structure of small molecules: review and progress. A response to the letter by the Cambridge Crystallographic Data Centre.

PubMed

Baldi, Pierre

2011-12-27

A response is presented to sentiments expressed in "Data-Driven High-Throughput Prediction of the 3-D Structure of Small Molecules: Review and Progress. A Response from The Cambridge Crystallographic Data Centre", recently published in the Journal of Chemical Information and Modeling, (1) which may give readers a misleading impression regarding significant impediments to scientific research posed by the CCDC.

Collaborative Core Research Program for Chemical-Biological Warfare Defense

DTIC Science & Technology

2015-01-04

Discovery through High Throughput Screening (HTS) and Fragment-Based Drug Design (FBDD...Discovery through High Throughput Screening (HTS) and Fragment-Based Drug Design (FBDD) Current pharmaceutical approaches involving drug discovery...structural analysis and docking program generally known as fragment based drug design (FBDD). The main advantage of using these approaches is that

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

High throughput platforms for structural genomics of integral membrane proteins.

PubMed

Mancia, Filippo; Love, James

2011-08-01

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

LSGermOPA, a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (Lactuca sativa L.) germplasm fingerprinting

USDA-ARS?s Scientific Manuscript database

We assessed the genetic diversity and population structure among 148 cultivated lettuce (Lactuca sativa L.) accessions using the high-throughput GoldenGate assay and 384 EST (Expressed Sequence Tag)-derived SNP (single nucleotide polymorphism) markers. A custom OPA (Oligo Pool All), LSGermOPA was fo...

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

PubMed

Chokhawala, Harshal A; Huang, Shengshu; Lau, Kam; Yu, Hai; Cheng, Jiansong; Thon, Vireak; Hurtado-Ziola, Nancy; Guerrero, Juan A; Varki, Ajit; Chen, Xi

2008-09-19

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.

A high-throughput approach to profile RNA structure.

PubMed

Delli Ponti, Riccardo; Marti, Stefanie; Armaos, Alexandros; Tartaglia, Gian Gaetano

2017-03-17

Here we introduce the Computational Recognition of Secondary Structure (CROSS) method to calculate the structural profile of an RNA sequence (single- or double-stranded state) at single-nucleotide resolution and without sequence length restrictions. We trained CROSS using data from high-throughput experiments such as Selective 2΄-Hydroxyl Acylation analyzed by Primer Extension (SHAPE; Mouse and HIV transcriptomes) and Parallel Analysis of RNA Structure (PARS; Human and Yeast transcriptomes) as well as high-quality NMR/X-ray structures (PDB database). The algorithm uses primary structure information alone to predict experimental structural profiles with >80% accuracy, showing high performances on large RNAs such as Xist (17 900 nucleotides; Area Under the ROC Curve AUC of 0.75 on dimethyl sulfate (DMS) experiments). We integrated CROSS in thermodynamics-based methods to predict secondary structure and observed an increase in their predictive power by up to 30%. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

DOE PAGES

Rames, Matthew; Yu, Yadong; Ren, Gang

2014-08-15

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electronmore » microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.« less

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

PubMed

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

High-throughput determination of RNA structure by proximity ligation.

PubMed

Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

2015-09-01

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

Solar fuels photoanode materials discovery by integrating high-throughput theory and experiment

DOE PAGES

Yan, Qimin; Yu, Jie; Suram, Santosh K.; ...

2017-03-06

The limited number of known low-band-gap photoelectrocatalytic materials poses a significant challenge for the generation of chemical fuels from sunlight. Here, using high-throughput ab initio theory with experiments in an integrated workflow, we find eight ternary vanadate oxide photoanodes in the target band-gap range (1.2-2.8 eV). Detailed analysis of these vanadate compounds reveals the key role of VO 4 structural motifs and electronic band-edge character in efficient photoanodes, initiating a genome for such materials and paving the way for a broadly applicable high-throughput-discovery and materials-by-design feedback loop. Considerably expanding the number of known photoelectrocatalysts for water oxidation, our study establishesmore » ternary metal vanadates as a prolific class of photoanodematerials for generation of chemical fuels from sunlight and demonstrates our high-throughput theory-experiment pipeline as a prolific approach to materials discovery.« less

Solar fuels photoanode materials discovery by integrating high-throughput theory and experiment

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

Yan, Qimin; Yu, Jie; Suram, Santosh K.

The limited number of known low-band-gap photoelectrocatalytic materials poses a significant challenge for the generation of chemical fuels from sunlight. Here, using high-throughput ab initio theory with experiments in an integrated workflow, we find eight ternary vanadate oxide photoanodes in the target band-gap range (1.2-2.8 eV). Detailed analysis of these vanadate compounds reveals the key role of VO 4 structural motifs and electronic band-edge character in efficient photoanodes, initiating a genome for such materials and paving the way for a broadly applicable high-throughput-discovery and materials-by-design feedback loop. Considerably expanding the number of known photoelectrocatalysts for water oxidation, our study establishesmore » ternary metal vanadates as a prolific class of photoanodematerials for generation of chemical fuels from sunlight and demonstrates our high-throughput theory-experiment pipeline as a prolific approach to materials discovery.« less

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

PubMed

Kuhn, Alexandre; Ong, Yao Min; Quake, Stephen R; Burkholder, William F

2015-07-08

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

Low-dose fixed-target serial synchrotron crystallography.

PubMed

Owen, Robin L; Axford, Danny; Sherrell, Darren A; Kuo, Anling; Ernst, Oliver P; Schulz, Eike C; Miller, R J Dwayne; Mueller-Werkmeister, Henrike M

2017-04-01

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

High throughput light absorber discovery, Part 2: Establishing structure–band gap energy relationships

DOE PAGES

Suram, Santosh K.; Newhouse, Paul F.; Zhou, Lan; ...

2016-09-23

Combinatorial materials science strategies have accelerated materials development in a variety of fields, and we extend these strategies to enable structure-property mapping for light absorber materials, particularly in high order composition spaces. High throughput optical spectroscopy and synchrotron X-ray diffraction are combined to identify the optical properties of Bi-V-Fe oxides, leading to the identification of Bi 4V 1.5Fe 0.5O 10.5 as a light absorber with direct band gap near 2.7 eV. Here, the strategic combination of experimental and data analysis techniques includes automated Tauc analysis to estimate band gap energies from the high throughput spectroscopy data, providing an automated platformmore » for identifying new optical materials.« less

High throughput light absorber discovery, Part 2: Establishing structure–band gap energy relationships

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

Suram, Santosh K.; Newhouse, Paul F.; Zhou, Lan

Combinatorial materials science strategies have accelerated materials development in a variety of fields, and we extend these strategies to enable structure-property mapping for light absorber materials, particularly in high order composition spaces. High throughput optical spectroscopy and synchrotron X-ray diffraction are combined to identify the optical properties of Bi-V-Fe oxides, leading to the identification of Bi 4V 1.5Fe 0.5O 10.5 as a light absorber with direct band gap near 2.7 eV. Here, the strategic combination of experimental and data analysis techniques includes automated Tauc analysis to estimate band gap energies from the high throughput spectroscopy data, providing an automated platformmore » for identifying new optical materials.« less

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

Hattrick-Simpers, Jason R.; Gregoire, John M.; Kusne, A. Gilad

With their ability to rapidly elucidate composition-structure-property relationships, high-throughput experimental studies have revolutionized how materials are discovered, optimized, and commercialized. It is now possible to synthesize and characterize high-throughput libraries that systematically address thousands of individual cuts of fabrication parameter space. An unresolved issue remains transforming structural characterization data into phase mappings. This difficulty is related to the complex information present in diffraction and spectroscopic data and its variation with composition and processing. Here, we review the field of automated phase diagram attribution and discuss the impact that emerging computational approaches will have in the generation of phase diagrams andmore » beyond.« less

Recent developments in software tools for high-throughput in vitro ADME support with high-resolution MS.

PubMed

Paiva, Anthony; Shou, Wilson Z

2016-08-01

The last several years have seen the rapid adoption of the high-resolution MS (HRMS) for bioanalytical support of high throughput in vitro ADME profiling. Many capable software tools have been developed and refined to process quantitative HRMS bioanalysis data for ADME samples with excellent performance. Additionally, new software applications specifically designed for quan/qual soft spot identification workflows using HRMS have greatly enhanced the quality and efficiency of the structure elucidation process for high throughput metabolite ID in early in vitro ADME profiling. Finally, novel approaches in data acquisition and compression, as well as tools for transferring, archiving and retrieving HRMS data, are being continuously refined to tackle the issue of large data file size typical for HRMS analyses.

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

PubMed

Reddy, Jithender G; Kumar, Dinesh; Hosur, Ramakrishna V

2015-02-01

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH – which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.

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.

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

PubMed

Jowhar, Ziad; Gudla, Prabhakar R; Shachar, Sigal; Wangsa, Darawalee; Russ, Jill L; Pegoraro, Gianluca; Ried, Thomas; Raznahan, Armin; Misteli, Tom

2018-06-01

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues. Published by Elsevier Inc.

Microscale High-Throughput Experimentation as an Enabling Technology in Drug Discovery: Application in the Discovery of (Piperidinyl)pyridinyl-1H-benzimidazole Diacylglycerol Acyltransferase 1 Inhibitors.

PubMed

Cernak, Tim; Gesmundo, Nathan J; Dykstra, Kevin; Yu, Yang; Wu, Zhicai; Shi, Zhi-Cai; Vachal, Petr; Sperbeck, Donald; He, Shuwen; Murphy, Beth Ann; Sonatore, Lisa; Williams, Steven; Madeira, Maria; Verras, Andreas; Reiter, Maud; Lee, Claire Heechoon; Cuff, James; Sherer, Edward C; Kuethe, Jeffrey; Goble, Stephen; Perrotto, Nicholas; Pinto, Shirly; Shen, Dong-Ming; Nargund, Ravi; Balkovec, James; DeVita, Robert J; Dreher, Spencer D

2017-05-11

Miniaturization and parallel processing play an important role in the evolution of many technologies. We demonstrate the application of miniaturized high-throughput experimentation methods to resolve synthetic chemistry challenges on the frontlines of a lead optimization effort to develop diacylglycerol acyltransferase (DGAT1) inhibitors. Reactions were performed on ∼1 mg scale using glass microvials providing a miniaturized high-throughput experimentation capability that was used to study a challenging S N Ar reaction. The availability of robust synthetic chemistry conditions discovered in these miniaturized investigations enabled the development of structure-activity relationships that ultimately led to the discovery of soluble, selective, and potent inhibitors of DGAT1.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now over 53,000 proteins structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact. PMID:19765976

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

An Extensible Schema-less Database Framework for Managing High-throughput Semi-Structured Documents

NASA Technical Reports Server (NTRS)

Maluf, David A.; Tran, Peter B.; La, Tracy; Clancy, Daniel (Technical Monitor)

2002-01-01

Object-Relational database management system is an integrated hybrid cooperative approach to combine the best practices of both the relational model utilizing SQL queries and the object oriented, semantic paradigm for supporting complex data creation. In this paper, a highly scalable, information on demand database framework, called NETMARK is introduced. NETMARK takes advantages of the Oracle 8i object-relational database using physical addresses data types for very efficient keyword searches of records for both context and content. NETMARK was originally developed in early 2000 as a research and development prototype to solve the vast amounts of unstructured and semi-structured documents existing within NASA enterprises. Today, NETMARK is a flexible, high throughput open database framework for managing, storing, and searching unstructured or semi structured arbitrary hierarchal models, XML and HTML.

High-throughput Molecular Simulations of MOFs for CO2 Separation: Opportunities and Challenges

NASA Astrophysics Data System (ADS)

Erucar, Ilknur; Keskin, Seda

2018-02-01

Metal organic frameworks (MOFs) have emerged as great alternatives to traditional nanoporous materials for CO2 separation applications. MOFs are porous materials that are formed by self-assembly of transition metals and organic ligands. The most important advantage of MOFs over well-known porous materials is the possibility to generate multiple materials with varying structural properties and chemical functionalities by changing the combination of metal centers and organic linkers during the synthesis. This leads to a large diversity of materials with various pore sizes and shapes that can be efficiently used for CO2 separations. Since the number of synthesized MOFs has already reached to several thousand, experimental investigation of each MOF at the lab-scale is not practical. High-throughput computational screening of MOFs is a great opportunity to identify the best materials for CO2 separation and to gain molecular-level insights into the structure-performance relationships. This type of knowledge can be used to design new materials with the desired structural features that can lead to extraordinarily high CO2 selectivities. In this mini-review, we focused on developments in high-throughput molecular simulations of MOFs for CO2 separations. After reviewing the current studies on this topic, we discussed the opportunities and challenges in the field and addressed the potential future developments.

Development of a sub-cm high resolution ion Doppler tomography diagnostics for fine structure measurement of guide field reconnection in TS-U

NASA Astrophysics Data System (ADS)

Tanabe, Hiroshi; Koike, Hideya; Hatano, Hironori; Hayashi, Takumi; Cao, Qinghong; Himeno, Shunichi; Kaneda, Taishi; Akimitsu, Moe; Sawada, Asuka; Ono, Yasushi

2017-10-01

A new type of high-throughput/high-resolution 96CH ion Doppler tomography diagnostics has been developed using ``multi-slit'' spectroscopy technique for detailed investigation of fine structure formation during high guide field magnetic reconnection. In the last three years, high field merging experiment in MAST pioneered new frontiers of reconnection heating: formation of highly peaked structure around X-point in high guide field condition (Bt > 0.3 T), outflow dissipation under the influence of better plasma confinement to form high temperature ring structure which aligns with closed flux surface of toroidal plasma, and interaction between ion and electron temperature profile during transport/confinement phase to form triple peak structure (τeiE 4 ms). To investigate more detailed mechanism with in-situ magnetic measurement, the university of Tokyo starts the upgrade of plasma parameters and spatial resolution of optical diagnostics as in MAST. Now, a new type of high-throughput/high-resolution 96CH ion Doppler tomography diagnostics system construction has been completed and it successfully resolved fine structure of ion heating downstream, aligned with closed flux surface formed by reconnected field. This work was supported by JSPS KAKENHI Grant Numbers 15H05750, 15K14279 and 17H04863.

DockoMatic: automated peptide analog creation for high throughput virtual screening.

PubMed

Jacob, Reed B; Bullock, Casey W; Andersen, Tim; McDougal, Owen M

2011-10-01

The purpose of this manuscript is threefold: (1) to describe an update to DockoMatic that allows the user to generate cyclic peptide analog structure files based on protein database (pdb) files, (2) to test the accuracy of the peptide analog structure generation utility, and (3) to evaluate the high throughput capacity of DockoMatic. The DockoMatic graphical user interface interfaces with the software program Treepack to create user defined peptide analogs. To validate this approach, DockoMatic produced cyclic peptide analogs were tested for three-dimensional structure consistency and binding affinity against four experimentally determined peptide structure files available in the Research Collaboratory for Structural Bioinformatics database. The peptides used to evaluate this new functionality were alpha-conotoxins ImI, PnIA, and their published analogs. Peptide analogs were generated by DockoMatic and tested for their ability to bind to X-ray crystal structure models of the acetylcholine binding protein originating from Aplysia californica. The results, consisting of more than 300 simulations, demonstrate that DockoMatic predicts the binding energy of peptide structures to within 3.5 kcal mol(-1), and the orientation of bound ligand compares to within 1.8 Å root mean square deviation for ligand structures as compared to experimental data. Evaluation of high throughput virtual screening capacity demonstrated that Dockomatic can collect, evaluate, and summarize the output of 10,000 AutoDock jobs in less than 2 hours of computational time, while 100,000 jobs requires approximately 15 hours and 1,000,000 jobs is estimated to take up to a week. Copyright © 2011 Wiley Periodicals, Inc.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

PubMed Central

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

PubMed Central

Ulrich, Nikea; Rosenberger, Abigail; Brislawn, Colin; Wright, Justin; Kessler, Collin; Toole, David; Solomon, Caroline; Strutt, Steven; McClure, Erin

2016-01-01

ABSTRACT Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. IMPORTANCE In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment. PMID:27060115

Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

PubMed

He, Feng; Phan, Duke H; Hogan, Sabine; Bailey, Robert; Becker, Gerald W; Narhi, Linda O; Razinkov, Vladimir I

2010-06-01

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy.

PubMed

Lai, Yiu Wai; Krause, Michael; Savan, Alan; Thienhaus, Sigurd; Koukourakis, Nektarios; Hofmann, Martin R; Ludwig, Alfred

2011-10-01

A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.

High-throughput syntheses of iron phosphite open frameworks in ionic liquids

NASA Astrophysics Data System (ADS)

Wang, Zhixiu; Mu, Ying; Wang, Yilin; Bing, Qiming; Su, Tan; Liu, Jingyao

2017-02-01

Three open-framework iron phosphites: Feп5(NH4)2(HPO3)6 (1), Feп2Fe♯(NH4)(HPO3)4 (2) and Fe♯2(HPO3)3 (3) have been synthesized under ionothermal conditions. How the different synthesis parameters, such as the gel concentrations, synthetic times, reaction temperatures and solvents affect the products have been monitored by using high-throughput approaches. Within each type of experiment, relevant products have been investigated. The optimal reaction conditions are obtained from a series of experiments by high-throughput approaches. All the structures are determined by single-crystal X-ray diffraction analysis and further characterized by PXRD, TGA and FTIR analyses. Magnetic study reveals that those three compounds show interesting magnetic behavior at low temperature.

High-Throughput Sequencing: A Roadmap Toward Community Ecology

PubMed Central

Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

2013-01-01

High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

Epigenetics and Epigenomics of Plants.

PubMed

Yadav, Chandra Bhan; Pandey, Garima; Muthamilarasan, Mehanathan; Prasad, Manoj

2018-01-23

The genetic material DNA in association with histone proteins forms the complex structure called chromatin, which is prone to undergo modification through certain epigenetic mechanisms including cytosine DNA methylation, histone modifications, and small RNA-mediated methylation. Alterations in chromatin structure lead to inaccessibility of genomic DNA to various regulatory proteins such as transcription factors, which eventually modulates gene expression. Advancements in high-throughput sequencing technologies have provided the opportunity to study the epigenetic mechanisms at genome-wide levels. Epigenomic studies using high-throughput technologies will widen the understanding of mechanisms as well as functions of regulatory pathways in plant genomes, which will further help in manipulating these pathways using genetic and biochemical approaches. This technology could be a potential research tool for displaying the systematic associations of genetic and epigenetic variations, especially in terms of cytosine methylation onto the genomic region in a specific cell or tissue. A comprehensive study of plant populations to correlate genotype to epigenotype and to phenotype, and also the study of methyl quantitative trait loci (QTL) or epiGWAS, is possible by using high-throughput sequencing methods, which will further accelerate molecular breeding programs for crop improvement. Graphical Abstract.

High performance hybrid magnetic structure for biotechnology applications

DOEpatents

Humphries, David E [El Cerrito, CA; Pollard, Martin J [El Cerrito, CA; Elkin, Christopher J [San Ramon, CA

2009-02-03

The present disclosure provides a high performance hybrid magnetic structure made from a combination of permanent magnets and ferromagnetic pole materials which are assembled in a predetermined array. The hybrid magnetic structure provides means for separation and other biotechnology applications involving holding, manipulation, or separation of magnetic or magnetizable molecular structures and targets. Also disclosed are further improvements to aspects of the hybrid magnetic structure, including additional elements and for adapting the use of the hybrid magnetic structure for use in biotechnology and high throughput processes.

Effect of PS-PVD production throughput on Si nanoparticles for negative electrode of lithium ion batteries

NASA Astrophysics Data System (ADS)

Ohta, R.; Fukada, K.; Tashiro, T.; Dougakiuchi, M.; Kambara, M.

2018-03-01

Silicon nanoparticles (Si-NPs) have been produced by plasma spray physical vapor deposition at throughput as high as 1 kg h-1 (17 g min-1) and the effect on the battery performance is investigated. When the Si powder feed-rate is changed from 1 to 17 g min-1, although the average primary particle size increases to 50 nm, the cycle capacity of the batteries using these Si-NPs is improved slightly owing to their less agglomerated structure. In contrast, when Ni is added to Si feedstock, the cycle capacity is improved at 1 g min-1 due to modified Si-NP structure having SiNi2 interface. Whereas, the batteries with the Si-NP produced at 17 g min-1 shows significant decrease in the cycle capacity because of the excess Ni silicide formation that is resulted from the elevated co-condensation point and the increased reaction area at high throughputs despite the constant Ni concentration in the feedstock.

AR-NE3A, a New Macromolecular Crystallography Beamline for Pharmaceutical Applications at the Photon Factory

NASA Astrophysics Data System (ADS)

Yamada, Yusuke; Hiraki, Masahiko; Sasajima, Kumiko; Matsugaki, Naohiro; Igarashi, Noriyuki; Amano, Yasushi; Warizaya, Masaichi; Sakashita, Hitoshi; Kikuchi, Takashi; Mori, Takeharu; Toyoshima, Akio; Kishimoto, Shunji; Wakatsuki, Soichi

2010-06-01

Recent advances in high-throughput techniques for macromolecular crystallography have highlighted the importance of structure-based drug design (SBDD), and the demand for synchrotron use by pharmaceutical researchers has increased. Thus, in collaboration with Astellas Pharma Inc., we have constructed a new high-throughput macromolecular crystallography beamline, AR-NE3A, which is dedicated to SBDD. At AR-NE3A, a photon flux up to three times higher than those at existing high-throughput beams at the Photon Factory, AR-NW12A and BL-5A, can be realized at the same sample positions. Installed in the experimental hutch are a high-precision diffractometer, fast-readout, high-gain CCD detector, and sample exchange robot capable of handling more than two hundred cryo-cooled samples stored in a Dewar. To facilitate high-throughput data collection required for pharmaceutical research, fully automated data collection and processing systems have been developed. Thus, sample exchange, centering, data collection, and data processing are automatically carried out based on the user's pre-defined schedule. Although Astellas Pharma Inc. has a priority access to AR-NE3A, the remaining beam time is allocated to general academic and other industrial users.

Fabrication of Gold-coated 3-D Woodpile Structures for Mid-IR Thermal Emitters

NASA Astrophysics Data System (ADS)

Li, Shengkai; Moridani, Amir; Kothari, Rohit; Lee, Jae-Hwang; Watkins, James

3-D metallic woodpile nanostructures possess enhancements in thermal radiation that are both wavelength and polarization specific and are promising for thermal-optical devices for various applications including thermal photovoltaics, self-cooling devices, and chemical and bio-sensors. However, current fabrication techniques for such structures are limited by slow speed, small area capability, the need for expensive facilities and, in general, are not suitable for high-throughput mass production. Here we demonstrate a new strategy for the fabrication of 3D metallic woodpile structures. Well-defined TiO2 woodpile structures were fabricated using a layer-by-layer nanoimprint method using TiO2 nanoparticle ink dispersions. The TiO2 woodpile was then coated with a high purity, conformal gold film via reactive deposition in supercritical carbon dioxide. The final gold-coated woodpile structures exhibit strong spectral and polarization specific thermal emission enhancements. The fabrication method demonstrated here is promising for high-throughput, low-cost preparation of 3D metallic woodpile structures and other 3D nanostructures. Center for Hierarchical Manufacturing, NSF.

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

PubMed

Inagaki, Soichi; Henry, Isabelle M; Lieberman, Meric C; Comai, Luca

2015-01-01

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.

A data set from flash X-ray imaging of carboxysomes

NASA Astrophysics Data System (ADS)

Hantke, Max F.; Hasse, Dirk; Ekeberg, Tomas; John, Katja; Svenda, Martin; Loh, Duane; Martin, Andrew V.; Timneanu, Nicusor; Larsson, Daniel S. D.; van der Schot, Gijs; Carlsson, Gunilla H.; Ingelman, Margareta; Andreasson, Jakob; Westphal, Daniel; Iwan, Bianca; Uetrecht, Charlotte; Bielecki, Johan; Liang, Mengning; Stellato, Francesco; Deponte, Daniel P.; Bari, Sadia; Hartmann, Robert; Kimmel, Nils; Kirian, Richard A.; Seibert, M. Marvin; Mühlig, Kerstin; Schorb, Sebastian; Ferguson, Ken; Bostedt, Christoph; Carron, Sebastian; Bozek, John D.; Rolles, Daniel; Rudenko, Artem; Foucar, Lutz; Epp, Sascha W.; Chapman, Henry N.; Barty, Anton; Andersson, Inger; Hajdu, Janos; Maia, Filipe R. N. C.

2016-08-01

Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.

The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

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

Smith, C.A.; Cohen, A.E.

2009-05-26

The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screenedmore » in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.« less

Multilength Scale Patterning of Functional Layers by Roll-to-Roll Ultraviolet-Light-Assisted Nanoimprint Lithography.

PubMed

Leitgeb, Markus; Nees, Dieter; Ruttloff, Stephan; Palfinger, Ursula; Götz, Johannes; Liska, Robert; Belegratis, Maria R; Stadlober, Barbara

2016-05-24

Top-down fabrication of nanostructures with high throughput is still a challenge. We demonstrate the fast (>10 m/min) and continuous fabrication of multilength scale structures by roll-to-roll UV-nanoimprint lithography on a 250 mm wide web. The large-area nanopatterning is enabled by a multicomponent UV-curable resist system (JRcure) with viscous, mechanical, and surface properties that are tunable over a wide range to either allow for usage as polymer stamp material or as imprint resist. The adjustable elasticity and surface chemistry of the resist system enable multistep self-replication of structured resist layers. Decisive for defect-free UV-nanoimprinting in roll-to-roll is the minimization of the surface energies of stamp and resist, and the stepwise reduction of the stiffness from one layer to the next is essential for optimizing the reproduction fidelity especially for nanoscale features. Accordingly, we demonstrate the continuous replication of 3D nanostructures and the high-throughput fabrication of multilength scale resist structures resulting in flexible polyethylenetherephtalate film rolls with superhydrophobic properties. Moreover, a water-soluble UV-imprint resist (JRlift) is introduced that enables residue-free nanoimprinting in roll-to-roll. Thereby we could demonstrate high-throughput fabrication of metallic patterns with only 200 nm line width.

High throughput imaging cytometer with acoustic focussing.

PubMed

Zmijan, Robert; Jonnalagadda, Umesh S; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn; Glynne-Jones, Peter

2015-10-31

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

Advancements in zebrafish applications for 21st century toxicology.

PubMed

Garcia, Gloria R; Noyes, Pamela D; Tanguay, Robert L

2016-05-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. Copyright © 2016 Elsevier Inc. All rights reserved.

Advancements in zebrafish applications for 21st century toxicology

PubMed Central

Garcia, Gloria R.; Noyes, Pamela D.; Tanguay, Robert L.

2016-01-01

The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. PMID:27016469

Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies

PubMed Central

Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G.V.T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.

2014-01-01

In this chapter, we concentrate on the production of high quality protein samples for NMR studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium, and outline our high-throughput strategies for producing high quality protein samples for nuclear magnetic resonance (NMR) studies. Our strategy is based on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6X-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (> 97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5,000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this paper describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research. PMID:21371586

High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

NASA Astrophysics Data System (ADS)

Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

2015-12-01

The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

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.

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

High-throughput combinatorial chemical bath deposition: The case of doping Cu (In, Ga) Se film with antimony

NASA Astrophysics Data System (ADS)

Yan, Zongkai; Zhang, Xiaokun; Li, Guang; Cui, Yuxing; Jiang, Zhaolian; Liu, Wen; Peng, Zhi; Xiang, Yong

2018-01-01

The conventional methods for designing and preparing thin film based on wet process remain a challenge due to disadvantages such as time-consuming and ineffective, which hinders the development of novel materials. Herein, we present a high-throughput combinatorial technique for continuous thin film preparation relied on chemical bath deposition (CBD). The method is ideally used to prepare high-throughput combinatorial material library with low decomposition temperatures and high water- or oxygen-sensitivity at relatively high-temperature. To check this system, a Cu(In, Ga)Se (CIGS) thin films library doped with 0-19.04 at.% of antimony (Sb) was taken as an example to evaluate the regulation of varying Sb doping concentration on the grain growth, structure, morphology and electrical properties of CIGS thin film systemically. Combined with the Energy Dispersive Spectrometer (EDS), X-ray Photoelectron Spectroscopy (XPS), automated X-ray Diffraction (XRD) for rapid screening and Localized Electrochemical Impedance Spectroscopy (LEIS), it was confirmed that this combinatorial high-throughput system could be used to identify the composition with the optimal grain orientation growth, microstructure and electrical properties systematically, through accurately monitoring the doping content and material composition. According to the characterization results, a Sb2Se3 quasi-liquid phase promoted CIGS film-growth model has been put forward. In addition to CIGS thin film reported here, the combinatorial CBD also could be applied to the high-throughput screening of other sulfide thin film material systems.

Neutron reflecting supermirror structure

DOEpatents

Wood, J.L.

1992-12-01

An improved neutron reflecting supermirror structure comprising a plurality of stacked sets of bilayers of neutron reflecting materials. The improved neutron reflecting supermirror structure is adapted to provide extremely good performance at high incidence angles, i.e. up to four time the critical angle of standard neutron mirror structures. The reflection of neutrons striking the supermirror structure at a high critical angle provides enhanced neutron throughput, and hence more efficient and economical use of neutron sources. 2 figs.

Neutron reflecting supermirror structure

DOEpatents

Wood, James L.

1992-01-01

An improved neutron reflecting supermirror structure comprising a plurality of stacked sets of bilayers of neutron reflecting materials. The improved neutron reflecting supermirror structure is adapted to provide extremely good performance at high incidence angles, i.e. up to four time the critical angle of standard neutron mirror structures. The reflection of neutrons striking the supermirror structure at a high critical angle provides enhanced neutron throughput, and hence more efficient and economical use of neutron sources.

Turbocharged molecular discovery of OLED emitters: from high-throughput quantum simulation to highly efficient TADF devices

NASA Astrophysics Data System (ADS)

Gómez-Bombarelli, Rafael; Aguilera-Iparraguirre, Jorge; Hirzel, Timothy D.; Ha, Dong-Gwang; Einzinger, Markus; Wu, Tony; Baldo, Marc A.; Aspuru-Guzik, Alán.

2016-09-01

Discovering new OLED emitters requires many experiments to synthesize candidates and test performance in devices. Large scale computer simulation can greatly speed this search process but the problem remains challenging enough that brute force application of massive computing power is not enough to successfully identify novel structures. We report a successful High Throughput Virtual Screening study that leveraged a range of methods to optimize the search process. The generation of candidate structures was constrained to contain combinatorial explosion. Simulations were tuned to the specific problem and calibrated with experimental results. Experimentalists and theorists actively collaborated such that experimental feedback was regularly utilized to update and shape the computational search. Supervised machine learning methods prioritized candidate structures prior to quantum chemistry simulation to prevent wasting compute on likely poor performers. With this combination of techniques, each multiplying the strength of the search, this effort managed to navigate an area of molecular space and identify hundreds of promising OLED candidate structures. An experimentally validated selection of this set shows emitters with external quantum efficiencies as high as 22%.

Custom Super-Resolution Microscope for the Structural Analysis of Nanostructures

DTIC Science & Technology

2018-05-29

research community. As part of our validation of the new design approach, we performed two - color imaging of pairs of adjacent oligo probes hybridized...nanostructures and biological targets. Our microscope features a large field of view and custom optics that facilitate 3D imaging and enhanced contrast in...our imaging throughput by creating two microscopy platforms for high-throughput, super-resolution materials characterization, with the AO set-up being

Quality control methodology for high-throughput protein-protein interaction screening.

PubMed

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

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

Optimal forwarding ratio on dynamical networks with heterogeneous mobility

NASA Astrophysics Data System (ADS)

Gan, Yu; Tang, Ming; Yang, Hanxin

2013-05-01

Since the discovery of non-Poisson statistics of human mobility trajectories, more attention has been paid to understand the role of these patterns in different dynamics. In this study, we first introduce the heterogeneous mobility of mobile agents into dynamical networks, and then investigate packet forwarding strategy on the heterogeneous dynamical networks. We find that the faster speed and the higher proportion of high-speed agents can enhance the network throughput and reduce the mean traveling time in random forwarding. A hierarchical structure in the dependence of high-speed is observed: the network throughput remains unchanged at small and large high-speed value. It is also interesting to find that a slightly preferential forwarding to high-speed agents can maximize the network capacity. Through theoretical analysis and numerical simulations, we show that the optimal forwarding ratio stems from the local structural heterogeneity of low-speed agents.

High-throughput materials discovery and development: breakthroughs and challenges in the mapping of the materials genome

NASA Astrophysics Data System (ADS)

Buongiorno Nardelli, Marco

High-Throughput Quantum-Mechanics computation of materials properties by ab initio methods has become the foundation of an effective approach to materials design, discovery and characterization. This data driven approach to materials science currently presents the most promising path to the development of advanced technological materials that could solve or mitigate important social and economic challenges of the 21st century. In particular, the rapid proliferation of computational data on materials properties presents the possibility to complement and extend materials property databases where the experimental data is lacking and difficult to obtain. Enhanced repositories such as AFLOWLIB open novel opportunities for structure discovery and optimization, including uncovering of unsuspected compounds, metastable structures and correlations between various properties. The practical realization of these opportunities depends almost exclusively on the the design of efficient algorithms for electronic structure simulations of realistic material systems beyond the limitations of the current standard theories. In this talk, I will review recent progress in theoretical and computational tools, and in particular, discuss the development and validation of novel functionals within Density Functional Theory and of local basis representations for effective ab-initio tight-binding schemes. Marco Buongiorno Nardelli is a pioneer in the development of computational platforms for theory/data/applications integration rooted in his profound and extensive expertise in the design of electronic structure codes and in his vision for sustainable and innovative software development for high-performance materials simulations. His research activities range from the design and discovery of novel materials for 21st century applications in renewable energy, environment, nano-electronics and devices, the development of advanced electronic structure theories and high-throughput techniques in materials genomics and computational materials design, to an active role as community scientific software developer (QUANTUM ESPRESSO, WanT, AFLOWpi)

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.

AR-NE3A, a New Macromolecular Crystallography Beamline for Pharmaceutical Applications at the Photon Factory

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

Yamada, Yusuke; Hiraki, Masahiko; Sasajima, Kumiko

2010-06-23

Recent advances in high-throughput techniques for macromolecular crystallography have highlighted the importance of structure-based drug design (SBDD), and the demand for synchrotron use by pharmaceutical researchers has increased. Thus, in collaboration with Astellas Pharma Inc., we have constructed a new high-throughput macromolecular crystallography beamline, AR-NE3A, which is dedicated to SBDD. At AR-NE3A, a photon flux up to three times higher than those at existing high-throughput beams at the Photon Factory, AR-NW12A and BL-5A, can be realized at the same sample positions. Installed in the experimental hutch are a high-precision diffractometer, fast-readout, high-gain CCD detector, and sample exchange robot capable ofmore » handling more than two hundred cryo-cooled samples stored in a Dewar. To facilitate high-throughput data collection required for pharmaceutical research, fully automated data collection and processing systems have been developed. Thus, sample exchange, centering, data collection, and data processing are automatically carried out based on the user's pre-defined schedule. Although Astellas Pharma Inc. has a priority access to AR-NE3A, the remaining beam time is allocated to general academic and other industrial users.« less

High-throughput analysis of T-DNA location and structure using sequence capture

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

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

High-throughput analysis of T-DNA location and structure using sequence capture

DOE PAGES

Inagaki, Soichi; Henry, Isabelle M.; Lieberman, Meric C.; ...

2015-10-07

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously,more » using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. As a result, our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.« less

An Extensible "SCHEMA-LESS" Database Framework for Managing High-Throughput Semi-Structured Documents

NASA Technical Reports Server (NTRS)

Maluf, David A.; Tran, Peter B.

2003-01-01

Object-Relational database management system is an integrated hybrid cooperative approach to combine the best practices of both the relational model utilizing SQL queries and the object-oriented, semantic paradigm for supporting complex data creation. In this paper, a highly scalable, information on demand database framework, called NETMARK, is introduced. NETMARK takes advantages of the Oracle 8i object-relational database using physical addresses data types for very efficient keyword search of records spanning across both context and content. NETMARK was originally developed in early 2000 as a research and development prototype to solve the vast amounts of unstructured and semistructured documents existing within NASA enterprises. Today, NETMARK is a flexible, high-throughput open database framework for managing, storing, and searching unstructured or semi-structured arbitrary hierarchal models, such as XML and HTML.

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

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

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

Using high-throughput literature mining to support read-across predictions of skin sensitization (WC)

EPA Science Inventory

Read-across predictions require high quality measured data for source analogues. These data are typically retrieved from structured databases, but biomedical literature data are often untapped because current literature mining approaches are resource intensive. Our high-throughpu...

Protein Structures Revealed at Record Pace

ScienceCinema

Hura, Greg

2017-12-11

The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

Protein Structures Revealed at Record Pace

ScienceCinema

Greg Hura

2017-12-09

The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

High throughput imaging cytometer with acoustic focussing† †Electronic supplementary information (ESI) available: High throughput imaging cytometer with acoustic focussing. See DOI: 10.1039/c5ra19497k Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

PubMed Central

Zmijan, Robert; Jonnalagadda, Umesh S.; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn

2015-01-01

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint. PMID:29456838

Towards High-Throughput, Simultaneous Characterization of Thermal and Thermoelectric Properties

NASA Astrophysics Data System (ADS)

Miers, Collier Stephen

The extension of thermoelectric generators to more general markets requires that the devices be affordable and practical (low $/Watt) to implement. A key challenge in this pursuit is the quick and accurate characterization of thermoelectric materials, which will allow researchers to tune and modify the material properties quickly. The goal of this thesis is to design and fabricate a high-throughput characterization system for the simultaneous characterization of thermal, electrical, and thermoelectric properties for device scale material samples. The measurement methodology presented in this thesis combines a custom designed measurement system created specifically for high-throughput testing with a novel device structure that permits simultaneous characterization of the material properties. The measurement system is based upon the 3o method for thermal conductivity measurements, with the addition of electrodes and voltage probes to measure the electrical conductivity and Seebeck coefficient. A device designed and optimized to permit the rapid characterization of thermoelectric materials is also presented. This structure is optimized to ensure 1D heat transfer within the sample, thus permitting rapid data analysis and fitting using a MATLAB script. Verification of the thermal portion of the system is presented using fused silica and sapphire materials for benchmarking. The fused silica samples yielded a thermal conductivity of 1.21 W/(m K), while a thermal conductivity of 31.2 W/(m K) was measured for the sapphire samples. The device and measurement system designed and developed in this thesis provide insight and serve as a foundation for the development of high throughput, simultaneous measurement platforms.

New Era of Studying RNA Secondary Structure and Its Influence on Gene Regulation in Plants.

PubMed

Yang, Xiaofei; Yang, Minglei; Deng, Hongjing; Ding, Yiliang

2018-01-01

The dynamic structure of RNA plays a central role in post-transcriptional regulation of gene expression such as RNA maturation, degradation, and translation. With the rise of next-generation sequencing, the study of RNA structure has been transformed from in vitro low-throughput RNA structure probing methods to in vivo high-throughput RNA structure profiling. The development of these methods enables incremental studies on the function of RNA structure to be performed, revealing new insights of novel regulatory mechanisms of RNA structure in plants. Genome-wide scale RNA structure profiling allows us to investigate general RNA structural features over 10s of 1000s of mRNAs and to compare RNA structuromes between plant species. Here, we provide a comprehensive and up-to-date overview of: (i) RNA structure probing methods; (ii) the biological functions of RNA structure; (iii) genome-wide RNA structural features corresponding to their regulatory mechanisms; and (iv) RNA structurome evolution in plants.

High-Throughput Characterization of Vapor-Deposited Organic Glasses

NASA Astrophysics Data System (ADS)

Dalal, Shakeel S.

Glasses are non-equilibrium materials which on short timescales behave like solids, and on long timescales betray their liquid-like structure. The most common way of preparing a glass is to cool the liquid faster than it can structurally rearrange. Until recently, most preparation schemes for a glass were considered to result in materials with undifferentiable structure and properties. This thesis utilizes a particular preparation method, physical vapor deposition, in order to prepare glasses of organic molecules with properties otherwise considered to be unobtainable. The glasses are characterized using spectroscopic ellipsometry, both as a dilatometric technique and as a reporter of molecular packing. The results reported here develop ellipsometry as a dilatometric technique on a pair of model glass formers, alpha,alpha,beta-trisnaphthylbenzene and indomethacin. It is found that the molecular orientation, as measured by birefringence, can be tuned by changing the substrate temperature during the deposition. In order to efficiently characterize the properties of vapor-deposited indomethacin as a function of substrate temperature, a high-throughput method is developed to capture the entire interesting range of substrate temperatures in just a few experiments. This high-throughput method is then leveraged to describe molecular mobility in vapor-deposited indomethacin. It is also used to demonstrate that the behavior of organic semiconducting molecules agrees with indomethacin quantitatively, and this agreement has implications for emerging technologies such as light-emitting diodes, photovoltaics and thin-film transistors made from organic molecules.

Correlating Oxygen Evolution Catalysts Activity and Electronic Structure by a High-Throughput Investigation of Ni1-y-zFeyCrzOx

PubMed Central

Schwanke, Christoph; Stein, Helge Sören; Xi, Lifei; Sliozberg, Kirill; Schuhmann, Wolfgang; Ludwig, Alfred; Lange, Kathrin M.

2017-01-01

High-throughput characterization by soft X-ray absorption spectroscopy (XAS) and electrochemical characterization is used to establish a correlation between electronic structure and catalytic activity of oxygen evolution reaction (OER) catalysts. As a model system a quasi-ternary materials library of Ni1-y-zFeyCrzOx was synthesized by combinatorial reactive magnetron sputtering, characterized by XAS, and an automated scanning droplet cell. The presence of Cr was found to increase the OER activity in the investigated compositional range. The electronic structure of NiII and CrIII remains unchanged over the investigated composition spread. At the Fe L-edge a linear combination of two spectra was observed. These spectra were assigned to FeIII in Oh symmetry and FeIII in Td symmetry. The ratio of FeIII Oh to FeIII Td increases with the amount of Cr and a correlation between the presence of the FeIII Oh and a high OER activity is found. PMID:28287134

Correlating Oxygen Evolution Catalysts Activity and Electronic Structure by a High-Throughput Investigation of Ni1-y-zFeyCrzOx

NASA Astrophysics Data System (ADS)

Schwanke, Christoph; Stein, Helge Sören; Xi, Lifei; Sliozberg, Kirill; Schuhmann, Wolfgang; Ludwig, Alfred; Lange, Kathrin M.

2017-03-01

High-throughput characterization by soft X-ray absorption spectroscopy (XAS) and electrochemical characterization is used to establish a correlation between electronic structure and catalytic activity of oxygen evolution reaction (OER) catalysts. As a model system a quasi-ternary materials library of Ni1-y-zFeyCrzOx was synthesized by combinatorial reactive magnetron sputtering, characterized by XAS, and an automated scanning droplet cell. The presence of Cr was found to increase the OER activity in the investigated compositional range. The electronic structure of NiII and CrIII remains unchanged over the investigated composition spread. At the Fe L-edge a linear combination of two spectra was observed. These spectra were assigned to FeIII in Oh symmetry and FeIII in Td symmetry. The ratio of FeIII Oh to FeIII Td increases with the amount of Cr and a correlation between the presence of the FeIII Oh and a high OER activity is found.

Semantically enabled and statistically supported biological hypothesis testing with tissue microarray databases

PubMed Central

2011-01-01

Background Although many biological databases are applying semantic web technologies, meaningful biological hypothesis testing cannot be easily achieved. Database-driven high throughput genomic hypothesis testing requires both of the capabilities of obtaining semantically relevant experimental data and of performing relevant statistical testing for the retrieved data. Tissue Microarray (TMA) data are semantically rich and contains many biologically important hypotheses waiting for high throughput conclusions. Methods An application-specific ontology was developed for managing TMA and DNA microarray databases by semantic web technologies. Data were represented as Resource Description Framework (RDF) according to the framework of the ontology. Applications for hypothesis testing (Xperanto-RDF) for TMA data were designed and implemented by (1) formulating the syntactic and semantic structures of the hypotheses derived from TMA experiments, (2) formulating SPARQLs to reflect the semantic structures of the hypotheses, and (3) performing statistical test with the result sets returned by the SPARQLs. Results When a user designs a hypothesis in Xperanto-RDF and submits it, the hypothesis can be tested against TMA experimental data stored in Xperanto-RDF. When we evaluated four previously validated hypotheses as an illustration, all the hypotheses were supported by Xperanto-RDF. Conclusions We demonstrated the utility of high throughput biological hypothesis testing. We believe that preliminary investigation before performing highly controlled experiment can be benefited. PMID:21342584

Probabilistic Assessment of High-Throughput Wireless Sensor Networks

PubMed Central

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

2016-01-01

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

Mass spectrometry-driven drug discovery for development of herbal medicine.

PubMed

Zhang, Aihua; Sun, Hui; Wang, Xijun

2018-05-01

Herbal medicine (HM) has made a major contribution to the drug discovery process with regard to identifying products compounds. Currently, more attention has been focused on drug discovery from natural compounds of HM. Despite the rapid advancement of modern analytical techniques, drug discovery is still a difficult and lengthy process. Fortunately, mass spectrometry (MS) can provide us with useful structural information for drug discovery, has been recognized as a sensitive, rapid, and high-throughput technology for advancing drug discovery from HM in the post-genomic era. It is essential to develop an efficient, high-quality, high-throughput screening method integrated with an MS platform for early screening of candidate drug molecules from natural products. We have developed a new chinmedomics strategy reliant on MS that is capable of capturing the candidate molecules, facilitating their identification of novel chemical structures in the early phase; chinmedomics-guided natural product discovery based on MS may provide an effective tool that addresses challenges in early screening of effective constituents of herbs against disease. This critical review covers the use of MS with related techniques and methodologies for natural product discovery, biomarker identification, and determination of mechanisms of action. It also highlights high-throughput chinmedomics screening methods suitable for lead compound discovery illustrated by recent successes. © 2016 Wiley Periodicals, Inc.

High-Throughput Image Analysis of Fibrillar Materials: A Case Study on Polymer Nanofiber Packing, Alignment, and Defects in Organic Field Effect Transistors.

PubMed

Persson, Nils E; Rafshoon, Joshua; Naghshpour, Kaylie; Fast, Tony; Chu, Ping-Hsun; McBride, Michael; Risteen, Bailey; Grover, Martha; Reichmanis, Elsa

2017-10-18

High-throughput discovery of process-structure-property relationships in materials through an informatics-enabled empirical approach is an increasingly utilized technique in materials research due to the rapidly expanding availability of data. Here, process-structure-property relationships are extracted for the nucleation, growth, and deposition of semiconducting poly(3-hexylthiophene) (P3HT) nanofibers used in organic field effect transistors, via high-throughput image analysis. This study is performed using an automated image analysis pipeline combining existing open-source software and new algorithms, enabling the rapid evaluation of structural metrics for images of fibrillar materials, including local orientational order, fiber length density, and fiber length distributions. We observe that microfluidic processing leads to fibers that pack with unusually high density, while sonication yields fibers that pack sparsely with low alignment. This is attributed to differences in their crystallization mechanisms. P3HT nanofiber packing during thin film deposition exhibits behavior suggesting that fibers are confined to packing in two-dimensional layers. We find that fiber alignment, a feature correlated with charge carrier mobility, is driven by increasing fiber length, and that shorter fibers tend to segregate to the buried dielectric interface during deposition, creating potentially performance-limiting defects in alignment. Another barrier to perfect alignment is the curvature of P3HT fibers; we propose a mechanistic simulation of fiber growth that reconciles both this curvature and the log-normal distribution of fiber lengths inherent to the fiber populations under consideration.

High-throughput screening for thermoelectric sulphides by using crystal structure features as descriptors

NASA Astrophysics Data System (ADS)

Zhang, Ruizhi; Du, Baoli; Chen, Kan; Reece, Mike; Materials Research Insititute Team

With the increasing computational power and reliable databases, high-throughput screening is playing a more and more important role in the search of new thermoelectric materials. Rather than the well established density functional theory (DFT) calculation based methods, we propose an alternative approach to screen for new TE materials: using crystal structural features as 'descriptors'. We show that a non-distorted transition metal sulphide polyhedral network can be a good descriptor for high power factor according to crystal filed theory. By using Cu/S containing compounds as an example, 1600+ Cu/S containing entries in the Inorganic Crystal Structure Database (ICSD) were screened, and of those 84 phases are identified as promising thermoelectric materials. The screening results are validated by both electronic structure calculations and experimental results from the literature. We also fabricated some new compounds to test our screening results. Another advantage of using crystal structure features as descriptors is that we can easily establish structural relationships between the identified phases. Based on this, two material design approaches are discussed: 1) High-pressure synthesis of metastable phase; 2) In-situ 2-phase composites with coherent interface. This work was supported by a Marie Curie International Incoming Fellowship of the European Community Human Potential Program.

Predicting hepatotoxicity using ToxCast in vitro bioactivity and chemical structure

EPA Science Inventory

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

What can one learn about material structure given a single first-principles calculation?

NASA Astrophysics Data System (ADS)

Rajen, Nicholas; Coh, Sinisa

2018-05-01

We extract a variable X from electron orbitals Ψn k and energies En k in the parent high-symmetry structure of a wide range of complex oxides: perovskites, rutiles, pyrochlores, and cristobalites. Even though calculation was done only in the parent structure, with no distortions, we show that X dictates material's true ground-state structure. We propose using Wannier functions to extract concealed variables such as X both for material structure prediction and for high-throughput approaches.

High-throughput search of ternary chalcogenides for p-type transparent electrodes

PubMed Central

Shi, Jingming; Cerqueira, Tiago F. T.; Cui, Wenwen; Nogueira, Fernando; Botti, Silvana; Marques, Miguel A. L.

2017-01-01

Delafossite crystals are fascinating ternary oxides that have demonstrated transparent conductivity and ambipolar doping. Here we use a high-throughput approach based on density functional theory to find delafossite and related layered phases of composition ABX2, where A and B are elements of the periodic table, and X is a chalcogen (O, S, Se, and Te). From the 15 624 compounds studied in the trigonal delafossite prototype structure, 285 are within 50 meV/atom from the convex hull of stability. These compounds are further investigated using global structural prediction methods to obtain their lowest-energy crystal structure. We find 79 systems not present in the materials project database that are thermodynamically stable and crystallize in the delafossite or in closely related structures. These novel phases are then characterized by calculating their band gaps and hole effective masses. This characterization unveils a large diversity of properties, ranging from normal metals, magnetic metals, and some candidate compounds for p-type transparent electrodes. PMID:28266587

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. Copyright © 2015 Zhou et al.

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

High-throughput metagenomic technologies for complex microbial community analysis. Open and closed formats

DOE PAGES

Zhou, Jizhong; He, Zhili; Yang, Yunfeng; ...

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 andmore » 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.« less

Depth-resolved incoherent and coherent wide-field high-content imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

So, Peter T.

2016-03-01

Recent advances in depth-resolved wide-field imaging technique has enabled many high throughput applications in biology and medicine. Depth resolved imaging of incoherent signals can be readily accomplished with structured light illumination or nonlinear temporal focusing. The integration of these high throughput systems with novel spectroscopic resolving elements further enable high-content information extraction. We will introduce a novel near common-path interferometer and demonstrate its uses in toxicology and cancer biology applications. The extension of incoherent depth-resolved wide-field imaging to coherent modality is non-trivial. Here, we will cover recent advances in wide-field 3D resolved mapping of refractive index, absorbance, and vibronic components in biological specimens.

Structural Genomics and Drug Discovery for Infectious Diseases

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

Anderson, W.F.

The application of structural genomics methods and approaches to proteins from organisms causing infectious diseases is making available the three dimensional structures of many proteins that are potential drug targets and laying the groundwork for structure aided drug discovery efforts. There are a number of structural genomics projects with a focus on pathogens that have been initiated worldwide. The Center for Structural Genomics of Infectious Diseases (CSGID) was recently established to apply state-of-the-art high throughput structural biology technologies to the characterization of proteins from the National Institute for Allergy and Infectious Diseases (NIAID) category A-C pathogens and organisms causing emerging,more » or re-emerging infectious diseases. The target selection process emphasizes potential biomedical benefits. Selected proteins include known drug targets and their homologs, essential enzymes, virulence factors and vaccine candidates. The Center also provides a structure determination service for the infectious disease scientific community. The ultimate goal is to generate a library of structures that are available to the scientific community and can serve as a starting point for further research and structure aided drug discovery for infectious diseases. To achieve this goal, the CSGID will determine protein crystal structures of 400 proteins and protein-ligand complexes using proven, rapid, highly integrated, and cost-effective methods for such determination, primarily by X-ray crystallography. High throughput crystallographic structure determination is greatly aided by frequent, convenient access to high-performance beamlines at third-generation synchrotron X-ray sources.« less

AELAS: Automatic ELAStic property derivations via high-throughput first-principles computation

NASA Astrophysics Data System (ADS)

Zhang, S. H.; Zhang, R. F.

2017-11-01

The elastic properties are fundamental and important for crystalline materials as they relate to other mechanical properties, various thermodynamic qualities as well as some critical physical properties. However, a complete set of experimentally determined elastic properties is only available for a small subset of known materials, and an automatic scheme for the derivations of elastic properties that is adapted to high-throughput computation is much demanding. In this paper, we present the AELAS code, an automated program for calculating second-order elastic constants of both two-dimensional and three-dimensional single crystal materials with any symmetry, which is designed mainly for high-throughput first-principles computation. Other derivations of general elastic properties such as Young's, bulk and shear moduli as well as Poisson's ratio of polycrystal materials, Pugh ratio, Cauchy pressure, elastic anisotropy and elastic stability criterion, are also implemented in this code. The implementation of the code has been critically validated by a lot of evaluations and tests on a broad class of materials including two-dimensional and three-dimensional materials, providing its efficiency and capability for high-throughput screening of specific materials with targeted mechanical properties. Program Files doi:http://dx.doi.org/10.17632/f8fwg4j9tw.1 Licensing provisions: BSD 3-Clause Programming language: Fortran Nature of problem: To automate the calculations of second-order elastic constants and the derivations of other elastic properties for two-dimensional and three-dimensional materials with any symmetry via high-throughput first-principles computation. Solution method: The space-group number is firstly determined by the SPGLIB code [1] and the structure is then redefined to unit cell with IEEE-format [2]. Secondly, based on the determined space group number, a set of distortion modes is automatically specified and the distorted structure files are generated. Afterwards, the total energy for each distorted structure is calculated by the first-principles codes, e.g. VASP [3]. Finally, the second-order elastic constants are determined from the quadratic coefficients of the polynomial fitting of the energies vs strain relationships and other elastic properties are accordingly derived. References [1] http://atztogo.github.io/spglib/. [2] A. Meitzler, H.F. Tiersten, A.W. Warner, D. Berlincourt, G.A. Couqin, F.S. Welsh III, IEEE standard on piezoelectricity, Society, 1988. [3] G. Kresse, J. Furthmüller, Phys. Rev. B 54 (1996) 11169.

Discovery of a novel general anesthetic chemotype using high-throughput screening.

PubMed

McKinstry-Wu, Andrew R; Bu, Weiming; Rai, Ganesha; Lea, Wendy A; Weiser, Brian P; Liang, David F; Simeonov, Anton; Jadhav, Ajit; Maloney, David J; Eckenhoff, Roderic G

2015-02-01

The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

76 FR 5182 - Center for Scientific Review; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-28

... Scientific Review Special Emphasis Panel, PAR-10-074: Technology Development for High-Throughput Structural...: Center for Scientific Review Special Emphasis Panel, Risk Prevention and Intervention Addictions...

ClusCo: clustering and comparison of protein models.

PubMed

Jamroz, Michal; Kolinski, Andrzej

2013-02-22

The development, optimization and validation of protein modeling methods require efficient tools for structural comparison. Frequently, a large number of models need to be compared with the target native structure. The main reason for the development of Clusco software was to create a high-throughput tool for all-versus-all comparison, because calculating similarity matrix is the one of the bottlenecks in the protein modeling pipeline. Clusco is fast and easy-to-use software for high-throughput comparison of protein models with different similarity measures (cRMSD, dRMSD, GDT_TS, TM-Score, MaxSub, Contact Map Overlap) and clustering of the comparison results with standard methods: K-means Clustering or Hierarchical Agglomerative Clustering. The application was highly optimized and written in C/C++, including the code for parallel execution on CPU and GPU, which resulted in a significant speedup over similar clustering and scoring computation programs.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

bcl::Cluster : A method for clustering biological molecules coupled with visualization in the Pymol Molecular Graphics System.

PubMed

Alexander, Nathan; Woetzel, Nils; Meiler, Jens

2011-02-01

Clustering algorithms are used as data analysis tools in a wide variety of applications in Biology. Clustering has become especially important in protein structure prediction and virtual high throughput screening methods. In protein structure prediction, clustering is used to structure the conformational space of thousands of protein models. In virtual high throughput screening, databases with millions of drug-like molecules are organized by structural similarity, e.g. common scaffolds. The tree-like dendrogram structure obtained from hierarchical clustering can provide a qualitative overview of the results, which is important for focusing detailed analysis. However, in practice it is difficult to relate specific components of the dendrogram directly back to the objects of which it is comprised and to display all desired information within the two dimensions of the dendrogram. The current work presents a hierarchical agglomerative clustering method termed bcl::Cluster. bcl::Cluster utilizes the Pymol Molecular Graphics System to graphically depict dendrograms in three dimensions. This allows simultaneous display of relevant biological molecules as well as additional information about the clusters and the members comprising them.

High-throughput interpretation of gene structure changes in human and nonhuman resequencing data, using ACE

USDA-ARS?s Scientific Manuscript database

We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE (“Assessing Changes to Exons”) converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detect...

On the possibility of using polycrystalline material in the development of structure-based generic assays

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

Allaire, Marc, E-mail: allaire@bnl.gov; Moiseeva, Natalia; Botez, Cristian E.

The correlation coefficients calculated between raw powder diffraction profiles can be used to identify ligand-bound/unbound states of lysozyme. The discovery of ligands that bind specifically to a targeted protein benefits from the development of generic assays for high-throughput screening of a library of chemicals. Protein powder diffraction (PPD) has been proposed as a potential method for use as a structure-based assay for high-throughput screening applications. Building on this effort, powder samples of bound/unbound states of soluble hen-egg white lysozyme precipitated with sodium chloride were compared. The correlation coefficients calculated between the raw diffraction profiles were consistent with the known bindingmore » properties of the ligands and suggested that the PPD approach can be used even prior to a full description using stereochemically restrained Rietveld refinement.« less

Combinatorial and High Throughput Discovery of High Temperature Piezoelectric Ceramics

DTIC Science & Technology

2011-10-10

the known candidate piezoelectric ferroelectric perovskites. Unlike most computational studies on crystal chemistry, where the starting point is some...studies on crystal chemistry, where the starting point is some form of electronic structure calculation, we use a data driven approach to initiate our...experimental measurements reported in the literature. Given that our models are based solely on crystal and electronic structure data and did not

ToxRefDB: Classifying ToxCast™ Phase I Chemicals Utilizing Structured Toxicity Information

EPA Science Inventory

There is an essential need for highly detailed chemicals classifications within the ToxCast™ research program. In order to develop predictive models and biological signatures utilizing high-throughput screening (HTS) and in vitro genomic data, relevant endpoints and toxicities m...

High-throughput SRCD using multi-well plates and its applications

NASA Astrophysics Data System (ADS)

Hussain, Rohanah; Jávorfi, Tamás; Rudd, Timothy R.; Siligardi, Giuliano

2016-12-01

The sample compartment for high-throughput synchrotron radiation circular dichroism (HT-SRCD) has been developed to satisfy an increased demand of protein characterisation in terms of folding and binding interaction properties not only in the traditional field of structural biology but also in the growing research area of material science with the potential to save time by 80%. As the understanding of protein behaviour in different solvent environments has increased dramatically the development of novel functions such as recombinant proteins modified to have different functions from harvesting solar energy to metabolonics for cleaning heavy and metal and organic molecule pollutions, there is a need to characterise speedily these system.

Entropy as a Gene-Like Performance Indicator Promoting Thermoelectric Materials.

PubMed

Liu, Ruiheng; Chen, Hongyi; Zhao, Kunpeng; Qin, Yuting; Jiang, Binbin; Zhang, Tiansong; Sha, Gang; Shi, Xun; Uher, Ctirad; Zhang, Wenqing; Chen, Lidong

2017-10-01

High-throughput explorations of novel thermoelectric materials based on the Materials Genome Initiative paradigm only focus on digging into the structure-property space using nonglobal indicators to design materials with tunable electrical and thermal transport properties. As the genomic units, following the biogene tradition, such indicators include localized crystal structural blocks in real space or band degeneracy at certain points in reciprocal space. However, this nonglobal approach does not consider how real materials differentiate from others. Here, this study successfully develops a strategy of using entropy as the global gene-like performance indicator that shows how multicomponent thermoelectric materials with high entropy can be designed via a high-throughput screening method. Optimizing entropy works as an effective guide to greatly improve the thermoelectric performance through either a significantly depressed lattice thermal conductivity down to its theoretical minimum value and/or via enhancing the crystal structure symmetry to yield large Seebeck coefficients. The entropy engineering using multicomponent crystal structures or other possible techniques provides a new avenue for an improvement of the thermoelectric performance beyond the current methods and approaches. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Software automation tools for increased throughput metabolic soft-spot identification in early drug discovery.

PubMed

Zelesky, Veronica; Schneider, Richard; Janiszewski, John; Zamora, Ismael; Ferguson, James; Troutman, Matthew

2013-05-01

The ability to supplement high-throughput metabolic clearance data with structural information defining the site of metabolism should allow design teams to streamline their synthetic decisions. However, broad application of metabolite identification in early drug discovery has been limited, largely due to the time required for data review and structural assignment. The advent of mass defect filtering and its application toward metabolite scouting paved the way for the development of software automation tools capable of rapidly identifying drug-related material in complex biological matrices. Two semi-automated commercial software applications, MetabolitePilot™ and Mass-MetaSite™, were evaluated to assess the relative speed and accuracy of structural assignments using data generated on a high-resolution MS platform. Review of these applications has demonstrated their utility in providing accurate results in a time-efficient manner, leading to acceleration of metabolite identification initiatives while highlighting the continued need for biotransformation expertise in the interpretation of more complex metabolic reactions.

On-the-fly machine-learning for high-throughput experiments: search for rare-earth-free permanent magnets

PubMed Central

Kusne, Aaron Gilad; Gao, Tieren; Mehta, Apurva; Ke, Liqin; Nguyen, Manh Cuong; Ho, Kai-Ming; Antropov, Vladimir; Wang, Cai-Zhuang; Kramer, Matthew J.; Long, Christian; Takeuchi, Ichiro

2014-01-01

Advanced materials characterization techniques with ever-growing data acquisition speed and storage capabilities represent a challenge in modern materials science, and new procedures to quickly assess and analyze the data are needed. Machine learning approaches are effective in reducing the complexity of data and rapidly homing in on the underlying trend in multi-dimensional data. Here, we show that by employing an algorithm called the mean shift theory to a large amount of diffraction data in high-throughput experimentation, one can streamline the process of delineating the structural evolution across compositional variations mapped on combinatorial libraries with minimal computational cost. Data collected at a synchrotron beamline are analyzed on the fly, and by integrating experimental data with the inorganic crystal structure database (ICSD), we can substantially enhance the accuracy in classifying the structural phases across ternary phase spaces. We have used this approach to identify a novel magnetic phase with enhanced magnetic anisotropy which is a candidate for rare-earth free permanent magnet. PMID:25220062

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

Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

PubMed Central

McKinstry-Wu, Andrew R.; Bu, Weiming; Rai, Ganesha; Lea, Wendy A.; Weiser, Brian P.; Liang, David F.; Simeonov, Anton; Jadhav, Ajit; Maloney, David J.; Eckenhoff, Roderic G.

2014-01-01

Background The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene (1-AMA) and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-AMA-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A-receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results From an initial chemical library of over 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-AMA binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based upon a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusions We demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype. PMID:25603205

EPAs DSSTox Chemical Database: A Resource for the Non-Targeted Testing Community (EPA NTA workshop)

EPA Science Inventory

EPA’s DSSTox database project, which includes coverage of the ToxCast and Tox21 high-throughput testing inventories, provides high-quality chemical-structure files for inventories of toxicological and environmental relevance. A feature of the DSSTox project, which differentiates ...

Holographic femtosecond laser processing and its application to biological materials (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hayasaki, Yoshio

2017-02-01

Femtosecond laser processing is a promising tool for fabricating novel and useful structures on the surfaces of and inside materials. An enormous number of pulse irradiation points will be required for fabricating actual structures with millimeter scale, and therefore, the throughput of femtosecond laser processing must be improved for practical adoption of this technique. One promising method to improve throughput is parallel pulse generation based on a computer-generated hologram (CGH) displayed on a spatial light modulator (SLM), a technique called holographic femtosecond laser processing. The holographic method has the advantages such as high throughput, high light use efficiency, and variable, instantaneous, and 3D patterning. Furthermore, the use of an SLM gives an ability to correct unknown imperfections of the optical system and inhomogeneity in a sample using in-system optimization of the CGH. Furthermore, the CGH can adaptively compensate in response to dynamic unpredictable mechanical movements, air and liquid disturbances, a shape variation and deformation of the target sample, as well as adaptive wavefront control for environmental changes. Therefore, it is a powerful tool for the fabrication of biological cells and tissues, because they have free form, variable, and deformable structures. In this paper, we present the principle and the experimental setup of holographic femtosecond laser processing, and the effective way for processing the biological sample. We demonstrate the femtosecond laser processing of biological materials and the processing properties.

High Throughput Biodegradation-Screening Test To Prioritize and Evaluate Chemical Biodegradability.

PubMed

Martin, Timothy J; Goodhead, Andrew K; Acharya, Kishor; Head, Ian M; Snape, Jason R; Davenport, Russell J

2017-06-20

Comprehensive assessment of environmental biodegradability of pollutants is limited by the use of low throughput systems. These are epitomized by the Organisation for Economic Cooperation and Development (OECD) Ready Biodegradability Tests (RBTs), where one sample from an environment may be used to assess a chemical's ability to readily biodegrade or persist universally in that environment. This neglects the considerable spatial and temporal microbial variation inherent in any environment. Inaccurate designations of biodegradability or persistence can occur as a result. RBTs are central in assessing the biodegradation fate of chemicals and inferring exposure concentrations in environmental risk assessments. We developed a colorimetric assay for the reliable quantification of suitable aromatic compounds in a high throughput biodegradation screening test (HT-BST). The HT-BST accurately differentiated and prioritized a range of structurally diverse aromatic compounds on the basis of their assigned relative biodegradabilities and quantitative structure-activity relationship (QSAR) model outputs. Approximately 20 000 individual biodegradation tests were performed, returning analogous results to conventional RBTs. The effect of substituent group structure and position on biodegradation potential demonstrated a significant correlation (P < 0.05) with Hammett's constant for substituents on position 3 of the phenol ring. The HT-BST may facilitate the rapid screening of 100 000 chemicals reportedly manufactured in Europe and reduce the need for higher-tier fate and effects tests.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

PubMed

Boosalis, Michael S; Sangerman, Jose I; White, Gary L; Wolf, Roman F; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H; Li, Biaoru; Pace, Betty S; Nouraie, Mehdi; Faller, Douglas V; Perrine, Susan P

2015-01-01

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

An open workflow to generate “MS Ready” structures and improve non-targeted mass spectrometry (ACS Fall 1 of 3)

EPA Science Inventory

High-throughput non-targeted analyses (NTA) rely on chemical reference databases for tentative identification of observed chemical features. Many of these databases and online resources incorporate chemical structure data not in a form that is readily observed by mass spectromet...

Software Voting in Asynchronous NMR (N-Modular Redundancy) Computer Structures.

DTIC Science & Technology

1983-05-06

added reliability is exchanged for increased system cost and decreased throughput. Some applications require extremely reliable systems, so the only...not the other way around. Although no systems proidc abstract voting yet. as more applications are written for NMR systems, the programmers are going...throughput goes down, the overhead goes up. Mathematically : Overhead= Non redundant Throughput- Actual Throughput (1) In this section, the actual throughput

Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease

PubMed Central

Zheng, Wei; Padia, Janak; Urban, Daniel J.; Jadhav, Ajit; Goker-Alpan, Ozlem; Simeonov, Anton; Goldin, Ehud; Auld, Douglas; LaMarca, Mary E.; Inglese, James; Austin, Christopher P.; Sidransky, Ellen

2007-01-01

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure–activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40–90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders. PMID:17670938

High mass throughput particle generation using multiple nozzle spraying

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

Pui, David Y. H.; Chen, Da-Ren

Spraying apparatus and methods that employ multiple nozzle structures for producing multiple sprays of particles, e.g., nanoparticles, for various applications, e.g., pharmaceuticals, are provided. For example, an electrospray dispensing device may include a plurality of nozzle structures, wherein each nozzle structure is separated from adjacent nozzle structures by an internozzle distance. Sprays of particles are established from the nozzle structures by creating a nonuniform electrical field between the nozzle structures and an electrode electrically isolated therefrom.

High mass throughput particle generation using multiple nozzle spraying

DOEpatents

Pui, David Y.H.; Chen, Da-Ren

2004-07-20

Spraying apparatus and methods that employ multiple nozzle structures for producing multiple sprays of particles, e.g., nanoparticles, for various applications, e.g., pharmaceuticals, are provided. For example, an electrospray dispensing device may include a plurality of nozzle structures, wherein each nozzle structure is separated from adjacent nozzle structures by an internozzle distance. Sprays of particles are established from the nozzle structures by creating a nonuniform electrical field between the nozzle structures and an electrode electrically isolated therefrom.

High mass throughput particle generation using multiple nozzle spraying

DOEpatents

Pui, David Y. H. [Plymouth, MN; Chen, Da-Ren [Creve Coeur, MO

2009-03-03

Spraying apparatus and methods that employ multiple nozzle structures for producing multiple sprays of particles, e.g., nanoparticles, for various applications, e.g., pharmaceuticals, are provided. For example, an electrospray dispensing device may include a plurality of nozzle structures, wherein each nozzle structure is separated from adjacent nozzle structures by an internozzle distance. Sprays of particles are established from the nozzle structures by creating a nonuniform electrical field between the nozzle structures and an electrode electrically isolated therefrom.

Control structures for high speed processors

NASA Technical Reports Server (NTRS)

Maki, G. K.; Mankin, R.; Owsley, P. A.; Kim, G. M.

1982-01-01

A special processor was designed to function as a Reed Solomon decoder with throughput data rate in the Mhz range. This data rate is significantly greater than is possible with conventional digital architectures. To achieve this rate, the processor design includes sequential, pipelined, distributed, and parallel processing. The processor was designed using a high level language register transfer language. The RTL can be used to describe how the different processes are implemented by the hardware. One problem of special interest was the development of dependent processes which are analogous to software subroutines. For greater flexibility, the RTL control structure was implemented in ROM. The special purpose hardware required approximately 1000 SSI and MSI components. The data rate throughput is 2.5 megabits/second. This data rate is achieved through the use of pipelined and distributed processing. This data rate can be compared with 800 kilobits/second in a recently proposed very large scale integration design of a Reed Solomon encoder.

Strategic and Operational Plan for Integrating Transcriptomics ...

EPA Pesticide Factsheets

Plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT; the details are in the attached slide presentation presentation on plans for incorporating high throughput transcriptomics into the current high throughput screening activities at NCCT, given at the OECD meeting on June 23, 2016

High-Throughput Experimental Approach Capabilities | Materials Science |

Science.gov Websites

NREL High-Throughput Experimental Approach Capabilities High-Throughput Experimental Approach by yellow and is for materials in the upper right sector. NREL's high-throughput experimental ,Te) and oxysulfide sputtering Combi-5: Nitrides and oxynitride sputtering We also have several non

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

PubMed Central

Sharlow, Elizabeth R.; Lyda, Todd A.; Dodson, Heidi C.; Mustata, Gabriela; Morris, Meredith T.; Leimgruber, Stephanie S.; Lee, Kuo-Hsiung; Kashiwada, Yoshiki; Close, David; Lazo, John S.; Morris, James C.

2010-01-01

Background The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay. Methodology/Principal Findings Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC50<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics. Conclusions/Significance The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome. PMID:20405000

Use of high-throughput and in vivo data to support read ...

EPA Pesticide Factsheets

Disrupting normal function of mitochondria can culminate in a variety of organ-level toxicities. A number of mechanisms - such as uncoupling of oxidative phosphorylation and inhibition of the electron transport chain - have been implicated in mitochondrial toxicity. The presence of mitochondrial toxicity has led to a number of drugs being withdrawn from the market highlighting the need to identify potential mitochondrial toxicants within the environment. High-throughput screening (HTS) assays provide a means of rapidly gathering toxicity data for a large number of chemicals; however, information as to the associated in vivo effect is typically unknown. The Adverse Outcome Pathway (AOP) concept provides a valuable scaffold onto which mechanistic data from different levels of biological organisation can be arranged.Information pertaining to mitochondrial toxicity from the U.S. EPA’s ToxCast program were integrated with rodent in vivo data from U.S. EPA’s ToxRefDB to connect the high throughput ToxCast assay results with potential adverse outcome data. Previously developed structural alerts were utilized to profile the chemicals with both in vitro mitochondrial toxicity and in vivo rodent data. Structural similarity guided by the toxicity profile as measured in the ToxCast assay battery was then used to group those chemicals which either were not tested in a mitochondrial toxicity assay or were not considered a “hit” and read-across was performed. Subsequen

From Lab to Fab: Developing a Nanoscale Delivery Tool for Scalable Nanomanufacturing

NASA Astrophysics Data System (ADS)

Safi, Asmahan A.

The emergence of nanomaterials with unique properties at the nanoscale over the past two decades carries a capacity to impact society and transform or create new industries ranging from nanoelectronics to nanomedicine. However, a gap in nanomanufacturing technologies has prevented the translation of nanomaterial into real-world commercialized products. Bridging this gap requires a paradigm shift in methods for fabricating structured devices with a nanoscale resolution in a repeatable fashion. This thesis explores the new paradigms for fabricating nanoscale structures devices and systems for high throughput high registration applications. We present a robust and scalable nanoscale delivery platform, the Nanofountain Probe (NFP), for parallel direct-write of functional materials. The design and microfabrication of NFP is presented. The new generation addresses the challenges of throughput, resolution and ink replenishment characterizing tip-based nanomanufacturing. To achieve these goals, optimized probe geometry is integrated to the process along with channel sealing and cantilever bending. The capabilities of the newly fabricated probes are demonstrated through two type of delivery: protein nanopatterning and single cell nanoinjection. The broad applications of the NFP for single cell delivery are investigated. An external microfluidic packaging is developed to enable delivery in liquid environment. The system is integrated to a combined atomic force microscope and inverted fluorescence microscope. Intracellular delivery is demonstrated by injecting a fluorescent dextran into Hela cells in vitro while monitoring the injection forces. Such developments enable in vitro cellular delivery for single cell studies and high throughput gene expression. The nanomanufacturing capabilities of NFPs are explored. Nanofabrication of carbon nanotube-based electronics presents all the manufacturing challenges characterizing of assembling nanomaterials precisely onto devices. The presented study combines top-down and bottom-approaches by integrating the catalyst patterning and carbon nanotube growth directly on structures. Large array of iron-rich catalyst are patterned on an substrate for subsequent carbon nanotubes synthesis. The dependence of probe geometry and substrate wetting is assessed by modeling and experimental studies. Finally preliminary results on synthesis of carbon nanotube by catalyst assisted chemical vapor deposition suggest increasing the catalyst yield is critical. Such work will enable high throughput nanomanufacturing of carbon nanotube based devices.

SVS: data and knowledge integration in computational biology.

PubMed

Zycinski, Grzegorz; Barla, Annalisa; Verri, Alessandro

2011-01-01

In this paper we present a framework for structured variable selection (SVS). The main concept of the proposed schema is to take a step towards the integration of two different aspects of data mining: database and machine learning perspective. The framework is flexible enough to use not only microarray data, but other high-throughput data of choice (e.g. from mass spectrometry, microarray, next generation sequencing). Moreover, the feature selection phase incorporates prior biological knowledge in a modular way from various repositories and is ready to host different statistical learning techniques. We present a proof of concept of SVS, illustrating some implementation details and describing current results on high-throughput microarray data.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

PubMed Central

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-01-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality. PMID:26121404

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

PubMed

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-08-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

PubMed Central

Lucanic, Mark; Garrett, Theo; Gill, Matthew S.; Lithgow, Gordon J.

2018-01-01

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening. PMID:29630057

High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.

PubMed

Myers, Stephanie M; Bawn, Ruth H; Bisset, Louise C; Blackburn, Timothy J; Cottyn, Betty; Molyneux, Lauren; Wong, Ai-Ching; Cano, Celine; Clegg, William; Harrington, Ross W; Leung, Hing; Rigoreau, Laurent; Vidot, Sandrine; Golding, Bernard T; Griffin, Roger J; Hammonds, Tim; Newell, David R; Hardcastle, Ian R

2016-08-08

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

A Low Power and High Throughput Self Synchronous FPGA Using 65nm CMOS with Throughput Optimization by Pipeline Alignment

NASA Astrophysics Data System (ADS)

Stefan Devlin, Benjamin; Nakura, Toru; Ikeda, Makoto; Asada, Kunihiro

We detail a self synchronous field programmable gate array (SSFPGA) with dual-pipeline (DP) architecture to conceal pre-charge time for dynamic logic, and its throughput optimization by using pipeline alignment implemented on benchmark circuits. A self synchronous LUT (SSLUT) consists of a three input tree-type structure with 8bits of SRAM for programming. A self synchronous switch box (SSSB) consists of both pass transistors and buffers to route signals, with 12bits of SRAM. One common block with one SSLUT and one SSSB occupies 2.2Mλ2 area with 35bits of SRAM, and the prototype SSFPGA with 34 × 30 (1020) blocks is designed and fabricated using 65nm CMOS. Measured results show at 1.2V 430MHz and 647MHz operation for a 3bit ripple carry adder, without and with throughput optimization, respectively. We find that using the proposed pipeline alignment techniques we can perform at maximum throughput of 647MHz in various benchmarks on the SSFPGA. We demonstrate up to 56.1 times throughput improvement with our pipeline alignment techniques. The pipeline alignment is carried out within the number of logic elements in the array and pipeline buffers in the switching matrix.

Emergence of a few distinct structures from a single formal structure type during high-throughput screening for stable compounds: The case of RbCuS and RbCuSe

NASA Astrophysics Data System (ADS)

Trimarchi, Giancarlo; Zhang, Xiuwen; DeVries Vermeer, Michael J.; Cantwell, Jacqueline; Poeppelmeier, Kenneth R.; Zunger, Alex

2015-10-01

Theoretical sorting of stable and synthesizable "missing compounds" from those that are unstable is a crucial step in the discovery of previously unknown functional materials. This active research area often involves high-throughput (HT) examination of the total energy of a given compound in a list of candidate formal structure types (FSTs), searching for those with the lowest energy within that list. While it is well appreciated that local relaxation methods based on a fixed list of structure types can lead to inaccurate geometries, this approach is widely used in HT studies because it produces answers faster than global optimization methods (that vary lattice vectors and atomic positions without local restrictions). We find, however, a different failure mode of the HT protocol: specific crystallographic classes of formal structure types each correspond to a series of chemically distinct "daughter structure types" (DSTs) that have the same space group but possess totally different local bonding configurations, including coordination types. Failure to include such DSTs in the fixed list of examined candidate structures used in contemporary high-throughput approaches can lead to qualitative misidentification of the stable bonding pattern, not just quantitative inaccuracies. In this work, we (i) clarify the understanding of the general DST-FST relationship, thus improving current discovery HT approaches, (ii) illustrate this failure mode for RbCuS and RbCuSe (the latter being a yet unreported compound and is predicted here) by developing a synthesis method and accelerated crystal-structure determination, and (iii) apply the genetic-algorithm-based global space-group optimization (GSGO) approach which is not vulnerable to the failure mode of HT searches of fixed lists, demonstrating a correct identification of the stable DST. The broad impact of items (i)-(iii) lies in the demonstrated predictive ability of a more comprehensive search strategy than what is currently used—use HT calculations as the preliminary broad screening followed by unbiased GSGO of the final candidates.

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

Sue, Shih-Che; Chang, Chi-Fon; Huang, Yao-Te; Chou, Ching-Yu; Huang, Tai-huang

2005-05-01

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.

Development and application of a fluorescent glucose uptake assay for the high-throughput screening of non-glycoside SGLT2 inhibitors.

PubMed

Wu, Szu-Huei; Yao, Chun-Hsu; Hsieh, Chieh-Jui; Liu, Yu-Wei; Chao, Yu-Sheng; Song, Jen-Shin; Lee, Jinq-Chyi

2015-07-10

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors are of current interest as a treatment for type 2 diabetes. Efforts have been made to discover phlorizin-related glycosides with good SGLT2 inhibitory activity. To increase structural diversity and better understand the role of non-glycoside SGLT2 inhibitors on glycemic control, we initiated a research program to identify non-glycoside hits from high-throughput screening. Here, we report the development of a novel, fluorogenic probe-based glucose uptake system based on a Cu(I)-catalyzed [3+2] cycloaddition. The safer processes and cheaper substances made the developed assay our first priority for large-scale primary screening as compared to the well-known [(14)C]-labeled α-methyl-D-glucopyranoside ([(14)C]-AMG) radioactive assay. This effort culminated in the identification of a benzimidazole, non-glycoside SGLT2 hit with an EC50 value of 0.62 μM by high-throughput screening of 41,000 compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

PubMed

Yang, Bin; Lamb, Michelle L; Zhang, Tao; Hennessy, Edward J; Grewal, Gurmit; Sha, Li; Zambrowski, Mark; Block, Michael H; Dowling, James E; Su, Nancy; Wu, Jiaquan; Deegan, Tracy; Mikule, Keith; Wang, Wenxian; Kaspera, Rüdiger; Chuaqui, Claudio; Chen, Huawei

2014-12-11

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

QR-on-a-chip: a computer-recognizable micro-pattern engraved microfluidic device for high-throughput image acquisition.

PubMed

Yun, Kyungwon; Lee, Hyunjae; Bang, Hyunwoo; Jeon, Noo Li

2016-02-21

This study proposes a novel way to achieve high-throughput image acquisition based on a computer-recognizable micro-pattern implemented on a microfluidic device. We integrated the QR code, a two-dimensional barcode system, onto the microfluidic device to simplify imaging of multiple ROIs (regions of interest). A standard QR code pattern was modified to arrays of cylindrical structures of polydimethylsiloxane (PDMS). Utilizing the recognition of the micro-pattern, the proposed system enables: (1) device identification, which allows referencing additional information of the device, such as device imaging sequences or the ROIs and (2) composing a coordinate system for an arbitrarily located microfluidic device with respect to the stage. Based on these functionalities, the proposed method performs one-step high-throughput imaging for data acquisition in microfluidic devices without further manual exploration and locating of the desired ROIs. In our experience, the proposed method significantly reduced the time for the preparation of an acquisition. We expect that the method will innovatively improve the prototype device data acquisition and analysis.

Transition-metal-free catalysts for the sustainable epoxidation of alkenes: from discovery to optimisation by means of high throughput experimentation.

PubMed

Lueangchaichaweng, Warunee; Geukens, Inge; Peeters, Annelies; Jarry, Benjamin; Launay, Franck; Bonardet, Jean-Luc; Jacobs, Pierre A; Pescarmona, Paolo P

2012-02-01

Transition-metal-free oxides were studied as heterogeneous catalysts for the sustainable epoxidation of alkenes with aqueous H₂O₂ by means of high throughput experimentation (HTE) techniques. A full-factorial HTE approach was applied in the various stages of the development of the catalysts: the synthesis of the materials, their screening as heterogeneous catalysts in liquid-phase epoxidation and the optimisation of the reaction conditions. Initially, the chemical composition of transition-metal-free oxides was screened, leading to the discovery of gallium oxide as a novel, active and selective epoxidation catalyst. On the basis of these results, the research line was continued with the study of structured porous aluminosilicates, gallosilicates and silica-gallia composites. In general, the gallium-based materials showed the best catalytic performances. This family of materials represents a promising class of heterogeneous catalysts for the sustainable epoxidation of alkenes and offers a valid alternative to the transition-metal heterogeneous catalysts commonly used in epoxidation. High throughput experimentation played an important role in promoting the development of these catalytic systems.

Biases in the Experimental Annotations of Protein Function and Their Effect on Our Understanding of Protein Function Space

PubMed Central

Schnoes, Alexandra M.; Ream, David C.; Thorman, Alexander W.; Babbitt, Patricia C.; Friedberg, Iddo

2013-01-01

The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the “few articles - many proteins” phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments. PMID:23737737

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

PubMed

Lagarde, Julien; Uszczynska-Ratajczak, Barbara; Carbonell, Silvia; Pérez-Lluch, Sílvia; Abad, Amaya; Davis, Carrie; Gingeras, Thomas R; Frankish, Adam; Harrow, Jennifer; Guigo, Roderic; Johnson, Rory

2017-12-01

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.

High-Throughput Non-Contact Vitrification of Cell-Laden Droplets Based on Cell Printing

NASA Astrophysics Data System (ADS)

Shi, Meng; Ling, Kai; Yong, Kar Wey; Li, Yuhui; Feng, Shangsheng; Zhang, Xiaohui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

2015-12-01

Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems.

High-Throughput Non-Contact Vitrification of Cell-Laden Droplets Based on Cell Printing

PubMed Central

Shi, Meng; Ling, Kai; Yong, Kar Wey; Li, Yuhui; Feng, Shangsheng; Zhang, Xiaohui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

2015-01-01

Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems. PMID:26655688

Aggregating Data for Computational Toxicology Applications ...

EPA Pesticide Factsheets

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built usi

SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

EPA Science Inventory

Abstract

Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

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

EPA Science Inventory

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

High-Throughput Analysis and Automation for Glycomics Studies.

PubMed

Shubhakar, Archana; Reiding, Karli R; Gardner, Richard A; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics-for example in Genome Wide Association Studies-to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.

L-edge spectroscopy of dilute, radiation-sensitive systems using a transition-edge-sensor array

NASA Astrophysics Data System (ADS)

Titus, Charles J.; Baker, Michael L.; Lee, Sang Jun; Cho, Hsiao-Mei; Doriese, William B.; Fowler, Joseph W.; Gaffney, Kelly; Gard, Johnathon D.; Hilton, Gene C.; Kenney, Chris; Knight, Jason; Li, Dale; Marks, Ronald; Minitti, Michael P.; Morgan, Kelsey M.; O'Neil, Galen C.; Reintsema, Carl D.; Schmidt, Daniel R.; Sokaras, Dimosthenis; Swetz, Daniel S.; Ullom, Joel N.; Weng, Tsu-Chien; Williams, Christopher; Young, Betty A.; Irwin, Kent D.; Solomon, Edward I.; Nordlund, Dennis

2017-12-01

We present X-ray absorption spectroscopy and resonant inelastic X-ray scattering (RIXS) measurements on the iron L-edge of 0.5 mM aqueous ferricyanide. These measurements demonstrate the ability of high-throughput transition-edge-sensor (TES) spectrometers to access the rich soft X-ray (100-2000 eV) spectroscopy regime for dilute and radiation-sensitive samples. Our low-concentration data are in agreement with high-concentration measurements recorded by grating spectrometers. These results show that soft-X-ray RIXS spectroscopy acquired by high-throughput TES spectrometers can be used to study the local electronic structure of dilute metal-centered complexes relevant to biology, chemistry, and catalysis. In particular, TES spectrometers have a unique ability to characterize frozen solutions of radiation- and temperature-sensitive samples.

Combinatorial materials synthesis and high-throughput screening: an integrated materials chip approach to mapping phase diagrams and discovery and optimization of functional materials.

PubMed

Xiang, X D

Combinatorial materials synthesis methods and high-throughput evaluation techniques have been developed to accelerate the process of materials discovery and optimization and phase-diagram mapping. Analogous to integrated circuit chips, integrated materials chips containing thousands of discrete different compositions or continuous phase diagrams, often in the form of high-quality epitaxial thin films, can be fabricated and screened for interesting properties. Microspot x-ray method, various optical measurement techniques, and a novel evanescent microwave microscope have been used to characterize the structural, optical, magnetic, and electrical properties of samples on the materials chips. These techniques are routinely used to discover/optimize and map phase diagrams of ferroelectric, dielectric, optical, magnetic, and superconducting materials.

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).

Direct Duplex Detection: An Emerging Tool in the RNA Structure Analysis Toolbox.

PubMed

Weidmann, Chase A; Mustoe, Anthony M; Weeks, Kevin M

2016-09-01

While a variety of powerful tools exists for analyzing RNA structure, identifying long-range and intermolecular base-pairing interactions has remained challenging. Recently, three groups introduced a high-throughput strategy that uses psoralen-mediated crosslinking to directly identify RNA-RNA duplexes in cells. Initial application of these methods highlights the preponderance of long-range structures within and between RNA molecules and their widespread structural dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

PubMed Central

Boosalis, Michael S.; Sangerman, Jose I.; White, Gary L.; Wolf, Roman F.; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H.; Li, Biaoru; Pace, Betty S.; Nouraie, Mehdi; Faller, Douglas V.; Perrine, Susan P.

2015-01-01

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies. PMID:26713848

Query3d: a new method for high-throughput analysis of functional residues in protein structures.

PubMed

Ausiello, Gabriele; Via, Allegra; Helmer-Citterich, Manuela

2005-12-01

The identification of local similarities between two protein structures can provide clues of a common function. Many different methods exist for searching for similar subsets of residues in proteins of known structure. However, the lack of functional and structural information on single residues, together with the low level of integration of this information in comparison methods, is a limitation that prevents these methods from being fully exploited in high-throughput analyses. Here we describe Query3d, a program that is both a structural DBMS (Database Management System) and a local comparison method. The method conserves a copy of all the residues of the Protein Data Bank annotated with a variety of functional and structural information. New annotations can be easily added from a variety of methods and known databases. The algorithm makes it possible to create complex queries based on the residues' function and then to compare only subsets of the selected residues. Functional information is also essential to speed up the comparison and the analysis of the results. With Query3d, users can easily obtain statistics on how many and which residues share certain properties in all proteins of known structure. At the same time, the method also finds their structural neighbours in the whole PDB. Programs and data can be accessed through the PdbFun web interface.

Query3d: a new method for high-throughput analysis of functional residues in protein structures

PubMed Central

Ausiello, Gabriele; Via, Allegra; Helmer-Citterich, Manuela

2005-01-01

Background The identification of local similarities between two protein structures can provide clues of a common function. Many different methods exist for searching for similar subsets of residues in proteins of known structure. However, the lack of functional and structural information on single residues, together with the low level of integration of this information in comparison methods, is a limitation that prevents these methods from being fully exploited in high-throughput analyses. Results Here we describe Query3d, a program that is both a structural DBMS (Database Management System) and a local comparison method. The method conserves a copy of all the residues of the Protein Data Bank annotated with a variety of functional and structural information. New annotations can be easily added from a variety of methods and known databases. The algorithm makes it possible to create complex queries based on the residues' function and then to compare only subsets of the selected residues. Functional information is also essential to speed up the comparison and the analysis of the results. Conclusion With Query3d, users can easily obtain statistics on how many and which residues share certain properties in all proteins of known structure. At the same time, the method also finds their structural neighbours in the whole PDB. Programs and data can be accessed through the PdbFun web interface. PMID:16351754

Data transfer nodes and demonstration of 100-400 Gbps wide area throughput using the Caltech SDN testbed

NASA Astrophysics Data System (ADS)

Mughal, A.; Newman, H.

2017-10-01

We review and demonstrate the design of efficient data transfer nodes (DTNs), from the perspective of the highest throughput over both local and wide area networks, as well as the highest performance per unit cost. A careful system-level design is required for the hardware, firmware, OS and software components. Furthermore, additional tuning of these components, and the identification and elimination of any remaining bottlenecks is needed once the system is assembled and commissioned, in order to obtain optimal performance. For high throughput data transfers, specialized software is used to overcome the traditional limits in performance caused by the OS, file system, file structures used, etc. Concretely, we will discuss and present the latest results using Fast Data Transfer (FDT), developed by Caltech. We present and discuss the design choices for three generations of Caltech DTNs. Their transfer capabilities range from 40 Gbps to 400 Gbps. Disk throughput is still the biggest challenge in the current generation of available hardware. However, new NVME drives combined with RDMA and a new NVME network fabric are expected to improve the overall data-transfer throughput and simultaneously reduce the CPU load on the end nodes.

Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa)

PubMed Central

Chavas, L. M. G.; Gumprecht, L.; Chapman, H. N.

2015-01-01

Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today's third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the goal of high-throughput structure determination with X-ray FELs. PMID:26798808

The US EPA ToxCast Program: Moving from Data Generation ...

EPA Pesticide Factsheets

The U.S. EPA ToxCast program is entering its tenth year. Significant learning and progress have occurred towards collection, analysis, and interpretation of the data. The library of ~1,800 chemicals has been subject to ongoing characterization (e.g., identity, purity, stability) and is unique in its scope, structural diversity, and use scenarios making it ideally suited to investigate the underlying molecular mechanisms of toxicity. The ~700 high-throughput in vitro assay endpoints cover 327 genes and 293 pathways as well as other integrated cellular processes and responses. The integrated analysis of high-throughput screening data has shown that most environmental and industrial chemicals are very non-selective in the biological targets they perturb, while a small subset of chemicals are relatively selective for specific biological targets. The selectivity of a chemical informs interpretation of the screening results while also guiding future mode-of-action or adverse outcome pathway approaches. Coupling the high-throughput in vitro assays with medium-throughput pharmacokinetic assays and reverse dosimetry allows conversion of the potency estimates to an administered dose. Comparison of the administered dose to human exposure provides a risk-based context. The lessons learned from this effort will be presented and discussed towards application to chemical safety decision making and the future of the computational toxicology program at the U.S. EPA. SOT pr

New Toxico-Cheminformatics & Computational Toxicology ...

EPA Pesticide Factsheets

EPA’s National Center for Computational Toxicology is building capabilities to support a new paradigm for toxicity screening and prediction. The DSSTox project is improving public access to quality structure-annotated chemical toxicity information in less summarized forms than traditionally employed in SAR modeling, and in ways that facilitate data-mining, and data read-across. The DSSTox Structure-Browser provides structure searchability across all published DSSTox toxicity-related inventory, and is enabling linkages between previously isolated toxicity data resources. As of early March 2008, the public DSSTox inventory has been integrated into PubChem, allowing a user to take full advantage of PubChem structure-activity and bioassay clustering features. The most recent DSSTox version of the Carcinogenic Potency Database file (CPDBAS) illustrates ways in which various summary definitions of carcinogenic activity can be employed in modeling and data mining. Phase I of the ToxCastTM project is generating high-throughput screening data from several hundred biochemical and cell-based assays for a set of 320 chemicals, mostly pesticide actives, with rich toxicology profiles. Incorporating and expanding traditional SAR concepts into this new high-throughput and data-rich world pose conceptual and practical challenges, but also holds great promise for improving predictive capabilities.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

PubMed

Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

2016-07-03

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

Predicting organ toxicity using in vitro bioactivity data and chemical structure

EPA Science Inventory

Animal testing alone cannot practically evaluate the health hazard posed by tens of thousands of environmental chemicals. Computational approaches together with high-throughput experimental data may provide more efficient means to predict chemical toxicity. Here, we use a superv...

High Throughput PBTK: Open-Source Data and Tools for ...

EPA Pesticide Factsheets

Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

Facile fabrication of nanofluidic diode membranes using anodic aluminium oxide

NASA Astrophysics Data System (ADS)

Wu, Songmei; Wildhaber, Fabien; Vazquez-Mena, Oscar; Bertsch, Arnaud; Brugger, Juergen; Renaud, Philippe

2012-08-01

Active control of ion transport plays important roles in chemical and biological analytical processes. Nanofluidic systems hold the promise for such control through electrostatic interaction between ions and channel surfaces. Most existing experiments rely on planar geometry where the nanochannels are generally very long and shallow with large aspect ratios. Based on this configuration the concepts of nanofluidic gating and rectification have been successfully demonstrated. However, device minimization and throughput scaling remain significant challenges. We report here an innovative and facile realization of hetero-structured Al2O3/SiO2 (Si) nanopore array membranes by using pattern transfer of self-organized nanopore structures of anodic aluminum oxide (AAO). Thanks to the opposite surface charge states of Al2O3 (positive) and SiO2 (negative), the membrane exhibits clear rectification of ion current in electrolyte solutions with very low aspect ratios compared to previous approaches. Our hetero-structured nanopore arrays provide a valuable platform for high throughput applications such as molecular separation, chemical processors and energy conversion.Active control of ion transport plays important roles in chemical and biological analytical processes. Nanofluidic systems hold the promise for such control through electrostatic interaction between ions and channel surfaces. Most existing experiments rely on planar geometry where the nanochannels are generally very long and shallow with large aspect ratios. Based on this configuration the concepts of nanofluidic gating and rectification have been successfully demonstrated. However, device minimization and throughput scaling remain significant challenges. We report here an innovative and facile realization of hetero-structured Al2O3/SiO2 (Si) nanopore array membranes by using pattern transfer of self-organized nanopore structures of anodic aluminum oxide (AAO). Thanks to the opposite surface charge states of Al2O3 (positive) and SiO2 (negative), the membrane exhibits clear rectification of ion current in electrolyte solutions with very low aspect ratios compared to previous approaches. Our hetero-structured nanopore arrays provide a valuable platform for high throughput applications such as molecular separation, chemical processors and energy conversion. Electronic supplementary information (ESI) available: Pattern transfer of local AAO mask into Si layers of different thickness; characterization of the Ag/AgCl electrodes and the cell constant; control experiments of mono-charged nanopore membranes; and simulation of ionic transport in nanofluidic diodes. See DOI: 10.1039/c2nr31243c

Neutron reflecting supermirror structure

DOEpatents

Wood, James L.

1992-01-01

An improved neutron reflecting supermirror structure comprising a plurality of stacked sets of bilayers of neutron reflecting materials. The improved neutron reflecting supermirror structure is adapted to provide extremely good performance at high incidence angles, i.e. up to four time the critical angle of standard neutron mirror structures. The reflection of neutrons striking the supermirror structure at a high critical angle provides enhanced neutron throughput, and hence more efficient and economical use of neutron sources. One layer of each set of bilayers consist of titanium, and the second layer of each set of bilayers consist of an alloy of nickel with carbon interstitially present in the nickel alloy.

Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

PubMed

Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L; Merrick, B Alex; Teng, Christina T; Tice, Raymond R

2015-10-01

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library

PubMed Central

Chen, Shiuan; Hsieh, Jui-Hua; Huang, Ruili; Sakamuru, Srilatha; Hsin, Li-Yu; Xia, Menghang; Shockley, Keith R.; Auerbach, Scott; Kanaya, Noriko; Lu, Hannah; Svoboda, Daniel; Witt, Kristine L.; Merrick, B. Alex; Teng, Christina T.; Tice, Raymond R.

2015-01-01

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase. PMID:26141389

DSSTox ToxCast and Tox21 Chemical Inventories: Laying the Foundation for the U.S. EPA’s Computational Toxicology Research Programs

EPA Science Inventory

High quality chemical structure inventories provide the foundation of the U.S. EPA’s ToxCast and Tox21 projects, which are employing high-throughput technologies to screen thousands of chemicals in hundreds of biochemical and cell-based assays, probing a wide diversity of targets...

Higher Throughput Calorimetry: Opportunities, Approaches and Challenges

PubMed Central

Recht, Michael I.; Coyle, Joseph E.; Bruce, Richard H.

2010-01-01

Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein-ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed. PMID:20888754

Fixing clearance as early as lead optimization using high throughput in vitro incubations in combination with exact mass detection and automatic structure elucidation of metabolites.

PubMed

Zimmerlin, Alfred; Kiffe, Michael

2013-01-01

New enabling MS technologies have made it possible to elucidate metabolic pathways present in ex vivo (blood, bile and/or urine) or in vitro (liver microsomes, hepatocytes and/or S9) samples. When investigating samples from high throughput assays the challenge that the user is facing now is to extract the appropriate information and compile it so that it is understandable to all. Medicinal chemist may then design the next generation of (better) drug candidates combining the needs for potency and metabolic stability and their synthetic creativity. This review focuses on the comparison of these enabling MS technologies and the IT tools developed for their interpretation.

A high-throughput screen for mitochondrial function reveals known and novel mitochondrial toxicants in a library of environmental agents

PubMed Central

Datta, Sandipan; Sahdeo, Sunil; Gray, Jennifer A.; Morriseau, Christophe; Hammock, Bruce D.; Cortopassi, Gino

2016-01-01

Mitochondrial toxicity is emerging as a major mechanism underlying serious human health consequences. This work performs a high-throughput screen (HTS) of 176 environmental chemicals for mitochondrial toxicity utilizing a previously reported biosensor platform. This established HTS confirmed known mitochondrial toxins and identified novel mitotochondrial uncouplers such as 2, 2′-Methylenebis(4-chlorophenol) and pentachlorophenol. It also identified a mitochondrial ‘structure activity relationship’ (SAR) in the sense that multiple environmental chlorophenols are mitochondrial inhibitors and uncouplers. This study demonstrates proof-of-concept that a mitochondrial HTS assay detects known and novel environmental mitotoxicants, and could be used to quickly evaluate human health risks from mitotoxicants in the environment. PMID:27717841

Integrated Analysis Platform: An Open-Source Information System for High-Throughput Plant Phenotyping1[C][W][OPEN

PubMed Central

Klukas, Christian; Chen, Dijun; Pape, Jean-Michel

2014-01-01

High-throughput phenotyping is emerging as an important technology to dissect phenotypic components in plants. Efficient image processing and feature extraction are prerequisites to quantify plant growth and performance based on phenotypic traits. Issues include data management, image analysis, and result visualization of large-scale phenotypic data sets. Here, we present Integrated Analysis Platform (IAP), an open-source framework for high-throughput plant phenotyping. IAP provides user-friendly interfaces, and its core functions are highly adaptable. Our system supports image data transfer from different acquisition environments and large-scale image analysis for different plant species based on real-time imaging data obtained from different spectra. Due to the huge amount of data to manage, we utilized a common data structure for efficient storage and organization of data for both input data and result data. We implemented a block-based method for automated image processing to extract a representative list of plant phenotypic traits. We also provide tools for build-in data plotting and result export. For validation of IAP, we performed an example experiment that contains 33 maize (Zea mays ‘Fernandez’) plants, which were grown for 9 weeks in an automated greenhouse with nondestructive imaging. Subsequently, the image data were subjected to automated analysis with the maize pipeline implemented in our system. We found that the computed digital volume and number of leaves correlate with our manually measured data in high accuracy up to 0.98 and 0.95, respectively. In summary, IAP provides a multiple set of functionalities for import/export, management, and automated analysis of high-throughput plant phenotyping data, and its analysis results are highly reliable. PMID:24760818

Finding the Stable Structures of N1-xWx with an Ab Initio High-Throughput Approach

DTIC Science & Technology

2015-05-26

W. These include borides , carbides, oxides, and other nitrides. We also invented many structures to mimic the random pattern of vacancies on both the...structures. These include nitrides, oxides, borides , and carbides, as well as supercells of standard structures with atoms removed to mimic the random patter...1930). [15] R. Kiessling and Y. H. Liu, Thermal stability of the chromium, iron, and tungsten borides in streaming ammonia and the existence of a new

DDBJ read annotation pipeline: a cloud computing-based pipeline for high-throughput analysis of next-generation sequencing data.

PubMed

Nagasaki, Hideki; Mochizuki, Takako; Kodama, Yuichi; Saruhashi, Satoshi; Morizaki, Shota; Sugawara, Hideaki; Ohyanagi, Hajime; Kurata, Nori; Okubo, Kousaku; Takagi, Toshihisa; Kaminuma, Eli; Nakamura, Yasukazu

2013-08-01

High-performance next-generation sequencing (NGS) technologies are advancing genomics and molecular biological research. However, the immense amount of sequence data requires computational skills and suitable hardware resources that are a challenge to molecular biologists. The DNA Data Bank of Japan (DDBJ) of the National Institute of Genetics (NIG) has initiated a cloud computing-based analytical pipeline, the DDBJ Read Annotation Pipeline (DDBJ Pipeline), for a high-throughput annotation of NGS reads. The DDBJ Pipeline offers a user-friendly graphical web interface and processes massive NGS datasets using decentralized processing by NIG supercomputers currently free of charge. The proposed pipeline consists of two analysis components: basic analysis for reference genome mapping and de novo assembly and subsequent high-level analysis of structural and functional annotations. Users may smoothly switch between the two components in the pipeline, facilitating web-based operations on a supercomputer for high-throughput data analysis. Moreover, public NGS reads of the DDBJ Sequence Read Archive located on the same supercomputer can be imported into the pipeline through the input of only an accession number. This proposed pipeline will facilitate research by utilizing unified analytical workflows applied to the NGS data. The DDBJ Pipeline is accessible at http://p.ddbj.nig.ac.jp/.

DDBJ Read Annotation Pipeline: A Cloud Computing-Based Pipeline for High-Throughput Analysis of Next-Generation Sequencing Data

PubMed Central

Nagasaki, Hideki; Mochizuki, Takako; Kodama, Yuichi; Saruhashi, Satoshi; Morizaki, Shota; Sugawara, Hideaki; Ohyanagi, Hajime; Kurata, Nori; Okubo, Kousaku; Takagi, Toshihisa; Kaminuma, Eli; Nakamura, Yasukazu

2013-01-01

High-performance next-generation sequencing (NGS) technologies are advancing genomics and molecular biological research. However, the immense amount of sequence data requires computational skills and suitable hardware resources that are a challenge to molecular biologists. The DNA Data Bank of Japan (DDBJ) of the National Institute of Genetics (NIG) has initiated a cloud computing-based analytical pipeline, the DDBJ Read Annotation Pipeline (DDBJ Pipeline), for a high-throughput annotation of NGS reads. The DDBJ Pipeline offers a user-friendly graphical web interface and processes massive NGS datasets using decentralized processing by NIG supercomputers currently free of charge. The proposed pipeline consists of two analysis components: basic analysis for reference genome mapping and de novo assembly and subsequent high-level analysis of structural and functional annotations. Users may smoothly switch between the two components in the pipeline, facilitating web-based operations on a supercomputer for high-throughput data analysis. Moreover, public NGS reads of the DDBJ Sequence Read Archive located on the same supercomputer can be imported into the pipeline through the input of only an accession number. This proposed pipeline will facilitate research by utilizing unified analytical workflows applied to the NGS data. The DDBJ Pipeline is accessible at http://p.ddbj.nig.ac.jp/. PMID:23657089

Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

EPA Science Inventory

Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

75 FR 9909 - National Institute of General Medical Sciences; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-04

... Sciences Special Emphasis Panel, Consortia for High-Throughput- Enabled Structural Biology Partnerships... Biomedical Research Support; 93.821, Cell Biology and Biophysics Research; 93.859, Pharmacology, Physiology, and Biological Chemistry Research; 93.862, Genetics and Developmental Biology Research; 93.88...

77 FR 6128 - National Institute of General Medical Sciences Notice of Closed Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-07

... Sciences Special Emphasis Panel; Review of application for High- Throughput-Enabled Structural Biology..., Cell Biology and Biophysics Research; 93.859, Pharmacology, Physiology, and Biological Chemistry Research; 93.862, Genetics and Developmental Biology Research; 93.88, Minority Access to Research Careers...

Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions.

PubMed

Hayden, Eric J

2016-08-15

RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

Hickey, Scott F; Hammond, Ming C

2014-03-20

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class. Copyright © 2014 Elsevier Ltd. All rights reserved.

The ESFRI Instruct Core Centre Frankfurt: automated high-throughput crystallization suited for membrane proteins and more.

PubMed

Thielmann, Yvonne; Koepke, Juergen; Michel, Hartmut

2012-06-01

Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

High-Throughput Characterization of Porous Materials Using Graphics Processing Units

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

Kim, Jihan; Martin, Richard L.; Rübel, Oliver

We have developed a high-throughput graphics processing units (GPU) code that can characterize a large database of crystalline porous materials. In our algorithm, the GPU is utilized to accelerate energy grid calculations where the grid values represent interactions (i.e., Lennard-Jones + Coulomb potentials) between gas molecules (i.e., CHmore » $$_{4}$$ and CO$$_{2}$$) and material's framework atoms. Using a parallel flood fill CPU algorithm, inaccessible regions inside the framework structures are identified and blocked based on their energy profiles. Finally, we compute the Henry coefficients and heats of adsorption through statistical Widom insertion Monte Carlo moves in the domain restricted to the accessible space. The code offers significant speedup over a single core CPU code and allows us to characterize a set of porous materials at least an order of magnitude larger than ones considered in earlier studies. For structures selected from such a prescreening algorithm, full adsorption isotherms can be calculated by conducting multiple grand canonical Monte Carlo simulations concurrently within the GPU.« less

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds.

PubMed

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-03-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing.

PubMed

Ma, Qiao; Qu, Yuanyuan; Shen, Wenli; Zhang, Zhaojing; Wang, Jingwei; Liu, Ziyan; Li, Duanxing; Li, Huijie; Zhou, Jiti

2015-03-01

In this study, Illumina high-throughput sequencing was used to reveal the community structures of nine coking wastewater treatment plants (CWWTPs) in China for the first time. The sludge systems exhibited a similar community composition at each taxonomic level. Compared to previous studies, some of the core genera in municipal wastewater treatment plants such as Zoogloea, Prosthecobacter and Gp6 were detected as minor species. Thiobacillus (20.83%), Comamonas (6.58%), Thauera (4.02%), Azoarcus (7.78%) and Rhodoplanes (1.42%) were the dominant genera shared by at least six CWWTPs. The percentages of autotrophic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were unexpectedly low, which were verified by both real-time PCR and fluorescence in situ hybridization analyses. Hierarchical clustering and canonical correspondence analysis indicated that operation mode, flow rate and temperature might be the key factors in community formation. This study provides new insights into our understanding of microbial community compositions and structures of CWWTPs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds

NASA Astrophysics Data System (ADS)

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-02-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

Application of ToxCast High-Throughput Screening and ...

EPA Pesticide Factsheets

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

A versatile and efficient high-throughput cloning tool for structural biology.

PubMed

Geertsma, Eric R; Dutzler, Raimund

2011-04-19

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

High-throughput continuous hydrothermal synthesis of an entire nanoceramic phase diagram.

PubMed

Weng, Xiaole; Cockcroft, Jeremy K; Hyett, Geoffrey; Vickers, Martin; Boldrin, Paul; Tang, Chiu C; Thompson, Stephen P; Parker, Julia E; Knowles, Jonathan C; Rehman, Ihtesham; Parkin, Ivan; Evans, Julian R G; Darr, Jawwad A

2009-01-01

A novel High-Throughput Continuous Hydrothermal (HiTCH) flow synthesis reactor was used to make directly and rapidly a 66-sample nanoparticle library (entire phase diagram) of nanocrystalline Ce(x)Zr(y)Y(z)O(2-delta) in less than 12 h. High resolution PXRD data were obtained for the entire heat-treated library (at 1000 degrees C/1 h) in less than a day using the new robotic beamline I11, located at Diamond Light Source (DLS). This allowed Rietveld-quality powder X-ray diffraction (PXRD) data collection of the entire 66-sample library in <1 day. Consequently, the authors rapidly mapped out phase behavior and sintering behaviors for the entire library. Out of the entire 66-sample heat-treated library, the PXRD data suggests that 43 possess the fluorite structure, of which 30 (out of 36) are ternary compositions. The speed, quantity and quality of data obtained by our new approach, offers an exciting new development which will allow structure-property relationships to be accessed for nanoceramics in much shorter time periods.

IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening

PubMed Central

Tidten-Luksch, Naomi; Grimaldi, Raffaella; Torrie, Leah S.; Frearson, Julie A.; Hunter, William N.; Brenk, Ruth

2012-01-01

CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens. PMID:22563402

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

PubMed

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

L-edge spectroscopy of dilute, radiation-sensitive systems using a transition-edge-sensor array

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

Titus, Charles J.; Baker, Michael L.; Lee, Sang Jun

Here, we present X-ray absorption spectroscopy and resonant inelastic X-ray scattering (RIXS) measurements on the iron L-edge of 0.5 mM aqueous ferricyanide. These measurements then demonstrate the ability of high-throughput transition-edge-sensor (TES) spectrometers to access the rich soft X-ray (100–2000 eV) spectroscopy regime for dilute and radiation-sensitive samples. Our low-concentration data are in agreement with high-concentration measurements recorded by grating spectrometers. These results show that soft-X-ray RIXS spectroscopy acquired by high-throughput TES spectrometers can be used to study the local electronic structure of dilute metal-centered complexes relevant to biology, chemistry, and catalysis. In particular, TES spectrometers have a unique abilitymore » to characterize frozen solutions of radiation- and temperature-sensitive samples.« less

L-edge spectroscopy of dilute, radiation-sensitive systems using a transition-edge-sensor array

DOE PAGES

Titus, Charles J.; Baker, Michael L.; Lee, Sang Jun; ...

2017-12-07

Here, we present X-ray absorption spectroscopy and resonant inelastic X-ray scattering (RIXS) measurements on the iron L-edge of 0.5 mM aqueous ferricyanide. These measurements then demonstrate the ability of high-throughput transition-edge-sensor (TES) spectrometers to access the rich soft X-ray (100–2000 eV) spectroscopy regime for dilute and radiation-sensitive samples. Our low-concentration data are in agreement with high-concentration measurements recorded by grating spectrometers. These results show that soft-X-ray RIXS spectroscopy acquired by high-throughput TES spectrometers can be used to study the local electronic structure of dilute metal-centered complexes relevant to biology, chemistry, and catalysis. In particular, TES spectrometers have a unique abilitymore » to characterize frozen solutions of radiation- and temperature-sensitive samples.« less

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

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.

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...

A high volume cost efficient production macrostructuring process. [for silicon solar cell surface treatment

NASA Technical Reports Server (NTRS)

Chitre, S. R.

1978-01-01

The paper presents an experimentally developed surface macro-structuring process suitable for high volume production of silicon solar cells. The process lends itself easily to automation for high throughput to meet low-cost solar array goals. The tetrahedron structure observed is 0.5 - 12 micron high. The surface has minimal pitting with virtually no or very few undeveloped areas across the surface. This process has been developed for (100) oriented as cut silicon. Chemi-etched, hydrophobic and lapped surfaces were successfully texturized. A cost analysis as per Samics is presented.

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs

PubMed Central

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G.; Rigoutsos, Isidore

2017-01-01

Abstract Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. PMID:28108659

A high-throughput fluorescence polarization assay for inhibitors of gyrase B.

PubMed

Glaser, Bryan T; Malerich, Jeremiah P; Duellman, Sarah J; Fong, Julie; Hutson, Christopher; Fine, Richard M; Keblansky, Boris; Tang, Mary J; Madrid, Peter B

2011-02-01

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

High-throughput screening of small-molecule adsorption in MOF-74

NASA Astrophysics Data System (ADS)

Thonhauser, T.; Canepa, P.

2014-03-01

Using high-throughput screening coupled with state-of-the-art van der Waals density functional theory, we investigate the adsorption properties of four important molecules, H2, CO2, CH4, and H2O in MOF-74-  with  = Be, Mg, Al, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Zr, Nb, Ru, Rh, Pd, La, W, Os, Ir, and Pt. We show that high-throughput techniques can aid in speeding up the development and refinement of effective materials for hydrogen storage, carbon capture, and gas separation. The exploration of the configurational adsorption space allows us to extract crucial information concerning, for example, the competition of water with CO2 for the adsorption binding sites. We find that only a few noble metals--Rh, Pd, Os, Ir, and Pt--favor the adsorption of CO2 and hence are potential candidates for effective carbon-capture materials. Our findings further reveal significant differences in the binding characteristics of H2, CO2, CH4, and H2O within the MOF structure, indicating that molecular blends can be successfully separated by these nano-porous materials. Supported by DOE DE-FG02-08ER46491.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Automated electrochemical synthesis and photoelectrochemical characterization of Zn1-xCo(x)O thin films for solar hydrogen production.

PubMed

Jaramillo, Thomas F; Baeck, Sung-Hyeon; Kleiman-Shwarsctein, Alan; Choi, Kyoung-Shin; Stucky, Galen D; McFarland, Eric W

2005-01-01

High-throughput electrochemical methods have been developed for the investigation of Zn1-xCo(x)O films for photoelectrochemical hydrogen production from water. A library of 120 samples containing 27 different compositions (0

Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays

PubMed Central

Aryee, Martin J.; Jaffe, Andrew E.; Corrada-Bravo, Hector; Ladd-Acosta, Christine; Feinberg, Andrew P.; Hansen, Kasper D.; Irizarry, Rafael A.

2014-01-01

Motivation: The recently released Infinium HumanMethylation450 array (the ‘450k’ array) provides a high-throughput assay to quantify DNA methylation (DNAm) at ∼450 000 loci across a range of genomic features. Although less comprehensive than high-throughput sequencing-based techniques, this product is more cost-effective and promises to be the most widely used DNAm high-throughput measurement technology over the next several years. Results: Here we describe a suite of computational tools that incorporate state-of-the-art statistical techniques for the analysis of DNAm data. The software is structured to easily adapt to future versions of the technology. We include methods for preprocessing, quality assessment and detection of differentially methylated regions from the kilobase to the megabase scale. We show how our software provides a powerful and flexible development platform for future methods. We also illustrate how our methods empower the technology to make discoveries previously thought to be possible only with sequencing-based methods. Availability and implementation: http://bioconductor.org/packages/release/bioc/html/minfi.html. Contact: khansen@jhsph.edu; rafa@jimmy.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24478339

GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies

PubMed Central

Zhang, Bing; Schmoyer, Denise; Kirov, Stefan; Snoddy, Jay

2004-01-01

Background Microarray and other high-throughput technologies are producing large sets of interesting genes that are difficult to analyze directly. Bioinformatics tools are needed to interpret the functional information in the gene sets. Results We have created a web-based tool for data analysis and data visualization for sets of genes called GOTree Machine (GOTM). This tool was originally intended to analyze sets of co-regulated genes identified from microarray analysis but is adaptable for use with other gene sets from other high-throughput analyses. GOTree Machine generates a GOTree, a tree-like structure to navigate the Gene Ontology Directed Acyclic Graph for input gene sets. This system provides user friendly data navigation and visualization. Statistical analysis helps users to identify the most important Gene Ontology categories for the input gene sets and suggests biological areas that warrant further study. GOTree Machine is available online at . Conclusion GOTree Machine has a broad application in functional genomic, proteomic and other high-throughput methods that generate large sets of interesting genes; its primary purpose is to help users sort for interesting patterns in gene sets. PMID:14975175

Finding the Stable Structures of WxN1-x with an ab-initio High-Throughput Approach

DTIC Science & Technology

2014-03-13

cubic boron nitride[4], carbonitrides,[5] and transition metal borides .[6, 7] Over the past several years there has been considerable theoretical...include ionic and covalent structures which seem chemically similar to W-N. These include borides , carbides, oxides, and other nitrides. In this paper we...metallic alloys, [23–27] we extended it to include over fifty new structures. These include nitrides, oxides, borides , and carbides. The important

High-Throughput, Adaptive FFT Architecture for FPGA-Based Spaceborne Data Processors

NASA Technical Reports Server (NTRS)

NguyenKobayashi, Kayla; Zheng, Jason X.; He, Yutao; Shah, Biren N.

2011-01-01

Exponential growth in microelectronics technology such as field-programmable gate arrays (FPGAs) has enabled high-performance spaceborne instruments with increasing onboard data processing capabilities. As a commonly used digital signal processing (DSP) building block, fast Fourier transform (FFT) has been of great interest in onboard data processing applications, which needs to strike a reasonable balance between high-performance (throughput, block size, etc.) and low resource usage (power, silicon footprint, etc.). It is also desirable to be designed so that a single design can be reused and adapted into instruments with different requirements. The Multi-Pass Wide Kernel FFT (MPWK-FFT) architecture was developed, in which the high-throughput benefits of the parallel FFT structure and the low resource usage of Singleton s single butterfly method is exploited. The result is a wide-kernel, multipass, adaptive FFT architecture. The 32K-point MPWK-FFT architecture includes 32 radix-2 butterflies, 64 FIFOs to store the real inputs, 64 FIFOs to store the imaginary inputs, complex twiddle factor storage, and FIFO logic to route the outputs to the correct FIFO. The inputs are stored in sequential fashion into the FIFOs, and the outputs of each butterfly are sequentially written first into the even FIFO, then the odd FIFO. Because of the order of the outputs written into the FIFOs, the depth of the even FIFOs, which are 768 each, are 1.5 times larger than the odd FIFOs, which are 512 each. The total memory needed for data storage, assuming that each sample is 36 bits, is 2.95 Mbits. The twiddle factors are stored in internal ROM inside the FPGA for fast access time. The total memory size to store the twiddle factors is 589.9Kbits. This FFT structure combines the benefits of high throughput from the parallel FFT kernels and low resource usage from the multi-pass FFT kernels with desired adaptability. Space instrument missions that need onboard FFT capabilities such as the proposed DESDynl, SWOT (Surface Water Ocean Topography), and Europa sounding radar missions would greatly benefit from this technology with significant reductions in non-recurring cost and risk.

High Throughput Transcriptomics: From screening to pathways

EPA Science Inventory

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

77 FR 68773 - FIFRA Scientific Advisory Panel; Notice of Public Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-16

... for physical chemical properties that cannot be easily tested in in vitro systems or stable enough for.... Quantitative structural-activity relationship (QSAR) models and estrogen receptor (ER) expert systems development. High-throughput data generation and analysis (expertise focused on how this methodology can be...

76 FR 62080 - National Institute of General Medical Sciences; Notice of Closed Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-06

... Sciences Special Emphasis Panel, Review of Applications for High- Throughput-Enabled Structural Biology... Support; 93.821, Cell Biology and Biophysics Research; 93.859, Pharmacology, Physiology, and Biological Chemistry Research; 93.862, Genetics and Developmental Biology Research; 93.88, Minority Access to Research...

High-throughput screening of chemicals as functional substitutes using structure-based classification models

EPA Science Inventory

Identifying chemicals that provide a specific function within a product, yet have minimal impact on the human body or environment, is the goal of most formulation chemists and engineers practicing green chemistry. We present a methodology to identify potential chemical functional...

Exploring the diversity of Listeria monocytogenes biofilm architecture by high-throughput confocal laser scanning microscopy and the predominance of the honeycomb-like morphotype.

PubMed

Guilbaud, Morgan; Piveteau, Pascal; Desvaux, Mickaël; Brisse, Sylvain; Briandet, Romain

2015-03-01

Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

High Throughput Experimental Materials Database

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

Zakutayev, Andriy; Perkins, John; Schwarting, Marcus

The mission of the High Throughput Experimental Materials Database (HTEM DB) is to enable discovery of new materials with useful properties by releasing large amounts of high-quality experimental data to public. The HTEM DB contains information about materials obtained from high-throughput experiments at the National Renewable Energy Laboratory (NREL).

Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.

PubMed

Forreryd, Andy; Johansson, Henrik; Albrekt, Ann-Sofie; Lindstedt, Malin

2014-05-16

Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skin sensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms--nCounter®, BioMark HD™ and OpenArray®--and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms. Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput. We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.

Fabrication of nanostructured transmissive optical devices on ITO-glass with UV1116 photoresist using high-energy electron beam lithography.

PubMed

Williams, Calum; Bartholomew, Richard; Rughoobur, Girish; Gordon, George S D; Flewitt, Andrew J; Wilkinson, Timothy D

2016-12-02

High-energy electron beam lithography for patterning nanostructures on insulating substrates can be challenging. For high resolution, conventional resists require large exposure doses and for reasonable throughput, using typical beam currents leads to charge dissipation problems. Here, we use UV1116 photoresist (Dow Chemical Company), designed for photolithographic technologies, with a relatively low area dose at a standard operating current (80 kV, 40-50 μC cm -2 , 1 nAs -1 ) to pattern over large areas on commercially coated ITO-glass cover slips. The minimum linewidth fabricated was ∼33 nm with 80 nm spacing; for isolated structures, ∼45 nm structural width with 50 nm separation. Due to the low beam dose, and nA current, throughput is high. This work highlights the use of UV1116 photoresist as an alternative to conventional e-beam resists on insulating substrates. To evaluate suitability, we fabricate a range of transmissive optical devices, that could find application for customized wire-grid polarisers and spectral filters for imaging, which operate based on the excitation of surface plasmon polaritons in nanosized geometries, with arrays encompassing areas ∼0.25 cm 2 .

Fabrication of nanostructured transmissive optical devices on ITO-glass with UV1116 photoresist using high-energy electron beam lithography

NASA Astrophysics Data System (ADS)

Williams, Calum; Bartholomew, Richard; Rughoobur, Girish; Gordon, George S. D.; Flewitt, Andrew J.; Wilkinson, Timothy D.

2016-12-01

High-energy electron beam lithography for patterning nanostructures on insulating substrates can be challenging. For high resolution, conventional resists require large exposure doses and for reasonable throughput, using typical beam currents leads to charge dissipation problems. Here, we use UV1116 photoresist (Dow Chemical Company), designed for photolithographic technologies, with a relatively low area dose at a standard operating current (80 kV, 40-50 μC cm-2, 1 nAs-1) to pattern over large areas on commercially coated ITO-glass cover slips. The minimum linewidth fabricated was ˜33 nm with 80 nm spacing; for isolated structures, ˜45 nm structural width with 50 nm separation. Due to the low beam dose, and nA current, throughput is high. This work highlights the use of UV1116 photoresist as an alternative to conventional e-beam resists on insulating substrates. To evaluate suitability, we fabricate a range of transmissive optical devices, that could find application for customized wire-grid polarisers and spectral filters for imaging, which operate based on the excitation of surface plasmon polaritons in nanosized geometries, with arrays encompassing areas ˜0.25 cm2.

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

EPA Science Inventory

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

Evaluation of Sequencing Approaches for High-Throughput Transcriptomics - (BOSC)

EPA Science Inventory

Whole-genome in vitro transcriptomics has shown the capability to identify mechanisms of action and estimates of potency for chemical-mediated effects in a toxicological framework, but with limited throughput and high cost. The generation of high-throughput global gene expression...

Mapper: high throughput maskless lithography

NASA Astrophysics Data System (ADS)

Kuiper, V.; Kampherbeek, B. J.; Wieland, M. J.; de Boer, G.; ten Berge, G. F.; Boers, J.; Jager, R.; van de Peut, T.; Peijster, J. J. M.; Slot, E.; Steenbrink, S. W. H. K.; Teepen, T. F.; van Veen, A. H. V.

2009-01-01

Maskless electron beam lithography, or electron beam direct write, has been around for a long time in the semiconductor industry and was pioneered from the mid-1960s onwards. This technique has been used for mask writing applications as well as device engineering and in some cases chip manufacturing. However because of its relatively low throughput compared to optical lithography, electron beam lithography has never been the mainstream lithography technology. To extend optical lithography double patterning, as a bridging technology, and EUV lithography are currently explored. Irrespective of the technical viability of both approaches, one thing seems clear. They will be expensive [1]. MAPPER Lithography is developing a maskless lithography technology based on massively-parallel electron-beam writing with high speed optical data transport for switching the electron beams. In this way optical columns can be made with a throughput of 10-20 wafers per hour. By clustering several of these columns together high throughputs can be realized in a small footprint. This enables a highly cost-competitive alternative to double patterning and EUV alternatives. In 2007 MAPPER obtained its Proof of Lithography milestone by exposing in its Demonstrator 45 nm half pitch structures with 110 electron beams in parallel, where all the beams where individually switched on and off [2]. In 2008 MAPPER has taken a next step in its development by building several tools. A new platform has been designed and built which contains a 300 mm wafer stage, a wafer handler and an electron beam column with 110 parallel electron beams. This manuscript describes the first patterning results with this 300 mm platform.

Genecentric: a package to uncover graph-theoretic structure in high-throughput epistasis data.

PubMed

Gallant, Andrew; Leiserson, Mark D M; Kachalov, Maxim; Cowen, Lenore J; Hescott, Benjamin J

2013-01-18

New technology has resulted in high-throughput screens for pairwise genetic interactions in yeast and other model organisms. For each pair in a collection of non-essential genes, an epistasis score is obtained, representing how much sicker (or healthier) the double-knockout organism will be compared to what would be expected from the sickness of the component single knockouts. Recent algorithmic work has identified graph-theoretic patterns in this data that can indicate functional modules, and even sets of genes that may occur in compensatory pathways, such as a BPM-type schema first introduced by Kelley and Ideker. However, to date, any algorithms for finding such patterns in the data were implemented internally, with no software being made publically available. Genecentric is a new package that implements a parallelized version of the Leiserson et al. algorithm (J Comput Biol 18:1399-1409, 2011) for generating generalized BPMs from high-throughput genetic interaction data. Given a matrix of weighted epistasis values for a set of double knock-outs, Genecentric returns a list of generalized BPMs that may represent compensatory pathways. Genecentric also has an extension, GenecentricGO, to query FuncAssociate (Bioinformatics 25:3043-3044, 2009) to retrieve GO enrichment statistics on generated BPMs. Python is the only dependency, and our web site provides working examples and documentation. We find that Genecentric can be used to find coherent functional and perhaps compensatory gene sets from high throughput genetic interaction data. Genecentric is made freely available for download under the GPLv2 from http://bcb.cs.tufts.edu/genecentric.

Genecentric: a package to uncover graph-theoretic structure in high-throughput epistasis data

PubMed Central

2013-01-01

Background New technology has resulted in high-throughput screens for pairwise genetic interactions in yeast and other model organisms. For each pair in a collection of non-essential genes, an epistasis score is obtained, representing how much sicker (or healthier) the double-knockout organism will be compared to what would be expected from the sickness of the component single knockouts. Recent algorithmic work has identified graph-theoretic patterns in this data that can indicate functional modules, and even sets of genes that may occur in compensatory pathways, such as a BPM-type schema first introduced by Kelley and Ideker. However, to date, any algorithms for finding such patterns in the data were implemented internally, with no software being made publically available. Results Genecentric is a new package that implements a parallelized version of the Leiserson et al. algorithm (J Comput Biol 18:1399-1409, 2011) for generating generalized BPMs from high-throughput genetic interaction data. Given a matrix of weighted epistasis values for a set of double knock-outs, Genecentric returns a list of generalized BPMs that may represent compensatory pathways. Genecentric also has an extension, GenecentricGO, to query FuncAssociate (Bioinformatics 25:3043-3044, 2009) to retrieve GO enrichment statistics on generated BPMs. Python is the only dependency, and our web site provides working examples and documentation. Conclusion We find that Genecentric can be used to find coherent functional and perhaps compensatory gene sets from high throughput genetic interaction data. Genecentric is made freely available for download under the GPLv2 from http://bcb.cs.tufts.edu/genecentric. PMID:23331614

High-throughput literature mining to support read-across ...

EPA Pesticide Factsheets

Building scientific confidence in the development and evaluation of read-across remains an ongoing challenge. Approaches include establishing systematic frameworks to identify sources of uncertainty and ways to address them. One source of uncertainty is related to characterizing biological similarity. Many research efforts are underway such as structuring mechanistic data in adverse outcome pathways and investigating the utility of high throughput (HT)/high content (HC) screening data. A largely untapped resource for read-across to date is the biomedical literature. This information has the potential to support read-across by facilitating the identification of valid source analogues with similar biological and toxicological profiles as well as providing the mechanistic understanding for any prediction made. A key challenge in using biomedical literature is to convert and translate its unstructured form into a computable format that can be linked to chemical structure. We developed a novel text-mining strategy to represent literature information for read across. Keywords were used to organize literature into toxicity signatures at the chemical level. These signatures were integrated with HT in vitro data and curated chemical structures. A rule-based algorithm assessed the strength of the literature relationship, providing a mechanism to rank and visualize the signature as literature ToxPIs (LitToxPIs). LitToxPIs were developed for over 6,000 chemicals for a varie

Automated glycopeptide analysis—review of current state and future directions

PubMed Central

Dallas, David C.; Martin, William F.; Hua, Serenus

2013-01-01

Glycosylation of proteins is involved in immune defense, cell–cell adhesion, cellular recognition and pathogen binding and is one of the most common and complex post-translational modifications. Science is still struggling to assign detailed mechanisms and functions to this form of conjugation. Even the structural analysis of glycoproteins—glycoproteomics—remains in its infancy due to the scarcity of high-throughput analytical platforms capable of determining glycopeptide composition and structure, especially platforms for complex biological mixtures. Glycopeptide composition and structure can be determined with high mass-accuracy mass spectrometry, particularly when combined with chromatographic separation, but the sheer volume of generated data necessitates computational software for interpretation. This review discusses the current state of glycopeptide assignment software—advances made to date and issues that remain to be addressed. The various software and algorithms developed so far provide important insights into glycoproteomics. However, there is currently no freely available software that can analyze spectral data in batch and unambiguously determine glycopeptide compositions for N- and O-linked glycopeptides from relevant biological sources such as human milk and serum. Few programs are capable of aiding in structural determination of the glycan component. To significantly advance the field of glycoproteomics, analytical software and algorithms are required that: (i) solve for both N- and O-linked glycopeptide compositions, structures and glycosites in biological mixtures; (ii) are high-throughput and process data in batches; (iii) can interpret mass spectral data from a variety of sources and (iv) are open source and freely available. PMID:22843980

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

PubMed Central

Osterman, Ilya A.; Komarova, Ekaterina S.; Shiryaev, Dmitry I.; Korniltsev, Ilya A.; Khven, Irina M.; Lukyanov, Dmitry A.; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Efremenkova, Olga V.; Ivanenkov, Yan A.; Bogdanov, Alexey A.; Dontsova, Olga A.

2016-01-01

In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals. PMID:27736765

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

PubMed

Tan, Wui Siew; Lewis, Christina L; Horelik, Nicholas E; Pregibon, Daniel C; Doyle, Patrick S; Yi, Hyunmin

2008-11-04

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

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

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

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

EPA Science Inventory

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

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

PubMed Central

Suzuki, Yasuhiro; Kagawa, Naoko; Fujino, Toru; Sumiya, Tsuyoshi; Andoh, Taichi; Ishikawa, Kumiko; Kimura, Rie; Kemmochi, Kiyokazu; Ohta, Tsutomu; Tanaka, Shigeo

2005-01-01

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. PMID:16009811

Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-secretase.

PubMed

Barrow, James C; Stauffer, Shaun R; Rittle, Kenneth E; Ngo, Phung L; Yang, ZhiQiang; Selnick, Harold G; Graham, Samuel L; Munshi, Sanjeev; McGaughey, Georgia B; Holloway, M Katharine; Simon, Adam J; Price, Eric A; Sankaranarayanan, Sethu; Colussi, Dennis; Tugusheva, Katherine; Lai, Ming-Tain; Espeseth, Amy S; Xu, Min; Huang, Qian; Wolfe, Abigail; Pietrak, Beth; Zuck, Paul; Levorse, Dorothy A; Hazuda, Daria; Vacca, Joseph P

2008-10-23

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Aggregating Data for Computational Toxicology Applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System

EPA Science Inventory

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for...

The Upgrade Programme for the Structural Biology beamlines at the European Synchrotron Radiation Facility - High throughput sample evaluation and automation

NASA Astrophysics Data System (ADS)

Theveneau, P.; Baker, R.; Barrett, R.; Beteva, A.; Bowler, M. W.; Carpentier, P.; Caserotto, H.; de Sanctis, D.; Dobias, F.; Flot, D.; Guijarro, M.; Giraud, T.; Lentini, M.; Leonard, G. A.; Mattenet, M.; McCarthy, A. A.; McSweeney, S. M.; Morawe, C.; Nanao, M.; Nurizzo, D.; Ohlsson, S.; Pernot, P.; Popov, A. N.; Round, A.; Royant, A.; Schmid, W.; Snigirev, A.; Surr, J.; Mueller-Dieckmann, C.

2013-03-01

Automation and advances in technology are the key elements in addressing the steadily increasing complexity of Macromolecular Crystallography (MX) experiments. Much of this complexity is due to the inter-and intra-crystal heterogeneity in diffraction quality often observed for crystals of multi-component macromolecular assemblies or membrane proteins. Such heterogeneity makes high-throughput sample evaluation an important and necessary tool for increasing the chances of a successful structure determination. The introduction at the ESRF of automatic sample changers in 2005 dramatically increased the number of samples that were tested for diffraction quality. This "first generation" of automation, coupled with advances in software aimed at optimising data collection strategies in MX, resulted in a three-fold increase in the number of crystal structures elucidated per year using data collected at the ESRF. In addition, sample evaluation can be further complemented using small angle scattering experiments on the newly constructed bioSAXS facility on BM29 and the micro-spectroscopy facility (ID29S). The construction of a second generation of automated facilities on the MASSIF (Massively Automated Sample Screening Integrated Facility) beam lines will build on these advances and should provide a paradigm shift in how MX experiments are carried out which will benefit the entire Structural Biology community.

Metabolic cancer biology: structural-based analysis of cancer as a metabolic disease, new sights and opportunities for disease treatment.

PubMed

Masoudi-Nejad, Ali; Asgari, Yazdan

2015-02-01

The cancer cell metabolism or the Warburg effect discovery goes back to 1924 when, for the first time Otto Warburg observed, in contrast to the normal cells, cancer cells have different metabolism. With the initiation of high throughput technologies and computational systems biology, cancer cell metabolism renaissances and many attempts were performed to revise the Warburg effect. The development of experimental and analytical tools which generate high-throughput biological data including lots of information could lead to application of computational models in biological discovery and clinical medicine especially for cancer. Due to the recent availability of tissue-specific reconstructed models, new opportunities in studying metabolic alteration in various kinds of cancers open up. Structural approaches at genome-scale levels seem to be suitable for developing diagnostic and prognostic molecular signatures, as well as in identifying new drug targets. In this review, we have considered these recent advances in structural-based analysis of cancer as a metabolic disease view. Two different structural approaches have been described here: topological and constraint-based methods. The ultimate goal of this type of systems analysis is not only the discovery of novel drug targets but also the development of new systems-based therapy strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.

EPA Project Updates: DSSTox and ToxCast Generating New ...

EPA Pesticide Factsheets

EPAs National Center for Computational Toxicology is building capabilities to support a new paradigm for toxicity screening and prediction. The DSSTox project is improving public access to quality structure-annotated chemical toxicity information in less summarized forms than traditionally employed in SAR modeling, and in ways that facilitate data-mining, and data read-across. The DSSTox Structure-Browser, launched in September 2007, provides structure searchability across all published DSSTox toxicity-related inventory, and is enabling linkages between previously isolated toxicity data resources. As of early March 2008, the public DSSTox inventory as been integrated into PubChem, allowing a user to take full advantage of PubChem structure-activity and bioassay clustering features. The most recent DSSTox version of Carcinogenic Potency Database file (CPDBAS) illustrates ways in which various summary definitions of carcinogenic activity can be employed in modeling and data mining. Phase I of the ToxCast project is generating high-throughput screening data from several hundred biochemical and cell-based assays for a set of 320 chemicals, mostly pesticide actives, with rich toxicology profiles. Incorporating and expanding traditional SAR Concepts into this new high-throughput and data-rich would pose conceptual and practical challenges, but also holds great promise for improving predictive capabilities. EPA's National Center for Computational Toxicology is bu

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

PubMed

Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C; Johnson, Michael E

2014-01-01

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development. Copyright © 2013. Published by Elsevier Ltd.

Facile fabrication of nanofluidic diode membranes using anodic aluminium oxide.

PubMed

Wu, Songmei; Wildhaber, Fabien; Vazquez-Mena, Oscar; Bertsch, Arnaud; Brugger, Juergen; Renaud, Philippe

2012-09-21

Active control of ion transport plays important roles in chemical and biological analytical processes. Nanofluidic systems hold the promise for such control through electrostatic interaction between ions and channel surfaces. Most existing experiments rely on planar geometry where the nanochannels are generally very long and shallow with large aspect ratios. Based on this configuration the concepts of nanofluidic gating and rectification have been successfully demonstrated. However, device minimization and throughput scaling remain significant challenges. We report here an innovative and facile realization of hetero-structured Al(2)O(3)/SiO(2) (Si) nanopore array membranes by using pattern transfer of self-organized nanopore structures of anodic aluminum oxide (AAO). Thanks to the opposite surface charge states of Al(2)O(3) (positive) and SiO(2) (negative), the membrane exhibits clear rectification of ion current in electrolyte solutions with very low aspect ratios compared to previous approaches. Our hetero-structured nanopore arrays provide a valuable platform for high throughput applications such as molecular separation, chemical processors and energy conversion.

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

DOE PAGES

Jung, Seung-Yong; Notton, Timothy; Fong, Erika; ...

2015-01-07

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min -1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

Quantitative description on structure–property relationships of Li-ion battery materials for high-throughput computations

PubMed Central

Wang, Youwei; Zhang, Wenqing; Chen, Lidong; Shi, Siqi; Liu, Jianjun

2017-01-01

Abstract Li-ion batteries are a key technology for addressing the global challenge of clean renewable energy and environment pollution. Their contemporary applications, for portable electronic devices, electric vehicles, and large-scale power grids, stimulate the development of high-performance battery materials with high energy density, high power, good safety, and long lifetime. High-throughput calculations provide a practical strategy to discover new battery materials and optimize currently known material performances. Most cathode materials screened by the previous high-throughput calculations cannot meet the requirement of practical applications because only capacity, voltage and volume change of bulk were considered. It is important to include more structure–property relationships, such as point defects, surface and interface, doping and metal-mixture and nanosize effects, in high-throughput calculations. In this review, we established quantitative description of structure–property relationships in Li-ion battery materials by the intrinsic bulk parameters, which can be applied in future high-throughput calculations to screen Li-ion battery materials. Based on these parameterized structure–property relationships, a possible high-throughput computational screening flow path is proposed to obtain high-performance battery materials. PMID:28458737

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53

PubMed Central

Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena

2016-01-01

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways. PMID:27124407

Deep sequencing in library selection projects: what insight does it bring?

PubMed

Glanville, J; D'Angelo, S; Khan, T A; Reddy, S T; Naranjo, L; Ferrara, F; Bradbury, A R M

2015-08-01

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Qgui: A high-throughput interface for automated setup and analysis of free energy calculations and empirical valence bond simulations in biological systems.

PubMed

Isaksen, Geir Villy; Andberg, Tor Arne Heim; Åqvist, Johan; Brandsdal, Bjørn Olav

2015-07-01

Structural information and activity data has increased rapidly for many protein targets during the last decades. In this paper, we present a high-throughput interface (Qgui) for automated free energy and empirical valence bond (EVB) calculations that use molecular dynamics (MD) simulations for conformational sampling. Applications to ligand binding using both the linear interaction energy (LIE) method and the free energy perturbation (FEP) technique are given using the estrogen receptor (ERα) as a model system. Examples of free energy profiles obtained using the EVB method for the rate-limiting step of the enzymatic reaction catalyzed by trypsin are also shown. In addition, we present calculation of high-precision Arrhenius plots to obtain the thermodynamic activation enthalpy and entropy with Qgui from running a large number of EVB simulations. Copyright © 2015 Elsevier Inc. All rights reserved.

Deep sequencing in library selection projects: what insight does it bring?

PubMed Central

Glanville, J; D’Angelo, S; Khan, T.A.; Reddy, S. T.; Naranjo, L.; Ferrara, F.; Bradbury, A.R.M.

2015-01-01

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology. PMID:26451649

Characterization of DNA-protein interactions using high-throughput sequencing data from pulldown experiments

NASA Astrophysics Data System (ADS)

Moreland, Blythe; Oman, Kenji; Curfman, John; Yan, Pearlly; Bundschuh, Ralf

Methyl-binding domain (MBD) protein pulldown experiments have been a valuable tool in measuring the levels of methylated CpG dinucleotides. Due to the frequent use of this technique, high-throughput sequencing data sets are available that allow a detailed quantitative characterization of the underlying interaction between methylated DNA and MBD proteins. Analyzing such data sets, we first found that two such proteins cannot bind closer to each other than 2 bp, consistent with structural models of the DNA-protein interaction. Second, the large amount of sequencing data allowed us to find rather weak but nevertheless clearly statistically significant sequence preferences for several bases around the required CpG. These results demonstrate that pulldown sequencing is a high-precision tool in characterizing DNA-protein interactions. This material is based upon work supported by the National Science Foundation under Grant No. DMR-1410172.

High-throughput screening (HTS) and modeling of the retinoid ...

EPA Pesticide Factsheets

Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Evaluating High Throughput Toxicokinetics and Toxicodynamics for IVIVE (WC10)

EPA Science Inventory

High-throughput screening (HTS) generates in vitro data for characterizing potential chemical hazard. TK models are needed to allow in vitro to in vivo extrapolation (IVIVE) to real world situations. The U.S. EPA has created a public tool (R package “httk” for high throughput tox...

High-throughput RAD-SNP genotyping for characterization of sugar beet genotypes

USDA-ARS?s Scientific Manuscript database

High-throughput SNP genotyping provides a rapid way of developing resourceful set of markers for delineating the genetic architecture and for effective species discrimination. In the presented research, we demonstrate a set of 192 SNPs for effective genotyping in sugar beet using high-throughput mar...

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

EPA Science Inventory

Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

A quantitative literature-curated gold standard for kinase-substrate pairs

PubMed Central

2011-01-01

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future. PMID:21492431

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

PubMed

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

2016-09-12

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

Outlook for Development of High-throughput Cryopreservation for Small-bodied Biomedical Model Fishes★

PubMed Central

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

2011-01-01

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. PMID:21440666

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

Discovery of imidazo[1,2-b]pyridazine derivatives as IKKbeta inhibitors. Part 1: Hit-to-lead study and structure-activity relationship.

PubMed

Shimizu, Hiroki; Tanaka, Shinji; Toki, Tadashi; Yasumatsu, Isao; Akimoto, Toshihiko; Morishita, Kaoru; Yamasaki, Tomonori; Yasukochi, Takanori; Iimura, Shin

2010-09-01

Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKbeta inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKbeta inhibitory activity and TNFalpha inhibitory activity in THP-1 cell increased. Also, these compounds showed high kinase selectivity. The structure-activity relationship was revealed and the interaction model of imidazo[1,2-b]pyridazine compounds with IKKbeta was constructed. Copyright 2010. Published by Elsevier Ltd.

Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays

PubMed Central

Yang, Jing; Mei, Ying; Hook, Andrew L.; Taylor, Michael; Urquhart, Andrew J.; Bogatyrev, Said R.; Langer, Robert; Anderson, Daniel G.; Davies, Martyn C.; Alexander, Morgan R.

2010-01-01

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterisation (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), x-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability of identifying surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis. PMID:20832108

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen.

PubMed

Nir, Oaz; Bakal, Chris; Perrimon, Norbert; Berger, Bonnie

2010-03-01

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.

TCP Throughput Profiles Using Measurements over Dedicated Connections

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

Rao, Nageswara S.; Liu, Qiang; Sen, Satyabrata

Wide-area data transfers in high-performance computing infrastructures are increasingly being carried over dynamically provisioned dedicated network connections that provide high capacities with no competing traffic. We present extensive TCP throughput measurements and time traces over a suite of physical and emulated 10 Gbps connections with 0-366 ms round-trip times (RTTs). Contrary to the general expectation, they show significant statistical and temporal variations, in addition to the overall dependencies on the congestion control mechanism, buffer size, and the number of parallel streams. We analyze several throughput profiles that have highly desirable concave regions wherein the throughput decreases slowly with RTTs, inmore » stark contrast to the convex profiles predicted by various TCP analytical models. We present a generic throughput model that abstracts the ramp-up and sustainment phases of TCP flows, which provides insights into qualitative trends observed in measurements across TCP variants: (i) slow-start followed by well-sustained throughput leads to concave regions; (ii) large buffers and multiple parallel streams expand the concave regions in addition to improving the throughput; and (iii) stable throughput dynamics, indicated by a smoother Poincare map and smaller Lyapunov exponents, lead to wider concave regions. These measurements and analytical results together enable us to select a TCP variant and its parameters for a given connection to achieve high throughput with statistical guarantees.« less

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

PubMed

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G; Rigoutsos, Isidore; Kirino, Yohei

2017-05-19

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Enhancing high throughput toxicology - development of putative adverse outcome pathways linking US EPA ToxCast screening targets to relevant apical hazards.

EPA Science Inventory

High throughput toxicology programs, such as ToxCast and Tox21, have provided biological effects data for thousands of chemicals at multiple concentrations. Compared to traditional, whole-organism approaches, high throughput assays are rapid and cost-effective, yet they generall...

Evaluation of High-Throughput Chemical Exposure Models via Analysis of Matched Environmental and Biological Media Measurements

EPA Science Inventory

The U.S. EPA, under its ExpoCast program, is developing high-throughput near-field modeling methods to estimate human chemical exposure and to provide real-world context to high-throughput screening (HTS) hazard data. These novel modeling methods include reverse methods to infer ...

The development of a general purpose ARM-based processing unit for the ATLAS TileCal sROD

NASA Astrophysics Data System (ADS)

Cox, M. A.; Reed, R.; Mellado, B.

2015-01-01

After Phase-II upgrades in 2022, the data output from the LHC ATLAS Tile Calorimeter will increase significantly. ARM processors are common in mobile devices due to their low cost, low energy consumption and high performance. It is proposed that a cost-effective, high data throughput Processing Unit (PU) can be developed by using several consumer ARM processors in a cluster configuration to allow aggregated processing performance and data throughput while maintaining minimal software design difficulty for the end-user. This PU could be used for a variety of high-level functions on the high-throughput raw data such as spectral analysis and histograms to detect possible issues in the detector at a low level. High-throughput I/O interfaces are not typical in consumer ARM System on Chips but high data throughput capabilities are feasible via the novel use of PCI-Express as the I/O interface to the ARM processors. An overview of the PU is given and the results for performance and throughput testing of four different ARM Cortex System on Chips are presented.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

PubMed Central

Xiao, Rong; Anderson, Stephen; Aramini, James; Belote, Rachel; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John K.; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Jiang, Mei; Kornhaber, Gregory J.; Lee, Dong Yup; Locke, Jessica Y.; Ma, Li-Chung; Maglaqui, Melissa; Mao, Lei; Mitra, Saheli; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Sharma, Seema; Shastry, Ritu; Swapna, G.V.T.; Tong, Saichu N.; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.; Acton, Thomas B.

2014-01-01

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities. PMID:20688167

Ligand screening systems for human glucose transporters as tools in drug discovery

NASA Astrophysics Data System (ADS)

Schmidl, Sina; Iancu, Cristina V.; Choe, Jun-yong; Oreb, Mislav

2018-05-01

Hexoses are the major source of energy and carbon skeletons for biosynthetic processes in all kingdoms of life. Their cellular uptake is mediated by specialized transporters, including glucose transporters (GLUT, SLC2 gene family). Malfunction or altered expression pattern of GLUTs in humans is associated with several widespread diseases including cancer, diabetes and severe metabolic disorders. Their high relevance in the medical area makes these transporters valuable drug targets and potential biomarkers. Nevertheless, the lack of a suitable high-throughput screening system has impeded the determination of compounds that would enable specific manipulation of GLUTs so far. Availability of structural data on several GLUTs enabled in silico ligand screening, though limited by the fact that only two major conformations of the transporters can be tested. Recently, convenient high-throughput microbial and cell-free screening systems have been developed. These remarkable achievements set the foundation for further and detailed elucidation of the molecular mechanisms of glucose transport and will also lead to great progress in the discovery of GLUT effectors as therapeutic agents. In this mini-review, we focus on recent efforts to identify potential GLUT-targeting drugs, based on a combination of structural biology and different assay systems.

Quantifying collagen orientation in breast tissue biopsies using SLIM (Conference Presentation)

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Okoro, Chukwuemeka; Balla, Andre; Toussaint, Kimani C.; Popescu, Gabriel

2017-02-01

Breast cancer is a major public health problem worldwide, being the most common type of cancer among women according to the World Health Organization (WHO). The WHO has further stressed the importance of an early determination of the disease course through prognostic markers. Recent studies have shown that the alignment of collagen fibers in tumor adjacent stroma correlate with poorer health outcomes in patients. Such studies have typically been carried out using Second-Harmonic Generation (SHG) microscopy. SHG images are very useful for quantifying collagen fiber orientation due their specificity to non-centrosymmetric structures in tissue, leading to high contrast in collagen rich areas. However, the imaging throughput in SHG microscopy is limited by its point scanning geometry. In this work, we show that SLIM, a wide-field high-throughput QPI technique, can be used to obtain the same information on collagen fiber orientation as is obtainable through SHG microscopy. We imaged a tissue microarray containing both benign and malignant cores using both SHG microscopy and SLIM. The cellular (non-collagenous) structures in the SLIM images were next segmented out using an algorithm developed in-house. Using the previously published Fourier Transform Second Harmonic Generation (FT-SHG) tool, the fiber orientations in SHG and segmented SLIM images were then quantified. The resulting histograms of fiber orientation angles showed that both SHG and SLIM generate similar measurements of collagen fiber orientation. The SLIM modality, however, can generate these results at much higher throughput due to its wide-field, whole-slide scanning capabilities.

Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

PubMed

Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

2016-11-01

High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Methods for fabrication of positional and compositionally controlled nanostructures on substrate

DOEpatents

Zhu, Ji; Grunes, Jeff; Choi, Yang-Kyu; Bokor, Jeffrey; Somorjai, Gabor

2013-07-16

Fabrication methods disclosed herein provide for a nanoscale structure or a pattern comprising a plurality of nanostructures of specific predetermined position, shape and composition, including nanostructure arrays having large area at high throughput necessary for industrial production. The resultant nanostracture patterns are useful for nanostructure arrays, specifically sensor and catalytic arrays.

Cheminformatics and Data Mining Approaches for Exploring the Alternatives Testing Landscsape: Case Studies in ToxCast and Skin Sensitization (BOSC CSS)

EPA Science Inventory

Cheminformatics approaches and structure-based rules are being used to evaluate and explore the ToxCast chemical landscape and associated high-throughput screening (HTS) data. We have shown that the library provides comprehensive coverage of the knowledge domains and target inven...

Supporting read-across predictions of chemical toxicity using high-throughput text-mining (ACS 2017 Spring meeting )

EPA Science Inventory

Read-across is a technique used to fill data gaps within chemical safety assessments. It is based on the premise that chemicals with similar structures are likely to have similar biological activities. Known information on the property of a chemical (source) is used to make a pre...

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

EPA Science Inventory

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

Community standards for genomic resources, genetic conservation, and data integration

Treesearch

Jill Wegrzyn; Meg Staton; Emily Grau; Richard Cronn; C. Dana Nelson

2017-01-01

Genetics and genomics are increasingly important in forestry management and conservation. Next generation sequencing can increase analytical power, but still relies on building on the structure of previously acquired data. Data standards and data sharing allow the community to maximize the analytical power of high throughput genomics data. The landscape of incomplete...

75 FR 51082 - Center for Scientific Review; Notice of Closed Meetings

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-18

...: Muscle Biology. Date: September 8, 2010. Time: 11 a.m. to 3 p.m. Agenda: To review and evaluate grant...: Technology Development for High-Throughput Structural Biology Research Review. Date: September 14-15, 2010...: Biology of Development and Aging Integrated Review Group; Development--1 Study Section. Date: September 30...

[Study on Microbial Diversity of Peri-implantitis Subgingival by High-throughput Sequencing].

PubMed

Li, Zhi-jie; Wang, Shao-guo; Li, Yue-hong; Tu, Dong-xiang; Liu, Shi-yun; Nie, Hong-bing; Li, Zhi-qiang; Zhang, Ju-mei

2015-07-01

To study microbial diversity of peri-implantitis subgingival with high-throughput sequencing, and investigate microbiological etiology of peri-implantitis. Subgingival plaques were sampled from the patients with peri-implantitis (D group) and non-peri-implantitis subjects (N group). The microbiological diversity of the subgingival plaques was detected by sequencing V4 region of 16S rRNA with Illumina Miseq platform. The diversity of the community structure was analyzed using Mothur software. A total of 156 507 gene sequences were detected in nine samples and 4 402 operational taxonomic units (OTUs) were found. Selenomonas, Pseudomonas, and Fusobacterium were dominant bacteria in D group, while Fusobacterium, Veillonella and Streptococcus were dominant bacteria in N group. Differences between peri-implantitis and non-peri-implantitis bacterial communities were observed at all phylogenetic levels by LEfSe, which was also found in PcoA test. The occurrence of peri-implantitis is not only related to periodontitis pathogenic microbe, but also related with the changes of oral microbial community structure. Treponema, Herbaspirillum, Butyricimonas and Phaeobacte may be closely related to the occurrence and development of peri-implantitis.

Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

PubMed

Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J; Panarese, Joseph D; Smith, Emery; Chase, Peter; Hodder, Peter; Niswender, Colleen; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

2015-01-15

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase

PubMed Central

Babaoglu, Kerim; Simeonov, Anton; Irwin, John J.; Nelson, Michael E.; Feng, Brian; Thomas, Craig J.; Cancian, Laura; Costi, M. Paola; Maltby, David A.; Jadhav, Ajit; Inglese, James; Austin, Christopher P.; Shoichet, Brian K.

2009-01-01

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate “hit lists”; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against β-lactamase using quantitative HTS (qHTS). Of the 1274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting β-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 µM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens. PMID:18333608

High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level

PubMed Central

Gong, Hui; Xu, Dongli; Yuan, Jing; Li, Xiangning; Guo, Congdi; Peng, Jie; Li, Yuxin; Schwarz, Lindsay A.; Li, Anan; Hu, Bihe; Xiong, Benyi; Sun, Qingtao; Zhang, Yalun; Liu, Jiepeng; Zhong, Qiuyuan; Xu, Tonghui; Zeng, Shaoqun; Luo, Qingming

2016-01-01

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. PMID:27374071

Automated recycling of chemistry for virtual screening and library design.

PubMed

Vainio, Mikko J; Kogej, Thierry; Raubacher, Florian

2012-07-23

An early stage drug discovery project needs to identify a number of chemically diverse and attractive compounds. These hit compounds are typically found through high-throughput screening campaigns. The diversity of the chemical libraries used in screening is therefore important. In this study, we describe a virtual high-throughput screening system called Virtual Library. The system automatically "recycles" validated synthetic protocols and available starting materials to generate a large number of virtual compound libraries, and allows for fast searches in the generated libraries using a 2D fingerprint based screening method. Virtual Library links the returned virtual hit compounds back to experimental protocols to quickly assess the synthetic accessibility of the hits. The system can be used as an idea generator for library design to enrich the screening collection and to explore the structure-activity landscape around a specific active compound.

A kinase-focused compound collection: compilation and screening strategy.

PubMed

Sun, Dongyu; Chuaqui, Claudio; Deng, Zhan; Bowes, Scott; Chin, Donovan; Singh, Juswinder; Cullen, Patrick; Hankins, Gretchen; Lee, Wen-Cherng; Donnelly, Jason; Friedman, Jessica; Josiah, Serene

2006-06-01

Lead identification by high-throughput screening of large compound libraries has been supplemented with virtual screening and focused compound libraries. To complement existing approaches for lead identification at Biogen Idec, a kinase-focused compound collection was designed, developed and validated. Two strategies were adopted to populate the compound collection: a ligand shape-based virtual screening and a receptor-based approach (structural interaction fingerprint). Compounds selected with the two approaches were cherry-picked from an existing high-throughput screening compound library, ordered from suppliers and supplemented with specific medicinal compounds from internal programs. Promising hits and leads have been generated from the kinase-focused compound collection against multiple kinase targets. The principle of the collection design and screening strategy was validated and the use of the kinase-focused compound collection for lead identification has been added to existing strategies.

High-throughput analysis of the protein sequence-stability landscape using a quantitative "yeast surface two-hybrid" system and fragment reconstitution

PubMed Central

Dutta, Sanjib; Koide, Akiko; Koide, Shohei

2008-01-01

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore the sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a "yeast surface two-hybrid" method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are predominantly recovered. Thus, it allows for the isolation of sequences that exhibits a desired level of stability. We identified over one hundred unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means to rapidly assess stability of many variants, to systematically assess contribution of different factors to protein stability and to enhance protein stability. PMID:18674545

Optical designs of reflection and refraction collection optics for a JT-60SA core Thomson scattering system

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

Tojo, H.; Hatae, T.; Hamano, T.

2013-09-15

Collection optics for core measurements in a JT-60SA Thomson scattering system were designed. The collection optics will be installed in a limited space and have a wide field of view and wide wavelength range. Two types of the optics are therefore suggested: refraction and reflection types. The reflection system, with a large primary mirror, avoids large chromatic aberrations. Because the size limit of the primary mirror and vignetting due to the secondary mirror affect the total collection throughput, conditions that provide the high throughput are found through an optimization. A refraction system with four lenses forming an Ernostar system ismore » also employed. The use of high-refractive-index glass materials enhances the freedom of the lens curvatures, resulting in suppression of the spherical and coma aberration. Moreover, sufficient throughput can be achieved, even with smaller lenses than that of a previous design given in [H. Tojo, T. Hatae, T. Sakuma, T. Hamano, K. Itami, Y. Aida, S. Suitoh, and D. Fujie, Rev. Sci. Instrum. 81, 10D539 (2010)]. The optical resolutions of the reflection and refraction systems are both sufficient for understanding the spatial structures in plasma. In particular, the spot sizes at the image of the optics are evaluated as ∼0.3 mm and ∼0.4 mm, respectively. The throughput for the two systems, including the pupil size and transmissivity, are also compared. The results show that good measurement accuracy (<10%) even at high electron temperatures (<30 keV) can be expected in the refraction system.« less

Optical designs of reflection and refraction collection optics for a JT-60SA core Thomson scattering system.

PubMed

Tojo, H; Hatae, T; Hamano, T; Sakuma, T; Itami, K

2013-09-01

Collection optics for core measurements in a JT-60SA Thomson scattering system were designed. The collection optics will be installed in a limited space and have a wide field of view and wide wavelength range. Two types of the optics are therefore suggested: refraction and reflection types. The reflection system, with a large primary mirror, avoids large chromatic aberrations. Because the size limit of the primary mirror and vignetting due to the secondary mirror affect the total collection throughput, conditions that provide the high throughput are found through an optimization. A refraction system with four lenses forming an Ernostar system is also employed. The use of high-refractive-index glass materials enhances the freedom of the lens curvatures, resulting in suppression of the spherical and coma aberration. Moreover, sufficient throughput can be achieved, even with smaller lenses than that of a previous design given in [H. Tojo, T. Hatae, T. Sakuma, T. Hamano, K. Itami, Y. Aida, S. Suitoh, and D. Fujie, Rev. Sci. Instrum. 81, 10D539 (2010)]. The optical resolutions of the reflection and refraction systems are both sufficient for understanding the spatial structures in plasma. In particular, the spot sizes at the image of the optics are evaluated as ~0.3 mm and ~0.4 mm, respectively. The throughput for the two systems, including the pupil size and transmissivity, are also compared. The results show that good measurement accuracy (<10%) even at high electron temperatures (<30 keV) can be expected in the refraction system.

Simulation modeling of high-throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing

PubMed Central

Hu, E; Liao, T. W.; Tiersch, T. R.

2013-01-01

Emerging commercial-level technology for aquatic sperm cryopreservation has not been modeled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high-throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: 1) predict the maximum output for 8-hr workday; 2) analyze the bottlenecks within the process, and 3) estimate operational costs when run for daily maximum output. High-throughput cryopreservation was divided into six major steps modeled with time, resources and logic structures. The modeled production processed 18 fish and produced 1164 ± 33 (mean ± SD) 0.5-ml straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the US and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation, and biomedical model fishes. PMID:25580079

Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

PubMed Central

Bokulich, Nicholas A.

2013-01-01

Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

ChemHTPS - A virtual high-throughput screening program suite for the chemical and materials sciences

NASA Astrophysics Data System (ADS)

Afzal, Mohammad Atif Faiz; Evangelista, William; Hachmann, Johannes

The discovery of new compounds, materials, and chemical reactions with exceptional properties is the key for the grand challenges in innovation, energy and sustainability. This process can be dramatically accelerated by means of the virtual high-throughput screening (HTPS) of large-scale candidate libraries. The resulting data can further be used to study the underlying structure-property relationships and thus facilitate rational design capability. This approach has been extensively used for many years in the drug discovery community. However, the lack of openly available virtual HTPS tools is limiting the use of these techniques in various other applications such as photovoltaics, optoelectronics, and catalysis. Thus, we developed ChemHTPS, a general-purpose, comprehensive and user-friendly suite, that will allow users to efficiently perform large in silico modeling studies and high-throughput analyses in these applications. ChemHTPS also includes a massively parallel molecular library generator which offers a multitude of options to customize and restrict the scope of the enumerated chemical space and thus tailor it for the demands of specific applications. To streamline the non-combinatorial exploration of chemical space, we incorporate genetic algorithms into the framework. In addition to implementing smarter algorithms, we also focus on the ease of use, workflow, and code integration to make this technology more accessible to the community.

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

PubMed

Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

2016-05-01

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

High-throughput design and optimization of fast lithium ion conductors by the combination of bond-valence method and density functional theory

NASA Astrophysics Data System (ADS)

Xiao, Ruijuan; Li, Hong; Chen, Liquan

2015-09-01

Looking for solid state electrolytes with fast lithium ion conduction is an important prerequisite for developing all-solid-state lithium secondary batteries. By combining the simulation techniques in different levels of accuracy, e.g. the bond-valence (BV) method and the density functional theory (DFT), a high-throughput design and optimization scheme is proposed for searching fast lithium ion conductors as candidate solid state electrolytes for lithium rechargeable batteries. The screening from more than 1000 compounds is performed through BV-based method, and the ability to predict reliable tendency of the Li+ migration energy barriers is confirmed by comparing with the results from DFT calculations. β-Li3PS4 is taken as a model system to demonstrate the application of this combination method in optimizing properties of solid electrolytes. By employing the high-throughput DFT simulations to more than 200 structures of the doping derivatives of β-Li3PS4, the effects of doping on the ionic conductivities in this material are predicted by the BV calculations. The O-doping scheme is proposed as a promising way to improve the kinetic properties of this materials, and the validity of the optimization is proved by the first-principles molecular dynamics (FPMD) simulations.

Sources of PCR-induced distortions in high-throughput sequencing data sets

PubMed Central

Kebschull, Justus M.; Zador, Anthony M.

2015-01-01

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

Aggregating Data for Computational Toxicology Applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System

PubMed Central

Judson, Richard S.; Martin, Matthew T.; Egeghy, Peter; Gangwal, Sumit; Reif, David M.; Kothiya, Parth; Wolf, Maritja; Cathey, Tommy; Transue, Thomas; Smith, Doris; Vail, James; Frame, Alicia; Mosher, Shad; Cohen Hubal, Elaine A.; Richard, Ann M.

2012-01-01

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built using open source tools and is freely available to download. This review describes the organization of the data repository and provides selected examples of use cases. PMID:22408426

Aggregating data for computational toxicology applications: The U.S. Environmental Protection Agency (EPA) Aggregated Computational Toxicology Resource (ACToR) System.

PubMed

Judson, Richard S; Martin, Matthew T; Egeghy, Peter; Gangwal, Sumit; Reif, David M; Kothiya, Parth; Wolf, Maritja; Cathey, Tommy; Transue, Thomas; Smith, Doris; Vail, James; Frame, Alicia; Mosher, Shad; Cohen Hubal, Elaine A; Richard, Ann M

2012-01-01

Computational toxicology combines data from high-throughput test methods, chemical structure analyses and other biological domains (e.g., genes, proteins, cells, tissues) with the goals of predicting and understanding the underlying mechanistic causes of chemical toxicity and for predicting toxicity of new chemicals and products. A key feature of such approaches is their reliance on knowledge extracted from large collections of data and data sets in computable formats. The U.S. Environmental Protection Agency (EPA) has developed a large data resource called ACToR (Aggregated Computational Toxicology Resource) to support these data-intensive efforts. ACToR comprises four main repositories: core ACToR (chemical identifiers and structures, and summary data on hazard, exposure, use, and other domains), ToxRefDB (Toxicity Reference Database, a compilation of detailed in vivo toxicity data from guideline studies), ExpoCastDB (detailed human exposure data from observational studies of selected chemicals), and ToxCastDB (data from high-throughput screening programs, including links to underlying biological information related to genes and pathways). The EPA DSSTox (Distributed Structure-Searchable Toxicity) program provides expert-reviewed chemical structures and associated information for these and other high-interest public inventories. Overall, the ACToR system contains information on about 400,000 chemicals from 1100 different sources. The entire system is built using open source tools and is freely available to download. This review describes the organization of the data repository and provides selected examples of use cases.

The Scottish Structural Proteomics Facility: targets, methods and outputs.

PubMed

Oke, Muse; Carter, Lester G; Johnson, Kenneth A; Liu, Huanting; McMahon, Stephen A; Yan, Xuan; Kerou, Melina; Weikart, Nadine D; Kadi, Nadia; Sheikh, Md Arif; Schmelz, Stefan; Dorward, Mark; Zawadzki, Michal; Cozens, Christopher; Falconer, Helen; Powers, Helen; Overton, Ian M; van Niekerk, C A Johannes; Peng, Xu; Patel, Prakash; Garrett, Roger A; Prangishvili, David; Botting, Catherine H; Coote, Peter J; Dryden, David T F; Barton, Geoffrey J; Schwarz-Linek, Ulrich; Challis, Gregory L; Taylor, Garry L; White, Malcolm F; Naismith, James H

2010-06-01

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

A high-throughput investigation of Fe-Cr-Al as a novel high-temperature coating for nuclear cladding materials.

PubMed

Bunn, Jonathan Kenneth; Fang, Randy L; Albing, Mark R; Mehta, Apurva; Kramer, Matthew J; Besser, Matthew F; Hattrick-Simpers, Jason R

2015-07-10

High-temperature alloy coatings that can resist oxidation are urgently needed as nuclear cladding materials to mitigate the danger of hydrogen explosions during meltdown. Here we apply a combination of computationally guided materials synthesis, high-throughput structural characterization and data analysis tools to investigate the feasibility of coatings from the Fe–Cr–Al alloy system. Composition-spread samples were synthesized to cover the region of the phase diagram previous bulk studies have identified as forming protective oxides. The metallurgical and oxide phase evolution were studied via in situ synchrotron glancing incidence x-ray diffraction at temperatures up to 690 K. A composition region with an Al concentration greater than 3.08 at%, and between 20.0 at% and 32.9 at% Cr showed the least overall oxide growth. Subsequently, a series of samples were deposited on stubs and their oxidation behavior at 1373 K was observed. The continued presence of a passivating oxide was confirmed in this region over a period of 6 h.

High-Throughput Printing Process for Flexible Electronics

NASA Astrophysics Data System (ADS)

Hyun, Woo Jin

Printed electronics is an emerging field for manufacturing electronic devices with low cost and minimal material waste for a variety of applications including displays, distributed sensing, smart packaging, and energy management. Moreover, its compatibility with roll-to-roll production formats and flexible substrates is desirable for continuous, high-throughput production of flexible electronics. Despite the promise, however, the roll-to-roll production of printed electronics is quite challenging due to web movement hindering accurate ink registration and high-fidelity printing. In this talk, I will present a promising strategy for roll-to-roll production using a novel printing process that we term SCALE (Self-aligned Capillarity-Assisted Lithography for Electronics). By utilizing capillarity of liquid inks on nano/micro-structured substrates, the SCALE process facilitates high-resolution and self-aligned patterning of electrically functional inks with greatly improved printing tolerance. I will show the fabrication of key building blocks (e.g. transistor, resistor, capacitor) for electronic circuits using the SCALE process on plastics.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

PubMed Central

Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

2016-01-01

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

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 range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.

Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations.

PubMed

Erickson, Heidi S

2012-09-28

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling. Copyright © 2012 John Wiley & Sons, Ltd.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

PubMed Central

Luo, Minjie; Cui, Xiangfeng; Fredman, David; Brookes, Anthony J.; Azaro, Marco A.; Greenawalt, Danielle M.; Hu, Guohong; Wang, Hui-Yun; Tereshchenko, Irina V.; Lin, Yong; Shentu, Yue; Gao, Richeng; Shen, Li; Li, Honghua

2009-01-01

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis. PMID:19384415

Genomic-assisted haplotype analysis and the development of high-throughput SNP markers for salinity tolerance in soybean

PubMed Central

Patil, Gunvant; Do, Tuyen; Vuong, Tri D.; Valliyodan, Babu; Lee, Jeong-Dong; Chaudhary, Juhi; Shannon, J. Grover; Nguyen, Henry T.

2016-01-01

Soil salinity is a limiting factor of crop yield. The soybean is sensitive to soil salinity, and a dominant gene, Glyma03g32900 is primarily responsible for salt-tolerance. The identification of high throughput and robust markers as well as the deployment of salt-tolerant cultivars are effective approaches to minimize yield loss under saline conditions. We utilized high quality (15x) whole-genome resequencing (WGRS) on 106 diverse soybean lines and identified three major structural variants and allelic variation in the promoter and genic regions of the GmCHX1 gene. The discovery of single nucleotide polymorphisms (SNPs) associated with structural variants facilitated the design of six KASPar assays. Additionally, haplotype analysis and pedigree tracking of 93 U.S. ancestral lines were performed using publically available WGRS datasets. Identified SNP markers were validated, and a strong correlation was observed between the genotype and salt treatment phenotype (leaf scorch, chlorophyll content and Na+ accumulation) using a panel of 104 soybean lines and, an interspecific bi-parental population (F8) from PI483463 x Hutcheson. These markers precisely identified salt-tolerant/sensitive genotypes (>91%), and different structural-variants (>98%). These SNP assays, supported by accurate phenotyping, haplotype analyses and pedigree tracking information, will accelerate marker-assisted selection programs to enhance the development of salt-tolerant soybean cultivars. PMID:26781337

Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges

PubMed Central

Montanez-Sauri, Sara I.; Beebe, David J.; Sung, Kyung Eun

2015-01-01

The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g. spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments. PMID:25274061

40 CFR Table 9 to Subpart Eeee of... - Continuous Compliance With Operating Limits-High Throughput Transfer Racks

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Continuous Compliance With Operating Limits-High Throughput Transfer Racks 9 Table 9 to Subpart EEEE of Part 63 Protection of Environment...—Continuous Compliance With Operating Limits—High Throughput Transfer Racks As stated in §§ 63.2378(a) and (b...

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

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

PubMed

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.

An Automated, High-Throughput Method for Interpreting the Tandem Mass Spectra of Glycosaminoglycans

NASA Astrophysics Data System (ADS)

Duan, Jiana; Jonathan Amster, I.

2018-05-01

The biological interactions between glycosaminoglycans (GAGs) and other biomolecules are heavily influenced by structural features of the glycan. The structure of GAGs can be assigned using tandem mass spectrometry (MS2), but analysis of these data, to date, requires manually interpretation, a slow process that presents a bottleneck to the broader deployment of this approach to solving biologically relevant problems. Automated interpretation remains a challenge, as GAG biosynthesis is not template-driven, and therefore, one cannot predict structures from genomic data, as is done with proteins. The lack of a structure database, a consequence of the non-template biosynthesis, requires a de novo approach to interpretation of the mass spectral data. We propose a model for rapid, high-throughput GAG analysis by using an approach in which candidate structures are scored for the likelihood that they would produce the features observed in the mass spectrum. To make this approach tractable, a genetic algorithm is used to greatly reduce the search-space of isomeric structures that are considered. The time required for analysis is significantly reduced compared to an approach in which every possible isomer is considered and scored. The model is coded in a software package using the MATLAB environment. This approach was tested on tandem mass spectrometry data for long-chain, moderately sulfated chondroitin sulfate oligomers that were derived from the proteoglycan bikunin. The bikunin data was previously interpreted manually. Our approach examines glycosidic fragments to localize SO3 modifications to specific residues and yields the same structures reported in literature, only much more quickly.

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test, comparison with pot grown plants and validation.

PubMed

Jeudy, Christian; Adrian, Marielle; Baussard, Christophe; Bernard, Céline; Bernaud, Eric; Bourion, Virginie; Busset, Hughes; Cabrera-Bosquet, Llorenç; Cointault, Frédéric; Han, Simeng; Lamboeuf, Mickael; Moreau, Delphine; Pivato, Barbara; Prudent, Marion; Trouvelot, Sophie; Truong, Hoai Nam; Vernoud, Vanessa; Voisin, Anne-Sophie; Wipf, Daniel; Salon, Christophe

2016-01-01

In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck. Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using "classic" growth conditions in pots. This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

PubMed Central

Roychowdhury, Sameek; Iyer, Matthew K.; Robinson, Dan R.; Lonigro, Robert J.; Wu, Yi-Mi; Cao, Xuhong; Kalyana-Sundaram, Shanker; Sam, Lee; Balbin, O. Alejandro; Quist, Michael J.; Barrette, Terrence; Everett, Jessica; Siddiqui, Javed; Kunju, Lakshmi P.; Navone, Nora; Araujo, John C.; Troncoso, Patricia; Logothetis, Christopher J.; Innis, Jeffrey W.; Smith, David C.; Lao, Christopher D.; Kim, Scott Y.; Roberts, J. Scott; Gruber, Stephen B.; Pienta, Kenneth J.; Talpaz, Moshe; Chinnaiyan, Arul M.

2012-01-01

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology. PMID:22133722

The Representation of Heart Development in the Gene Ontology

PubMed Central

Khodiyar, Varsha K.; Hill, David P.; Howe, Doug; Berardini, Tanya Z.; Tweedie, Susan; Talmud, Philippa J.; Breckenridge, Ross; Bhattarcharya, Shoumo; Riley, Paul; Scambler, Peter; Lovering, Ruth C.

2012-01-01

An understanding of heart development is critical in any systems biology approach to cardiovascular disease. The interpretation of data generated from high-throughput technologies (such as microarray and proteomics) is also essential to this approach. However, characterizing the role of genes in the processes underlying heart development and cardiovascular disease involves the non-trivial task of data analysis and integration of previous knowledge. The Gene Ontology (GO) Consortium provides structured controlled biological vocabularies that are used to summarize previous functional knowledge for gene products across all species. One aspect of GO describes biological processes, such as development and signaling. In order to support high-throughput cardiovascular research, we have initiated an effort to fully describe heart development in GO; expanding the number of GO terms describing heart development from 12 to over 280. This new ontology describes heart morphogenesis, the differentiation of specific cardiac cell types, and the involvement of signaling pathways in heart development and aligns GO with the current views of the heart development research community and its representation in the literature. This extension of GO allows gene product annotators to comprehensively capture the genetic program leading to the developmental progression of the heart. This will enable users to integrate heart development data across species, resulting in the comprehensive retrieval of information about this subject. The revised GO structure, combined with gene product annotations, should improve the interpretation of data from high-throughput methods in a variety of cardiovascular research areas, including heart development, congenital cardiac disease, and cardiac stem cell research. Additionally, we invite the heart development community to contribute to the expansion of this important dataset for the benefit of future research in this area. PMID:21419760

Using high throughput sequencing to explore the biodiversity in oral bacterial communities.

PubMed

Diaz, P I; Dupuy, A K; Abusleme, L; Reese, B; Obergfell, C; Choquette, L; Dongari-Bagtzoglou, A; Peterson, D E; Terzi, E; Strausbaugh, L D

2012-06-01

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns. © 2012 John Wiley & Sons A/S.

Leaf-rolling in maize crops: from leaf scoring to canopy-level measurements for phenotyping

PubMed Central

Madec, Simon; Irfan, Kamran; Lopez, Jeremy; Comar, Alexis; Hemmerlé, Matthieu; Dutartre, Dan; Praud, Sebastien; Tixier, Marie Helene

2018-01-01

Abstract Leaf rolling in maize crops is one of the main plant reactions to water stress that can be visually scored in the field. However, leaf-scoring techniques do not meet the high-throughput requirements needed by breeders for efficient phenotyping. Consequently, this study investigated the relationship between leaf-rolling scores and changes in canopy structure that can be determined by high-throughput remote-sensing techniques. Experiments were conducted in 2015 and 2016 on maize genotypes subjected to water stress. Leaf-rolling was scored visually over the whole day around the flowering stage. Concurrent digital hemispherical photographs were taken to evaluate the impact of leaf-rolling on canopy structure using the computed fraction of intercepted diffuse photosynthetically active radiation, FIPARdif. The results showed that leaves started to roll due to water stress around 09:00 h and leaf-rolling reached its maximum around 15:00 h (the photoperiod was about 05:00–20:00 h). In contrast, plants maintained under well-watered conditions did not show any significant rolling during the same day. A canopy-level index of rolling (CLIR) is proposed to quantify the diurnal changes in canopy structure induced by leaf-rolling. It normalizes for the differences in FIPARdif between genotypes observed in the early morning when leaves are unrolled, as well as for yearly effects linked to environmental conditions. Leaf-level rolling score was very strongly correlated with changes in canopy structure as described by the CLIR (r2=0.86, n=370). The daily time course of rolling was characterized using the amplitude of variation, and the rate and the timing of development computed at both the leaf and canopy levels. Results obtained from eight genotypes common between the two years of experiments showed that the amplitude of variation of the CLIR was the more repeatable trait (Spearman coefficient ρ=0.62) as compared to the rate (ρ=0.29) and the timing of development (ρ=0.33). The potential of these findings for the development of a high-throughput method for determining leaf-rolling based on aerial drone observations are considered. PMID:29617837

The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration.

PubMed

Scafaro, Andrew P; Negrini, A Clarissa A; O'Leary, Brendan; Rashid, F Azzahra Ahmad; Hayes, Lucy; Fan, Yuzhen; Zhang, You; Chochois, Vincent; Badger, Murray R; Millar, A Harvey; Atkin, Owen K

2017-01-01

Mitochondrial respiration in the dark ( R dark ) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R dark is essential for agronomic and ecological studies. However, currently methods used to measure R dark in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O 2 consumption rates. The fluorophore technique was compared with O 2 -electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. The high-throughput fluorophore system provided stable measurements of R dark in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R dark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R dark through dissection and simultaneous measurements of above- and below-ground organs. Variation in absolute R dark was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R dark to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R dark on multiple samples simultaneously, irrespective of plant or tissue type.

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

High-throughput sample adaptive offset hardware architecture for high-efficiency video coding

NASA Astrophysics Data System (ADS)

Zhou, Wei; Yan, Chang; Zhang, Jingzhi; Zhou, Xin

2018-03-01

A high-throughput hardware architecture for a sample adaptive offset (SAO) filter in the high-efficiency video coding video coding standard is presented. First, an implementation-friendly and simplified bitrate estimation method of rate-distortion cost calculation is proposed to reduce the computational complexity in the mode decision of SAO. Then, a high-throughput VLSI architecture for SAO is presented based on the proposed bitrate estimation method. Furthermore, multiparallel VLSI architecture for in-loop filters, which integrates both deblocking filter and SAO filter, is proposed. Six parallel strategies are applied in the proposed in-loop filters architecture to improve the system throughput and filtering speed. Experimental results show that the proposed in-loop filters architecture can achieve up to 48% higher throughput in comparison with prior work. The proposed architecture can reach a high-operating clock frequency of 297 MHz with TSMC 65-nm library and meet the real-time requirement of the in-loop filters for 8 K × 4 K video format at 132 fps.

EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments.

PubMed

Zhou, Bailing; Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu; Yang, Yuedong; Zhou, Yaoqi; Wang, Jihua

2018-01-04

Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments

PubMed Central

Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu

2018-01-01

Abstract Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. PMID:28985416

In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening.

PubMed

Gea, An; Stringano, Elisabetta; Brown, Ron H; Mueller-Harvey, Irene

2011-01-26

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin ( Onobrychis viciifolia ) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6-113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

NASA Astrophysics Data System (ADS)

Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

2014-07-01

Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

Graph theoretic analysis of protein interaction networks of eukaryotes

NASA Astrophysics Data System (ADS)

Goh, K.-I.; Kahng, B.; Kim, D.

2005-11-01

Owing to the recent progress in high-throughput experimental techniques, the datasets of large-scale protein interactions of prototypical multicellular species, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, have been assayed. The datasets are obtained mainly by using the yeast hybrid method, which contains false-positive and false-negative simultaneously. Accordingly, while it is desirable to test such datasets through further wet experiments, here we invoke recent developed network theory to test such high-throughput datasets in a simple way. Based on the fact that the key biological processes indispensable to maintaining life are conserved across eukaryotic species, and the comparison of structural properties of the protein interaction networks (PINs) of the two species with those of the yeast PIN, we find that while the worm and yeast PIN datasets exhibit similar structural properties, the current fly dataset, though most comprehensively screened ever, does not reflect generic structural properties correctly as it is. The modularity is suppressed and the connectivity correlation is lacking. Addition of interologs to the current fly dataset increases the modularity and enhances the occurrence of triangular motifs as well. The connectivity correlation function of the fly, however, remains distinct under such interolog additions, for which we present a possible scenario through an in silico modeling.

Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension.

PubMed

Su, Hao; Yan, Ji; Xu, Jian; Fan, Xi-Zhen; Sun, Xian-Lin; Chen, Kang-Yu

2015-08-01

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH. In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products. In the pipeline, the stepwise high-throughput virtual screening, quantitative structure-activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol. With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 μM, respectively. Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.

Template-based structure modeling of protein-protein interactions

PubMed Central

Szilagyi, Andras; Zhang, Yang

2014-01-01

The structure of protein-protein complexes can be constructed by using the known structure of other protein complexes as a template. The complex structure templates are generally detected either by homology-based sequence alignments or, given the structure of monomer components, by structure-based comparisons. Critical improvements have been made in recent years by utilizing interface recognition and by recombining monomer and complex template libraries. Encouraging progress has also been witnessed in genome-wide applications of template-based modeling, with modeling accuracy comparable to high-throughput experimental data. Nevertheless, bottlenecks exist due to the incompleteness of the proteinprotein complex structure library and the lack of methods for distant homologous template identification and full-length complex structure refinement. PMID:24721449

NMR in the SPINE Structural Proteomics project.

PubMed

Ab, E; Atkinson, A R; Banci, L; Bertini, I; Ciofi-Baffoni, S; Brunner, K; Diercks, T; Dötsch, V; Engelke, F; Folkers, G E; Griesinger, C; Gronwald, W; Günther, U; Habeck, M; de Jong, R N; Kalbitzer, H R; Kieffer, B; Leeflang, B R; Loss, S; Luchinat, C; Marquardsen, T; Moskau, D; Neidig, K P; Nilges, M; Piccioli, M; Pierattelli, R; Rieping, W; Schippmann, T; Schwalbe, H; Travé, G; Trenner, J; Wöhnert, J; Zweckstetter, M; Kaptein, R

2006-10-01

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

Antibody Epitope Analysis to Investigate Folded Structure, Allosteric Conformation, and Evolutionary Lineage of Proteins.

PubMed

Wong, Sienna; Jin, J-P

2017-01-01

Study of folded structure of proteins provides insights into their biological functions, conformational dynamics and molecular evolution. Current methods of elucidating folded structure of proteins are laborious, low-throughput, and constrained by various limitations. Arising from these methods is the need for a sensitive, quantitative, rapid and high-throughput method not only analysing the folded structure of proteins, but also to monitor dynamic changes under physiological or experimental conditions. In this focused review, we outline the foundation and limitations of current protein structure-determination methods prior to discussing the advantages of an emerging antibody epitope analysis for applications in structural, conformational and evolutionary studies of proteins. We discuss the application of this method using representative examples in monitoring allosteric conformation of regulatory proteins and the determination of the evolutionary lineage of related proteins and protein isoforms. The versatility of the method described herein is validated by the ability to modulate a variety of assay parameters to meet the needs of the user in order to monitor protein conformation. Furthermore, the assay has been used to clarify the lineage of troponin isoforms beyond what has been depicted by sequence homology alone, demonstrating the nonlinear evolutionary relationship between primary structure and tertiary structure of proteins. The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Causal Inferences from Mining ToxCast Data and the Biomedical Literature for Molecular Pathways and Cellular Processes in Cleft Palate (SOT)

EPA Science Inventory

Sixty-five chemicals in the ToxCast high-throughput screening (HTS) dataset have been linked to cleft palate based on data from ToxRefDB (rat or rabbit prenatal developmental toxicity studies) or from literature reports. These compounds are structurally diverse and thus likely to...

Genetic diversity and structure in Mamey [P. sapota (Jacq.) H.E. Moore & Stearn] by using microsatellite markers

USDA-ARS?s Scientific Manuscript database

The tropical plant Pouteria sapota (Jacq.) is known for its edible fruits that contain unique carotenoids, as well as for the fungitoxic, anti-inflamatory and anti-oxidant activity of extracts from its bark, leaves and roots, though its genetics is unknown. We did high-throughput sequencing of micr...

A re-sequencing based assessment of genomic heterogeneity and fast neutron-induced deletions in a common bean cultivar

USDA-ARS?s Scientific Manuscript database

A small fast neutron mutant population has been established from Phaseolus vulgaris cv. Red Hawk. We leveraged the available P. vulgaris genome sequence and high throughput next generation DNA sequencing to examine the genomic structure of five Phaseolus vulgaris cv. Red Hawk fast neutron mutants wi...

High throughput light absorber discovery, Part 1: An algorithm for automated tauc analysis

DOE PAGES

Suram, Santosh K.; Newhouse, Paul F.; Gregoire, John M.

2016-09-23

High-throughput experimentation provides efficient mapping of composition-property relationships, and its implementation for the discovery of optical materials enables advancements in solar energy and other technologies. In a high throughput pipeline, automated data processing algorithms are often required to match experimental throughput, and we present an automated Tauc analysis algorithm for estimating band gap energies from optical spectroscopy data. The algorithm mimics the judgment of an expert scientist, which is demonstrated through its application to a variety of high throughput spectroscopy data, including the identification of indirect or direct band gaps in Fe 2O 3, Cu 2V 2O 7, and BiVOmore » 4. Here, the applicability of the algorithm to estimate a range of band gap energies for various materials is demonstrated by a comparison of direct-allowed band gaps estimated by expert scientists and by automated algorithm for 60 optical spectra.« less

Ultra-High Throughput Synthesis of Nanoparticles with Homogeneous Size Distribution Using a Coaxial Turbulent Jet Mixer

PubMed Central

2015-01-01

High-throughput production of nanoparticles (NPs) with controlled quality is critical for their clinical translation into effective nanomedicines for diagnostics and therapeutics. Here we report a simple and versatile coaxial turbulent jet mixer that can synthesize a variety of NPs at high throughput up to 3 kg/d, while maintaining the advantages of homogeneity, reproducibility, and tunability that are normally accessible only in specialized microscale mixing devices. The device fabrication does not require specialized machining and is easy to operate. As one example, we show reproducible, high-throughput formulation of siRNA-polyelectrolyte polyplex NPs that exhibit effective gene knockdown but exhibit significant dependence on batch size when formulated using conventional methods. The coaxial turbulent jet mixer can accelerate the development of nanomedicines by providing a robust and versatile platform for preparation of NPs at throughputs suitable for in vivo studies, clinical trials, and industrial-scale production. PMID:24824296

Scalable Manufacturing of Plasmonic Nanodisk Dimers and Cusp Nanostructures using Salting-out Quenching Method and Colloidal Lithography

PubMed Central

Juluri, Bala Krishna; Chaturvedi, Neetu; Hao, Qingzhen; Lu, Mengqian; Velegol, Darrell; Jensen, Lasse; Huang, Tony Jun

2014-01-01

Localization of large electric fields in plasmonic nanostructures enables various processes such as single molecule detection, higher harmonic light generation, and control of molecular fluorescence and absorption. High-throughput, simple nanofabrication techniques are essential for implementing plasmonic nanostructures with large electric fields for practical applications. In this article we demonstrate a scalable, rapid, and inexpensive fabrication method based on the salting-out quenching technique and colloidal lithography for the fabrication of two types of nanostructures with large electric field: nanodisk dimers and cusp nanostructures. Our technique relies on fabricating polystyrene doublets from single beads by controlled aggregation and later using them as soft masks to fabricate metal nanodisk dimers and nanocusp structures. Both of these structures have a well-defined geometry for the localization of large electric fields comparable to structures fabricated by conventional nanofabrication techniques. We also show that various parameters in the fabrication process can be adjusted to tune the geometry of the final structures and control their plasmonic properties. With advantages in throughput, cost, and geometric tunability, our fabrication method can be valuable in many applications that require plasmonic nanostructures with large electric fields. PMID:21692473

Hadoop and friends - first experience at CERN with a new platform for high throughput analysis steps

NASA Astrophysics Data System (ADS)

Duellmann, D.; Surdy, K.; Menichetti, L.; Toebbicke, R.

2017-10-01

The statistical analysis of infrastructure metrics comes with several specific challenges, including the fairly large volume of unstructured metrics from a large set of independent data sources. Hadoop and Spark provide an ideal environment in particular for the first steps of skimming rapidly through hundreds of TB of low relevance data to find and extract the much smaller data volume that is relevant for statistical analysis and modelling. This presentation will describe the new Hadoop service at CERN and the use of several of its components for high throughput data aggregation and ad-hoc pattern searches. We will describe the hardware setup used, the service structure with a small set of decoupled clusters and the first experience with co-hosting different applications and performing software upgrades. We will further detail the common infrastructure used for data extraction and preparation from continuous monitoring and database input sources.

Automated Phase Segmentation for Large-Scale X-ray Diffraction Data Using a Graph-Based Phase Segmentation (GPhase) Algorithm.

PubMed

Xiong, Zheng; He, Yinyan; Hattrick-Simpers, Jason R; Hu, Jianjun

2017-03-13

The creation of composition-processing-structure relationships currently represents a key bottleneck for data analysis for high-throughput experimental (HTE) material studies. Here we propose an automated phase diagram attribution algorithm for HTE data analysis that uses a graph-based segmentation algorithm and Delaunay tessellation to create a crystal phase diagram from high throughput libraries of X-ray diffraction (XRD) patterns. We also propose the sample-pair based objective evaluation measures for the phase diagram prediction problem. Our approach was validated using 278 diffraction patterns from a Fe-Ga-Pd composition spread sample with a prediction precision of 0.934 and a Matthews Correlation Coefficient score of 0.823. The algorithm was then applied to the open Ni-Mn-Al thin-film composition spread sample to obtain the first predicted phase diagram mapping for that sample.

Droplet barcoding for single cell transcriptomics applied to embryonic stem cells

PubMed Central

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-01-01

Summary It has long been the dream of biologists to map gene expression at the single cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility of these high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. PMID:26000487

High throughput dual-wavelength temperature distribution imaging via compressive imaging

NASA Astrophysics Data System (ADS)

Yao, Xu-Ri; Lan, Ruo-Ming; Liu, Xue-Feng; Zhu, Ge; Zheng, Fu; Yu, Wen-Kai; Zhai, Guang-Jie

2018-03-01

Thermal imaging is an essential tool in a wide variety of research areas. In this work we demonstrate high-throughput double-wavelength temperature distribution imaging using a modified single-pixel camera without the requirement of a beam splitter (BS). A digital micro-mirror device (DMD) is utilized to display binary masks and split the incident radiation, which eliminates the necessity of a BS. Because the spatial resolution is dictated by the DMD, this thermal imaging system has the advantage of perfect spatial registration between the two images, which limits the need for the pixel registration and fine adjustments. Two bucket detectors, which measures the total light intensity reflected from the DMD, are employed in this system and yield an improvement in the detection efficiency of the narrow-band radiation. A compressive imaging algorithm is utilized to achieve under-sampling recovery. A proof-of-principle experiment was presented to demonstrate the feasibility of this structure.

Reverse Ecology: from systems to environments and back.

PubMed

Levy, Roie; Borenstein, Elhanan

2012-01-01

The structure of complex biological systems reflects not only their function but also the environments in which they evolved and are adapted to. Reverse Ecology-an emerging new frontier in Evolutionary Systems Biology-aims to extract this information and to obtain novel insights into an organism's ecology. The Reverse Ecology framework facilitates the translation of high-throughput genomic data into large-scale ecological data, and has the potential to transform ecology into a high-throughput field. In this chapter, we describe some of the pioneering work in Reverse Ecology, demonstrating how system-level analysis of complex biological networks can be used to predict the natural habitats of poorly characterized microbial species, their interactions with other species, and universal patterns governing the adaptation of organisms to their environments. We further present several studies that applied Reverse Ecology to elucidate various aspects of microbial ecology, and lay out exciting future directions and potential future applications in biotechnology, biomedicine, and ecological engineering.

A high-throughput technique for determining grain boundary character non-destructively in microstructures with through-thickness grains

DOE PAGES

Seita, Matteo; Volpi, Marco; Patala, Srikanth; ...

2016-06-24

Grain boundaries (GBs) govern many properties of polycrystalline materials. However, because of their structural variability, our knowledge of GB constitutive relations is still very limited. We present a novel method to characterise the complete crystallography of individual GBs non-destructively, with high-throughput, and using commercially available tools. This method combines electron diffraction, optical reflectance and numerical image analysis to determine all five crystallographic parameters of numerous GBs in samples with through-thickness grains. We demonstrate the technique by measuring the crystallographic character of about 1,000 individual GBs in aluminum in a single run. Our method enables cost- and time-effective assembly of crystallography–propertymore » databases for thousands of individual GBs. Furthermore, such databases are essential for identifying GB constitutive relations and for predicting GB-related behaviours of polycrystalline solids.« less

Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers.

PubMed

Egorov, Evgeny S; Merzlyak, Ekaterina M; Shelenkov, Andrew A; Britanova, Olga V; Sharonov, George V; Staroverov, Dmitriy B; Bolotin, Dmitriy A; Davydov, Alexey N; Barsova, Ekaterina; Lebedev, Yuriy B; Shugay, Mikhail; Chudakov, Dmitriy M

2015-06-15

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications. Copyright © 2015 by The American Association of Immunologists, Inc.

Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging

PubMed Central

Stoltzfus, Caleb R.; Rebane, Aleksander

2016-01-01

We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2. PMID:27231620

3D-SURFER: software for high-throughput protein surface comparison and analysis

PubMed Central

La, David; Esquivel-Rodríguez, Juan; Venkatraman, Vishwesh; Li, Bin; Sael, Lee; Ueng, Stephen; Ahrendt, Steven; Kihara, Daisuke

2009-01-01

Summary: We present 3D-SURFER, a web-based tool designed to facilitate high-throughput comparison and characterization of proteins based on their surface shape. As each protein is effectively represented by a vector of 3D Zernike descriptors, comparison times for a query protein against the entire PDB take, on an average, only a couple of seconds. The web interface has been designed to be as interactive as possible with displays showing animated protein rotations, CATH codes and structural alignments using the CE program. In addition, geometrically interesting local features of the protein surface, such as pockets that often correspond to ligand binding sites as well as protrusions and flat regions can also be identified and visualized. Availability: 3D-SURFER is a web application that can be freely accessed from: http://dragon.bio.purdue.edu/3d-surfer Contact: dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19759195

3D-SURFER: software for high-throughput protein surface comparison and analysis.

PubMed

La, David; Esquivel-Rodríguez, Juan; Venkatraman, Vishwesh; Li, Bin; Sael, Lee; Ueng, Stephen; Ahrendt, Steven; Kihara, Daisuke

2009-11-01

We present 3D-SURFER, a web-based tool designed to facilitate high-throughput comparison and characterization of proteins based on their surface shape. As each protein is effectively represented by a vector of 3D Zernike descriptors, comparison times for a query protein against the entire PDB take, on an average, only a couple of seconds. The web interface has been designed to be as interactive as possible with displays showing animated protein rotations, CATH codes and structural alignments using the CE program. In addition, geometrically interesting local features of the protein surface, such as pockets that often correspond to ligand binding sites as well as protrusions and flat regions can also be identified and visualized. 3D-SURFER is a web application that can be freely accessed from: http://dragon.bio.purdue.edu/3d-surfer dkihara@purdue.edu Supplementary data are available at Bioinformatics online.

Hit to lead account of the discovery of bisbenzamide and related ureidobenzamide inhibitors of Rho kinase.

PubMed

Morwick, Tina; Büttner, Frank H; Cywin, Charles L; Dahmann, Georg; Hickey, Eugene; Jakes, Scott; Kaplita, Paul; Kashem, Mohammed A; Kerr, Steven; Kugler, Stanley; Mao, Wang; Marshall, Daniel; Paw, Zofia; Shih, Cheng-Kon; Wu, Frank; Young, Erick

2010-01-28

A highly selective series of bisbenzamide inhibitors of Rho-associated coiled-coil forming protein kinase (ROCK) and a related ureidobenzamide series, both identified by high throughput screening (HTS), are described. Details of the hit validation and lead generation process, including structure-activity relationship (SAR) studies, a selectivity assessment, target-independent profiling (TIP) results, and an analysis of functional activity using a rat aortic ring assay are discussed.

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

PubMed

Li, Fumin; Wang, Jun; Jenkins, Rand

2016-05-01

There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development. Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects. High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.

High-content profiling of cell responsiveness to graded substrates based on combinyatorially variant polymers.

PubMed

Liu, Er; Treiser, Matthew D; Patel, Hiral; Sung, Hak-Joon; Roskov, Kristen E; Kohn, Joachim; Becker, Matthew L; Moghe, Prabhas V

2009-08-01

We have developed a novel approach combining high information and high throughput analysis to characterize cell adhesive responses to biomaterial substrates possessing gradients in surface topography. These gradients were fabricated by subjecting thin film blends of tyrosine-derived polycarbonates, i.e. poly(DTE carbonate) and poly(DTO carbonate) to a gradient temperature annealing protocol. Saos-2 cells engineered with a green fluorescent protein (GFP) reporter for farnesylation (GFP-f) were cultured on the gradient substrates to assess the effects of nanoscale surface topology and roughness that arise during the phase separation process on cell attachment and adhesion strength. The high throughput imaging approach allowed us to rapidly identify the "global" and "high content" structure-property relationships between cell adhesion and biomaterial properties such as polymer chemistry and topography. This study found that cell attachment and spreading increased monotonically with DTE content and were significantly elevated at the position with intermediate regions corresponding to the highest "gradient" of surface roughness, while GFP-f farnesylation intensity descriptors were sensitively altered by surface roughness, even in cells with comparable levels of spreading.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials

PubMed Central

Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

2008-01-01

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

Identification of functional modules using network topology and high-throughput data.

PubMed

Ulitsky, Igor; Shamir, Ron

2007-01-26

With the advent of systems biology, biological knowledge is often represented today by networks. These include regulatory and metabolic networks, protein-protein interaction networks, and many others. At the same time, high-throughput genomics and proteomics techniques generate very large data sets, which require sophisticated computational analysis. Usually, separate and different analysis methodologies are applied to each of the two data types. An integrated investigation of network and high-throughput information together can improve the quality of the analysis by accounting simultaneously for topological network properties alongside intrinsic features of the high-throughput data. We describe a novel algorithmic framework for this challenge. We first transform the high-throughput data into similarity values, (e.g., by computing pairwise similarity of gene expression patterns from microarray data). Then, given a network of genes or proteins and similarity values between some of them, we seek connected sub-networks (or modules) that manifest high similarity. We develop algorithms for this problem and evaluate their performance on the osmotic shock response network in S. cerevisiae and on the human cell cycle network. We demonstrate that focused, biologically meaningful and relevant functional modules are obtained. In comparison with extant algorithms, our approach has higher sensitivity and higher specificity. We have demonstrated that our method can accurately identify functional modules. Hence, it carries the promise to be highly useful in analysis of high throughput data.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

PubMed

Nakamura, Akira; Ohtsuka, Jun; Kashiwagi, Tatsuki; Numoto, Nobutaka; Hirota, Noriyuki; Ode, Takahiro; Okada, Hidehiko; Nagata, Koji; Kiyohara, Motosuke; Suzuki, Ei-Ichiro; Kita, Akiko; Wada, Hitoshi; Tanokura, Masaru

2016-02-26

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for "difficult" proteins, such as membrane proteins and supermolecular complexes.

Rational Methods for the Selection of Diverse Screening Compounds

PubMed Central

Huggins, David J.; Venkitaraman, Ashok R.; Spring, David R.

2016-01-01

Traditionally a pursuit of large pharmaceutical companies, high-throughput screening assays are becoming increasingly common within academic and government laboratories. This shift has been instrumental in enabling projects that have not been commercially viable, such as chemical probe discovery and screening against high risk targets. Once an assay has been prepared and validated, it must be fed with screening compounds. Crafting a successful collection of small molecules for screening poses a significant challenge. An optimized collection will minimize false positives whilst maximizing hit rates of compounds that are amenable to lead generation and optimization. Without due consideration of the relevant protein targets and the downstream screening assays, compound filtering and selection can fail to explore the great extent of chemical diversity and eschew valuable novelty. Herein, we discuss the different factors to be considered and methods that may be employed when assembling a structurally diverse compound screening collection. Rational methods for selecting diverse chemical libraries are essential for their effective use in high-throughput screens. PMID:21261294

An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

PubMed

Zhai, Yufeng; Chen, Kaisheng; Zhong, Yang; Zhou, Bin; Ainscow, Edward; Wu, Ying-Ta; Zhou, Yingyao

2016-09-01

The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-QC led to a noticeably stronger structure-activity relationship. The method was further tested in several independent HTS runs, where QC results were sampled for experimental validation. Significantly increased hit confirmation rates were obtained after the QC steps, confirming that the proposed method was effective in enriching true-positive hits. An implementation of the algorithm is available to the screening community. © 2016 Society for Laboratory Automation and Screening.

High-Throughput Top-Down and Bottom-Up Processes for Forming Single-Nanotube Based Architectures for 3D Electronics

NASA Technical Reports Server (NTRS)

Kaul, Anupama B.; Megerian, Krikor G.; von Allmen, Paul; Kowalczyk, Robert; Baron, Richard

2009-01-01

We have developed manufacturable approaches to form single, vertically aligned carbon nanotubes, where the tubes are centered precisely, and placed within a few hundred nm of 1-1.5 micron deep trenches. These wafer-scale approaches were enabled by chemically amplified resists and inductively coupled Cryo-etchers for forming the 3D nanoscale architectures. The tube growth was performed using dc plasma-enhanced chemical vapor deposition (PECVD), and the materials used for the pre-fabricated 3D architectures were chemically and structurally compatible with the high temperature (700 C) PECVD synthesis of our tubes, in an ammonia and acetylene ambient. Tube characteristics were also engineered to some extent, by adjusting growth parameters, such as Ni catalyst thickness, pressure and plasma power during growth. Such scalable, high throughput top-down fabrication techniques, combined with bottom-up tube synthesis, should accelerate the development of PECVD tubes for applications such as interconnects, nano-electromechanical (NEMS), sensors or 3D electronics in general.

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

PubMed

Simm, Jaak; Klambauer, Günter; Arany, Adam; Steijaert, Marvin; Wegner, Jörg Kurt; Gustin, Emmanuel; Chupakhin, Vladimir; Chong, Yolanda T; Vialard, Jorge; Buijnsters, Peter; Velter, Ingrid; Vapirev, Alexander; Singh, Shantanu; Carpenter, Anne E; Wuyts, Roel; Hochreiter, Sepp; Moreau, Yves; Ceulemans, Hugo

2018-05-17

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening.

PubMed

Li, Gang; Chen, Qiang; Li, Junjun; Hu, Xiaojian; Zhao, Jianlong

2010-06-01

A centrifuge-based microfluidic system has been developed that enables automated high-throughput and low-volume protein crystallizations. In this system, protein solution was automatically and accurately metered and dispensed into nanoliter-sized multiple reaction chambers, and it was mixed with various types of precipitants using a combination of capillary effect and centrifugal force. It has the advantages of simple fabrication, easy operation, and extremely low waste. To demonstrate the feasibility of this system, we constructed a chip containing 24 units and used it to perform lysozyme and cyan fluorescent protein (CyPet) crystallization trials. The results demonstrate that high-quality crystals can be grown and harvested from such a nanoliter-volume microfluidic system. Compared to other microfluidic technologies for protein crystallization, this microfluidic system allows zero waste, simple structure and convenient operation, which suggests that our microfluidic disk can be applied not only to protein crystallization, but also to the miniaturization of various biochemical reactions requiring precise nanoscale control.

High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

PubMed Central

Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

2010-01-01

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

High-Throughput Phenotyping and QTL Mapping Reveals the Genetic Architecture of Maize Plant Growth.

PubMed

Zhang, Xuehai; Huang, Chenglong; Wu, Di; Qiao, Feng; Li, Wenqiang; Duan, Lingfeng; Wang, Ke; Xiao, Yingjie; Chen, Guoxing; Liu, Qian; Xiong, Lizhong; Yang, Wanneng; Yan, Jianbing

2017-03-01

With increasing demand for novel traits in crop breeding, the plant research community faces the challenge of quantitatively analyzing the structure and function of large numbers of plants. A clear goal of high-throughput phenotyping is to bridge the gap between genomics and phenomics. In this study, we quantified 106 traits from a maize ( Zea mays ) recombinant inbred line population ( n = 167) across 16 developmental stages using the automatic phenotyping platform. Quantitative trait locus (QTL) mapping with a high-density genetic linkage map, including 2,496 recombinant bins, was used to uncover the genetic basis of these complex agronomic traits, and 988 QTLs have been identified for all investigated traits, including three QTL hotspots. Biomass accumulation and final yield were predicted using a combination of dissected traits in the early growth stage. These results reveal the dynamic genetic architecture of maize plant growth and enhance ideotype-based maize breeding and prediction. © 2017 American Society of Plant Biologists. All Rights Reserved.

High-Throughput Phenotyping and QTL Mapping Reveals the Genetic Architecture of Maize Plant Growth1[OPEN

PubMed Central

Huang, Chenglong; Wu, Di; Qiao, Feng; Li, Wenqiang; Duan, Lingfeng; Wang, Ke; Xiao, Yingjie; Chen, Guoxing; Liu, Qian; Yang, Wanneng

2017-01-01

With increasing demand for novel traits in crop breeding, the plant research community faces the challenge of quantitatively analyzing the structure and function of large numbers of plants. A clear goal of high-throughput phenotyping is to bridge the gap between genomics and phenomics. In this study, we quantified 106 traits from a maize (Zea mays) recombinant inbred line population (n = 167) across 16 developmental stages using the automatic phenotyping platform. Quantitative trait locus (QTL) mapping with a high-density genetic linkage map, including 2,496 recombinant bins, was used to uncover the genetic basis of these complex agronomic traits, and 988 QTLs have been identified for all investigated traits, including three QTL hotspots. Biomass accumulation and final yield were predicted using a combination of dissected traits in the early growth stage. These results reveal the dynamic genetic architecture of maize plant growth and enhance ideotype-based maize breeding and prediction. PMID:28153923

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering.

PubMed

Groen, Nathalie; Guvendiren, Murat; Rabitz, Herschel; Welsh, William J; Kohn, Joachim; de Boer, Jan

2016-04-01

The research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. In this opinion paper, we postulate that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field. Copyright © 2016. Published by Elsevier Ltd.

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.

PubMed

Scott, Daniel J; Kummer, Lutz; Egloff, Pascal; Bathgate, Ross A D; Plückthun, Andreas

2014-11-01

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (II) Dephytylated derivatives.

PubMed

Chen, Kewei; Ríos, José Julián; Roca, María; Pérez-Gálvez, Antonio

2015-09-18

Dephytylated chlorophylls (chlorophyllides and pheophorbides) are the starting point of the chlorophyll catabolism in green tissues, components of the chlorophyll pattern in storage/processed food vegetables, as well as the favoured structural arrangement for chlorophyll absorption. In addition, dephytylated native chlorophylls are prone to several modifications of their structure yielding pyro-, 13(2)-hydroxy- and 15(1)-hydroxy-lactone derivatives. Despite of these outstanding remarks only few of them have been analysed by MS(n). Besides new protocols for obtaining standards, we have developed a new high throughput methodology able to determine the fragmentation pathway of 16 dephytylated chlorophyll derivatives, elucidating the structures of the new product ions and new mechanisms of fragmentation. The new methodology combines, by first time, high resolution time-of-flight mass spectrometry and powerful post-processing software. Native chlorophyllides and pheophorbides mainly exhibit product ions that involve the fragmentation of D ring, as well as additional exclusive product ions. The introduction of an oxygenated function at E ring enhances the progress of fragmentation reactions through the β-keto ester group, developing also exclusive product ions for 13(2)-hydroxy derivatives and for 15(1)-hydroxy-lactone ones. Consequently, while MS(2)-based reactions of phytylated chlorophyll derivatives point to fragmentations at the phytyl and propionic chains, dephytylated chlorophyll derivatives behave different as the absence of phytyl makes β-keto ester group and E ring more prone to fragmentation. Proposals of the key reaction mechanisms underlying the origin of new product ions have been made. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Throughput Phenotyping of Plant Height: Comparing Unmanned Aerial Vehicles and Ground LiDAR Estimates.

PubMed

Madec, Simon; Baret, Fred; de Solan, Benoît; Thomas, Samuel; Dutartre, Dan; Jezequel, Stéphane; Hemmerlé, Matthieu; Colombeau, Gallian; Comar, Alexis

2017-01-01

The capacity of LiDAR and Unmanned Aerial Vehicles (UAVs) to provide plant height estimates as a high-throughput plant phenotyping trait was explored. An experiment over wheat genotypes conducted under well watered and water stress modalities was conducted. Frequent LiDAR measurements were performed along the growth cycle using a phénomobile unmanned ground vehicle. UAV equipped with a high resolution RGB camera was flying the experiment several times to retrieve the digital surface model from structure from motion techniques. Both techniques provide a 3D dense point cloud from which the plant height can be estimated. Plant height first defined as the z -value for which 99.5% of the points of the dense cloud are below. This provides good consistency with manual measurements of plant height (RMSE = 3.5 cm) while minimizing the variability along each microplot. Results show that LiDAR and structure from motion plant height values are always consistent. However, a slight under-estimation is observed for structure from motion techniques, in relation with the coarser spatial resolution of UAV imagery and the limited penetration capacity of structure from motion as compared to LiDAR. Very high heritability values ( H 2 > 0.90) were found for both techniques when lodging was not present. The dynamics of plant height shows that it carries pertinent information regarding the period and magnitude of the plant stress. Further, the date when the maximum plant height is reached was found to be very heritable ( H 2 > 0.88) and a good proxy of the flowering stage. Finally, the capacity of plant height as a proxy for total above ground biomass and yield is discussed.

High-Throughput Phenotyping of Plant Height: Comparing Unmanned Aerial Vehicles and Ground LiDAR Estimates

PubMed Central

Madec, Simon; Baret, Fred; de Solan, Benoît; Thomas, Samuel; Dutartre, Dan; Jezequel, Stéphane; Hemmerlé, Matthieu; Colombeau, Gallian; Comar, Alexis

2017-01-01

The capacity of LiDAR and Unmanned Aerial Vehicles (UAVs) to provide plant height estimates as a high-throughput plant phenotyping trait was explored. An experiment over wheat genotypes conducted under well watered and water stress modalities was conducted. Frequent LiDAR measurements were performed along the growth cycle using a phénomobile unmanned ground vehicle. UAV equipped with a high resolution RGB camera was flying the experiment several times to retrieve the digital surface model from structure from motion techniques. Both techniques provide a 3D dense point cloud from which the plant height can be estimated. Plant height first defined as the z-value for which 99.5% of the points of the dense cloud are below. This provides good consistency with manual measurements of plant height (RMSE = 3.5 cm) while minimizing the variability along each microplot. Results show that LiDAR and structure from motion plant height values are always consistent. However, a slight under-estimation is observed for structure from motion techniques, in relation with the coarser spatial resolution of UAV imagery and the limited penetration capacity of structure from motion as compared to LiDAR. Very high heritability values (H2> 0.90) were found for both techniques when lodging was not present. The dynamics of plant height shows that it carries pertinent information regarding the period and magnitude of the plant stress. Further, the date when the maximum plant height is reached was found to be very heritable (H2> 0.88) and a good proxy of the flowering stage. Finally, the capacity of plant height as a proxy for total above ground biomass and yield is discussed. PMID:29230229

40 CFR Table 3 to Subpart Eeee of... - Operating Limits-High Throughput Transfer Racks

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 12 2010-07-01 2010-07-01 true Operating Limits-High Throughput Transfer Racks 3 Table 3 to Subpart EEEE of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION... Throughput Transfer Racks As stated in § 63.2346(e), you must comply with the operating limits for existing...

Synthetic Molecular Evolution of Membrane-Active Peptides

NASA Astrophysics Data System (ADS)

Wimley, William

The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

PubMed

Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

2016-02-01

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

NASA Astrophysics Data System (ADS)

Soundararajan, Venky; Aravamudan, Murali

2014-12-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action - inspired by the Google page rank algorithm that unearths most ``globally connected'' websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs - up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site - encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows.

Microfabrication of a High-Throughput Nanochannel Delivery/Filtration System

NASA Technical Reports Server (NTRS)

Ferrari, Mauro; Liu, Xuewu; Grattoni, Alessandro; Fine, Daniel; Hosali, Sharath; Goodall, Randi; Medema, Ryan; Hudson, Lee

2011-01-01

A microfabrication process is proposed to produce a nanopore membrane for continuous passive drug release to maintain constant drug concentrations in the patient s blood throughout the delivery period. Based on silicon microfabrication technology, the dimensions of the nanochannel area, as well as microchannel area, can be precisely controlled, thus providing a steady, constant drug release rate within an extended time period. The multilayered nanochannel structures extend the limit of release rate range of a single-layer nanochannel system, and allow a wide range of pre-defined porosity to achieve any arbitrary drug release rate using any preferred nanochannel size. This membrane system could also be applied to molecular filtration or isolation. In this case, the nanochannel length can be reduced to the nanofabrication limit, i.e., 10s of nm. The nanochannel delivery system membrane is composed of a sandwich of a thin top layer, the horizontal nanochannels, and a thicker bottom wafer. The thin top layer houses an array of microchannels that offers the inlet port for diffusing molecules. It also works as a lid for the nanochannels by providing the channels a top surface. The nanochannels are fabricated by a sacrificial layer technique that obtains smooth surfaces and precisely controlled dimensions. The structure of this nanopore membrane is optimized to yield high mechanical strength and high throughput.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

PubMed

Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

2014-09-01

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

Drosophila melanogaster as a High-Throughput Model for Host-Microbiota Interactions.

PubMed

Trinder, Mark; Daisley, Brendan A; Dube, Josh S; Reid, Gregor

2017-01-01

Microbiota research often assumes that differences in abundance and identity of microorganisms have unique influences on host physiology. To test this concept mechanistically, germ-free mice are colonized with microbial communities to assess causation. Due to the cost, infrastructure challenges, and time-consuming nature of germ-free mouse models, an alternative approach is needed to investigate host-microbial interactions. Drosophila melanogaster (fruit flies) can be used as a high throughput in vivo screening model of host-microbiome interactions as they are affordable, convenient, and replicable. D. melanogaster were essential in discovering components of the innate immune response to pathogens. However, axenic D. melanogaster can easily be generated for microbiome studies without the need for ethical considerations. The simplified microbiota structure enables researchers to evaluate permutations of how each microbial species within the microbiota contribute to host phenotypes of interest. This enables the possibility of thorough strain-level analysis of host and microbial properties relevant to physiological outcomes. Moreover, a wide range of mutant D. melanogaster strains can be affordably obtained from public stock centers. Given this, D. melanogaster can be used to identify candidate mechanisms of host-microbe symbioses relevant to pathogen exclusion, innate immunity modulation, diet, xenobiotics, and probiotic/prebiotic properties in a high throughput manner. This perspective comments on the most promising areas of microbiota research that could immediately benefit from using the D. melanogaster model.

Ultra High Throughput Screening of Natural Product Extracts to Identify Pro-apoptotic Inhibitors of Bcl-2 Family Proteins

PubMed Central

Hassig, Christian A.; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W.; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J.; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E.; Brown, Susan G.; Baire, Beeraiah; Michel, Andrew R.; Hoye, Thomas R.; Francis, Subhashree; Georg, Gunda I.; Walters, Michael A.; Divlianska, Daniela B.; Roth, Gregory P.; Wright, Amy E.; Reed, John C.

2015-01-01

Anti-apoptotic Bcl-2 family proteins are validated cancer targets comprised of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). While several isoform-selective inhibitors have been developed using structure-based design or high throughput screening (HTS) of synthetic chemical libraries, no large scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six anti-apoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally-relevant PPIs. The screens were conducted in 1,536-well format and displayed satisfactory overall HTS statistics, with Z’-factor values ranging from 0.72 to 0.83, and a hit confirmation rate between 16-64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra high throughput screening using natural product sources and highlight some of the challenges associated with this approach. PMID:24870016

Evaluation of a High Throughput Starch Analysis Optimised for Wood

PubMed Central

Bellasio, Chandra; Fini, Alessio; Ferrini, Francesco

2014-01-01

Starch is the most important long-term reserve in trees, and the analysis of starch is therefore useful source of physiological information. Currently published protocols for wood starch analysis impose several limitations, such as long procedures and a neutralization step. The high-throughput standard protocols for starch analysis in food and feed represent a valuable alternative. However, they have not been optimised or tested with woody samples. These have particular chemical and structural characteristics, including the presence of interfering secondary metabolites, low reactivity of starch, and low starch content. In this study, a standard method for starch analysis used for food and feed (AOAC standard method 996.11) was optimised to improve precision and accuracy for the analysis of starch in wood. Key modifications were introduced in the digestion conditions and in the glucose assay. The optimised protocol was then evaluated through 430 starch analyses of standards at known starch content, matrix polysaccharides, and wood collected from three organs (roots, twigs, mature wood) of four species (coniferous and flowering plants). The optimised protocol proved to be remarkably precise and accurate (3%), suitable for a high throughput routine analysis (35 samples a day) of specimens with a starch content between 40 mg and 21 µg. Samples may include lignified organs of coniferous and flowering plants and non-lignified organs, such as leaves, fruits and rhizomes. PMID:24523863

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Compound Transfer by Acoustic Droplet Ejection Promotes Quality and Efficiency in Ultra-High-Throughput Screening Campaigns.

PubMed

Dawes, Timothy D; Turincio, Rebecca; Jones, Steven W; Rodriguez, Richard A; Gadiagellan, Dhireshan; Thana, Peter; Clark, Kevin R; Gustafson, Amy E; Orren, Linda; Liimatta, Marya; Gross, Daniel P; Maurer, Till; Beresini, Maureen H

2016-02-01

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed. © 2015 Society for Laboratory Automation and Screening.

An Automated High-Throughput System to Fractionate Plant Natural Products for Drug Discovery

PubMed Central

Tu, Ying; Jeffries, Cynthia; Ruan, Hong; Nelson, Cynthia; Smithson, David; Shelat, Anang A.; Brown, Kristin M.; Li, Xing-Cong; Hester, John P.; Smillie, Troy; Khan, Ikhlas A.; Walker, Larry; Guy, Kip; Yan, Bing

2010-01-01

The development of an automated, high-throughput fractionation procedure to prepare and analyze natural product libraries for drug discovery screening is described. Natural products obtained from plant materials worldwide were extracted and first prefractionated on polyamide solid-phase extraction cartridges to remove polyphenols, followed by high-throughput automated fractionation, drying, weighing, and reformatting for screening and storage. The analysis of fractions with UPLC coupled with MS, PDA and ELSD detectors provides information that facilitates characterization of compounds in active fractions. Screening of a portion of fractions yielded multiple assay-specific hits in several high-throughput cellular screening assays. This procedure modernizes the traditional natural product fractionation paradigm by seamlessly integrating automation, informatics, and multimodal analytical interrogation capabilities. PMID:20232897

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

PubMed Central

Wang, Zhu; Zhang, Xu-Xiang; Lu, Xin; Liu, Bo; Li, Yan; Long, Chao; Li, Aimin

2014-01-01

Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment. PMID:25420093

Novel organosilicone materials and patterning techniques for nanoimprint lithography

NASA Astrophysics Data System (ADS)

Pina, Carlos Alberto

Nanoimprint Lithography (NIL) is a high-throughput patterning technique that allows the fabrication of nanostructures with great precision. It has been listed on the International Technology Roadmap for Semiconductors (ITRS) as a candidate technology for future generation Si chip manufacturing. In nanoimprint Lithography a resist material, e.g. a thermoplastic polymer, is placed in contact with a mold and then mechanically deformed under an applied load to transfer the nano-features on the mold surface into the resist. The success of NIL relies heavily in the capability of fabricating nanostructures on different types of materials. Thus, a key factor for NIL implementation in industrial settings is the development of advanced materials suitable as the nanoimprint resist. This dissertation focuses on the engineering of new polymer materials suitable as NIL resist. A variety of silicone-based polymer precursors were synthesized and formulated for NIL applications. High throughput and high yield nanopatterning was successfully achieved. Furthermore, additional capabilities of the developed materials were explored for a range of NIL applications such as their use as flexible, UV-transparent stamps and silicon compatible etching layers. Finally, new strategies were investigated to expand the NIL potentiality. High throughput, non-residual layer imprinting was achieved with the newly developed resist materials. In addition, several strategies were designed for the precise control of nanoscale size patterned structures with multifunctional resist systems by post-imprinting modification of the pattern size. These developments provide NIL with a new set of tools for a variety of additional important applications.

High-throughput protein analysis integrating bioinformatics and experimental assays

PubMed Central

del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

2004-01-01

The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit

PubMed Central

Pronk, Sander; Páll, Szilárd; Schulz, Roland; Larsson, Per; Bjelkmar, Pär; Apostolov, Rossen; Shirts, Michael R.; Smith, Jeremy C.; Kasson, Peter M.; van der Spoel, David; Hess, Berk; Lindahl, Erik

2013-01-01

Motivation: Molecular simulation has historically been a low-throughput technique, but faster computers and increasing amounts of genomic and structural data are changing this by enabling large-scale automated simulation of, for instance, many conformers or mutants of biomolecules with or without a range of ligands. At the same time, advances in performance and scaling now make it possible to model complex biomolecular interaction and function in a manner directly testable by experiment. These applications share a need for fast and efficient software that can be deployed on massive scale in clusters, web servers, distributed computing or cloud resources. Results: Here, we present a range of new simulation algorithms and features developed during the past 4 years, leading up to the GROMACS 4.5 software package. The software now automatically handles wide classes of biomolecules, such as proteins, nucleic acids and lipids, and comes with all commonly used force fields for these molecules built-in. GROMACS supports several implicit solvent models, as well as new free-energy algorithms, and the software now uses multithreading for efficient parallelization even on low-end systems, including windows-based workstations. Together with hand-tuned assembly kernels and state-of-the-art parallelization, this provides extremely high performance and cost efficiency for high-throughput as well as massively parallel simulations. Availability: GROMACS is an open source and free software available from http://www.gromacs.org. Contact: erik.lindahl@scilifelab.se Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23407358

Purinergic P2X(7) receptor antagonists: Chemistry and fundamentals of biological screening.

PubMed

Gunosewoyo, Hendra; Coster, Mark J; Bennett, Maxwell R; Kassiou, Michael

2009-07-15

The purinergic P2X(7) receptor is a unique member of the ATP-gated P2X family. This receptor has been implicated in numerous diseases and many structurally diverse ligands have been discovered via high throughput screening. This perspective will attempt to highlight some of the most recent key findings in both the biology and chemistry.

Identification of novel IP receptor agonists using historical ligand biased chemical arrays.

PubMed

McKeown, Stephen C; Charlton, Steven J; Cox, Brian; Fitch, Helen; Howson, Christopher D; Leblanc, Catherine; Meyer, Arndt; Rosethorne, Elizabeth M; Stanley, Emily

2014-05-15

By considering published structural information we have designed high throughput biaryl lipophilic acid arrays leveraging facile chemistry to expedite their synthesis. We rapidly identified multiple hits which were of suitable IP agonist potency. These relatively simple and strategically undecorated molecules present an ideal opportunity for optimization towards our target candidate profile. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors

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

Shin, Youseung; Chen, Weiming; Habel, Jeff

2009-09-14

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 1: High-Throughput Screening and Preliminary Exploration.

PubMed

Sartori, Luca; Mercurio, Ciro; Amigoni, Federica; Cappa, Anna; Fagá, Giovanni; Fattori, Raimondo; Legnaghi, Elena; Ciossani, Giuseppe; Mattevi, Andrea; Meroni, Giuseppe; Moretti, Loris; Cecatiello, Valentina; Pasqualato, Sebastiano; Romussi, Alessia; Thaler, Florian; Trifiró, Paolo; Villa, Manuela; Vultaggio, Stefania; Botrugno, Oronza A; Dessanti, Paola; Minucci, Saverio; Zagarrí, Elisa; Carettoni, Daniele; Iuzzolino, Lucia; Varasi, Mario; Vianello, Paola

2017-03-09

Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC 50 , thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC 50 = 2.9 μM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC 50 (0.162 μM), capable of inhibiting the target in cells.

A Fair Contention Access Scheme for Low-Priority Traffic in Wireless Body Area Networks

PubMed Central

Sajeel, Muhammad; Bashir, Faisal; Asfand-e-yar, Muhammad; Tauqir, Muhammad

2017-01-01

Recently, wireless body area networks (WBANs) have attracted significant consideration in ubiquitous healthcare. A number of medium access control (MAC) protocols, primarily derived from the superframe structure of the IEEE 802.15.4, have been proposed in literature. These MAC protocols aim to provide quality of service (QoS) by prioritizing different traffic types in WBANs. A contention access period (CAP)with high contention in priority-based MAC protocols can result in higher number of collisions and retransmissions. During CAP, traffic classes with higher priority are dominant over low-priority traffic; this has led to starvation of low-priority traffic, thus adversely affecting WBAN throughput, delay, and energy consumption. Hence, this paper proposes a traffic-adaptive priority-based superframe structure that is able to reduce contention in the CAP period, and provides a fair chance for low-priority traffic. Simulation results in ns-3 demonstrate that the proposed MAC protocol, called traffic- adaptive priority-based MAC (TAP-MAC), achieves low energy consumption, high throughput, and low latency compared to the IEEE 802.15.4 standard, and the most recent priority-based MAC protocol, called priority-based MAC protocol (PA-MAC). PMID:28832495

Multiple e-pharmacophore modelling pooled with high-throughput virtual screening, docking and molecular dynamics simulations to discover potential inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH).

PubMed

Saxena, Shalini; Durgam, Laxman; Guruprasad, Lalitha

2018-05-14

Development of new antimalarial drugs continues to be of huge importance because of the resistance of malarial parasite towards currently used drugs. Due to the reliance of parasite on glycolysis for energy generation, glycolytic enzymes have played important role as potential targets for the development of new drugs. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a key enzyme for energy generation of malarial parasites and is considered to be a potential antimalarial target. Presently, there are nearly 15 crystal structures bound with inhibitors and substrate that are available in the protein data bank (PDB). In the present work, we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 1.4 × 10 2 -1.3 × 10 6  nM efficacy and optimized the pharmacophore based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput virtual screening (HTVS) combined with molecular docking, ADME predictions and molecular dynamics simulation led to the identification of 20 potential compounds which could be further developed as novel inhibitors for PfLDH.

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

PubMed

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

2016-01-01

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

[Morphometry of pulmonary tissue: From manual to high throughput automation].

PubMed

Sallon, C; Soulet, D; Tremblay, Y

2017-12-01

Weibel's research has shown that any alteration of the pulmonary structure has effects on function. This demonstration required a quantitative analysis of lung structures called morphometry. This is possible thanks to stereology, a set of methods based on principles of geometry and statistics. His work has helped to better understand the morphological harmony of the lung, which is essential for its proper functioning. An imbalance leads to pathophysiology such as chronic obstructive pulmonary disease in adults and bronchopulmonary dysplasia in neonates. It is by studying this imbalance that new therapeutic approaches can be developed. These advances are achievable only through morphometric analytical methods, which are increasingly precise and focused, in particular thanks to the high-throughput automation of these methods. This review makes a comparison between an automated method that we developed in the laboratory and semi-manual methods of morphometric analyzes. The automation of morphometric measurements is a fundamental asset in the study of pulmonary pathophysiology because it is an assurance of robustness, reproducibility and speed. This tool will thus contribute significantly to the acceleration of the race for the development of new drugs. Copyright © 2017 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Capturing anharmonicity in a lattice thermal conductivity model for high-throughput predictions

DOE PAGES

Miller, Samuel A.; Gorai, Prashun; Ortiz, Brenden R.; ...

2017-01-06

High-throughput, low-cost, and accurate predictions of thermal properties of new materials would be beneficial in fields ranging from thermal barrier coatings and thermoelectrics to integrated circuits. To date, computational efforts for predicting lattice thermal conductivity (κ L) have been hampered by the complexity associated with computing multiple phonon interactions. In this work, we develop and validate a semiempirical model for κ L by fitting density functional theory calculations to experimental data. Experimental values for κ L come from new measurements on SrIn 2O 4, Ba 2SnO 4, Cu 2ZnSiTe 4, MoTe 2, Ba 3In 2O 6, Cu 3TaTe 4, SnO,more » and InI as well as 55 compounds from across the published literature. Here, to capture the anharmonicity in phonon interactions, we incorporate a structural parameter that allows the model to predict κ L within a factor of 1.5 of the experimental value across 4 orders of magnitude in κ L values and over a diverse chemical and structural phase space, with accuracy similar to or better than that of computationally more expensive models.« less

Ultra-high frequency ultrasound biomicroscopy and high throughput cardiovascular phenotyping in a large scale mouse mutagenesis screen

NASA Astrophysics Data System (ADS)

Liu, Xiaoqin; Francis, Richard; Tobita, Kimimasa; Kim, Andy; Leatherbury, Linda; Lo, Cecilia W.

2013-02-01

Ultrasound biomicroscopy (UBM) is ideally suited for phenotyping fetal mice for congenital heart disease (CHD), as imaging can be carried out noninvasively to provide both hemodynamic and structural information essential for CHD diagnosis. Using the UBM (Vevo 2100; 40Hz) in conjunction with the clinical ultrasound system (Acuson Sequioa C512; 15Hz), we developed a two-step screening protocol to scan thousands fetuses derived from ENU mutagenized pedigrees. A wide spectrum of CHD was detected by the UBM, which were subsequently confirmed with follow-up necropsy and histopathology examination with episcopic fluorescence image capture. CHD observed included outflow anomalies, left/right heart obstructive lesions, septal/valvular defects and cardiac situs anomalies. Meanwhile, various extracardiac defects were found, such as polydactyly, craniofacial defects, exencephaly, omphalocele-cleft palate, most of which were associated with cardiac defects. Our analyses showed the UBM was better at assessing cardiac structure and blood flow profiles, while conventional ultrasound allowed higher throughput low-resolution screening. Our study showed the integration of conventional clinical ultrasound imaging with the UBM for fetal mouse cardiovascular phenotyping can maximize the detection and recovery of CHD mutants.

Triangle network motifs predict complexes by complementing high-error interactomes with structural information.

PubMed

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-06-27

A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN.

Triangle network motifs predict complexes by complementing high-error interactomes with structural information

PubMed Central

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-01-01

Background A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. Results We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Conclusion Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN. PMID:19558694

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High-Throughput Flow

DTIC Science & Technology

2012-08-01

techniques and STEAM imager. It couples the high-speed capability of the STEAM imager and differential phase contrast imaging of DIC / Nomarski microscopy...On 10 TPE chips, we obtained 9 homogenous and strong bonds, the failed bond being due to operator error and presence of air bubbles in the TPE...instruments, structural dynamics, and microelectromechanical systems (MEMS) via laser-scanning surface vibrometry , and observation of biomechanical motility

A generalized method for high throughput in-situ experiment data analysis: An example of battery materials exploration

NASA Astrophysics Data System (ADS)

Aoun, Bachir; Yu, Cun; Fan, Longlong; Chen, Zonghai; Amine, Khalil; Ren, Yang

2015-04-01

A generalized method is introduced to extract critical information from series of ranked correlated data. The method is generally applicable to all types of spectra evolving as a function of any arbitrary parameter. This approach is based on correlation functions and statistical scedasticity formalism. Numerous challenges in analyzing high throughput experimental data can be tackled using the herein proposed method. We applied this method to understand the reactivity pathway and formation mechanism of a Li-ion battery cathode material during high temperature synthesis using in-situ high-energy X-ray diffraction. We demonstrate that Pearson's correlation function can easily unravel all major phase transition and, more importantly, the minor structural changes which cannot be revealed by conventionally inspecting the series of diffraction patterns. Furthermore, a two-dimensional (2D) reactivity pattern calculated as the scedasticity along all measured reciprocal space of all successive diffraction pattern pairs unveils clearly the structural evolution path and the active areas of interest during the synthesis. The methods described here can be readily used for on-the-fly data analysis during various in-situ operando experiments in order to quickly evaluate and optimize experimental conditions, as well as for post data analysis and large data mining where considerable amount of data hinders the feasibility of the investigation through point-by-point inspection.

Making big sense from big data in toxicology by read-across.

PubMed

Hartung, Thomas

2016-01-01

Modern information technologies have made big data available in safety sciences, i.e., extremely large data sets that may be analyzed only computationally to reveal patterns, trends and associations. This happens by (1) compilation of large sets of existing data, e.g., as a result of the European REACH regulation, (2) the use of omics technologies and (3) systematic robotized testing in a high-throughput manner. All three approaches and some other high-content technologies leave us with big data--the challenge is now to make big sense of these data. Read-across, i.e., the local similarity-based intrapolation of properties, is gaining momentum with increasing data availability and consensus on how to process and report it. It is predominantly applied to in vivo test data as a gap-filling approach, but can similarly complement other incomplete datasets. Big data are first of all repositories for finding similar substances and ensure that the available data is fully exploited. High-content and high-throughput approaches similarly require focusing on clusters, in this case formed by underlying mechanisms such as pathways of toxicity. The closely connected properties, i.e., structural and biological similarity, create the confidence needed for predictions of toxic properties. Here, a new web-based tool under development called REACH-across, which aims to support and automate structure-based read-across, is presented among others.

An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

PubMed Central

Meirson, Tomer; Samson, Abraham O; Gil-Henn, Hava

2017-01-01

The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK) was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. PMID:28572720

Data-Driven High-Throughput Prediction of the 3D Structure of Small Molecules: Review and Progress

PubMed Central

Andronico, Alessio; Randall, Arlo; Benz, Ryan W.; Baldi, Pierre

2011-01-01

Accurate prediction of the 3D structure of small molecules is essential in order to understand their physical, chemical, and biological properties including how they interact with other molecules. Here we survey the field of high-throughput methods for 3D structure prediction and set up new target specifications for the next generation of methods. We then introduce COSMOS, a novel data-driven prediction method that utilizes libraries of fragment and torsion angle parameters. We illustrate COSMOS using parameters extracted from the Cambridge Structural Database (CSD) by analyzing their distribution and then evaluating the system’s performance in terms of speed, coverage, and accuracy. Results show that COSMOS represents a significant improvement when compared to the state-of-the-art, particularly in terms of coverage of complex molecular structures, including metal-organics. COSMOS can predict structures for 96.4% of the molecules in the CSD [99.6% organic, 94.6% metal-organic] whereas the widely used commercial method CORINA predicts structures for 68.5% [98.5% organic, 51.6% metal-organic]. On the common subset of molecules predicted by both methods COSMOS makes predictions with an average speed per molecule of 0.15s [0.10s organic, 0.21s metal-organic], and an average RMSD of 1.57Å [1.26Å organic, 1.90Å metal-organic], and CORINA makes predictions with an average speed per molecule of 0.13s [0.18s organic, 0.08s metal-organic], and an average RMSD of 1.60Å [1.13Å organic, 2.11Å metal-organic]. COSMOS is available through the ChemDB chemoinformatics web portal at: http://cdb.ics.uci.edu/. PMID:21417267

The interdependence between screening methods and screening libraries.

PubMed

Shelat, Anang A; Guy, R Kiplin

2007-06-01

The most common methods for discovery of chemical compounds capable of manipulating biological function involves some form of screening. The success of such screens is highly dependent on the chemical materials - commonly referred to as libraries - that are assayed. Classic methods for the design of screening libraries have depended on knowledge of target structure and relevant pharmacophores for target focus, and on simple count-based measures to assess other properties. The recent proliferation of two novel screening paradigms, structure-based screening and high-content screening, prompts a profound rethink about the ideal composition of small-molecule screening libraries. We suggest that currently utilized libraries are not optimal for addressing new targets by high-throughput screening, or complex phenotypes by high-content screening.

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

PubMed

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

2015-01-01

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

Structure-guided wavelength tuning in far-red fluorescent proteins

PubMed Central

Ng, Ho-Leung; Lin, Michael Z.

2017-01-01

In recent years, protein engineers have succeeded in tuning the excitation spectra of natural fluorescent proteins from green wavelengths into orange and red wavelengths, resulting in the creation of a series of fluorescent proteins with emission in the far-red portions of the optical spectrum. These results have arisen from the synergistic combination of structural knowledge of fluorescent proteins, chemical intuition, and high-throughput screening methods. Here we review structural features found in autocatalytic far-red fluorescent proteins, and discuss how they add to our understanding of the biophysical mechanisms of wavelength tuning in biological chromophores. PMID:27468111

On the design of turbo codes

NASA Technical Reports Server (NTRS)

Divsalar, D.; Pollara, F.

1995-01-01

In this article, we design new turbo codes that can achieve near-Shannon-limit performance. The design criterion for random interleavers is based on maximizing the effective free distance of the turbo code, i.e., the minimum output weight of codewords due to weight-2 input sequences. An upper bound on the effective free distance of a turbo code is derived. This upper bound can be achieved if the feedback connection of convolutional codes uses primitive polynomials. We review multiple turbo codes (parallel concatenation of q convolutional codes), which increase the so-called 'interleaving gain' as q and the interleaver size increase, and a suitable decoder structure derived from an approximation to the maximum a posteriori probability decision rule. We develop new rate 1/3, 2/3, 3/4, and 4/5 constituent codes to be used in the turbo encoder structure. These codes, for from 2 to 32 states, are designed by using primitive polynomials. The resulting turbo codes have rates b/n (b = 1, 2, 3, 4 and n = 2, 3, 4, 5, 6), and include random interleavers for better asymptotic performance. These codes are suitable for deep-space communications with low throughput and for near-Earth communications where high throughput is desirable. The performance of these codes is within 1 dB of the Shannon limit at a bit-error rate of 10(exp -6) for throughputs from 1/15 up to 4 bits/s/Hz.

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

PubMed Central

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

2016-01-01

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

An image analysis toolbox for high-throughput C. elegans assays

PubMed Central

Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H.; Riklin-Raviv, Tammy; Conery, Annie L.; O’Rourke, Eyleen J.; Sokolnicki, Katherine L.; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E.; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M.; Carpenter, Anne E.

2012-01-01

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available via the open-source CellProfiler project and enables objective scoring of whole-animal high-throughput image-based assays of C. elegans for the study of diverse biological pathways relevant to human disease. PMID:22522656

High-throughput, image-based screening of pooled genetic variant libraries

PubMed Central

Emanuel, George; Moffitt, Jeffrey R.; Zhuang, Xiaowei

2018-01-01

Image-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants. PMID:29083401

Experimental Design for Combinatorial and High Throughput Materials Development

NASA Astrophysics Data System (ADS)

Cawse, James N.

2002-12-01

In the past decade, combinatorial and high throughput experimental methods have revolutionized the pharmaceutical industry, allowing researchers to conduct more experiments in a week than was previously possible in a year. Now high throughput experimentation is rapidly spreading from its origins in the pharmaceutical world to larger industrial research establishments such as GE and DuPont, and even to smaller companies and universities. Consequently, researchers need to know the kinds of problems, desired outcomes, and appropriate patterns for these new strategies. Editor James Cawse's far-reaching study identifies and applies, with specific examples, these important new principles and techniques. Experimental Design for Combinatorial and High Throughput Materials Development progresses from methods that are now standard, such as gradient arrays, to mathematical developments that are breaking new ground. The former will be particularly useful to researchers entering the field, while the latter should inspire and challenge advanced practitioners. The book's contents are contributed by leading researchers in their respective fields. Chapters include: -High Throughput Synthetic Approaches for the Investigation of Inorganic Phase Space -Combinatorial Mapping of Polymer Blends Phase Behavior -Split-Plot Designs -Artificial Neural Networks in Catalyst Development -The Monte Carlo Approach to Library Design and Redesign This book also contains over 200 useful charts and drawings. Industrial chemists, chemical engineers, materials scientists, and physicists working in combinatorial and high throughput chemistry will find James Cawse's study to be an invaluable resource.

Present and future of membrane protein structure determination by electron crystallography.

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing

PubMed Central

Chandran, Anandhakumar; Syed, Junetha; Taylor, Rhys D.; Kashiwazaki, Gengo; Sato, Shinsuke; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2016-01-01

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2 showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing. PMID:27098039

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

PubMed

Van Dolah, F M; Finley, E L; Haynes, B L; Doucette, G J; Moeller, P D; Ramsdell, J S

1994-01-01

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.

Advances in structural and functional analysis of membrane proteins by electron crystallography

PubMed Central

Wisedchaisri, Goragot; Reichow, Steve L.; Gonen, Tamir

2011-01-01

Summary Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. PMID:22000511

Advances in structural and functional analysis of membrane proteins by electron crystallography.

PubMed

Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir

2011-10-12

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. Copyright © 2011 Elsevier Ltd. All rights reserved.

High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast

EPA Science Inventory

High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

MAPPER: high-throughput maskless lithography

NASA Astrophysics Data System (ADS)

Wieland, M. J.; de Boer, G.; ten Berge, G. F.; Jager, R.; van de Peut, T.; Peijster, J. J. M.; Slot, E.; Steenbrink, S. W. H. K.; Teepen, T. F.; van Veen, A. H. V.; Kampherbeek, B. J.

2009-03-01

Maskless electron beam lithography, or electron beam direct write, has been around for a long time in the semiconductor industry and was pioneered from the mid-1960s onwards. This technique has been used for mask writing applications as well as device engineering and in some cases chip manufacturing. However because of its relatively low throughput compared to optical lithography, electron beam lithography has never been the mainstream lithography technology. To extend optical lithography double patterning, as a bridging technology, and EUV lithography are currently explored. Irrespective of the technical viability of both approaches, one thing seems clear. They will be expensive [1]. MAPPER Lithography is developing a maskless lithography technology based on massively-parallel electron-beam writing with high speed optical data transport for switching the electron beams. In this way optical columns can be made with a throughput of 10-20 wafers per hour. By clustering several of these columns together high throughputs can be realized in a small footprint. This enables a highly cost-competitive alternative to double patterning and EUV alternatives. In 2007 MAPPER obtained its Proof of Lithography milestone by exposing in its Demonstrator 45 nm half pitch structures with 110 electron beams in parallel, where all the beams where individually switched on and off [2]. In 2008 MAPPER has taken a next step in its development by building several tools. The objective of building these tools is to involve semiconductor companies to be able to verify tool performance in their own environment. To enable this, the tools will have a 300 mm wafer stage in addition to a 110-beam optics column. First exposures at 45 nm half pitch resolution have been performed and analyzed. On the same wafer it is observed that all beams print and based on analysis of 11 beams the CD for the different patterns is within 2.2 nm from target and the CD uniformity for the different patterns is better than 2.8 nm.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.

The use of high-throughput screening techniques to evaluate mitochondrial toxicity.

PubMed

Wills, Lauren P

2017-11-01

Toxicologists and chemical regulators depend on accurate and effective methods to evaluate and predict the toxicity of thousands of current and future compounds. Robust high-throughput screening (HTS) experiments have the potential to efficiently test large numbers of chemical compounds for effects on biological pathways. HTS assays can be utilized to examine chemical toxicity across multiple mechanisms of action, experimental models, concentrations, and lengths of exposure. Many agricultural, industrial, and pharmaceutical chemicals classified as harmful to human and environmental health exert their effects through the mechanism of mitochondrial toxicity. Mitochondrial toxicants are compounds that cause a decrease in the number of mitochondria within a cell, and/or decrease the ability of mitochondria to perform normal functions including producing adenosine triphosphate (ATP) and maintaining cellular homeostasis. Mitochondrial dysfunction can lead to apoptosis, necrosis, altered metabolism, muscle weakness, neurodegeneration, decreased organ function, and eventually disease or death of the whole organism. The development of HTS techniques to identify mitochondrial toxicants will provide extensive databases with essential connections between mechanistic mitochondrial toxicity and chemical structure. Computational and bioinformatics approaches can be used to evaluate compound databases for specific chemical structures associated with toxicity, with the goal of developing quantitative structure-activity relationship (QSAR) models and mitochondrial toxicophores. Ultimately these predictive models will facilitate the identification of mitochondrial liabilities in consumer products, industrial compounds, pharmaceuticals and environmental hazards. Copyright © 2017 Elsevier B.V. All rights reserved.

Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

PubMed Central

Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; Tiedje, James M.

2016-01-01

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation. PMID:27313569

High-Throughput Screening of Vascular Endothelium-Destructive or Protective Microenvironments: Cooperative Actions of Extracellular Matrix Composition, Stiffness, and Structure.

PubMed

Ding, Yonghui; Floren, Michael; Tan, Wei

2017-06-01

Pathological modification of the subendothelial extracellular matrix (ECM) has closely been associated with endothelial activation and subsequent cardiovascular disease progression. To understand regulatory mechanisms of these matrix modifications, the majority of previous efforts have focused on the modulation of either chemical composition or matrix stiffness on 2D smooth surfaces without simultaneously probing their cooperative effects on endothelium function on in vivo like 3D fibrous matrices. To this end, a high-throughput, combinatorial microarray platform on 2D and 3D hydrogel settings to resemble the compositions, stiffness, and structure of healthy and diseased subendothelial ECM has been established, and further their respective and combined effects on endothelial attachment, proliferation, inflammation, and junctional integrity have been investigated. For the first time, the results demonstrate that 3D fibrous structure resembling native ECM is a critical endothelium-protective microenvironmental factor by maintaining the stable, quiescent endothelium with strong resistance to proinflammatory stimuli. It is also revealed that matrix stiffening, in concert with chemical compositions resembling diseased ECM, particularly collagen III, could aggravate activation of nuclear factor kappa B, disruption of endothelium integrity, and susceptibility to proinflammatory stimuli. This study elucidates cooperative effects of various microenvironmental factors on endothelial activation and sheds light on new in vitro model for cardiovascular diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation

PubMed Central

Le Dily, François; Baù, Davide; Pohl, Andy; Vicent, Guillermo P.; Serra, François; Soronellas, Daniel; Castellano, Giancarlo; Wright, Roni H.G.; Ballare, Cecilia; Filion, Guillaume; Marti-Renom, Marc A.

2014-01-01

The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as “regulons” to enable spatially proximal genes to be coordinately transcribed in response to hormones. PMID:25274727

Nanomanufacturing : nano-structured materials made layer-by-layer.

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

Cox, James V.; Cheng, Shengfeng; Grest, Gary Stephen

Large-scale, high-throughput production of nano-structured materials (i.e. nanomanufacturing) is a strategic area in manufacturing, with markets projected to exceed $1T by 2015. Nanomanufacturing is still in its infancy; process/product developments are costly and only touch on potential opportunities enabled by growing nanoscience discoveries. The greatest promise for high-volume manufacturing lies in age-old coating and imprinting operations. For materials with tailored nm-scale structure, imprinting/embossing must be achieved at high speeds (roll-to-roll) and/or over large areas (batch operation) with feature sizes less than 100 nm. Dispersion coatings with nanoparticles can also tailor structure through self- or directed-assembly. Layering films structured with thesemore » processes have tremendous potential for efficient manufacturing of microelectronics, photovoltaics and other topical nano-structured devices. This project is designed to perform the requisite R and D to bring Sandia's technology base in computational mechanics to bear on this scale-up problem. Project focus is enforced by addressing a promising imprinting process currently being commercialized.« less

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Colour-barcoded magnetic microparticles for multiplexed bioassays.

PubMed

Lee, Howon; Kim, Junhoi; Kim, Hyoki; Kim, Jiyun; Kwon, Sunghoon

2010-09-01

Encoded particles have a demonstrated value for multiplexed high-throughput bioassays such as drug discovery and clinical diagnostics. In diverse samples, the ability to use a large number of distinct identification codes on assay particles is important to increase throughput. Proper handling schemes are also needed to readout these codes on free-floating probe microparticles. Here we create vivid, free-floating structural coloured particles with multi-axis rotational control using a colour-tunable magnetic material and a new printing method. Our colour-barcoded magnetic microparticles offer a coding capacity easily into the billions with distinct magnetic handling capabilities including active positioning for code readouts and active stirring for improved reaction kinetics in microscale environments. A DNA hybridization assay is done using the colour-barcoded magnetic microparticles to demonstrate multiplexing capabilities.

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

PubMed

Yang, Darren; Wong, Wesley P

2018-01-01

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

High throughput single cell counting in droplet-based microfluidics.

PubMed

Lu, Heng; Caen, Ouriel; Vrignon, Jeremy; Zonta, Eleonora; El Harrak, Zakaria; Nizard, Philippe; Baret, Jean-Christophe; Taly, Valérie

2017-05-02

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

High-Throughput Sequencing of Germline and Tumor From Men with Early-Onset Metastatic Prostate Cancer

DTIC Science & Technology

2016-12-01

AWARD NUMBER: W81XWH-13-1-0371 TITLE: High-Throughput Sequencing of Germline and Tumor From Men with Early- Onset Metastatic Prostate Cancer...DATES COVERED 30 Sep 2013 - 29 Sep 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER High-Throughput Sequencing of Germline and Tumor From Men with...presenting with metastatic prostate cancer at a young age (before age 60 years). Whole exome sequencing identified a panel of germline variants that have

Solvent influence upon structure & throughput of poly vinyledene fluoride thin film nano-patterns by imprint lithography

NASA Astrophysics Data System (ADS)

Sankar, M. S. Ravi; Gangineni, R. B.

2018-04-01

This work aims at understanding the solvent influence upon the throughput and structure of poly vinyledene fluoride (PVDF)nano-patterned films. The PVDF thin films are deposited by spin coating method using Dimethylsulfoxide (DMSO), Tetrahydrofuran (THF) and 2-butanone solvents. The nano-patterns are realized by imprinting SONY 700 MB CD aluminum constructions on PVDF thin filmsusing imprint lithography technique under ambient annealing temperature and pressure. Surface morphology &imprint pattern transfer quality is evaluated with Atomic force microscopy (AFM). Raman spectroscopy is used for evaluating the structural evolutions with respect to solvent & patterning.

HPC AND GRID COMPUTING FOR INTEGRATIVE BIOMEDICAL RESEARCH

PubMed Central

Kurc, Tahsin; Hastings, Shannon; Kumar, Vijay; Langella, Stephen; Sharma, Ashish; Pan, Tony; Oster, Scott; Ervin, David; Permar, Justin; Narayanan, Sivaramakrishnan; Gil, Yolanda; Deelman, Ewa; Hall, Mary; Saltz, Joel

2010-01-01

Integrative biomedical research projects query, analyze, and integrate many different data types and make use of datasets obtained from measurements or simulations of structure and function at multiple biological scales. With the increasing availability of high-throughput and high-resolution instruments, the integrative biomedical research imposes many challenging requirements on software middleware systems. In this paper, we look at some of these requirements using example research pattern templates. We then discuss how middleware systems, which incorporate Grid and high-performance computing, could be employed to address the requirements. PMID:20107625

Selected approaches for rational drug design and high throughput screening to identify anti-cancer molecules.

PubMed

Hedvat, Michael; Emdad, Luni; Das, Swadesh K; Kim, Keetae; Dasgupta, Santanu; Thomas, Shibu; Hu, Bin; Zhu, Shan; Dash, Rupesh; Quinn, Bridget A; Oyesanya, Regina A; Kegelman, Timothy P; Sokhi, Upneet K; Sarkar, Siddik; Erdogan, Eda; Menezes, Mitchell E; Bhoopathi, Praveen; Wang, Xiang-Yang; Pomper, Martin G; Wei, Jun; Wu, Bainan; Stebbins, John L; Diaz, Paul W; Reed, John C; Pellecchia, Maurizio; Sarkar, Devanand; Fisher, Paul B

2012-11-01

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Quantitative Developments of Biomolecular Databases, Measurement Methodology, and Comprehensive Transport Models for Bioanalytical Microfluidics

DTIC Science & Technology

2006-10-01

Gibbs, E. M., Fletterick, R. J., Day, Y. S. N., Myszka, D. G., and Rath, V. L. (2002) “Structure-activity analysis of the purine-binding site of human ...Rich, R. L., Day, Y. S. N., Morton, T. A., and Myszka, D. G., (2001) “High- resolution and high-throughput protocols for measuring drug/ human serum...entire text) 1. Attard, P., Images of nanobubbles on hydrophobic surfaces and their interactions. Phys. Rev. Lett., 2001. 87. 2. Ottino, J.M

Design and evaluation of 1,7-naphthyridones as novel KDM5 inhibitors.

PubMed

Labadie, Sharada S; Dragovich, Peter S; Cummings, Richard T; Deshmukh, Gauri; Gustafson, Amy; Han, Ning; Harmange, Jean-Christophe; Kiefer, James R; Li, Yue; Liang, Jun; Liederer, Bianca M; Liu, Yichin; Manieri, Wanda; Mao, Wiefeng; Murray, Lesley; Ortwine, Daniel F; Trojer, Patrick; VanderPorten, Erica; Vinogradova, Maia; Wen, Li

2016-09-15

Features from a high throughput screening (HTS) hit and a previously reported scaffold were combined to generate 1,7-naphthyridones as novel KDM5 enzyme inhibitors with nanomolar potencies. These molecules exhibited high selectivity over the related KDM4C and KDM2B isoforms. An X-ray co-crystal structure of a representative molecule bound to KDM5A showed that these inhibitors are competitive with the co-substrate (2-oxoglutarate or 2-OG). Copyright © 2016 Elsevier Ltd. All rights reserved.

Toward large-area roll-to-roll printed nanophotonic sensors

NASA Astrophysics Data System (ADS)

Karioja, Pentti; Hiltunen, Jussi; Aikio, Sanna M.; Alajoki, Teemu; Tuominen, Jarkko; Hiltunen, Marianne; Siitonen, Samuli; Kontturi, Ville; Böhlen, Karl; Hauser, Rene; Charlton, Martin; Boersma, Arjen; Lieberzeit, Peter; Felder, Thorsten; Eustace, David; Haskal, Eliav

2014-05-01

Polymers have become an important material group in fabricating discrete photonic components and integrated optical devices. This is due to their good properties: high optical transmittance, versatile processability at relative low temperatures and potential for low-cost production. Recently, nanoimprinting or nanoimprint lithography (NIL) has obtained a plenty of research interest. In NIL, a mould is pressed against a substrate coated with a moldable material. After deformation of the material, the mold is separated and a replica of the mold is formed. Compared with conventional lithographic methods, imprinting is simple to carry out, requires less-complicated equipment and can provide high-resolution with high throughput. Nanoimprint lithography has shown potential to become a method for low-cost and high-throughput fabrication of nanostructures. We show the development process of nano-structured, large-area multi-parameter sensors using Photonic Crystal (PC) and Surface Enhanced Raman Scattering (SERS) methodologies for environmental and pharmaceutical applications. We address these challenges by developing roll-to-roll (R2R) UV-nanoimprint fabrication methods. Our development steps are the following: Firstly, the proof of concept structures are fabricated by the use of wafer-level processes in Si-based materials. Secondly, the master molds of successful designs are fabricated, and they are used to transfer the nanophotonic structures into polymer materials using sheet-level UV-nanoimprinting. Thirdly, the sheet-level nanoimprinting processes are transferred to roll-to-roll fabrication. In order to enhance roll-to-roll manufacturing capabilities, silicone-based polymer material development was carried out. In the different development phases, Photonic Crystal and SERS sensor structures with increasing complexities were fabricated using polymer materials in order to enhance sheet-level and roll-to-roll manufacturing processes. In addition, chemical and molecular imprint (MIP) functionalization methods were applied in the sensor demonstrators. In this paper, the process flow in fabricating large-area nanophotonic structures by the use of sheet-level and roll-to-roll UV- nanoimprinting is reported.

High-throughput sequencing methods to study neuronal RNA-protein interactions.

PubMed

Ule, Jernej

2009-12-01

UV-cross-linking and RNase protection, combined with high-throughput sequencing, have provided global maps of RNA sites bound by individual proteins or ribosomes. Using a stringent purification protocol, UV-CLIP (UV-cross-linking and immunoprecipitation) was able to identify intronic and exonic sites bound by splicing regulators in mouse brain tissue. Ribosome profiling has been used to quantify ribosome density on budding yeast mRNAs under different environmental conditions. Post-transcriptional regulation in neurons requires high spatial and temporal precision, as is evident from the role of localized translational control in synaptic plasticity. It remains to be seen if the high-throughput methods can be applied quantitatively to study the dynamics of RNP (ribonucleoprotein) remodelling in specific neuronal populations during the neurodegenerative process. It is certain, however, that applications of new biochemical techniques followed by high-throughput sequencing will continue to provide important insights into the mechanisms of neuronal post-transcriptional regulation.

Mix-and-diffuse serial synchrotron crystallography

DOE PAGES

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio; ...

2017-10-09

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

Mix-and-diffuse serial synchrotron crystallography

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

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

Evaluation of Compatibility of ToxCast High-Throughput/High-Content Screening Assays with Engineered Nanomaterials

EPA Science Inventory

High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

Aminopyrazinamides: novel and specific GyrB inhibitors that kill replicating and nonreplicating Mycobacterium tuberculosis.

PubMed

Shirude, Pravin S; Madhavapeddi, Prashanti; Tucker, Julie A; Murugan, Kannan; Patil, Vikas; Basavarajappa, Halesha; Raichurkar, Anandkumar V; Humnabadkar, Vaishali; Hussein, Syeed; Sharma, Sreevalli; Ramya, V K; Narayan, Chandan B; Balganesh, Tanjore S; Sambandamurthy, Vasan K

2013-03-15

Aminopyrazinamides originated from a high throughput screen targeting the Mycobacterium smegmatis (Msm) GyrB ATPase. This series displays chemical tractability, robust structure-activity relationship, and potent antitubercular activity. The crystal structure of Msm GyrB in complex with one of the aminopyrazinamides revealed promising attributes of specificity against other broad spectrum pathogens and selectivity against eukaryotic kinases due to novel interactions at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides display excellent mycobacterial kill under in vitro, intracellular, and hypoxic conditions.

Natural genetic variability reduces recalcitrance in poplar

DOE PAGES

Bhagia, Samarthya; Muchero, Wellington; Kumar, Rajeev; ...

2016-05-20

Here, lignin content and structure are known to affect recalcitrance of lignocellulosic biomass to chemical/biochemical conversion. Previously, we identified rare Populus trichocarpa natural variants with significantly reduced lignin content. Because reduced lignin content may lower recalcitrance, 18 rare variants along with 4 comparators, and BESC standard Populus was analyzed for composition of structural carbohydrates and lignin. Sugar yields from these plants were measured at 5 process conditions: one for just enzymatic hydrolysis without pretreatment and four via our combined high-throughput hot water pretreatment and co-hydrolysis (HTPH) technique.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes. PMID:29250096

Relationship between the Decomposition Process of Coarse Woody Debris and Fungal Community Structure as Detected by High-Throughput Sequencing in a Deciduous Broad-Leaved Forest in Japan

PubMed Central

Yamashita, Satoshi; Masuya, Hayato; Abe, Shin; Masaki, Takashi; Okabe, Kimiko

2015-01-01

We examined the relationship between the community structure of wood-decaying fungi, detected by high-throughput sequencing, and the decomposition rate using 13 years of data from a forest dynamics plot. For molecular analysis and wood density measurements, drill dust samples were collected from logs and stumps of Fagus and Quercus in the plot. Regression using a negative exponential model between wood density and time since death revealed that the decomposition rate of Fagus was greater than that of Quercus. The residual between the expected value obtained from the regression curve and the observed wood density was used as a decomposition rate index. Principal component analysis showed that the fungal community compositions of both Fagus and Quercus changed with time since death. Principal component analysis axis scores were used as an index of fungal community composition. A structural equation model for each wood genus was used to assess the effect of fungal community structure traits on the decomposition rate and how the fungal community structure was determined by the traits of coarse woody debris. Results of the structural equation model suggested that the decomposition rate of Fagus was affected by two fungal community composition components: one that was affected by time since death and another that was not affected by the traits of coarse woody debris. In contrast, the decomposition rate of Quercus was not affected by coarse woody debris traits or fungal community structure. These findings suggest that, in the case of Fagus coarse woody debris, the fungal community structure is related to the decomposition process of its host substrate. Because fungal community structure is affected partly by the decay stage and wood density of its substrate, these factors influence each other. Further research on interactive effects is needed to improve our understanding of the relationship between fungal community structure and the woody debris decomposition process. PMID:26110605

Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model

NASA Astrophysics Data System (ADS)

Mondal, Sudip; Hegarty, Evan; Martin, Chris; Gökçe, Sertan Kutal; Ghorashian, Navid; Ben-Yakar, Adela

2016-10-01

Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ~4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ~100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ~1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

The Scottish Structural Proteomics Facility: targets, methods and outputs

PubMed Central

Oke, Muse; Carter, Lester G.; Johnson, Kenneth A.; Liu, Huanting; McMahon, Stephen A.; Yan, Xuan; Kerou, Melina; Weikart, Nadine D.; Kadi, Nadia; Sheikh, Md. Arif; Schmelz, Stefan; Dorward, Mark; Zawadzki, Michal; Cozens, Christopher; Falconer, Helen; Powers, Helen; Overton, Ian M.; van Niekerk, C. A. Johannes; Peng, Xu; Patel, Prakash; Garrett, Roger A.; Prangishvili, David; Botting, Catherine H.; Coote, Peter J.; Dryden, David T. F.; Barton, Geoffrey J.; Schwarz-Linek, Ulrich; Challis, Gregory L.; Taylor, Garry L.; White, Malcolm F.

2010-01-01

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology. Electronic supplementary material The online version of this article (doi:10.1007/s10969-010-9090-y) contains supplementary material, which is available to authorized users. PMID:20419351

ToxCast Dashboard

EPA Pesticide Factsheets

The ToxCast Dashboard helps users examine high-throughput assay data to inform chemical safety decisions. To date, it has data on over 9,000 chemicals and information from more than 1,000 high-throughput assay endpoint components.

RapidTox Dashboard

EPA Pesticide Factsheets

The ToxCast Dashboard helps users examine high-throughput assay data to inform chemical safety decisions. To date, it has data on over 9,000 chemicals and information from more than 1,000 high-throughput assay endpoint components.

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

PubMed Central

Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Duan, Lingfeng; Chen, Guoxing; Jiang, Ni; Fang, Wei; Feng, Hui; Xie, Weibo; Lian, Xingming; Wang, Gongwei; Luo, Qingming; Zhang, Qifa; Liu, Qian; Xiong, Lizhong

2014-01-01

Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits. PMID:25295980

A high-quality annotated transcriptome of swine peripheral blood

USDA-ARS?s Scientific Manuscript database

Background: High throughput gene expression profiling assays of peripheral blood are widely used in biomedicine, as well as in animal genetics and physiology research. Accurate, comprehensive, and precise interpretation of such high throughput assays relies on well-characterized reference genomes an...

The Grapevine and Wine Microbiome: Insights from High-Throughput Amplicon Sequencing

PubMed Central

Morgan, Horatio H.; du Toit, Maret; Setati, Mathabatha E.

2017-01-01

From the time when microbial activity in wine fermentation was first demonstrated, the microbial ecology of the vineyard, grape, and wine has been extensively investigated using culture-based methods. However, the last 2 decades have been characterized by an important change in the approaches used for microbial examination, due to the introduction of DNA-based community fingerprinting methods such as DGGE, SSCP, T-RFLP, and ARISA. These approaches allowed for the exploration of microbial community structures without the need to cultivate, and have been extensively applied to decipher the microbial populations associated with the grapevine as well as the microbial dynamics throughout grape berry ripening and wine fermentation. These techniques are well-established for the rapid more sensitive profiling of microbial communities; however, they often do not provide direct taxonomic information and possess limited ability to detect the presence of rare taxa and taxa with low abundance. Consequently, the past 5 years have seen an upsurge in the application of high-throughput sequencing methods for the in-depth assessment of the grapevine and wine microbiome. Although a relatively new approach in wine sciences, these methods reveal a considerably greater diversity than previously reported, and identified several species that had not yet been reported. The aim of the current review is to highlight the contribution of high-throughput next generation sequencing and metagenomics approaches to vineyard microbial ecology especially unraveling the influence of vineyard management practices on microbial diversity. PMID:28553266

Epigenetic regulation of gene expression in cancer: techniques, resources and analysis

PubMed Central

Kagohara, Luciane T; Stein-O’Brien, Genevieve L; Kelley, Dylan; Flam, Emily; Wick, Heather C; Danilova, Ludmila V; Easwaran, Hariharan; Favorov, Alexander V; Qian, Jiang; Gaykalova, Daria A; Fertig, Elana J

2018-01-01

Abstract Cancer is a complex disease, driven by aberrant activity in numerous signaling pathways in even individual malignant cells. Epigenetic changes are critical mediators of these functional changes that drive and maintain the malignant phenotype. Changes in DNA methylation, histone acetylation and methylation, noncoding RNAs, posttranslational modifications are all epigenetic drivers in cancer, independent of changes in the DNA sequence. These epigenetic alterations were once thought to be crucial only for the malignant phenotype maintenance. Now, epigenetic alterations are also recognized as critical for disrupting essential pathways that protect the cells from uncontrolled growth, longer survival and establishment in distant sites from the original tissue. In this review, we focus on DNA methylation and chromatin structure in cancer. The precise functional role of these alterations is an area of active research using emerging high-throughput approaches and bioinformatics analysis tools. Therefore, this review also describes these high-throughput measurement technologies, public domain databases for high-throughput epigenetic data in tumors and model systems and bioinformatics algorithms for their analysis. Advances in bioinformatics data that combine these epigenetic data with genomics data are essential to infer the function of specific epigenetic alterations in cancer. These integrative algorithms are also a focus of this review. Future studies using these emerging technologies will elucidate how alterations in the cancer epigenome cooperate with genetic aberrations during tumor initiation and progression. This deeper understanding is essential to future studies with epigenetics biomarkers and precision medicine using emerging epigenetic therapies. PMID:28968850

Evaluating High-Throughput Ab Initio Gene Finders to Discover Proteins Encoded in Eukaryotic Pathogen Genomes Missed by Laboratory Techniques

PubMed Central

Goodswen, Stephen J.; Kennedy, Paul J.; Ellis, John T.

2012-01-01

Next generation sequencing technology is advancing genome sequencing at an unprecedented level. By unravelling the code within a pathogen’s genome, every possible protein (prior to post-translational modifications) can theoretically be discovered, irrespective of life cycle stages and environmental stimuli. Now more than ever there is a great need for high-throughput ab initio gene finding. Ab initio gene finders use statistical models to predict genes and their exon-intron structures from the genome sequence alone. This paper evaluates whether existing ab initio gene finders can effectively predict genes to deduce proteins that have presently missed capture by laboratory techniques. An aim here is to identify possible patterns of prediction inaccuracies for gene finders as a whole irrespective of the target pathogen. All currently available ab initio gene finders are considered in the evaluation but only four fulfil high-throughput capability: AUGUSTUS, GeneMark_hmm, GlimmerHMM, and SNAP. These gene finders require training data specific to a target pathogen and consequently the evaluation results are inextricably linked to the availability and quality of the data. The pathogen, Toxoplasma gondii, is used to illustrate the evaluation methods. The results support current opinion that predicted exons by ab initio gene finders are inaccurate in the absence of experimental evidence. However, the results reveal some patterns of inaccuracy that are common to all gene finders and these inaccuracies may provide a focus area for future gene finder developers. PMID:23226328

Representing high throughput expression profiles via perturbation barcodes reveals compound targets.

PubMed

Filzen, Tracey M; Kutchukian, Peter S; Hermes, Jeffrey D; Li, Jing; Tudor, Matthew

2017-02-01

High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound's high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data.

Representing high throughput expression profiles via perturbation barcodes reveals compound targets

PubMed Central

Kutchukian, Peter S.; Li, Jing; Tudor, Matthew

2017-01-01

High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound’s high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data. PMID:28182661

RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

PubMed

Fu, Jiaqi; Fernandez, Daniel; Ferrer, Marc; Titus, Steven A; Buehler, Eugen; Lal-Nag, Madhu A

2017-06-01

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.

The Grapevine and Wine Microbiome: Insights from High-Throughput Amplicon Sequencing.

PubMed

Morgan, Horatio H; du Toit, Maret; Setati, Mathabatha E

2017-01-01

From the time when microbial activity in wine fermentation was first demonstrated, the microbial ecology of the vineyard, grape, and wine has been extensively investigated using culture-based methods. However, the last 2 decades have been characterized by an important change in the approaches used for microbial examination, due to the introduction of DNA-based community fingerprinting methods such as DGGE, SSCP, T-RFLP, and ARISA. These approaches allowed for the exploration of microbial community structures without the need to cultivate, and have been extensively applied to decipher the microbial populations associated with the grapevine as well as the microbial dynamics throughout grape berry ripening and wine fermentation. These techniques are well-established for the rapid more sensitive profiling of microbial communities; however, they often do not provide direct taxonomic information and possess limited ability to detect the presence of rare taxa and taxa with low abundance. Consequently, the past 5 years have seen an upsurge in the application of high-throughput sequencing methods for the in-depth assessment of the grapevine and wine microbiome. Although a relatively new approach in wine sciences, these methods reveal a considerably greater diversity than previously reported, and identified several species that had not yet been reported. The aim of the current review is to highlight the contribution of high-throughput next generation sequencing and metagenomics approaches to vineyard microbial ecology especially unraveling the influence of vineyard management practices on microbial diversity.

Accurate high-throughput structure mapping and prediction with transition metal ion FRET

PubMed Central

Yu, Xiaozhen; Wu, Xiongwu; Bermejo, Guillermo A.; Brooks, Bernard R.; Taraska, Justin W.

2013-01-01

Mapping the landscape of a protein’s conformational space is essential to understanding its functions and regulation. The limitations of many structural methods have made this process challenging for most proteins. Here, we report that transition metal ion FRET (tmFRET) can be used in a rapid, highly parallel screen, to determine distances from multiple locations within a protein at extremely low concentrations. The distances generated through this screen for the protein Maltose Binding Protein (MBP) match distances from the crystal structure to within a few angstroms. Furthermore, energy transfer accurately detects structural changes during ligand binding. Finally, fluorescence-derived distances can be used to guide molecular simulations to find low energy states. Our results open the door to rapid, accurate mapping and prediction of protein structures at low concentrations, in large complex systems, and in living cells. PMID:23273426

Kaleidoscopic imaging patterns of complex structures fabricated by laser-induced deformation

PubMed Central

Zhang, Haoran; Yang, Fengyou; Dong, Jianjie; Du, Lena; Wang, Chuang; Zhang, Jianming; Guo, Chuan Fei; Liu, Qian

2016-01-01

Complex surface structures have stimulated a great deal of interests due to many potential applications in surface devices. However, in the fabrication of complex surface micro-/nanostructures, there are always great challenges in precise design, or good controllability, or low cost, or high throughput. Here, we present a route for the accurate design and highly controllable fabrication of surface quasi-three-dimensional (quasi-3D) structures based on a thermal deformation of simple two-dimensional laser-induced patterns. A complex quasi-3D structure, coaxially nested convex–concave microlens array, as an example, demonstrates our capability of design and fabrication of surface elements with this method. Moreover, by using only one relief mask with the convex–concave microlens structure, we have gotten hundreds of target patterns at different imaging planes, offering a cost-effective solution for mass production in lithography and imprinting, and portending a paradigm in quasi-3D manufacturing. PMID:27910852

HTSeq--a Python framework to work with high-throughput sequencing data.

PubMed

Anders, Simon; Pyl, Paul Theodor; Huber, Wolfgang

2015-01-15

A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed. We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes. HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq. © The Author 2014. Published by Oxford University Press.

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

PubMed

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-05-21

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Ultrahigh-throughput exfoliation of graphite into pristine 'single-layer' graphene using microwaves and molecularly engineered ionic liquids.

PubMed

Matsumoto, Michio; Saito, Yusuke; Park, Chiyoung; Fukushima, Takanori; Aida, Takuzo

2015-09-01

Graphene has shown much promise as an organic electronic material but, despite recent achievements in the production of few-layer graphene, the quantitative exfoliation of graphite into pristine single-layer graphene has remained one of the main challenges in developing practical devices. Recently, reduced graphene oxide has been recognized as a non-feasible alternative to graphene owing to variable defect types and levels, and attention is turning towards reliable methods for the high-throughput exfoliation of graphite. Here we report that microwave irradiation of graphite suspended in molecularly engineered oligomeric ionic liquids allows for ultrahigh-efficiency exfoliation (93% yield) with a high selectivity (95%) towards 'single-layer' graphene (that is, with thicknesses <1 nm) in a short processing time (30 minutes). The isolated graphene sheets show negligible structural deterioration. They are also readily redispersible in oligomeric ionic liquids up to ~100 mg ml(-1), and form physical gels in which an anisotropic orientation of graphene sheets, once induced by a magnetic field, is maintained.

Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques.

PubMed

Khan, Ferdous; Tare, Rahul S; Kanczler, Janos M; Oreffo, Richard O C; Bradley, Mark

2010-03-01

A combination of high-throughput material formulation and microarray techniques were synergistically applied for the efficient analysis of the biological functionality of 135 binary polymer blends. This allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth in both in vitro and in vivo applications. The blended polymeric materials were developed from commercially available, inexpensive and well characterised biodegradable polymers, which on their own lacked both the structural requirements of a scaffold material and, critically, the ability to facilitate cell growth. Blends identified here proved excellent templates for cell attachment, and in addition, a number of blends displayed remarkable bone-like architecture and facilitated bone regeneration by providing 3D biomimetic scaffolds for skeletal cell growth and osteogenic differentiation. This study demonstrates a unique strategy to generate and identify innovative materials with widespread application in cell biology as well as offering a new reparative platform strategy applicable to skeletal tissues. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Application of multivariate statistical techniques in microbial ecology

PubMed Central

Paliy, O.; Shankar, V.

2016-01-01

Recent advances in high-throughput methods of molecular analyses have led to an explosion of studies generating large scale ecological datasets. Especially noticeable effect has been attained in the field of microbial ecology, where new experimental approaches provided in-depth assessments of the composition, functions, and dynamic changes of complex microbial communities. Because even a single high-throughput experiment produces large amounts of data, powerful statistical techniques of multivariate analysis are well suited to analyze and interpret these datasets. Many different multivariate techniques are available, and often it is not clear which method should be applied to a particular dataset. In this review we describe and compare the most widely used multivariate statistical techniques including exploratory, interpretive, and discriminatory procedures. We consider several important limitations and assumptions of these methods, and we present examples of how these approaches have been utilized in recent studies to provide insight into the ecology of the microbial world. Finally, we offer suggestions for the selection of appropriate methods based on the research question and dataset structure. PMID:26786791

The Gene Expression Omnibus Database.

PubMed

Clough, Emily; Barrett, Tanya

2016-01-01

The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome-protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/.

The Gene Expression Omnibus database

PubMed Central

Clough, Emily; Barrett, Tanya

2016-01-01

The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

Advances in the Study of Heart Development and Disease Using Zebrafish

PubMed Central

Brown, Daniel R.; Samsa, Leigh Ann; Qian, Li; Liu, Jiandong

2016-01-01

Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications. PMID:27335817

The high-throughput synthesis and phase characterisation of amphiphiles: a sweet case study.

PubMed

Feast, George C; Hutt, Oliver E; Mulet, Xavier; Conn, Charlotte E; Drummond, Calum J; Savage, G Paul

2014-03-03

A new method for the discovery of amphiphiles by using high-throughput (HT) methods to synthesise and characterise a library of galactose- and glucose-containing amphiphilic compounds is presented. The copper-catalysed azide–alkyne cycloaddition (CuAAC) “click” reaction between azide-tethered simple sugars and alkyne-substituted hydrophobic tails was employed to synthesise a library of compounds with systematic variations in chain length and unsaturation in a 24-vial array format. The liquid–crystalline phase behaviour was characterised in a HT manner by using synchrotron small-angle X-ray scattering (SSAXS). The observed structural variation with respect to chain parameters, including chain length and degree of unsaturation, is discussed, as well as hydration effects and degree of hydrogen bonding between head groups. The validity of our HT screening approach was verified by resynthesising a short-chain glucose amphiphile. A separate phase analysis of this compound confirmed the presence of numerous lyotropic liquid–crystalline phases.

Computer applications making rapid advances in high throughput microbial proteomics (HTMP).

PubMed

Anandkumar, Balakrishna; Haga, Steve W; Wu, Hui-Fen

2014-02-01

The last few decades have seen the rise of widely-available proteomics tools. From new data acquisition devices, such as MALDI-MS and 2DE to new database searching softwares, these new products have paved the way for high throughput microbial proteomics (HTMP). These tools are enabling researchers to gain new insights into microbial metabolism, and are opening up new areas of study, such as protein-protein interactions (interactomics) discovery. Computer software is a key part of these emerging fields. This current review considers: 1) software tools for identifying the proteome, such as MASCOT or PDQuest, 2) online databases of proteomes, such as SWISS-PROT, Proteome Web, or the Proteomics Facility of the Pathogen Functional Genomics Resource Center, and 3) software tools for applying proteomic data, such as PSI-BLAST or VESPA. These tools allow for research in network biology, protein identification, functional annotation, target identification/validation, protein expression, protein structural analysis, metabolic pathway engineering and drug discovery.

Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

Thermoelectric Performance and Defect Chemistry in n-Type Zintl KGaSb 4

DOE PAGES

Ortiz, Brenden R.; Gorai, Prashun; Stevanovic, Vladan; ...

2017-05-08

The rise of high-throughput calculations has accelerated the discovery of promising classes of thermoelectric materials. In prior work, we identified the n-type Zintl pnictides as one such material class. To date, however, a lack of detailed defect calculations and chemical intuition has led the community to investigate p-type Zintls almost exclusively. Here, we investigate the synthesis, thermoelectric properties, and defect structure of the complex Zintl KGaSb 4. We find that KGaSb 4 is successfully doped n-type with Ba and has the potential for p-type doping with Zn. Our calculations reveal the fundamental defect structure in KGaSb 4 that enables n-typemore » and p-type doping. We find that Ba doped KGaSb4 exhibits high electronic mobility (~50 cm 2V -1s -1) and near minimum lattice thermal conductivity (<0.5 Wm -1K -1) at 400 °C. Samples doped with 1.5% Ba achieve zT > 0.9 at 400 °C, promising for a previously unstudied material. Here, we also briefly investigate the series of alloys between KGaSb 4 and KAlSb 4, finding that a full solid solution exists. Altogether our work reinforces motivation for the exploration of n-type Zintl materials, especially in tandem with high-throughput defect calculations to inform selection of effective dopants and systems amenable to n-type transport.« less

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

PubMed Central

Soundararajan, Venky; Aravamudan, Murali

2014-01-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action – inspired by the Google page rank algorithm that unearths most “globally connected” websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs – up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site – encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows. PMID:25465236

A generalized method for high throughput in-situ experiment data analysis: An example of battery materials exploration

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

Aoun, Bachir; Yu, Cun; Fan, Longlong

A generalized method is introduced to extract critical information from series of ranked correlated data. The method is generally applicable to all types of spectra evolving as a function of any arbitrary parameter. This approach is based on correlation functions and statistical scedasticity formalism. Numerous challenges in analyzing high throughput experimental data can be tackled using the herein proposed method. We applied this method to understand the reactivity pathway and formation mechanism of a Li-ion battery cathode material during high temperature synthesis using in-situ highenergy X-ray diffraction. We demonstrate that Pearson's correlation function can easily unravel all major phase transitionmore » and, more importantly, the minor structural changes which cannot be revealed by conventionally inspecting the series of diffraction patterns. Furthermore, a two-dimensional (2D) reactivity pattern calculated as the scedasticity along all measured reciprocal space of all successive diffraction pattern pairs unveils clearly the structural evolution path and the active areas of interest during the synthesis. The methods described here can be readily used for on-the-fly data analysis during various in-situ operando experiments in order to quickly evaluate and optimize experimental conditions, as well as for post data analysis and large data mining where considerable amount of data hinders the feasibility of the investigation through point-by-point inspection.« less

Structure-activity relationships for a series of compounds that inhibit aggregation of the Alzheimer's peptide, Aβ42.

PubMed

McKoy, Angela F; Chen, Jermont; Schupbach, Trudi; Hecht, Michael H

2014-11-01

Inhibiting aggregation of the amyloid-beta (Aβ) peptide may be an effective strategy for combating Alzheimer's disease. As the high-resolution structure of the toxic Aβ aggregate is unknown, rational design of small molecule inhibitors is not possible, and inhibitors are best isolated by high-throughput screening. We applied high-throughput screening to a collection of 65,000 compounds to identify compound D737 as an inhibitor of Aβ aggregation. D737 diminished the formation of oligomers and fibrils, and reduced Aβ42-induced cytotoxicity. Most importantly, D737 increased the life span and locomotive ability of transgenic flies in a Drosophila melanogaster model of Alzheimer's disease (J Biol Chem, 287, 2012, 38992). To explore the chemical features that make D737 an effective inhibitor of Aβ42 aggregation and toxicity, we tested a small collection of eleven analogues of D737. Overall, the ability of a compound to inhibit Aβ aggregation was a good predictor of its efficacy in prolonging the life span and locomotive ability of transgenic flies expressing human Aβ42 in the central nervous system. Two compounds (D744 and D830) with fluorine substitutions on an aromatic ring were effective inhibitors of Aβ42 aggregation and increased the longevity of transgenic flies beyond that observed for the parent compound, D737. © 2014 John Wiley & Sons A/S.

Automated, high-throughput platform for protein solubility screening using a split-GFP system

PubMed Central

Listwan, Pawel; Terwilliger, Thomas C.

2010-01-01

Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are “difficult to handle” as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216–228) can be detected in vivo and in vitro using the engineered GFP 1–10 “detector” fragment (amino acids 1–215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries. PMID:19039681

High-throughput estimation of incident light, light interception and radiation-use efficiency of thousands of plants in a phenotyping platform.

PubMed

Cabrera-Bosquet, Llorenç; Fournier, Christian; Brichet, Nicolas; Welcker, Claude; Suard, Benoît; Tardieu, François

2016-10-01

Light interception and radiation-use efficiency (RUE) are essential components of plant performance. Their genetic dissections require novel high-throughput phenotyping methods. We have developed a suite of methods to evaluate the spatial distribution of incident light, as experienced by hundreds of plants in a glasshouse, by simulating sunbeam trajectories through glasshouse structures every day of the year; the amount of light intercepted by maize (Zea mays) plants via a functional-structural model using three-dimensional (3D) reconstructions of each plant placed in a virtual scene reproducing the canopy in the glasshouse; and RUE, as the ratio of plant biomass to intercepted light. The spatial variation of direct and diffuse incident light in the glasshouse (up to 24%) was correctly predicted at the single-plant scale. Light interception largely varied between maize lines that differed in leaf angles (nearly stable between experiments) and area (highly variable between experiments). Estimated RUEs varied between maize lines, but were similar in two experiments with contrasting incident light. They closely correlated with measured gas exchanges. The methods proposed here identified reproducible traits that might be used in further field studies, thereby opening up the way for large-scale genetic analyses of the components of plant performance. © 2016 INRA New Phytologist © 2016 New Phytologist Trust.

Thermoelectric Performance and Defect Chemistry in n-Type Zintl KGaSb 4

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

Ortiz, Brenden R.; Gorai, Prashun; Stevanovic, Vladan

The rise of high-throughput calculations has accelerated the discovery of promising classes of thermoelectric materials. In prior work, we identified the n-type Zintl pnictides as one such material class. To date, however, a lack of detailed defect calculations and chemical intuition has led the community to investigate p-type Zintls almost exclusively. Here, we investigate the synthesis, thermoelectric properties, and defect structure of the complex Zintl KGaSb 4. We find that KGaSb 4 is successfully doped n-type with Ba and has the potential for p-type doping with Zn. Our calculations reveal the fundamental defect structure in KGaSb 4 that enables n-typemore » and p-type doping. We find that Ba doped KGaSb4 exhibits high electronic mobility (~50 cm 2V -1s -1) and near minimum lattice thermal conductivity (<0.5 Wm -1K -1) at 400 °C. Samples doped with 1.5% Ba achieve zT > 0.9 at 400 °C, promising for a previously unstudied material. Here, we also briefly investigate the series of alloys between KGaSb 4 and KAlSb 4, finding that a full solid solution exists. Altogether our work reinforces motivation for the exploration of n-type Zintl materials, especially in tandem with high-throughput defect calculations to inform selection of effective dopants and systems amenable to n-type transport.« less

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

PubMed Central

Gil, Geun-Cheol; Iliff, Bryce; Cerny, Ron; Velander, William H.; Van Cott, Kevin E.

2010-01-01

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method. PMID:20586471

A nanoforest structure for practical surface-enhanced Raman scattering substrates

NASA Astrophysics Data System (ADS)

Seol, Myeong-Lok; Choi, Sung-Jin; Baek, David J.; Park, Tae Jung; Ahn, Jae-Hyuk; Lee, Sang Yup; Choi, Yang-Kyu

2012-03-01

A nanoforest structure for surface-enhanced Raman scattering (SERS) active substrates is fabricated and analyzed. The detailed morphology of the resulting structure can be easily controlled by modifying the process parameters such as initial gold layer thickness and etching time. The applicability of the nanoforest substrate as a label-free SERS immunosensor is demonstrated using influenza A virus subtype H1N1. Selective binding of the H1N1 surface antigen and the anti-H1 antibody is directly detected by the SERS signal differences. Simple fabrication and high throughput with strong in-plane hot-spots imply that the nanoforest structure can be a practical sensing component of a chip-based SERS sensing system.

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, ...

Rapid and high throughput fabrication of high temperature stable structures through PDMS transfer printing

NASA Astrophysics Data System (ADS)

Hohenberger, Erik; Freitag, Nathan; Korampally, Venumadhav

2017-07-01

We report on a facile and low cost fabrication approach for structures—gratings and enclosed nanochannels, through simple solution processed chemistries in conjunction with nanotransfer printing techniques. The ink formulation primarily consisting of an organosilicate polymeric network with a small percentage of added 3-aminopropyl triethoxysilane crosslinker allows one to obtain robust structures that are not only stable towards high temperature processing steps as high as 550 °C but also exhibit exceptional stability against a host of organic solvent washes. No discernable structure distortion was observed compared to the as-printed structures (room temperature processed) when printed structures were subjected to temperatures as high as 550 °C. We further demonstrate the applicability of this technique towards the fabrication of more complex nanostructures such as enclosed channels through a double transfer method, leveraging the exceptional room temperature cross-linking ability of the printed structures and their subsequent resistance to dissolution in organic solvent washes. The exceptional temperature and physico-chemical stability of the nanotransfer printed structures makes this a useful fabrication tool that may be applied as is, or integrated with conventional lithographic techniques for the large area fabrication of functional nanostructures and devices.

High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China.

PubMed

Wei, Yu-Jie; Wu, Yun; Yan, Yin-Zhuo; Zou, Wan; Xue, Jie; Ma, Wen-Rui; Wang, Wei; Tian, Ge; Wang, Li-Ye

2018-01-01

In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine.

Strain Prioritization for Natural Product Discovery by a High-Throughput Real-Time PCR Method

PubMed Central

2015-01-01

Natural products offer unmatched chemical and structural diversity compared to other small-molecule libraries, but traditional natural product discovery programs are not sustainable, demanding too much time, effort, and resources. Here we report a strain prioritization method for natural product discovery. Central to the method is the application of real-time PCR, targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds in a high-throughput format. The practicality and effectiveness of the method were showcased by prioritizing 1911 actinomycete strains for diterpenoid discovery. A total of 488 potential diterpenoid producers were identified, among which six were confirmed as platensimycin and platencin dual producers and one as a viguiepinol and oxaloterpin producer. While the method as described is most appropriate to prioritize strains for discovering specific natural products, variations of this method should be applicable to the discovery of other classes of natural products. Applications of genome sequencing and genome mining to the high-priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered. PMID:25238028

High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China

PubMed Central

Yan, Yin-zhuo; Zou, Wan; Ma, Wen-rui; Wang, Wei; Tian, Ge; Wang, Li-ye

2018-01-01

In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine. PMID:29565999

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

Siol, Sebastian; Dhakal, Tara P.; Gudavalli, Ganesh S.

High-throughput computational and experimental techniques have been used in the past to accelerate the discovery of new promising solar cell materials. An important part of the development of novel thin film solar cell technologies, that is still considered a bottleneck for both theory and experiment, is the search for alternative interfacial contact (buffer) layers. The research and development of contact materials is difficult due to the inherent complexity that arises from its interactions at the interface with the absorber. A promising alternative to the commonly used CdS buffer layer in thin film solar cells that contain absorbers with lower electronmore » affinity can be found in ..beta..-In2S3. However, the synthesis conditions for the sputter deposition of this material are not well-established. Here, In2S3 is investigated as a solar cell contact material utilizing a high-throughput combinatorial screening of the temperature-flux parameter space, followed by a number of spatially resolved characterization techniques. It is demonstrated that, by tuning the sulfur partial pressure, phase pure ..beta..-In2S3 could be deposited using a broad range of substrate temperatures between 500 degrees C and ambient temperature. Combinatorial photovoltaic device libraries with Al/ZnO/In2S3/Cu2ZnSnS4/Mo/SiO2 structure were built at optimal processing conditions to investigate the feasibility of the sputtered In2S3 buffer layers and of an accelerated optimization of the device structure. The performance of the resulting In2S3/Cu2ZnSnS4 photovoltaic devices is on par with CdS/Cu2ZnSnS4 reference solar cells with similar values for short circuit currents and open circuit voltages, despite the overall quite low efficiency of the devices (-2%). Overall, these results demonstrate how a high-throughput experimental approach can be used to accelerate the development of contact materials and facilitate the optimization of thin film solar cell devices.« less

Normalization and standardization of electronic health records for high-throughput phenotyping: the SHARPn consortium

PubMed Central

Pathak, Jyotishman; Bailey, Kent R; Beebe, Calvin E; Bethard, Steven; Carrell, David S; Chen, Pei J; Dligach, Dmitriy; Endle, Cory M; Hart, Lacey A; Haug, Peter J; Huff, Stanley M; Kaggal, Vinod C; Li, Dingcheng; Liu, Hongfang; Marchant, Kyle; Masanz, James; Miller, Timothy; Oniki, Thomas A; Palmer, Martha; Peterson, Kevin J; Rea, Susan; Savova, Guergana K; Stancl, Craig R; Sohn, Sunghwan; Solbrig, Harold R; Suesse, Dale B; Tao, Cui; Taylor, David P; Westberg, Les; Wu, Stephen; Zhuo, Ning; Chute, Christopher G

2013-01-01

Research objective To develop scalable informatics infrastructure for normalization of both structured and unstructured electronic health record (EHR) data into a unified, concept-based model for high-throughput phenotype extraction. Materials and methods Software tools and applications were developed to extract information from EHRs. Representative and convenience samples of both structured and unstructured data from two EHR systems—Mayo Clinic and Intermountain Healthcare—were used for development and validation. Extracted information was standardized and normalized to meaningful use (MU) conformant terminology and value set standards using Clinical Element Models (CEMs). These resources were used to demonstrate semi-automatic execution of MU clinical-quality measures modeled using the Quality Data Model (QDM) and an open-source rules engine. Results Using CEMs and open-source natural language processing and terminology services engines—namely, Apache clinical Text Analysis and Knowledge Extraction System (cTAKES) and Common Terminology Services (CTS2)—we developed a data-normalization platform that ensures data security, end-to-end connectivity, and reliable data flow within and across institutions. We demonstrated the applicability of this platform by executing a QDM-based MU quality measure that determines the percentage of patients between 18 and 75 years with diabetes whose most recent low-density lipoprotein cholesterol test result during the measurement year was <100 mg/dL on a randomly selected cohort of 273 Mayo Clinic patients. The platform identified 21 and 18 patients for the denominator and numerator of the quality measure, respectively. Validation results indicate that all identified patients meet the QDM-based criteria. Conclusions End-to-end automated systems for extracting clinical information from diverse EHR systems require extensive use of standardized vocabularies and terminologies, as well as robust information models for storing, discovering, and processing that information. This study demonstrates the application of modular and open-source resources for enabling secondary use of EHR data through normalization into standards-based, comparable, and consistent format for high-throughput phenotyping to identify patient cohorts. PMID:24190931

GiNA, an efficient and high-throughput software for horticultural phenotyping

USDA-ARS?s Scientific Manuscript database

Traditional methods for trait phenotyping have been a bottleneck for research in many crop species due to their intensive labor, high cost, complex implementation, lack of reproducibility and propensity to subjective bias. Recently, multiple high-throughput phenotyping platforms have been developed,...

LS-align: an atom-level, flexible ligand structural alignment algorithm for high-throughput virtual screening.

PubMed

Hu, Jun; Liu, Zi; Yu, Dong-Jun; Zhang, Yang

2018-02-15

Sequence-order independent structural comparison, also called structural alignment, of small ligand molecules is often needed for computer-aided virtual drug screening. Although many ligand structure alignment programs are proposed, most of them build the alignments based on rigid-body shape comparison which cannot provide atom-specific alignment information nor allow structural variation; both abilities are critical to efficient high-throughput virtual screening. We propose a novel ligand comparison algorithm, LS-align, to generate fast and accurate atom-level structural alignments of ligand molecules, through an iterative heuristic search of the target function that combines inter-atom distance with mass and chemical bond comparisons. LS-align contains two modules of Rigid-LS-align and Flexi-LS-align, designed for rigid-body and flexible alignments, respectively, where a ligand-size independent, statistics-based scoring function is developed to evaluate the similarity of ligand molecules relative to random ligand pairs. Large-scale benchmark tests are performed on prioritizing chemical ligands of 102 protein targets involving 1,415,871 candidate compounds from the DUD-E (Database of Useful Decoys: Enhanced) database, where LS-align achieves an average enrichment factor (EF) of 22.0 at the 1% cutoff and the AUC score of 0.75, which are significantly higher than other state-of-the-art methods. Detailed data analyses show that the advanced performance is mainly attributed to the design of the target function that combines structural and chemical information to enhance the sensitivity of recognizing subtle difference of ligand molecules and the introduces of structural flexibility that help capture the conformational changes induced by the ligand-receptor binding interactions. These data demonstrate a new avenue to improve the virtual screening efficiency through the development of sensitive ligand structural alignments. http://zhanglab.ccmb.med.umich.edu/LS-align/. njyudj@njust.edu.cn or zhng@umich.edu. Supplementary data are available at Bioinformatics online.

High-throughput quantification of hydroxyproline for determination of collagen.

PubMed

Hofman, Kathleen; Hall, Bronwyn; Cleaver, Helen; Marshall, Susan

2011-10-15

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline. Copyright © 2011 Elsevier Inc. All rights reserved.

High-Throughput Method for Strontium Isotope Analysis by Multi-Collector-Inductively Coupled Plasma-Mass Spectrometer

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

Wall, Andrew J.; Capo, Rosemary C.; Stewart, Brian W.

2016-09-22

This technical report presents the details of the Sr column configuration and the high-throughput Sr separation protocol. Data showing the performance of the method as well as the best practices for optimizing Sr isotope analysis by MC-ICP-MS is presented. Lastly, this report offers tools for data handling and data reduction of Sr isotope results from the Thermo Scientific Neptune software to assist in data quality assurance, which help avoid issues of data glut associated with high sample throughput rapid analysis.

High-Throughput Method for Strontium Isotope Analysis by Multi-Collector-Inductively Coupled Plasma-Mass Spectrometer

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

Hakala, Jacqueline Alexandra

2016-11-22

This technical report presents the details of the Sr column configuration and the high-throughput Sr separation protocol. Data showing the performance of the method as well as the best practices for optimizing Sr isotope analysis by MC-ICP-MS is presented. Lastly, this report offers tools for data handling and data reduction of Sr isotope results from the Thermo Scientific Neptune software to assist in data quality assurance, which help avoid issues of data glut associated with high sample throughput rapid analysis.

A Memory Efficient Network Encryption Scheme

NASA Astrophysics Data System (ADS)

El-Fotouh, Mohamed Abo; Diepold, Klaus

In this paper, we studied the two widely used encryption schemes in network applications. Shortcomings have been found in both schemes, as these schemes consume either more memory to gain high throughput or low memory with low throughput. The need has aroused for a scheme that has low memory requirements and in the same time possesses high speed, as the number of the internet users increases each day. We used the SSM model [1], to construct an encryption scheme based on the AES. The proposed scheme possesses high throughput together with low memory requirements.