Evolutionary Origins of Cancer Driver Genes and Implications for Cancer Prognosis.

PubMed

Chu, Xin-Yi; Jiang, Ling-Han; Zhou, Xiong-Hui; Cui, Ze-Jia; Zhang, Hong-Yu

2017-07-14

The cancer atavistic theory suggests that carcinogenesis is a reverse evolution process. It is thus of great interest to explore the evolutionary origins of cancer driver genes and the relevant mechanisms underlying the carcinogenesis. Moreover, the evolutionary features of cancer driver genes could be helpful in selecting cancer biomarkers from high-throughput data. In this study, through analyzing the cancer endogenous molecular networks, we revealed that the subnetwork originating from eukaryota could control the unlimited proliferation of cancer cells, and the subnetwork originating from eumetazoa could recapitulate the other hallmarks of cancer. In addition, investigations based on multiple datasets revealed that cancer driver genes were enriched in genes originating from eukaryota, opisthokonta, and eumetazoa. These results have important implications for enhancing the robustness of cancer prognosis models through selecting the gene signatures by the gene age information.

Constructing phylogenetic trees using interacting pathways.

PubMed

Wan, Peng; Che, Dongsheng

2013-01-01

Phylogenetic trees are used to represent evolutionary relationships among biological species or organisms. The construction of phylogenetic trees is based on the similarities or differences of their physical or genetic features. Traditional approaches of constructing phylogenetic trees mainly focus on physical features. The recent advancement of high-throughput technologies has led to accumulation of huge amounts of biological data, which in turn changed the way of biological studies in various aspects. In this paper, we report our approach of building phylogenetic trees using the information of interacting pathways. We have applied hierarchical clustering on two domains of organisms-eukaryotes and prokaryotes. Our preliminary results have shown the effectiveness of using the interacting pathways in revealing evolutionary relationships.

A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing.

PubMed

Oono, Ryoko

2017-01-01

High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions 'how and why are communities different?' This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences.

A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing

PubMed Central

2017-01-01

High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions ‘how and why are communities different?’ This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences. PMID:29253889

Universality and predictability in molecular quantitative genetics.

PubMed

Nourmohammad, Armita; Held, Torsten; Lässig, Michael

2013-12-01

Molecular traits, such as gene expression levels or protein binding affinities, are increasingly accessible to quantitative measurement by modern high-throughput techniques. Such traits measure molecular functions and, from an evolutionary point of view, are important as targets of natural selection. We review recent developments in evolutionary theory and experiments that are expected to become building blocks of a quantitative genetics of molecular traits. We focus on universal evolutionary characteristics: these are largely independent of a trait's genetic basis, which is often at least partially unknown. We show that universal measurements can be used to infer selection on a quantitative trait, which determines its evolutionary mode of conservation or adaptation. Furthermore, universality is closely linked to predictability of trait evolution across lineages. We argue that universal trait statistics extends over a range of cellular scales and opens new avenues of quantitative evolutionary systems biology. Copyright © 2013. Published by Elsevier Ltd.

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.

High-throughput sequencing data clarify evolutionary relationships among North American Vitis species and improve identification in USDA Vitis germplasm collections

USDA-ARS?s Scientific Manuscript database

Grapes are one of the most economically important berry crops worldwide, with the vast majority of production derived from the domesticated Eurasian species Vitis vinifera. Expansion of production into new areas, development of new cultivars, and concerns about adapting grapevines for changing clima...

SICLE: a high-throughput tool for extracting evolutionary relationships from phylogenetic trees.

PubMed

DeBlasio, Dan F; Wisecaver, Jennifer H

2016-01-01

We present the phylogeny analysis software SICLE (Sister Clade Extractor), an easy-to-use, high-throughput tool to describe the nearest neighbors to a node of interest in a phylogenetic tree as well as the support value for the relationship. The application is a command line utility that can be embedded into a phylogenetic analysis pipeline or can be used as a subroutine within another C++ program. As a test case, we applied this new tool to the published phylome of Salinibacter ruber, a species of halophilic Bacteriodetes, identifying 13 unique sister relationships to S. ruber across the 4,589 gene phylogenies. S. ruber grouped with bacteria, most often other Bacteriodetes, in the majority of phylogenies, but 91 phylogenies showed a branch-supported sister association between S. ruber and Archaea, an evolutionarily intriguing relationship indicative of horizontal gene transfer. This test case demonstrates how SICLE makes it possible to summarize the phylogenetic information produced by automated phylogenetic pipelines to rapidly identify and quantify the possible evolutionary relationships that merit further investigation. SICLE is available for free for noncommercial use at http://eebweb.arizona.edu/sicle/.

MIPS plant genome information resources.

PubMed

Spannagl, Manuel; Haberer, Georg; Ernst, Rebecca; Schoof, Heiko; Mayer, Klaus F X

2007-01-01

The Munich Institute for Protein Sequences (MIPS) has been involved in maintaining plant genome databases since the Arabidopsis thaliana genome project. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable data sets for model plant genomes as a backbone against which experimental data, for example from high-throughput functional genomics, can be organized and evaluated. In addition, model genomes also form a scaffold for comparative genomics, and much can be learned from genome-wide evolutionary studies.

NASA's Evolutionary Xenon Thruster (NEXT) Project Qualification Propellant Throughput Milestone: Performance, Erosion, and Thruster Service Life Prediction After 450 kg

NASA Technical Reports Server (NTRS)

Herman, Daniel A.

2010-01-01

The NASA s Evolutionary Xenon Thruster (NEXT) program is tasked with significantly improving and extending the capabilities of current state-of-the-art NSTAR thruster. The service life capability of the NEXT ion thruster is being assessed by thruster wear test and life-modeling of critical thruster components, such as the ion optics and cathodes. The NEXT Long-Duration Test (LDT) was initiated to validate and qualify the NEXT thruster propellant throughput capability. The NEXT thruster completed the primary goal of the LDT; namely to demonstrate the project qualification throughput of 450 kg by the end of calendar year 2009. The NEXT LDT has demonstrated 28,500 hr of operation and processed 466 kg of xenon throughput--more than double the throughput demonstrated by the NSTAR flight-spare. Thruster performance changes have been consistent with a priori predictions. Thruster erosion has been minimal and consistent with the thruster service life assessment, which predicts the first failure mode at greater than 750 kg throughput. The life-limiting failure mode for NEXT is predicted to be loss of structural integrity of the accelerator grid due to erosion by charge-exchange ions.

Diverse papillomaviruses identified in Weddell seals.

PubMed

Smeele, Zoe E; Burns, Jennifer M; Van Doorsaler, Koenraad; Fontenele, Rafaela S; Waits, Kara; Stainton, Daisy; Shero, Michelle R; Beltran, Roxanne S; Kirkham, Amy L; Berngartt, Rachel; Kraberger, Simona; Varsani, Arvind

2018-04-01

Papillomaviridae is a diverse family of circular, double-stranded DNA (dsDNA) viruses that infect a broad range of mammalian, avian and fish hosts. While papillomaviruses have been characterized most extensively in humans, the study of non-human papillomaviruses has contributed greatly to our understanding of their pathogenicity and evolution. Using high-throughput sequencing approaches, we identified 7 novel papillomaviruses from vaginal swabs collected from 81 adult female Weddell seals (Leptonychotes weddellii) in the Ross Sea of Antarctica between 2014-2017. These seven papillomavirus genomes were amplified from seven individual seals, and six of the seven genomes represented novel species with distinct evolutionary lineages. This highlights the diversity of papillomaviruses among the relatively small number of Weddell seal samples tested. Viruses associated with large vertebrates are poorly studied in Antarctica, and this study adds information about papillomaviruses associated with Weddell seals and contributes to our understanding of the evolutionary history of papillomaviruses.

Observing Clonal Dynamics across Spatiotemporal Axes: A Prelude to Quantitative Fitness Models for Cancer.

PubMed

McPherson, Andrew W; Chan, Fong Chun; Shah, Sohrab P

2018-02-01

The ability to accurately model evolutionary dynamics in cancer would allow for prediction of progression and response to therapy. As a prelude to quantitative understanding of evolutionary dynamics, researchers must gather observations of in vivo tumor evolution. High-throughput genome sequencing now provides the means to profile the mutational content of evolving tumor clones from patient biopsies. Together with the development of models of tumor evolution, reconstructing evolutionary histories of individual tumors generates hypotheses about the dynamics of evolution that produced the observed clones. In this review, we provide a brief overview of the concepts involved in predicting evolutionary histories, and provide a workflow based on bulk and targeted-genome sequencing. We then describe the application of this workflow to time series data obtained for transformed and progressed follicular lymphomas (FL), and contrast the observed evolutionary dynamics between these two subtypes. We next describe results from a spatial sampling study of high-grade serous (HGS) ovarian cancer, propose mechanisms of disease spread based on the observed clonal mixtures, and provide examples of diversification through subclonal acquisition of driver mutations and convergent evolution. Finally, we state implications of the techniques discussed in this review as a necessary but insufficient step on the path to predictive modelling of disease dynamics. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Smartphones in ecology and evolution: a guide for the app-rehensive.

PubMed

Teacher, Amber G F; Griffiths, David J; Hodgson, David J; Inger, Richard

2013-12-01

Smartphones and their apps (application software) are now used by millions of people worldwide and represent a powerful combination of sensors, information transfer, and computing power that deserves better exploitation by ecological and evolutionary researchers. We outline the development process for research apps, provide contrasting case studies for two new research apps, and scan the research horizon to suggest how apps can contribute to the rapid collection, interpretation, and dissemination of data in ecology and evolutionary biology. We emphasize that the usefulness of an app relies heavily on the development process, recommend that app developers are engaged with the process at the earliest possible stage, and commend efforts to create open-source software scaffolds on which customized apps can be built by nonexperts. We conclude that smartphones and their apps could replace many traditional handheld sensors, calculators, and data storage devices in ecological and evolutionary research. We identify their potential use in the high-throughput collection, analysis, and storage of complex ecological information.

Status of the NASA's Evolutionary Xenon Thruster (NEXT) Long-Duration Test After 30,352 Hours of Operation

NASA Technical Reports Server (NTRS)

Herman, Daniel A.

2010-01-01

The NASA s Evolutionary Xenon Thruster (NEXT) program is tasked with significantly improving and extending the capabilities of current state-of-the-art NSTAR thruster. The service life capability of the NEXT ion thruster is being assessed by thruster wear test and life-modeling of critical thruster components, such as the ion optics and cathodes. The NEXT Long-Duration Test (LDT) was initiated to validate and qualify the NEXT thruster propellant throughput capability. The NEXT thruster completed the primary goal of the LDT; namely to demonstrate the project qualification throughput of 450 kg by the end of calendar year 2009. The NEXT LDT has demonstrated 30,352 hr of operation and processed 490 kg of xenon throughput--surpassing the NSTAR Extended Life Test hours demonstrated and more than double the throughput demonstrated by the NSTAR flight-spare. Thruster performance changes have been consistent with a priori predictions. Thruster erosion has been minimal and consistent with the thruster service life assessment, which predicts the first failure mode at greater than 750 kg throughput. The life-limiting failure mode for NEXT is predicted to be loss of structural integrity of the accelerator grid due to erosion by charge-exchange ions.

A High-Throughput Pipeline for the Design of Real-Time PCR Signatures

DTIC Science & Technology

2010-06-23

evolutionary relationships among the causative agents of melioidosis and glanders , Burkholderia pseudomallei and Burkholderia mallei . J Clin Microbiol... Burkholderia mallei with respect to Burkholderia pseudomallei. B. mallei and B. pseudomallei are closely related pathogens that cause different diseases... glanders and melioidosis, respectively [16]. B. mallei is believed to have been clonally evolved from B. pseudomallei [17], with a significantly

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli.

PubMed

McCloskey, Douglas; Xu, Julia; Schrübbers, Lars; Christensen, Hanne B; Herrgård, Markus J

2018-04-25

Fast metabolite quantification methods are required for high throughput screening of microbial strains obtained by combinatorial or evolutionary engineering approaches. In this study, a rapid RIP-LC-MS/MS (RapidRIP) method for high-throughput quantitative metabolomics was developed and validated that was capable of quantifying 102 metabolites from central, amino acid, energy, nucleotide, and cofactor metabolism in less than 5 minutes. The method was shown to have comparable sensitivity and resolving capability as compared to a full length RIP-LC-MS/MS method (FullRIP). The RapidRIP method was used to quantify the metabolome of seven industrial strains of E. coli revealing significant differences in glycolytic, pentose phosphate, TCA cycle, amino acid, and energy and cofactor metabolites were found. These differences translated to statistically and biologically significant differences in thermodynamics of biochemical reactions between strains that could have implications when choosing a host for bioprocessing. Copyright © 2018. Published by Elsevier Inc.

Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent Decarboxylase Genes.

PubMed

Kumar, Rahul

2016-01-01

Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC) enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

PubMed Central

James, Karen E.; Schneider, Harald; Ansell, Stephen W.; Evers, Margaret; Robba, Lavinia; Uszynski, Grzegorz; Pedersen, Niklas; Newton, Angela E.; Russell, Stephen J.; Vogel, Johannes C.; Kilian, Andrzej

2008-01-01

Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography. PMID:18301759

Complete Chloroplast Genome of the Multifunctional Crop Globe Artichoke and Comparison with Other Asteraceae

PubMed Central

Curci, Pasquale L.; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G.; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for “specific barcode” purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants. PMID:25774672

Complete chloroplast genome of the multifunctional crop globe artichoke and comparison with other Asteraceae.

PubMed

Curci, Pasquale L; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants.

Relative evolutionary rate inference in HyPhy with LEISR.

PubMed

Spielman, Stephanie J; Kosakovsky Pond, Sergei L

2018-01-01

We introduce LEISR (Likehood Estimation of Individual Site Rates, pronounced "laser"), a tool to infer relative evolutionary rates from protein and nucleotide data, implemented in HyPhy. LEISR is based on the popular Rate4Site (Pupko et al., 2002) approach for inferring relative site-wise evolutionary rates, primarily from protein data. We extend the original method for more general use in several key ways: (i) we increase the support for nucleotide data with additional models, (ii) we allow for datasets of arbitrary size, (iii) we support analysis of site-partitioned datasets to correct for the presence of recombination breakpoints, (iv) we produce rate estimates at all sites rather than at just a subset of sites, and (v) we implemented LEISR as MPI-enabled to support rapid, high-throughput analysis. LEISR is available in HyPhy starting with version 2.3.8, and it is accessible as an option in the HyPhy analysis menu ("Relative evolutionary rate inference"), which calls the HyPhy batchfile LEISR.bf.

Mission Advantages of NEXT: Nasa's Evolutionary Xenon Thruster

NASA Technical Reports Server (NTRS)

Oleson, Steven; Gefert, Leon; Benson, Scott; Patterson, Michael; Noca, Muriel; Sims, Jon

2002-01-01

With the demonstration of the NSTAR propulsion system on the Deep Space One mission, the range of the Discovery class of NASA missions can now be expanded. NSTAR lacks, however, sufficient performance for many of the more challenging Office of Space Science (OSS) missions. Recent studies have shown that NASA's Evolutionary Xenon Thruster (NEXT) ion propulsion system is the best choice for many exciting potential OSS missions including outer planet exploration and inner solar system sample returns. The NEXT system provides the higher power, higher specific impulse, and higher throughput required by these science missions.

A high density physical map of chromosome 1BL supports evolutionary studies, map-based cloning and sequencing in wheat

PubMed Central

2013-01-01

Background As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning. Results Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome. Conclusions Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing. PMID:23800011

Evolutionary Patterns and Processes: Lessons from Ancient DNA.

PubMed

Leonardi, Michela; Librado, Pablo; Der Sarkissian, Clio; Schubert, Mikkel; Alfarhan, Ahmed H; Alquraishi, Saleh A; Al-Rasheid, Khaled A S; Gamba, Cristina; Willerslev, Eske; Orlando, Ludovic

2017-01-01

Ever since its emergence in 1984, the field of ancient DNA has struggled to overcome the challenges related to the decay of DNA molecules in the fossil record. With the recent development of high-throughput DNA sequencing technologies and molecular techniques tailored to ultra-damaged templates, it has now come of age, merging together approaches in phylogenomics, population genomics, epigenomics, and metagenomics. Leveraging on complete temporal sample series, ancient DNA provides direct access to the most important dimension in evolution—time, allowing a wealth of fundamental evolutionary processes to be addressed at unprecedented resolution. This review taps into the most recent findings in ancient DNA research to present analyses of ancient genomic and metagenomic data.

Evolutionary Patterns and Processes: Lessons from Ancient DNA

PubMed Central

Leonardi, Michela; Librado, Pablo; Der Sarkissian, Clio; Schubert, Mikkel; Alfarhan, Ahmed H.; Alquraishi, Saleh A.; Al-Rasheid, Khaled A. S.; Gamba, Cristina; Willerslev, Eske

2017-01-01

Abstract Ever since its emergence in 1984, the field of ancient DNA has struggled to overcome the challenges related to the decay of DNA molecules in the fossil record. With the recent development of high-throughput DNA sequencing technologies and molecular techniques tailored to ultra-damaged templates, it has now come of age, merging together approaches in phylogenomics, population genomics, epigenomics, and metagenomics. Leveraging on complete temporal sample series, ancient DNA provides direct access to the most important dimension in evolution—time, allowing a wealth of fundamental evolutionary processes to be addressed at unprecedented resolution. This review taps into the most recent findings in ancient DNA research to present analyses of ancient genomic and metagenomic data. PMID:28173586

Characterizing ncRNAs in Human Pathogenic Protists Using High-Throughput Sequencing Technology

PubMed Central

Collins, Lesley Joan

2011-01-01

ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases. PMID:22303390

Post-Test Inspection of NASA's Evolutionary Xenon Thruster Long-Duration Test Hardware: Discharge and Neutralizer Cathodes

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Soulas, George C.

2016-01-01

The NEXT Long-Duration Test is part of a comprehensive thruster service life assessment intended to demonstrate overall throughput capability, validate service life models, quantify wear rates as a function of time and operating condition, and identify any unknown life-limiting mechanisms. The test was voluntarily terminated in April 2014 after demonstrating 51,184 hours of high-voltage operation, 918 kg of propellant throughput, and 35.5 MN-s of total impulse. The post-test inspection of the thruster hardware began shortly afterwards with a combination of non-destructive and destructive analysis techniques, and is presently nearing completion. This presentation presents relevant results of the post-test inspection for both discharge and neutralizer cathodes.

High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA.

PubMed

Wang, Wenqin; Messing, Joachim

2011-01-01

Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

High-Throughput Sequencing of Three Lemnoideae (Duckweeds) Chloroplast Genomes from Total DNA

PubMed Central

Wang, Wenqin; Messing, Joachim

2011-01-01

Background Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. Methods We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. Conclusions This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power. PMID:21931804

When Reputation Enforces Evolutionary Cooperation in Unreliable MANETs.

PubMed

Tang, Changbing; Li, Ang; Li, Xiang

2015-10-01

In self-organized mobile ad hoc networks (MANETs), network functions rely on cooperation of self-interested nodes, where a challenge is to enforce their mutual cooperation. In this paper, we study cooperative packet forwarding in a one-hop unreliable channel which results from loss of packets and noisy observation of transmissions. We propose an indirect reciprocity framework based on evolutionary game theory, and enforce cooperation of packet forwarding strategies in both structured and unstructured MANETs. Furthermore, we analyze the evolutionary dynamics of cooperative strategies and derive the threshold of benefit-to-cost ratio to guarantee the convergence of cooperation. The numerical simulations verify that the proposed evolutionary game theoretic solution enforces cooperation when the benefit-to-cost ratio of the altruistic exceeds the critical condition. In addition, the network throughput performance of our proposed strategy in structured MANETs is measured, which is in close agreement with that of the full cooperative strategy.

Analyzing endocrine system conservation and evolution.

PubMed

Bonett, Ronald M

2016-08-01

Analyzing variation in rates of evolution can provide important insights into the factors that constrain trait evolution, as well as those that promote diversification. Metazoan endocrine systems exhibit apparent variation in evolutionary rates of their constituent components at multiple levels, yet relatively few studies have quantified these patterns and analyzed them in a phylogenetic context. This may be in part due to historical and current data limitations for many endocrine components and taxonomic groups. However, recent technological advancements such as high-throughput sequencing provide the opportunity to collect large-scale comparative data sets for even non-model species. Such ventures will produce a fertile data landscape for evolutionary analyses of nucleic acid and amino acid based endocrine components. Here I summarize evolutionary rate analyses that can be applied to categorical and continuous endocrine traits, and also those for nucleic acid and protein-based components. I emphasize analyses that could be used to test whether other variables (e.g., ecology, ontogenetic timing of expression, etc.) are related to patterns of rate variation and endocrine component diversification. The application of phylogenetic-based rate analyses to comparative endocrine data will greatly enhance our understanding of the factors that have shaped endocrine system evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolutionary combinatorial chemistry, a novel tool for SAR studies on peptide transport across the blood-brain barrier. Part 2. Design, synthesis and evaluation of a first generation of peptides.

PubMed

Teixidó, Meritxell; Belda, Ignasi; Zurita, Esther; Llorà, Xavier; Fabre, Myriam; Vilaró, Senén; Albericio, Fernando; Giralt, Ernest

2005-12-01

The use of high-throughput methods in drug discovery allows the generation and testing of a large number of compounds, but at the price of providing redundant information. Evolutionary combinatorial chemistry combines the selection and synthesis of biologically active compounds with artificial intelligence optimization methods, such as genetic algorithms (GA). Drug candidates for the treatment of central nervous system (CNS) disorders must overcome the blood-brain barrier (BBB). This paper reports a new genetic algorithm that searches for the optimal physicochemical properties for peptide transport across the blood-brain barrier. A first generation of peptides has been generated and synthesized. Due to the high content of N-methyl amino acids present in most of these peptides, their syntheses were especially challenging due to over-incorporations, deletions and DKP formations. Distinct fragmentation patterns during peptide cleavage have been identified. The first generation of peptides has been studied by evaluation techniques such as immobilized artificial membrane chromatography (IAMC), a cell-based assay, log Poctanol/water calculations, etc. Finally, a second generation has been proposed. (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

PubMed

Wang, Meng; Li, Sijin; Zhao, Huimin

2016-01-01

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

Human Genomic Loci Important in Common Infectious Diseases: Role of High-Throughput Sequencing and Genome-Wide Association Studies

PubMed Central

Sserwadda, Ivan; Amujal, Marion; Namatovu, Norah

2018-01-01

HIV/AIDS, tuberculosis (TB), and malaria are 3 major global public health threats that undermine development in many resource-poor settings. Recently, the notion that positive selection during epidemics or longer periods of exposure to common infectious diseases may have had a major effect in modifying the constitution of the human genome is being interrogated at a large scale in many populations around the world. This positive selection from infectious diseases increases power to detect associations in genome-wide association studies (GWASs). High-throughput sequencing (HTS) has transformed both the management of infectious diseases and continues to enable large-scale functional characterization of host resistance/susceptibility alleles and loci; a paradigm shift from single candidate gene studies. Application of genome sequencing technologies and genomics has enabled us to interrogate the host-pathogen interface for improving human health. Human populations are constantly locked in evolutionary arms races with pathogens; therefore, identification of common infectious disease-associated genomic variants/markers is important in therapeutic, vaccine development, and screening susceptible individuals in a population. This review describes a range of host-pathogen genomic loci that have been associated with disease susceptibility and resistant patterns in the era of HTS. We further highlight potential opportunities for these genetic markers. PMID:29755620

The evolution of tumour phylogenetics: principles and practice

PubMed Central

Schwartz, Russell; Schäffer, Alejandro A.

2018-01-01

Rapid advances in high-throughput sequencing and a growing realization of the importance of evolutionary theory to cancer genomics have led to a proliferation of phylogenetic studies of tumour progression. These studies have yielded not only new insights but also a plethora of experimental approaches, sometimes reaching conflicting or poorly supported conclusions. Here, we consider this body of work in light of the key computational principles underpinning phylogenetic inference, with the goal of providing practical guidance on the design and analysis of scientifically rigorous tumour phylogeny studies. We survey the range of methods and tools available to the researcher, their key applications, and the various unsolved problems, closing with a perspective on the prospects and broader implications of this field. PMID:28190876

The evolution of tumour phylogenetics: principles and practice.

PubMed

Schwartz, Russell; Schäffer, Alejandro A

2017-04-01

Rapid advances in high-throughput sequencing and a growing realization of the importance of evolutionary theory to cancer genomics have led to a proliferation of phylogenetic studies of tumour progression. These studies have yielded not only new insights but also a plethora of experimental approaches, sometimes reaching conflicting or poorly supported conclusions. Here, we consider this body of work in light of the key computational principles underpinning phylogenetic inference, with the goal of providing practical guidance on the design and analysis of scientifically rigorous tumour phylogeny studies. We survey the range of methods and tools available to the researcher, their key applications, and the various unsolved problems, closing with a perspective on the prospects and broader implications of this field.

Decentralized Grid Scheduling with Evolutionary Fuzzy Systems

NASA Astrophysics Data System (ADS)

Fölling, Alexander; Grimme, Christian; Lepping, Joachim; Papaspyrou, Alexander

In this paper, we address the problem of finding workload exchange policies for decentralized Computational Grids using an Evolutionary Fuzzy System. To this end, we establish a non-invasive collaboration model on the Grid layer which requires minimal information about the participating High Performance and High Throughput Computing (HPC/HTC) centers and which leaves the local resource managers completely untouched. In this environment of fully autonomous sites, independent users are assumed to submit their jobs to the Grid middleware layer of their local site, which in turn decides on the delegation and execution either on the local system or on remote sites in a situation-dependent, adaptive way. We find for different scenarios that the exchange policies show good performance characteristics not only with respect to traditional metrics such as average weighted response time and utilization, but also in terms of robustness and stability in changing environments.

A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa.

PubMed

Guimaraes, S; Pruvost, M; Daligault, J; Stoetzel, E; Bennett, E A; Côté, N M-L; Nicolas, V; Lalis, A; Denys, C; Geigl, E-M; Grange, T

2017-05-01

We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology. © 2016 John Wiley & Sons Ltd.

Plastid Phylogenomics Resolve Deep Relationships among Eupolypod II Ferns with Rapid Radiation and Rate Heterogeneity

PubMed Central

Wei, Ran; Yan, Yue-Hong; Harris, AJ; Kang, Jong-Soo; Shen, Hui; Zhang, Xian-Chun

2017-01-01

Abstract The eupolypods II ferns represent a classic case of evolutionary radiation and, simultaneously, exhibit high substitution rate heterogeneity. These factors have been proposed to contribute to the contentious resolutions among clades within this fern group in multilocus phylogenetic studies. We investigated the deep phylogenetic relationships of eupolypod II ferns by sampling all major families and using 40 plastid genomes, or plastomes, of which 33 were newly sequenced with next-generation sequencing technology. We performed model-based analyses to evaluate the diversity of molecular evolutionary rates for these ferns. Our plastome data, with more than 26,000 informative characters, yielded good resolution for deep relationships within eupolypods II and unambiguously clarified the position of Rhachidosoraceae and the monophyly of Athyriaceae. Results of rate heterogeneity analysis revealed approximately 33 significant rate shifts in eupolypod II ferns, with the most heterogeneous rates (both accelerations and decelerations) occurring in two phylogenetically difficult lineages, that is, the Rhachidosoraceae–Aspleniaceae and Athyriaceae clades. These observations support the hypothesis that rate heterogeneity has previously constrained the deep phylogenetic resolution in eupolypods II. According to the plastome data, we propose that 14 chloroplast markers are particularly phylogenetically informative for eupolypods II both at the familial and generic levels. Our study demonstrates the power of a character-rich plastome data set and high-throughput sequencing for resolving the recalcitrant lineages, which have undergone rapid evolutionary radiation and dramatic changes in substitution rates. PMID:28854625

Environmental microbiology through the lens of high-throughput DNA sequencing: synopsis of current platforms and bioinformatics approaches.

PubMed

Logares, Ramiro; Haverkamp, Thomas H A; Kumar, Surendra; Lanzén, Anders; Nederbragt, Alexander J; Quince, Christopher; Kauserud, Håvard

2012-10-01

The incursion of High-Throughput Sequencing (HTS) in environmental microbiology brings unique opportunities and challenges. HTS now allows a high-resolution exploration of the vast taxonomic and metabolic diversity present in the microbial world, which can provide an exceptional insight on global ecosystem functioning, ecological processes and evolution. This exploration has also economic potential, as we will have access to the evolutionary innovation present in microbial metabolisms, which could be used for biotechnological development. HTS is also challenging the research community, and the current bottleneck is present in the data analysis side. At the moment, researchers are in a sequence data deluge, with sequencing throughput advancing faster than the computer power needed for data analysis. However, new tools and approaches are being developed constantly and the whole process could be depicted as a fast co-evolution between sequencing technology, informatics and microbiologists. In this work, we examine the most popular and recently commercialized HTS platforms as well as bioinformatics methods for data handling and analysis used in microbial metagenomics. This non-exhaustive review is intended to serve as a broad state-of-the-art guide to researchers expanding into this rapidly evolving field. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed Central

Richter, Ingrid; Fidler, Andrew E.

2014-01-01

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

A genome-wide perspective on the evolutionary history of enigmatic wolf-like canids

PubMed Central

vonHoldt, Bridgett M.; Pollinger, John P.; Earl, Dent A.; Knowles, James C.; Boyko, Adam R.; Parker, Heidi; Geffen, Eli; Pilot, Malgorzata; Jedrzejewski, Wlodzimierz; Jedrzejewska, Bogumila; Sidorovich, Vadim; Greco, Claudia; Randi, Ettore; Musiani, Marco; Kays, Roland; Bustamante, Carlos D.; Ostrander, Elaine A.; Novembre, John; Wayne, Robert K.

2011-01-01

High-throughput genotyping technologies developed for model species can potentially increase the resolution of demographic history and ancestry in wild relatives. We use a SNP genotyping microarray developed for the domestic dog to assay variation in over 48K loci in wolf-like species worldwide. Despite the high mobility of these large carnivores, we find distinct hierarchical population units within gray wolves and coyotes that correspond with geographic and ecologic differences among populations. Further, we test controversial theories about the ancestry of the Great Lakes wolf and red wolf using an analysis of haplotype blocks across all 38 canid autosomes. We find that these enigmatic canids are highly admixed varieties derived from gray wolves and coyotes, respectively. This divergent genomic history suggests that they do not have a shared recent ancestry as proposed by previous researchers. Interspecific hybridization, as well as the process of evolutionary divergence, may be responsible for the observed phenotypic distinction of both forms. Such admixture complicates decisions regarding endangered species restoration and protection. PMID:21566151

EAPhy: A Flexible Tool for High-throughput Quality Filtering of Exon-alignments and Data Processing for Phylogenetic Methods.

PubMed

Blom, Mozes P K

2015-08-05

Recently developed molecular methods enable geneticists to target and sequence thousands of orthologous loci and infer evolutionary relationships across the tree of life. Large numbers of genetic markers benefit species tree inference but visual inspection of alignment quality, as traditionally conducted, is challenging with thousands of loci. Furthermore, due to the impracticality of repeated visual inspection with alternative filtering criteria, the potential consequences of using datasets with different degrees of missing data remain nominally explored in most empirical phylogenomic studies. In this short communication, I describe a flexible high-throughput pipeline designed to assess alignment quality and filter exonic sequence data for subsequent inference. The stringency criteria for alignment quality and missing data can be adapted based on the expected level of sequence divergence. Each alignment is automatically evaluated based on the stringency criteria specified, significantly reducing the number of alignments that require visual inspection. By developing a rapid method for alignment filtering and quality assessment, the consistency of phylogenetic estimation based on exonic sequence alignments can be further explored across distinct inference methods, while accounting for different degrees of missing data.

International Barcode of Life: Focus on big biodiversity in South Africa.

PubMed

Adamowicz, Sarah J; Hollingsworth, Peter M; Ratnasingham, Sujeevan; van der Bank, Michelle

2017-11-01

Participants in the 7th International Barcode of Life Conference (Kruger National Park, South Africa, 20-24 November 2017) share the latest findings in DNA barcoding research and its increasingly diversified applications. Here, we review prevailing trends synthesized from among 429 invited and contributed abstracts, which are collated in this open-access special issue of Genome. Hosted for the first time on the African continent, the 7th Conference places special emphasis on the evolutionary origins, biogeography, and conservation of African flora and fauna. Within Africa and elsewhere, DNA barcoding and related techniques are being increasingly used for wildlife forensics and for the validation of commercial products, such as medicinal plants and seafood species. A striking trend of the conference is the dramatic rise of studies on environmental DNA (eDNA) and on diverse uses of high-throughput sequencing techniques. Emerging techniques in these areas are opening new avenues for environmental biomonitoring, managing species-at-risk and invasive species, and revealing species interaction networks in unprecedented detail. Contributors call for the development of validated community standards for high-throughput sequence data generation and analysis, to enable the full potential of these methods to be realized for understanding and managing biodiversity on a global scale.

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian

PubMed Central

2014-01-01

Background The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. Description We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215–364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. Conclusions The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a “non-model system.” PMID:24467778

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian.

PubMed

Stefanik, Derek J; Lubinski, Tristan J; Granger, Brian R; Byrd, Allyson L; Reitzel, Adam M; DeFilippo, Lukas; Lorenc, Allison; Finnerty, John R

2014-01-28

The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215-364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a "non-model system."

High-throughput physical mapping of chromosomes using automated in situ hybridization.

PubMed

George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

2012-06-28

Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

Accurate prediction of protein-protein interactions by integrating potential evolutionary information embedded in PSSM profile and discriminative vector machine classifier.

PubMed

Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Li, Li-Ping; Huang, De-Shuang; Yan, Gui-Ying; Nie, Ru; Huang, Yu-An

2017-04-04

Identification of protein-protein interactions (PPIs) is of critical importance for deciphering the underlying mechanisms of almost all biological processes of cell and providing great insight into the study of human disease. Although much effort has been devoted to identifying PPIs from various organisms, existing high-throughput biological techniques are time-consuming, expensive, and have high false positive and negative results. Thus it is highly urgent to develop in silico methods to predict PPIs efficiently and accurately in this post genomic era. In this article, we report a novel computational model combining our newly developed discriminative vector machine classifier (DVM) and an improved Weber local descriptor (IWLD) for the prediction of PPIs. Two components, differential excitation and orientation, are exploited to build evolutionary features for each protein sequence. The main characteristics of the proposed method lies in introducing an effective feature descriptor IWLD which can capture highly discriminative evolutionary information from position-specific scoring matrixes (PSSM) of protein data, and employing the powerful and robust DVM classifier. When applying the proposed method to Yeast and H. pylori data sets, we obtained excellent prediction accuracies as high as 96.52% and 91.80%, respectively, which are significantly better than the previous methods. Extensive experiments were then performed for predicting cross-species PPIs and the predictive results were also pretty promising. To further validate the performance of the proposed method, we compared it with the state-of-the-art support vector machine (SVM) classifier on Human data set. The experimental results obtained indicate that our method is highly effective for PPIs prediction and can be taken as a supplementary tool for future proteomics research.

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins

PubMed Central

Wilburn, Damien B.; Swanson, Willie J.

2015-01-01

Sexual reproduction and the exchange of genetic information are essential biological processes for species across all branches of the tree of life. Over the last four decades, biochemists have continued to identify many of the factors that facilitate reproduction, but the molecular mechanisms that mediate this process continue to elude us. However, a recurring observation in this research has been the rapid evolution of reproductive proteins. In animals, the competing interests of males and females often result in arms race dynamics between pairs of interacting proteins. This phenomenon has been observed in all stages of reproduction, including pheromones, seminal fluid components, and gamete recognition proteins. In this article, we review how the integration of evolutionary theory with biochemical experiments can be used to study interacting reproductive proteins. Examples are included from both model and non-model organisms, and recent studies are highlighted for their use of state-of-the-art genomic and proteomic techniques. Significance Despite decades of research, our understanding of the molecular mechanisms that mediate fertilization remain poorly characterized. To date, molecular evolutionary studies on both model and non-model organisms have provided some of the best inferences to elucidating the molecular underpinnings of animal reproduction. This review article details how biochemical and evolutionary experiments have jointly enhanced the field for 40 years, and how recent work using high-throughput genomic and proteomic techniques have shed additional insights into this crucial biological process. PMID:26074353

Evolutionary genomics of animal personality.

PubMed

van Oers, Kees; Mueller, Jakob C

2010-12-27

Research on animal personality can be approached from both a phenotypic and a genetic perspective. While using a phenotypic approach one can measure present selection on personality traits and their combinations. However, this approach cannot reconstruct the historical trajectory that was taken by evolution. Therefore, it is essential for our understanding of the causes and consequences of personality diversity to link phenotypic variation in personality traits with polymorphisms in genomic regions that code for this trait variation. Identifying genes or genome regions that underlie personality traits will open exciting possibilities to study natural selection at the molecular level, gene-gene and gene-environment interactions, pleiotropic effects and how gene expression shapes personality phenotypes. In this paper, we will discuss how genome information revealed by already established approaches and some more recent techniques such as high-throughput sequencing of genomic regions in a large number of individuals can be used to infer micro-evolutionary processes, historical selection and finally the maintenance of personality trait variation. We will do this by reviewing recent advances in molecular genetics of animal personality, but will also use advanced human personality studies as case studies of how molecular information may be used in animal personality research in the near future.

A new era in palaeomicrobiology: prospects for ancient dental calculus as a long-term record of the human oral microbiome.

PubMed

Warinner, Christina; Speller, Camilla; Collins, Matthew J

2015-01-19

The field of palaeomicrobiology is dramatically expanding thanks to recent advances in high-throughput biomolecular sequencing, which allows unprecedented access to the evolutionary history and ecology of human-associated and environmental microbes. Recently, human dental calculus has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, preserving not only microbial and host biomolecules but also dietary and environmental debris. Modern investigations of native human microbiota have demonstrated that the human microbiome plays a central role in health and chronic disease, raising questions about changes in microbial ecology, diversity and function through time. This paper explores the current state of ancient oral microbiome research and discusses successful applications, methodological challenges and future possibilities in elucidating the intimate evolutionary relationship between humans and their microbes.

A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica.

PubMed

Wagner, James M; Liu, Leqian; Yuan, Shuo-Fu; Venkataraman, Maya V; Abate, Adam R; Alper, Hal S

2018-04-23

Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

PubMed Central

Buenrostro, Jason D.; Chircus, Lauren M.; Araya, Carlos L.; Layton, Curtis J.; Chang, Howard Y.; Snyder, Michael P.; Greenleaf, William J.

2015-01-01

RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of MS2 coat protein to >107 RNA targets generated on a flow-cell surface by in situ transcription and inter-molecular tethering of RNA to DNA. We decompose the binding energy contributions from primary and secondary RNA structure, finding that differences in affinity are often driven by sequence-specific changes in association rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis, and a long-hypothesized structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNAMaP) relationships across molecular variants. PMID:24727714

Transcriptome-Based Differentiation of Closely-Related Miscanthus Lines

DOE PAGES

Chouvarine, Philippe; Cooksey, Amanda M.; McCarthy, Fiona M.; ...

2012-01-10

Distinguishing between individuals is critical to those conducting animal/plant breeding, food safety/quality research, diagnostic and clinical testing, and evolutionary biology studies. Classical genetic identification studies are based on marker polymorphisms, but polymorphism-based techniques are time and labor intensive and often cannot distinguish between closely related individuals. Illumina sequencing technologies provide the detailed sequence data required for rapid and efficient differentiation of related species, lines/cultivars, and individuals in a cost-effective manner. Here we describe the use of Illumina high-throughput exome sequencing, coupled with SNP mapping, as a rapid means of distinguishing between related cultivars of the lignocellulosic bioenergy crop giant miscanthusmore » (Miscanthus6giganteus). We provide the first exome sequence database for Miscanthus species complete with Gene Ontology (GO) functional annotations."« less

Urban Evolutionary Ecology and the Potential Benefits of Implementing Genomics.

PubMed

Schell, Christopher J

2018-02-14

Urban habitats are quickly becoming exceptional models to address adaptation under rapid environmental change, given the expansive temporal and spatial scales with which anthropogenic landscape conversion occurs. Urban ecologists in the last 10-15 years have done an extraordinary job of highlighting phenotypic patterns that correspond with urban living, as well as delineating urban population structure using traditional genetic markers. The underpinning genetic mechanisms that govern those phenotypic patterns, however, are less well established. Moreover, the power of traditional molecular studies is constrained by the number of markers being evaluated, which limits the potential to assess fine-scale population structure potentially common in urban areas. With the recent proliferation of low-cost, high-throughput sequencing methods, we can begin to address an emerging question in urban ecology: are species adapted to local optima within cities or are they expressing latent phenotypic plasticity? Here, I provide a comprehensive review of previous urban ecological studies, with special focus on the molecular ecology and phenotypic adjustments documented in urban terrestrial and amphibious fauna. I subsequently pinpoint areas in the literature that could benefit from a genomic investigation and briefly discuss the suitability of specific techniques in addressing eco-evolutionary questions within urban ecology. Though many challenges exist with implementing genomics into urban ecology, such studies provide an exceptional opportunity to advance our understanding of eco-evolutionary processes in metropolitan areas. © The American Genetic Association 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Using High-Throughput Sequencing to Leverage Surveillance of Genetic Diversity and Oseltamivir Resistance: A Pilot Study during the 2009 Influenza A(H1N1) Pandemic

PubMed Central

Téllez-Sosa, Juan; Rodríguez, Mario Henry; Gómez-Barreto, Rosa E.; Valdovinos-Torres, Humberto; Hidalgo, Ana Cecilia; Cruz-Hervert, Pablo; Luna, René Santos; Carrillo-Valenzo, Erik; Ramos, Celso; García-García, Lourdes; Martínez-Barnetche, Jesús

2013-01-01

Background Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The “deep sequencing” approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples. Methodology and Principal Findings We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n  =  299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance. Conclusions NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases. PMID:23843978

Signatures of Rapid Evolution in Urban and Rural Transcriptomes of White-Footed Mice (Peromyscus leucopus) in the New York Metropolitan Area

PubMed Central

Harris, Stephen E.; Munshi-South, Jason; Obergfell, Craig; O’Neill, Rachel

2013-01-01

Urbanization is a major cause of ecological degradation around the world, and human settlement in large cities is accelerating. New York City (NYC) is one of the oldest and most urbanized cities in North America, but still maintains 20% vegetation cover and substantial populations of some native wildlife. The white-footed mouse, Peromyscus leucopus , is a common resident of NYC’s forest fragments and an emerging model system for examining the evolutionary consequences of urbanization. In this study, we developed transcriptomic resources for urban P . leucopus to examine evolutionary changes in protein-coding regions for an exemplar “urban adapter.” We used Roche 454 GS FLX+ high throughput sequencing to derive transcriptomes from multiple tissues from individuals across both urban and rural populations. From these data, we identified 31,015 SNPs and several candidate genes potentially experiencing positive selection in urban populations of P . leucopus . These candidate genes are involved in xenobiotic metabolism, innate immune response, demethylation activity, and other important biological phenomena in novel urban environments. This study is one of the first to report candidate genes exhibiting signatures of directional selection in divergent urban ecosystems. PMID:24015321

A field ornithologist’s guide to genomics: Practical considerations for ecology and conservation

USGS Publications Warehouse

Oyler-McCance, Sara J.; Oh, Kevin; Langin, Kathryn; Aldridge, Cameron L.

2016-01-01

Vast improvements in sequencing technology have made it practical to simultaneously sequence millions of nucleotides distributed across the genome, opening the door for genomic studies in virtually any species. Ornithological research stands to benefit in three substantial ways. First, genomic methods enhance our ability to parse and simultaneously analyze both neutral and non-neutral genomic regions, thus providing insight into adaptive evolution and divergence. Second, the sheer quantity of sequence data generated by current sequencing platforms allows increased precision and resolution in analyses. Third, high-throughput sequencing can benefit applications that focus on a small number of loci that are otherwise prohibitively expensive, time-consuming, and technically difficult using traditional sequencing methods. These advances have improved our ability to understand evolutionary processes like speciation and local adaptation, but they also offer many practical applications in the fields of population ecology, migration tracking, conservation planning, diet analyses, and disease ecology. This review provides a guide for field ornithologists interested in incorporating genomic approaches into their research program, with an emphasis on techniques related to ecology and conservation. We present a general overview of contemporary genomic approaches and methods, as well as important considerations when selecting a genomic technique. We also discuss research questions that are likely to benefit from utilizing high-throughput sequencing instruments, highlighting select examples from recent avian studies.

High-Throughput DNA sequencing of ancient wood.

PubMed

Wagner, Stefanie; Lagane, Frédéric; Seguin-Orlando, Andaine; Schubert, Mikkel; Leroy, Thibault; Guichoux, Erwan; Chancerel, Emilie; Bech-Hebelstrup, Inger; Bernard, Vincent; Billard, Cyrille; Billaud, Yves; Bolliger, Matthias; Croutsch, Christophe; Čufar, Katarina; Eynaud, Frédérique; Heussner, Karl Uwe; Köninger, Joachim; Langenegger, Fabien; Leroy, Frédéric; Lima, Christine; Martinelli, Nicoletta; Momber, Garry; Billamboz, André; Nelle, Oliver; Palomo, Antoni; Piqué, Raquel; Ramstein, Marianne; Schweichel, Roswitha; Stäuble, Harald; Tegel, Willy; Terradas, Xavier; Verdin, Florence; Plomion, Christophe; Kremer, Antoine; Orlando, Ludovic

2018-03-01

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high-throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long-term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human-induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro-evolutionary response of trees to climate change and human forest management. © 2018 John Wiley & Sons Ltd.

In silico modelling of directed evolution: Implications for experimental design and stepwise evolution.

PubMed

Wedge, David C; Rowe, William; Kell, Douglas B; Knowles, Joshua

2009-03-07

We model the process of directed evolution (DE) in silico using genetic algorithms. Making use of the NK fitness landscape model, we analyse the effects of mutation rate, crossover and selection pressure on the performance of DE. A range of values of K, the epistatic interaction of the landscape, are considered, and high- and low-throughput modes of evolution are compared. Our findings suggest that for runs of or around ten generations' duration-as is typical in DE-there is little difference between the way in which DE needs to be configured in the high- and low-throughput regimes, nor across different degrees of landscape epistasis. In all cases, a high selection pressure (but not an extreme one) combined with a moderately high mutation rate works best, while crossover provides some benefit but only on the less rugged landscapes. These genetic algorithms were also compared with a "model-based approach" from the literature, which uses sequential fixing of the problem parameters based on fitting a linear model. Overall, we find that purely evolutionary techniques fare better than do model-based approaches across all but the smoothest landscapes.

Evolutionary scalpels for dissecting tumor ecosystems

PubMed Central

Rosenbloom, Daniel I. S.; Camara, Pablo G.; Chu, Tim; Rabadan, Raul

2017-01-01

Amidst the growing literature on cancer genomics and intratumor heterogeneity, essential principles in evolutionary biology recur time and time again. Here we use these principles to guide the reader through major advances in cancer research, highlighting issues of “hit hard, hit early” treatment strategies, drug resistance, and metastasis. We distinguish between two frameworks for understanding heterogeneous tumors, both of which can inform treatment strategies: (1) The tumor as diverse ecosystem, a Darwinian population of sometimes-competing, sometimes-cooperating cells; (2) The tumor as tightly integrated, self-regulating organ, which may hijack developmental signals to restore functional heterogeneity after treatment. While the first framework dominates literature on cancer evolution, the second framework enjoys support as well. Throughout this review, we illustrate how mathematical models inform understanding of tumor progression and treatment outcomes. Connecting models to genomic data faces computational and technical hurdles, but high-throughput single-cell technologies show promise to clear these hurdles. PMID:27923679

A new era in palaeomicrobiology: prospects for ancient dental calculus as a long-term record of the human oral microbiome

PubMed Central

Warinner, Christina; Speller, Camilla; Collins, Matthew J.

2015-01-01

The field of palaeomicrobiology is dramatically expanding thanks to recent advances in high-throughput biomolecular sequencing, which allows unprecedented access to the evolutionary history and ecology of human-associated and environmental microbes. Recently, human dental calculus has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, preserving not only microbial and host biomolecules but also dietary and environmental debris. Modern investigations of native human microbiota have demonstrated that the human microbiome plays a central role in health and chronic disease, raising questions about changes in microbial ecology, diversity and function through time. This paper explores the current state of ancient oral microbiome research and discusses successful applications, methodological challenges and future possibilities in elucidating the intimate evolutionary relationship between humans and their microbes. PMID:25487328

Transcriptome-based differentiation of closely-related Miscanthus lines.

PubMed

Chouvarine, Philippe; Cooksey, Amanda M; McCarthy, Fiona M; Ray, David A; Baldwin, Brian S; Burgess, Shane C; Peterson, Daniel G

2012-01-01

Distinguishing between individuals is critical to those conducting animal/plant breeding, food safety/quality research, diagnostic and clinical testing, and evolutionary biology studies. Classical genetic identification studies are based on marker polymorphisms, but polymorphism-based techniques are time and labor intensive and often cannot distinguish between closely related individuals. Illumina sequencing technologies provide the detailed sequence data required for rapid and efficient differentiation of related species, lines/cultivars, and individuals in a cost-effective manner. Here we describe the use of Illumina high-throughput exome sequencing, coupled with SNP mapping, as a rapid means of distinguishing between related cultivars of the lignocellulosic bioenergy crop giant miscanthus (Miscanthus × giganteus). We provide the first exome sequence database for Miscanthus species complete with Gene Ontology (GO) functional annotations. A SNP comparative analysis of rhizome-derived cDNA sequences was successfully utilized to distinguish three Miscanthus × giganteus cultivars from each other and from other Miscanthus species. Moreover, the resulting phylogenetic tree generated from SNP frequency data parallels the known breeding history of the plants examined. Some of the giant miscanthus plants exhibit considerable sequence divergence. Here we describe an analysis of Miscanthus in which high-throughput exome sequencing was utilized to differentiate between closely related genotypes despite the current lack of a reference genome sequence. We functionally annotated the exome sequences and provide resources to support Miscanthus systems biology. In addition, we demonstrate the use of the commercial high-performance cloud computing to do computational GO annotation.

RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus

PubMed Central

Lenz, Tobias L; Eizaguirre, Christophe; Becker, Sven; Reusch, Thorsten BH

2009-01-01

Background In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes. Results We present a genotyping protocol that uses Reference Strand-mediated Conformation Analysis (RSCA), optimised for recently duplicated MHC class IIB genes that are typical for many fish and bird species, including the three-spined stickleback, Gasterosteus aculeatus. In addition we use a comprehensive plasmid library of MHC class IIB alleles to determine the nucleotide sequence of alleles represented by RSCA allele peaks. Verification of the RSCA typing by cloning and sequencing demonstrates high congruency between both methods and provides new insight into the polymorphism of classical stickleback MHC genes. Analysis of the plasmid library additionally reveals the high resolution and reproducibility of the RSCA technique. Conclusion This new RSCA genotyping protocol offers a fast, but sensitive and reliable way to determine the MHC allele repertoire of three-spined sticklebacks. It therefore provides a valuable tool to employ this highly polymorphic and adaptive marker in future high-throughput studies of host-parasite co-evolution and ecological speciation in this emerging model organism. PMID:19291291

Physics and evolution of thermophilic adaptation.

PubMed

Berezovsky, Igor N; Shakhnovich, Eugene I

2005-09-06

Analysis of structures and sequences of several hyperthermostable proteins from various sources reveals two major physical mechanisms of their thermostabilization. The first mechanism is "structure-based," whereby some hyperthermostable proteins are significantly more compact than their mesophilic homologues, while no particular interaction type appears to cause stabilization; rather, a sheer number of interactions is responsible for thermostability. Other hyperthermostable proteins employ an alternative, "sequence-based" mechanism of their thermal stabilization. They do not show pronounced structural differences from mesophilic homologues. Rather, a small number of apparently strong interactions is responsible for high thermal stability of these proteins. High-throughput comparative analysis of structures and complete genomes of several hyperthermophilic archaea and bacteria revealed that organisms develop diverse strategies of thermophilic adaptation by using, to a varying degree, two fundamental physical mechanisms of thermostability. The choice of a particular strategy depends on the evolutionary history of an organism. Proteins from organisms that originated in an extreme environment, such as hyperthermophilic archaea (Pyrococcus furiosus), are significantly more compact and more hydrophobic than their mesophilic counterparts. Alternatively, organisms that evolved as mesophiles but later recolonized a hot environment (Thermotoga maritima) relied in their evolutionary strategy of thermophilic adaptation on "sequence-based" mechanism of thermostability. We propose an evolutionary explanation of these differences based on physical concepts of protein designability.

Identification of structural variation in mouse genomes.

PubMed

Keane, Thomas M; Wong, Kim; Adams, David J; Flint, Jonathan; Reymond, Alexandre; Yalcin, Binnaz

2014-01-01

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Experimental Evolution as a High-Throughput Screen for Genetic Adaptations.

PubMed

Cooper, Vaughn S

2018-06-27

Experimental evolution is a method in which populations of organisms, often microbes, are founded by one or more ancestors of known genotype and then propagated under controlled conditions to study the evolutionary process. These evolving populations are influenced by all population genetic forces, including selection, mutation, drift, and recombination, and the relative contributions of these forces may be seen as mysterious. Here, I describe why the outcomes of experimental evolution should be viewed with greater certainty because the force of selection typically dominates. Importantly, any mutant rising rapidly to high frequency in large populations must have acquired adaptive traits in the selective environment. Sequencing the genomes of these mutants can identify genes or pathways that contribute to an adaptation. I review the logic and simple mathematics why this evolve-and-resequence approach is a powerful way to find the mutations or mutation combinations that best increase fitness in any new environment. Copyright © 2018 Cooper.

Microbial Ecology and Evolution in the Acid Mine Drainage Model System.

PubMed

Huang, Li-Nan; Kuang, Jia-Liang; Shu, Wen-Sheng

2016-07-01

Acid mine drainage (AMD) is a unique ecological niche for acid- and toxic-metals-adapted microorganisms. These low-complexity systems offer a special opportunity for the ecological and evolutionary analyses of natural microbial assemblages. The last decade has witnessed an unprecedented interest in the study of AMD communities using 16S rRNA high-throughput sequencing and community genomic and postgenomic methodologies, significantly advancing our understanding of microbial diversity, community function, and evolution in acidic environments. This review describes new data on AMD microbial ecology and evolution, especially dynamics of microbial diversity, community functions, and population genomes, and further identifies gaps in our current knowledge that future research, with integrated applications of meta-omics technologies, will fill. Copyright © 2016 Elsevier Ltd. All rights reserved.

Denoising DNA deep sequencing data—high-throughput sequencing errors and their correction

PubMed Central

Laehnemann, David; Borkhardt, Arndt

2016-01-01

Characterizing the errors generated by common high-throughput sequencing platforms and telling true genetic variation from technical artefacts are two interdependent steps, essential to many analyses such as single nucleotide variant calling, haplotype inference, sequence assembly and evolutionary studies. Both random and systematic errors can show a specific occurrence profile for each of the six prominent sequencing platforms surveyed here: 454 pyrosequencing, Complete Genomics DNA nanoball sequencing, Illumina sequencing by synthesis, Ion Torrent semiconductor sequencing, Pacific Biosciences single-molecule real-time sequencing and Oxford Nanopore sequencing. There is a large variety of programs available for error removal in sequencing read data, which differ in the error models and statistical techniques they use, the features of the data they analyse, the parameters they determine from them and the data structures and algorithms they use. We highlight the assumptions they make and for which data types these hold, providing guidance which tools to consider for benchmarking with regard to the data properties. While no benchmarking results are included here, such specific benchmarks would greatly inform tool choices and future software development. The development of stand-alone error correctors, as well as single nucleotide variant and haplotype callers, could also benefit from using more of the knowledge about error profiles and from (re)combining ideas from the existing approaches presented here. PMID:26026159

Status of NASA's Evolutionary Xenon Thruster (NEXT) Long-Duration Test as of 50,000 h and 900 kg Throughput

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2015-01-01

The NASA's Evolutionary Xenon Thruster (NEXT) project is developing the next-generation solar electric propulsion ion propulsion system with significant enhancements beyond the state-of-the-art NASA Solar Electric Propulsion Technology Application Readiness (NSTAR) ion propulsion system in order to provide future NASA science missions with enhanced propulsion capabilities. As part of a comprehensive thruster service life assessment, the NEXT Long-Duration Test (LDT) was initiated in June 2005 to demonstrate throughput capability and validate thruster service life modeling. The NEXT LDT exceeded its original qualification throughput requirement of 450 kg in December 2009. To date, the NEXT LDT has set records for electric propulsion lifetime and has demonstrated 50,170 h of operation, processed 902 kg of propellant, and delivered 34.9 MN-s of total impulse. The NEXT thruster design mitigated several life-limiting mechanisms encountered in the NSTAR design, dramatically increasing service life capability. Various component erosion rates compare favorably to the pretest predictions based upon semi-empirical ion thruster models. The NEXT LDT either met or exceeded all of its original goals regarding lifetime demonstration, performance and wear characterization, and modeling validation. In light of recent budget constraints and to focus on development of other components of the NEXT ion propulsion system, a voluntary termination procedure for the NEXT LDT began in April 2013. As part of this termination procedure, a comprehensive post-test performance characterization was conducted across all operating conditions of the NEXT throttle table. These measurements were found to be consistent with prior data that show minimal degradation of performance over the thruster's 50 kh lifetime. Repair of various diagnostics within the test facility is presently planned while keeping the thruster under high vacuum conditions. These diagnostics will provide additional critical information on the current state of the thruster, in regards to performance and wear, prior to destructive post-test analyses performed on the thruster under atmosphere conditions.

Status of NASA's Evolutionary Xenon Thruster (NEXT) Long-Duration Test as of 50,000 h and 900 kg Throughput

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2013-01-01

The NASA's Evolutionary Xenon Thruster (NEXT) project is developing the next-generation solar electric propulsion ion propulsion system with significant enhancements beyond the state-of-the-art NASA Solar Electric Propulsion Technology Application Readiness (NSTAR) ion propulsion system in order to provide future NASA science missions with enhanced propulsion capabilities. As part of a comprehensive thruster service life assessment, the NEXT Long-Duration Test (LDT) was initiated in June 2005 to demonstrate throughput capability and validate thruster service life modeling. The NEXT LDT exceeded its original qualification throughput requirement of 450 kg in December 2009. To date, the NEXT LDT has set records for electric propulsion lifetime and has demonstrated 50,170 hours of operation, processed 902 kg of propellant, and delivered 34.9 MN-s of total impulse. The NEXT thruster design mitigated several life-limiting mechanisms encountered in the NSTAR design, dramatically increasing service life capability. Various component erosion rates compare favorably to the pretest predictions based upon semi-empirical ion thruster models. The NEXT LDT either met or exceeded all of its original goals regarding lifetime demonstration, performance and wear characterization, and modeling validation. In light of recent budget constraints and to focus on development of other components of the NEXT ion propulsion system, a voluntary termination procedure for the NEXT LDT began in April 2013. As part of this termination procedure, a comprehensive post-test performance characterization was conducted across all operating conditions of the NEXT throttle table. These measurements were found to be consistent with prior data that show minimal degradation of performance over the thruster's 50 kh lifetime. Repair of various diagnostics within the test facility is presently planned while keeping the thruster under high vacuum conditions. These diagnostics will provide additional critical information on the current state of the thruster, in regards to performance and wear, prior to destructive post-test analyses performed on the thruster under atmosphere conditions.

Human Y chromosome copy number variation in the next generation sequencing era and beyond.

PubMed

Massaia, Andrea; Xue, Yali

2017-05-01

The human Y chromosome provides a fertile ground for structural rearrangements owing to its haploidy and high content of repeated sequences. The methodologies used for copy number variation (CNV) studies have developed over the years. Low-throughput techniques based on direct observation of rearrangements were developed early on, and are still used, often to complement array-based or sequencing approaches which have limited power in regions with high repeat content and specifically in the presence of long, identical repeats, such as those found in human sex chromosomes. Some specific rearrangements have been investigated for decades; because of their effects on fertility, or their outstanding evolutionary features, the interest in these has not diminished. However, following the flourishing of large-scale genomics, several studies have investigated CNVs across the whole chromosome. These studies sometimes employ data generated within large genomic projects such as the DDD study or the 1000 Genomes Project, and often survey large samples of healthy individuals without any prior selection. Novel technologies based on sequencing long molecules and combinations of technologies, promise to stimulate the study of Y-CNVs in the immediate future.

Lectin engineering, a molecular evolutionary approach to expanding the lectin utilities.

PubMed

Hu, Dan; Tateno, Hiroaki; Hirabayashi, Jun

2015-04-27

In the post genomic era, glycomics--the systematic study of all glycan structures of a given cell or organism--has emerged as an indispensable technology in various fields of biology and medicine. Lectins are regarded as "decipherers of glycans", being useful reagents for their structural analysis, and have been widely used in glycomic studies. However, the inconsistent activity and availability associated with the plant-derived lectins that comprise most of the commercially available lectins, and the limit in the range of glycan structures covered, have necessitated the development of innovative tools via engineering of lectins on existing scaffolds. This review will summarize the current state of the art of lectin engineering and highlight recent technological advances in this field. The key issues associated with the strategy of lectin engineering including selection of template lectin, construction of a mutagenesis library, and high-throughput screening methods are discussed.

Development, characterization, and cross-amplification of 16 microsatellite primers for Atriplex tatarica (Amaranthaceae)1

PubMed Central

Kondrysová, Eva; Krak, Karol; Mandák, Bohumil

2017-01-01

Premise of the study: Microsatellite primers were developed to characterize the genetic diversity and structure of the annual herb Atriplex tatarica (Amaranthaceae) and to facilitate ecological and evolutionary studies of A. tatarica and its relatives. Methods and Results: Sixteen novel microsatellite primers were developed for A. tatarica based on high-throughput sequencing of enriched libraries. All markers were polymorphic, with the number of alleles per locus ranging from three to 25 and observed and expected heterozygosity ranging from 0.08 to 0.74 and 0.10 to 0.87, respectively. In addition, some of these loci were successfully amplified and showed polymorphisms in four Atriplex and seven Chenopodium species. Conclusions: The microsatellite markers published here will be useful in assessing genetic diversity, structure, and gene flow within and across populations of A. tatarica, as well as in other species of Atriplex and the related genus Chenopodium. PMID:29188148

Genome-wide scans for loci under selection in humans

PubMed Central

2005-01-01

Natural selection, which can be defined as the differential contribution of genetic variants to future generations, is the driving force of Darwinian evolution. Identifying regions of the human genome that have been targets of natural selection is an important step in clarifying human evolutionary history and understanding how genetic variation results in phenotypic diversity, it may also facilitate the search for complex disease genes. Technological advances in high-throughput DNA sequencing and single nucleotide polymorphism genotyping have enabled several genome-wide scans of natural selection to be undertaken. Here, some of the observations that are beginning to emerge from these studies will be reviewed, including evidence for geographically restricted selective pressures (ie local adaptation) and a relationship between genes subject to natural selection and human disease. In addition, the paper will highlight several important problems that need to be addressed in future genome-wide studies of natural selection. PMID:16004726

Integrating protein structural dynamics and evolutionary analysis with Bio3D.

PubMed

Skjærven, Lars; Yao, Xin-Qiu; Scarabelli, Guido; Grant, Barry J

2014-12-10

Popular bioinformatics approaches for studying protein functional dynamics include comparisons of crystallographic structures, molecular dynamics simulations and normal mode analysis. However, determining how observed displacements and predicted motions from these traditionally separate analyses relate to each other, as well as to the evolution of sequence, structure and function within large protein families, remains a considerable challenge. This is in part due to the general lack of tools that integrate information of molecular structure, dynamics and evolution. Here, we describe the integration of new methodologies for evolutionary sequence, structure and simulation analysis into the Bio3D package. This major update includes unique high-throughput normal mode analysis for examining and contrasting the dynamics of related proteins with non-identical sequences and structures, as well as new methods for quantifying dynamical couplings and their residue-wise dissection from correlation network analysis. These new methodologies are integrated with major biomolecular databases as well as established methods for evolutionary sequence and comparative structural analysis. New functionality for directly comparing results derived from normal modes, molecular dynamics and principal component analysis of heterogeneous experimental structure distributions is also included. We demonstrate these integrated capabilities with example applications to dihydrofolate reductase and heterotrimeric G-protein families along with a discussion of the mechanistic insight provided in each case. The integration of structural dynamics and evolutionary analysis in Bio3D enables researchers to go beyond a prediction of single protein dynamics to investigate dynamical features across large protein families. The Bio3D package is distributed with full source code and extensive documentation as a platform independent R package under a GPL2 license from http://thegrantlab.org/bio3d/ .

Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone

PubMed Central

Maamary, Peter G.; Ben Zakour, Nouri L.; Cole, Jason N.; Hollands, Andrew; Aziz, Ramy K.; Barnett, Timothy C.; Cork, Amanda J.; Henningham, Anna; Sanderson-Smith, Martina; McArthur, Jason D.; Venturini, Carola; Gillen, Christine M.; Kirk, Joshua K.; Johnson, Dwight R.; Taylor, William L.; Kaplan, Edward L.; Kotb, Malak; Nizet, Victor; Beatson, Scott A.; Walker, Mark J.

2012-01-01

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.—Maamary, P. G., Ben Zakour, N. L., Cole, J. N., Hollands, A., Aziz, R. K., Barnett, T. C., Cork, A. J., Henningham, A., Sanderson-Smith, M., McArthur, J. D., Venturini, C., Gillen, C. M., Kirk, J. K., Johnson, D. R., Taylor, W. L., Kaplan, E. L., Kotb, M., Nizet, V., Beatson, S. A., Walker, M. J. Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone. PMID:22878963

Extendibility of chemically amplified resists: another brick wall?

NASA Astrophysics Data System (ADS)

Hinsberg, William D.; Houle, Frances A.; Sanchez, Martha I.; Hoffnagle, John A.; Wallraff, Gregory M.; Medeiros, David R.; Gallatin, Gregg M.; Cobb, Jonathan L.

2003-06-01

The chemically amplified resist concept, first described two decades past and originally targeted for the 1000 nm device generation, has proved to have remarkable versatility. The semiconductor industry has come to rely on the properties of CA resists to achieve high resolution, high aspect ratio imaging accompanied by the high throughput that stems from their catalytic imaging mechanism. As the industry maps the evolution of lithographic technology to the 20 nm regime, it is appropriate to review the factors that control the performance of CA resists, and examine whether the traditional evolutionary path of materials refinement will provide materials capable of supporting device manufacturing at those dimensions. The impacts of image blur, line-edge roughness and shot noise on the ability to image CA resists at nanoscale dimensions will be discussed.

Neutral Theory and Rapidly Evolving Viral Pathogens.

PubMed

Frost, Simon D W; Magalis, Brittany Rife; Kosakovsky Pond, Sergei L

2018-06-01

The evolution of viral pathogens is shaped by strong selective forces that are exerted during jumps to new hosts, confrontations with host immune responses and antiviral drugs, and numerous other processes. However, while undeniably strong and frequent, adaptive evolution is largely confined to small parts of information-packed viral genomes, and the majority of observed variation is effectively neutral. The predictions and implications of the neutral theory have proven immensely useful in this context, with applications spanning understanding within-host population structure, tracing the origins and spread of viral pathogens, predicting evolutionary dynamics, and modeling the emergence of drug resistance. We highlight the multiple ways in which the neutral theory has had an impact, which has been accelerated in the age of high-throughput, high-resolution genomics.

Biosequence Similarity Search on the Mercury System

PubMed Central

Krishnamurthy, Praveen; Buhler, Jeremy; Chamberlain, Roger; Franklin, Mark; Gyang, Kwame; Jacob, Arpith; Lancaster, Joseph

2007-01-01

Biosequence similarity search is an important application in modern molecular biology. Search algorithms aim to identify sets of sequences whose extensional similarity suggests a common evolutionary origin or function. The most widely used similarity search tool for biosequences is BLAST, a program designed to compare query sequences to a database. Here, we present the design of BLASTN, the version of BLAST that searches DNA sequences, on the Mercury system, an architecture that supports high-volume, high-throughput data movement off a data store and into reconfigurable hardware. An important component of application deployment on the Mercury system is the functional decomposition of the application onto both the reconfigurable hardware and the traditional processor. Both the Mercury BLASTN application design and its performance analysis are described. PMID:18846267

Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip.

PubMed

Tehran, Domenico Azarnia; Pirazzini, Marco

2018-05-10

Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of the genus Clostridium and, on the basis of their different immunological properties, were classified as seven distinct types of toxin. BoNT classification remained stagnant for the last 50 years until, via bioinformatics and high-throughput sequencing techniques, dozens of BoNT variants, novel serotypes as well as BoNT-like toxins within non-clostridial species have been discovered. Here, we discuss how the now “booming field” of botulinum neurotoxin may shed light on their evolutionary origin and open exciting avenues for future therapeutic applications.

Deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions

PubMed Central

2014-01-01

Deep sequencing harnesses the high throughput nature of next generation sequencing technologies to generate population samples, treating information contained in individual reads as meaningful. Here, we review applications of deep sequencing to pathogen evolution. Pioneering deep sequencing studies from the virology literature are discussed, such as whole genome Roche-454 sequencing analyses of the dynamics of the rapidly mutating pathogens hepatitis C virus and HIV. Extension of the deep sequencing approach to bacterial populations is then discussed, including the impacts of emerging sequencing technologies. While it is clear that deep sequencing has unprecedented potential for assessing the genetic structure and evolutionary history of pathogen populations, bioinformatic challenges remain. We summarise current approaches to overcoming these challenges, in particular methods for detecting low frequency variants in the context of sequencing error and reconstructing individual haplotypes from short reads. PMID:24428920

Phylogenetic Conflict in Bears Identified by Automated Discovery of Transposable Element Insertions in Low-Coverage Genomes

PubMed Central

Gallus, Susanne; Janke, Axel

2017-01-01

Abstract Phylogenetic reconstruction from transposable elements (TEs) offers an additional perspective to study evolutionary processes. However, detecting phylogenetically informative TE insertions requires tedious experimental work, limiting the power of phylogenetic inference. Here, we analyzed the genomes of seven bear species using high-throughput sequencing data to detect thousands of TE insertions. The newly developed pipeline for TE detection called TeddyPi (TE detection and discovery for Phylogenetic Inference) identified 150,513 high-quality TE insertions in the genomes of ursine and tremarctine bears. By integrating different TE insertion callers and using a stringent filtering approach, the TeddyPi pipeline produced highly reliable TE insertion calls, which were confirmed by extensive in vitro validation experiments. Analysis of single nucleotide substitutions in the flanking regions of the TEs shows that these substitutions correlate with the phylogenetic signal from the TE insertions. Our phylogenomic analyses show that TEs are a major driver of genomic variation in bears and enabled phylogenetic reconstruction of a well-resolved species tree, despite strong signals for incomplete lineage sorting and introgression. The analyses show that the Asiatic black, sun, and sloth bear form a monophyletic clade, in which phylogenetic incongruence originates from incomplete lineage sorting. TeddyPi is open source and can be adapted to various TE and structural variation callers. The pipeline makes it possible to confidently extract thousands of TE insertions even from low-coverage genomes (∼10×) of nonmodel organisms. This opens new possibilities for biologists to study phylogenies and evolutionary processes as well as rates and patterns of (retro-)transposition and structural variation. PMID:28985298

Successful development of microsatellite markers in a challenging species: the horizontal borer Austroplatypus incompertus (Coleoptera: Curculionidae).

PubMed

Smith, S; Joss, T; Stow, A

2011-10-01

The analysis of microsatellite loci has allowed significant advances in evolutionary biology and pest management. However, until very recently, the potential benefits have been compromised by the high costs of developing these neutral markers. High-throughput sequencing provides a solution to this problem. We describe the development of 13 microsatellite markers for the eusocial ambrosia beetle, Austroplatypus incompertus, a significant pest of forests in southeast Australia. The frequency of microsatellite repeats in the genome of A. incompertus was determined to be low, and previous attempts at microsatellite isolation using a traditional genomic library were problematic. Here, we utilised two protocols, microsatellite-enriched genomic library construction and high-throughput 454 sequencing and characterised 13 loci which were polymorphic among 32 individuals. Numbers of alleles per locus ranged from 2 to 17, and observed and expected heterozygosities from 0.344 to 0.767 and from 0.507 to 0.860, respectively. These microsatellites have the resolution required to analyse fine-scale colony and population genetic structure. Our work demonstrates the utility of next-generation 454 sequencing as a method for rapid and cost-effective acquisition of microsatellites where other techniques have failed, or for taxa where marker development has historically been both complicated and expensive.

Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library.

PubMed

Sánchez, Cecilia Castaño; Smith, Timothy P L; Wiedmann, Ralph T; Vallejo, Roger L; Salem, Mohamed; Yao, Jianbo; Rexroad, Caird E

2009-11-25

To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts. The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

Expression and phylogenetic analyses reveal paralogous lineages of putatively classical and non-classical MHC-I genes in three sparrow species (Passer).

PubMed

Drews, Anna; Strandh, Maria; Råberg, Lars; Westerdahl, Helena

2017-06-26

The Major Histocompatibility Complex (MHC) plays a central role in immunity and has been given considerable attention by evolutionary ecologists due to its associations with fitness-related traits. Songbirds have unusually high numbers of MHC class I (MHC-I) genes, but it is not known whether all are expressed and equally important for immune function. Classical MHC-I genes are highly expressed, polymorphic and present peptides to T-cells whereas non-classical MHC-I genes have lower expression, are more monomorphic and do not present peptides to T-cells. To get a better understanding of the highly duplicated MHC genes in songbirds, we studied gene expression in a phylogenetic framework in three species of sparrows (house sparrow, tree sparrow and Spanish sparrow), using high-throughput sequencing. We hypothesize that sparrows could have classical and non-classical genes, as previously indicated though never tested using gene expression. The phylogenetic analyses reveal two distinct types of MHC-I alleles among the three sparrow species, one with high and one with low level of polymorphism, thus resembling classical and non-classical genes, respectively. All individuals had both types of alleles, but there was copy number variation both within and among the sparrow species. However, the number of highly polymorphic alleles that were expressed did not vary between species, suggesting that the structural genomic variation is counterbalanced by conserved gene expression. Overall, 50% of the MHC-I alleles were expressed in sparrows. Expression of the highly polymorphic alleles was very variable, whereas the alleles with low polymorphism had uniformly low expression. Interestingly, within an individual only one or two alleles from the polymorphic genes were highly expressed, indicating that only a single copy of these is highly expressed. Taken together, the phylogenetic reconstruction and the analyses of expression suggest that sparrows have both classical and non-classical MHC-I genes, and that the evolutionary origin of these genes predate the split of the three investigated sparrow species 7 million years ago. Because only the classical MHC-I genes are involved in antigen presentation, the function of different MHC-I genes should be considered in future ecological and evolutionary studies of MHC-I in sparrows and other songbirds.

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

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus

PubMed Central

Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations. PMID:26180540

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus.

PubMed

Grunert, Steffen; Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations.

Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase.

PubMed

Obexer, Richard; Godina, Alexei; Garrabou, Xavier; Mittl, Peer R E; Baker, David; Griffiths, Andrew D; Hilvert, Donald

2017-01-01

Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >10 9 rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.

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.

PSOFuzzySVM-TMH: identification of transmembrane helix segments using ensemble feature space by incorporated fuzzy support vector machine.

PubMed

Hayat, Maqsood; Tahir, Muhammad

2015-08-01

Membrane protein is a central component of the cell that manages intra and extracellular processes. Membrane proteins execute a diversity of functions that are vital for the survival of organisms. The topology of transmembrane proteins describes the number of transmembrane (TM) helix segments and its orientation. However, owing to the lack of its recognized structures, the identification of TM helix and its topology through experimental methods is laborious with low throughput. In order to identify TM helix segments reliably, accurately, and effectively from topogenic sequences, we propose the PSOFuzzySVM-TMH model. In this model, evolutionary based information position specific scoring matrix and discrete based information 6-letter exchange group are used to formulate transmembrane protein sequences. The noisy and extraneous attributes are eradicated using an optimization selection technique, particle swarm optimization, from both feature spaces. Finally, the selected feature spaces are combined in order to form ensemble feature space. Fuzzy-support vector Machine is utilized as a classification algorithm. Two benchmark datasets, including low and high resolution datasets, are used. At various levels, the performance of the PSOFuzzySVM-TMH model is assessed through 10-fold cross validation test. The empirical results reveal that the proposed framework PSOFuzzySVM-TMH outperforms in terms of classification performance in the examined datasets. It is ascertained that the proposed model might be a useful and high throughput tool for academia and research community for further structure and functional studies on transmembrane proteins.

Application of Solar Electric Propulsion to a Comet Surface Sample Return Mission

NASA Technical Reports Server (NTRS)

Cupples, Mike; Coverstone, Victoria; Woo, Byoungsam

2004-01-01

Current NSTAR (planned for the Discovery Mission: Dawn) and NASA's Evolutionary Xenon Thruster based propulsion systems were compared for a comet surface sample return mission to Tempe1 1. Mission and systems analyses were conducted over a range of array power for each propulsion system with an array of 12 kW EOL at 1 AU chosen for a baseline. Engine configurations investigated for NSTAR included 4 operational engines with 1 spare and 5 operational engines with 1 spare. The NEXT configuration investigated included 2 operational engines plus 1 spare, with performance estimated for high thrust and high Isp throttling modes. Figures of merit for this comparison include Solar Electric Propulsion dry mass, average engine throughput, and net non-propulsion payload returned to Earth flyby.

Metaorganisms as the new frontier.

PubMed

Bosch, Thomas C G; McFall-Ngai, Margaret J

2011-09-01

Because it appears that almost all organisms are part of an interdependent metaorganism, an understanding of the underlying host-microbe species associations, and of evolution and molecular underpinnings, has become the new frontier in zoology. The availability of novel high-throughput sequencing methods, together with the conceptual understanding that advances mostly originate at the intersection of traditional disciplinary boundaries, enable biologists to dissect the mechanisms that control the interdependent associations of species. In this review article, we outline some of the issues in inter-species interactions, present two case studies illuminating the necessity of interfacial research when addressing complex and fundamental zoological problems, and show that an interdisciplinary approach that seeks to understand co-evolved multi-species relationships will connect genomes, phenotypes, ecosystems and the evolutionary forces that have shaped them. We hope that this article inspires other collaborations of a similar nature on the diverse landscape commonly referred to as "zoology". Copyright © 2011 Elsevier GmbH. All rights reserved.

Minimal metabolic pathway structure is consistent with associated biomolecular interactions

PubMed Central

Bordbar, Aarash; Nagarajan, Harish; Lewis, Nathan E; Latif, Haythem; Ebrahim, Ali; Federowicz, Stephen; Schellenberger, Jan; Palsson, Bernhard O

2014-01-01

Pathways are a universal paradigm for functionally describing cellular processes. Even though advances in high-throughput data generation have transformed biology, the core of our biological understanding, and hence data interpretation, is still predicated on human-defined pathways. Here, we introduce an unbiased, pathway structure for genome-scale metabolic networks defined based on principles of parsimony that do not mimic canonical human-defined textbook pathways. Instead, these minimal pathways better describe multiple independent pathway-associated biomolecular interaction datasets suggesting a functional organization for metabolism based on parsimonious use of cellular components. We use the inherent predictive capability of these pathways to experimentally discover novel transcriptional regulatory interactions in Escherichia coli metabolism for three transcription factors, effectively doubling the known regulatory roles for Nac and MntR. This study suggests an underlying and fundamental principle in the evolutionary selection of pathway structures; namely, that pathways may be minimal, independent, and segregated. PMID:24987116

Computational Study of the Genomic and Epigenomic Phenomena

NASA Astrophysics Data System (ADS)

Yang, Wenjing

Biological systems are perhaps the ultimate complex systems, uniquely capable of processing and communicating information, reproducing in their lifetimes, and adapting in evolutionary time scales. My dissertation research focuses on using computational approaches to understand the biocomplexity manifested in the multitude of length scales and time scales. At the molecular and cellular level, central to the complex behavior of a biological system is the regulatory network. My research study focused on epigenetics, which is essential for multicellular organisms to establish cellular identity during development or in response to intracellular and environmental stimuli. My computational study of epigenomics is greatly facilitated by recent advances in high-throughput sequencing technology, which enables high-resolution snapshots of epigenomes and transcriptomes. Using human CD4+ T cell as a model system, the dynamical changes in epigenome and transcriptome pertinent to T cell activation were investigated at the genome scale. Going beyond traditional focus on transcriptional regulation, I provided evidences that post-transcriptional regulation may serve as a major component of the regulatory network. In addition, I explored alternative polyadenylation, another novel aspect of gene regulation, and how it cross-talks with the local chromatin structure. As the renowned theoretical biologist Theodosius Dobzhansky said eloquently, "Nothing in biology makes sense except in the light of evolution''. To better understand this ubiquitous driving force in the biological world, I went beyond molecular events in a single organism, and investigated the dynamical changes of population structure along the evolutionary time scale. To this end, we used HIV virus population dynamics in the host immune system as a model system. The evolution of HIV viral population plays a key role in AIDS immunopathogenesis with its exceptionally high mutation rate. However, the theoretical studies of the effect of recombination have been rather limited. Given the phylogenetic and experimental evidences for the high recombination rate and its important role in HIV evolution and epidemics, I established a mathematical model to study the effect of recombination, and explored the complex behavior of this dynamics system.

Large-Scale Comparative Phenotypic and Genomic Analyses Reveal Ecological Preferences of Shewanella Species and Identify Metabolic Pathways Conserved at the Genus Level ▿ †

PubMed Central

Rodrigues, Jorge L. M.; Serres, Margrethe H.; Tiedje, James M.

2011-01-01

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects. PMID:21642407

Phylogenetic Conflict in Bears Identified by Automated Discovery of Transposable Element Insertions in Low-Coverage Genomes.

PubMed

Lammers, Fritjof; Gallus, Susanne; Janke, Axel; Nilsson, Maria A

2017-10-01

Phylogenetic reconstruction from transposable elements (TEs) offers an additional perspective to study evolutionary processes. However, detecting phylogenetically informative TE insertions requires tedious experimental work, limiting the power of phylogenetic inference. Here, we analyzed the genomes of seven bear species using high-throughput sequencing data to detect thousands of TE insertions. The newly developed pipeline for TE detection called TeddyPi (TE detection and discovery for Phylogenetic Inference) identified 150,513 high-quality TE insertions in the genomes of ursine and tremarctine bears. By integrating different TE insertion callers and using a stringent filtering approach, the TeddyPi pipeline produced highly reliable TE insertion calls, which were confirmed by extensive in vitro validation experiments. Analysis of single nucleotide substitutions in the flanking regions of the TEs shows that these substitutions correlate with the phylogenetic signal from the TE insertions. Our phylogenomic analyses show that TEs are a major driver of genomic variation in bears and enabled phylogenetic reconstruction of a well-resolved species tree, despite strong signals for incomplete lineage sorting and introgression. The analyses show that the Asiatic black, sun, and sloth bear form a monophyletic clade, in which phylogenetic incongruence originates from incomplete lineage sorting. TeddyPi is open source and can be adapted to various TE and structural variation callers. The pipeline makes it possible to confidently extract thousands of TE insertions even from low-coverage genomes (∼10×) of nonmodel organisms. This opens new possibilities for biologists to study phylogenies and evolutionary processes as well as rates and patterns of (retro-)transposition and structural variation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The pipid root.

PubMed

Bewick, Adam J; Chain, Frédéric J J; Heled, Joseph; Evans, Ben J

2012-12-01

The estimation of phylogenetic relationships is an essential component of understanding evolution. Accurate phylogenetic estimation is difficult, however, when internodes are short and old, when genealogical discordance is common due to large ancestral effective population sizes or ancestral population structure, and when homoplasy is prevalent. Inference of divergence times is also hampered by unknown and uneven rates of evolution, the incomplete fossil record, uncertainty in relationships between fossil and extant lineages, and uncertainty in the age of fossils. Ideally, these challenges can be overcome by developing large "phylogenomic" data sets and by analyzing them with methods that accommodate features of the evolutionary process, such as genealogical discordance, recurrent substitution, recombination, ancestral population structure, gene flow after speciation among sampled and unsampled taxa, and variation in evolutionary rates. In some phylogenetic problems, it is possible to use information that is independent of fossils, such as the geological record, to identify putative triggers for diversification whose associated estimated divergence times can then be compared a posteriori with estimated relationships and ages of fossils. The history of diversification of pipid frog genera Pipa, Hymenochirus, Silurana, and Xenopus, for instance, is characterized by many of these evolutionary and analytical challenges. These frogs diversified dozens of millions of years ago, they have a relatively rich fossil record, their distributions span continental plates with a well characterized geological record of ancient connectivity, and there is considerable disagreement across studies in estimated evolutionary relationships. We used high throughput sequencing and public databases to generate a large phylogenomic data set with which we estimated evolutionary relationships using multilocus coalescence methods. We collected sequence data from Pipa, Hymenochirus, Silurana, and Xenopus and the outgroup taxon Rhinophrynus dorsalis from coding sequence of 113 autosomal regions, averaging ∼300 bp in length (range: 102-1695 bp) and also a portion of the mitochondrial genome. Analysis of these data using multiple approaches recovers strong support for the ((Xenopus, Silurana)(Pipa, Hymenochirus)) topology, and geologically calibrated divergence time estimates that are consistent with estimated ages and phylogenetic affinities of many fossils. These results provide new insights into the biogeography and chronology of pipid diversification during the breakup of Gondwanaland and illustrate how phylogenomic data may be necessary to tackle tough problems in molecular systematics. [Coalescence; gene tree; high-throughout sequencing; lineage sorting; pipid; species tree; Xenopus.].

MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.

PubMed

Kumar, Sudhir; Stecher, Glen; Tamura, Koichiro

2016-07-01

We present the latest version of the Molecular Evolutionary Genetics Analysis (Mega) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, Mega has been optimized for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of thousands of sequences in Mega The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit Mega is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OS X. The command line Mega is available as native applications for Windows, Linux, and Mac OS X. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

NASA's Evolutionary Xenon Thruster (NEXT) Long-Duration Test as of 736 kg of Propellant Throughput

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2012-01-01

The NASA s Evolutionary Xenon Thruster (NEXT) program is developing the next-generation solar-electric ion propulsion system with significant enhancements beyond the state-of-the-art NASA Solar Electric Propulsion Technology Application Readiness (NSTAR) ion propulsion system to provide future NASA science missions with enhanced mission capabilities. A Long-Duration Test (LDT) was initiated in June 2005 to validate the thruster service life modeling and to qualify the thruster propellant throughput capability. The thruster has set electric propulsion records for the longest operating duration, highest propellant throughput, and most total impulse demonstrated. At the time of this publication, the NEXT LDT has surpassed 42,100 h of operation, processed more than 736 kg of xenon propellant, and demonstrated greater than 28.1 MN s total impulse. Thruster performance has been steady with negligible degradation. The NEXT thruster design has mitigated several lifetime limiting mechanisms encountered in the NSTAR design, including the NSTAR first failure mode, thereby drastically improving thruster capabilities. Component erosion rates and the progression of the predicted life-limiting erosion mechanism for the thruster compare favorably to pretest predictions based upon semi-empirical ion thruster models used in the thruster service life assessment. Service life model validation has been accomplished by the NEXT LDT. Assuming full-power operation until test article failure, the models and extrapolated erosion data predict penetration of the accelerator grid grooves after more than 45,000 hours of operation while processing over 800 kg of xenon propellant. Thruster failure due to degradation of the accelerator grid structural integrity is expected after groove penetration.

NASA's Evolutionary Xenon Thruster (NEXT) Long-Duration Test as of 736 kg of Propellant Throughput

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2012-01-01

The NASA s Evolutionary Xenon Thruster (NEXT) program is developing the next-generation solar-electric ion propulsion system with significant enhancements beyond the state-of-the-art NASA Solar Electric Propulsion Technology Application Readiness (NSTAR) ion propulsion system to provide future NASA science missions with enhanced mission capabilities. A Long-Duration Test (LDT) was initiated in June 2005 to validate the thruster service life modeling and to qualify the thruster propellant throughput capability. The thruster has set electric propulsion records for the longest operating duration, highest propellant throughput, and most total impulse demonstrated. At the time of this publication, the NEXT LDT has surpassed 42,100 h of operation, processed more than 736 kg of xenon propellant, and demonstrated greater than 28.1 MN s total impulse. Thruster performance has been steady with negligible degradation. The NEXT thruster design has mitigated several lifetime limiting mechanisms encountered in the NSTAR design, including the NSTAR first failure mode, thereby drastically improving thruster capabilities. Component erosion rates and the progression of the predicted life-limiting erosion mechanism for the thruster compare favorably to pretest predictions based upon semi-empirical ion thruster models used in the thruster service life assessment. Service life model validation has been accomplished by the NEXT LDT. Assuming full-power operation until test article failure, the models and extrapolated erosion data predict penetration of the accelerator grid grooves after more than 45,000 hours of operation while processing over 800 kg of xenon propellant. Thruster failure due to degradation of the accelerator grid structural integrity is expected after

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.

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

Visualization of genome signatures of eukaryote genomes by batch-learning self-organizing map with a special emphasis on Drosophila genomes.

PubMed

Abe, Takashi; Hamano, Yuta; Ikemura, Toshimichi

2014-01-01

A strategy of evolutionary studies that can compare vast numbers of genome sequences is becoming increasingly important with the remarkable progress of high-throughput DNA sequencing methods. We previously established a sequence alignment-free clustering method "BLSOM" for di-, tri-, and tetranucleotide compositions in genome sequences, which can characterize sequence characteristics (genome signatures) of a wide range of species. In the present study, we generated BLSOMs for tetra- and pentanucleotide compositions in approximately one million sequence fragments derived from 101 eukaryotes, for which almost complete genome sequences were available. BLSOM recognized phylotype-specific characteristics (e.g., key combinations of oligonucleotide frequencies) in the genome sequences, permitting phylotype-specific clustering of the sequences without any information regarding the species. In our detailed examination of 12 Drosophila species, the correlation between their phylogenetic classification and the classification on the BLSOMs was observed to visualize oligonucleotides diagnostic for species-specific clustering.

Parallel changes of taxonomic interaction networks in lacustrine bacterial communities induced by a polymetallic perturbation

PubMed Central

Laplante, Karine; Sébastien, Boutin; Derome, Nicolas

2013-01-01

Heavy metals released by anthropogenic activities such as mining trigger profound changes to bacterial communities. In this study we used 16S SSU rRNA gene high-throughput sequencing to characterize the impact of a polymetallic perturbation and other environmental parameters on taxonomic networks within five lacustrine bacterial communities from sites located near Rouyn-Noranda, Quebec, Canada. The results showed that community equilibrium was disturbed in terms of both diversity and structure. Moreover, heavy metals, especially cadmium combined with water acidity, induced parallel changes among sites via the selection of resistant OTUs (Operational Taxonomic Unit) and taxonomic dominance perturbations favoring the Alphaproteobacteria. Furthermore, under a similar selective pressure, covariation trends between phyla revealed conservation and parallelism within interphylum interactions. Our study sheds light on the importance of analyzing communities not only from a phylogenetic perspective but also including a quantitative approach to provide significant insights into the evolutionary forces that shape the dynamic of the taxonomic interaction networks in bacterial communities. PMID:23789031

Update of the NEXT Ion Thruster Service Life Assessment with Post-Test Correlation to the Long Duration Test

NASA Technical Reports Server (NTRS)

Yim, John T.; Soulas, George C.; Shastry, Rohit; Choi, Maria; Mackey, Jonathan A.; Sarver-Verhey, Timothy R.

2017-01-01

The service life assessment for NASA's Evolutionary Xenon Thruster is updated to incorporate the results from the successful and voluntarily early completion of the 51,184 hour long duration test which demonstrated 918 kg of total xenon throughput. The results of the numerous post-test investigations including destructive interrogations have been assessed against all of the critical known and suspected failure mechanisms to update the life and throughput expectations for each major component. Analysis results of two of the most acute failure mechanisms, namely pit-and-groove erosion and aperture enlargement of the accelerator grid, are not updated in this work but will be published at a future time after analysis completion.

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.

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

Genomic Tools for Evolution and Conservation in the Chimpanzee: Pan troglodytes ellioti Is a Genetically Distinct Population

PubMed Central

Myers, Simon; Hellenthal, Garrett; Nerrienet, Eric; Bontrop, Ronald E.; Freeman, Colin; Donnelly, Peter; Mundy, Nicholas I.

2012-01-01

In spite of its evolutionary significance and conservation importance, the population structure of the common chimpanzee, Pan troglodytes, is still poorly understood. An issue of particular controversy is whether the proposed fourth subspecies of chimpanzee, Pan troglodytes ellioti, from parts of Nigeria and Cameroon, is genetically distinct. Although modern high-throughput SNP genotyping has had a major impact on our understanding of human population structure and demographic history, its application to ecological, demographic, or conservation questions in non-human species has been extremely limited. Here we apply these tools to chimpanzee population structure, using ∼700 autosomal SNPs derived from chimpanzee genomic data and a further ∼100 SNPs from targeted re-sequencing. We demonstrate conclusively the existence of P. t. ellioti as a genetically distinct subgroup. We show that there is clear differentiation between the verus, troglodytes, and ellioti populations at the SNP and haplotype level, on a scale that is greater than that separating continental human populations. Further, we show that only a small set of SNPs (10–20) is needed to successfully assign individuals to these populations. Tellingly, use of only mitochondrial DNA variation to classify individuals is erroneous in 4 of 54 cases, reinforcing the dangers of basing demographic inference on a single locus and implying that the demographic history of the species is more complicated than that suggested analyses based solely on mtDNA. In this study we demonstrate the feasibility of developing economical and robust tests of individual chimpanzee origin as well as in-depth studies of population structure. These findings have important implications for conservation strategies and our understanding of the evolution of chimpanzees. They also act as a proof-of-principle for the use of cheap high-throughput genomic methods for ecological questions. PMID:22396655

Genomic tools for evolution and conservation in the chimpanzee: Pan troglodytes ellioti is a genetically distinct population.

PubMed

Bowden, Rory; MacFie, Tammie S; Myers, Simon; Hellenthal, Garrett; Nerrienet, Eric; Bontrop, Ronald E; Freeman, Colin; Donnelly, Peter; Mundy, Nicholas I

2012-01-01

In spite of its evolutionary significance and conservation importance, the population structure of the common chimpanzee, Pan troglodytes, is still poorly understood. An issue of particular controversy is whether the proposed fourth subspecies of chimpanzee, Pan troglodytes ellioti, from parts of Nigeria and Cameroon, is genetically distinct. Although modern high-throughput SNP genotyping has had a major impact on our understanding of human population structure and demographic history, its application to ecological, demographic, or conservation questions in non-human species has been extremely limited. Here we apply these tools to chimpanzee population structure, using ∼700 autosomal SNPs derived from chimpanzee genomic data and a further ∼100 SNPs from targeted re-sequencing. We demonstrate conclusively the existence of P. t. ellioti as a genetically distinct subgroup. We show that there is clear differentiation between the verus, troglodytes, and ellioti populations at the SNP and haplotype level, on a scale that is greater than that separating continental human populations. Further, we show that only a small set of SNPs (10-20) is needed to successfully assign individuals to these populations. Tellingly, use of only mitochondrial DNA variation to classify individuals is erroneous in 4 of 54 cases, reinforcing the dangers of basing demographic inference on a single locus and implying that the demographic history of the species is more complicated than that suggested analyses based solely on mtDNA. In this study we demonstrate the feasibility of developing economical and robust tests of individual chimpanzee origin as well as in-depth studies of population structure. These findings have important implications for conservation strategies and our understanding of the evolution of chimpanzees. They also act as a proof-of-principle for the use of cheap high-throughput genomic methods for ecological questions.

High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

PubMed

De Barba, M; Miquel, C; Lobréaux, S; Quenette, P Y; Swenson, J E; Taberlet, P

2017-05-01

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. © 2016 John Wiley & Sons Ltd.

The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes.

PubMed

Studer, Anthony J; Schnable, James C; Weissmann, Sarit; Kolbe, Allison R; McKain, Michael R; Shao, Ying; Cousins, Asaph B; Kellogg, Elizabeth A; Brutnell, Thomas P

2016-10-28

Comparisons between C 3 and C 4 grasses often utilize C 3 species from the subfamilies Ehrhartoideae or Pooideae and C 4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C 3 panicoid grass Dichanthelium oligosanthes from the independent C 4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C 3 and C 4 grasses. We report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C 3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C 4 photosynthesis. The phylogenetic location of D. oligosanthes makes it an ideal C 3 plant for comparative analysis of C 4 evolution in the panicoid grasses. This genome will not only provide a better C 3 species for comparisons with C 4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.

The draft genome of the C 3 panicoid grass species Dichanthelium oligosanthes

DOE PAGES

Studer, Anthony J.; Schnable, James C.; Weissmann, Sarit; ...

2016-10-28

Comparisons between C 3 and C 4 grasses often utilize C 3 species from the subfamilies Ehrhartoideae or Pooideae and C 4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C 3 panicoid grass Dichanthelium oligosanthes from the independent C 4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C 3 and C 4 grasses. Here, wemore » report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C 3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C 4 photosynthesis. In conclusion, the phylogenetic location of D. oligosanthes makes it an ideal C 3 plant for comparative analysis of C 4 evolution in the panicoid grasses. This genome will not only provide a better C 3 species for comparisons with C 4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.« less

The draft genome of the C 3 panicoid grass species Dichanthelium oligosanthes

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

Studer, Anthony J.; Schnable, James C.; Weissmann, Sarit

Comparisons between C 3 and C 4 grasses often utilize C 3 species from the subfamilies Ehrhartoideae or Pooideae and C 4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C 3 panicoid grass Dichanthelium oligosanthes from the independent C 4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C 3 and C 4 grasses. Here, wemore » report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C 3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C 4 photosynthesis. In conclusion, the phylogenetic location of D. oligosanthes makes it an ideal C 3 plant for comparative analysis of C 4 evolution in the panicoid grasses. This genome will not only provide a better C 3 species for comparisons with C 4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.« less

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.

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.

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

Reconstruction of a composite comparative map composed of ten legume genomes.

PubMed

Lee, Chaeyoung; Yu, Dongwoon; Choi, Hong-Kyu; Kim, Ryan W

2017-01-01

The Fabaceae (legume family) is the third largest and the second of agricultural importance among flowering plant groups. In this study, we report the reconstruction of a composite comparative map composed of ten legume genomes, including seven species from the galegoid clade ( Medicago truncatula , Medicago sativa , Lens culinaris, Pisum sativum , Lotus japonicus , Cicer arietinum , Vicia faba ) and three species from the phaseoloid clade ( Vigna radiata , Phaseolus vulgaris , Glycine max ). To accomplish this comparison, a total of 209 cross-species gene-derived markers were employed. The comparative analysis resulted in a single extensive genetic/genomic network composed of 93 chromosomes or linkage groups, from which 110 synteny blocks and other evolutionary events (e.g., 13 inversions) were identified. This comparative map also allowed us to deduce several large scale evolutionary events, such as chromosome fusion/fission, with which might explain differences in chromosome numbers among compared species or between the two clades. As a result, useful properties of cross-species genic markers were re-verified as an efficient tool for cross-species translation of genomic information, and similar approaches, combined with a high throughput bioinformatic marker design program, should be effective for applying the knowledge of trait-associated genes to other important crop species for breeding purposes. Here, we provide a basic comparative framework for the ten legume species, and expect to be usefully applied towards the crop improvement in legume breeding.

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

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.

Ion channels in key marine invertebrates; their diversity and potential for applications in biotechnology.

PubMed

Brown, Euan R; Piscopo, Stefania

2011-01-01

Of the intra-membrane proteins, the class that comprises voltage and ligand-gated ion channels represents the major substrate whereby signals pass between and within cells in all organisms. It has been presumed that vertebrate and particularly mammalian ion channels represent the apex of evolutionary complexity and diversity and much effort has been focused on understanding their function. However, the recent availability of cheap high throughput genome sequencing has massively broadened and deepened the quality of information across phylogeny and is radically changing this view. Here we review current knowledge on such channels in key marine invertebrates where physiological evidence is backed up by molecular sequences and expression/functional studies. As marine invertebrates represent a much greater range of phyla than terrestrial vertebrates and invertebrates together, we argue that these animals represent a highly divergent, though relatively underused source of channel novelty. As ion channels are exquisitely selective sensors for voltage and ligands, their potential and actual applications in biotechnology are manifold. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Complete sequence and comparative analysis of the chloroplast genome of Plinia trunciflora

PubMed Central

Eguiluz, Maria; Yuyama, Priscila Mary; Guzman, Frank; Rodrigues, Nureyev Ferreira; Margis, Rogerio

2017-01-01

Abstract Plinia trunciflora is a Brazilian native fruit tree from the Myrtaceae family, also known as jaboticaba. This species has great potential by its fruit production. Due to the high content of essential oils in their leaves and of anthocyanins in the fruits, there is also an increasing interest by the pharmaceutical industry. Nevertheless, there are few studies focusing on its molecular biology and genetic characterization. We herein report the complete chloroplast (cp) genome of P. trunciflora using high-throughput sequencing and compare it to other previously sequenced Myrtaceae genomes. The cp genome of P. trunciflora is 159,512 bp in size, comprising inverted repeats of 26,414 bp and single-copy regions of 88,097 bp (LSC) and 18,587 bp (SSC). The genome contains 111 single-copy genes (77 protein-coding, 30 tRNA and four rRNA genes). Phylogenetic analysis using 57 cp protein-coding genes demonstrated that P. trunciflora, Eugenia uniflora and Acca sellowiana form a cluster with closer relationship to Syzygium cumini than with Eucalyptus. The complete cp sequence reported here can be used in evolutionary and population genetics studies, contributing to resolve the complex taxonomy of this species and fill the gap in genetic characterization. PMID:29111566

MultiPhyl: a high-throughput phylogenomics webserver using distributed computing

PubMed Central

Keane, Thomas M.; Naughton, Thomas J.; McInerney, James O.

2007-01-01

With the number of fully sequenced genomes increasing steadily, there is greater interest in performing large-scale phylogenomic analyses from large numbers of individual gene families. Maximum likelihood (ML) has been shown repeatedly to be one of the most accurate methods for phylogenetic construction. Recently, there have been a number of algorithmic improvements in maximum-likelihood-based tree search methods. However, it can still take a long time to analyse the evolutionary history of many gene families using a single computer. Distributed computing refers to a method of combining the computing power of multiple computers in order to perform some larger overall calculation. In this article, we present the first high-throughput implementation of a distributed phylogenetics platform, MultiPhyl, capable of using the idle computational resources of many heterogeneous non-dedicated machines to form a phylogenetics supercomputer. MultiPhyl allows a user to upload hundreds or thousands of amino acid or nucleotide alignments simultaneously and perform computationally intensive tasks such as model selection, tree searching and bootstrapping of each of the alignments using many desktop machines. The program implements a set of 88 amino acid models and 56 nucleotide maximum likelihood models and a variety of statistical methods for choosing between alternative models. A MultiPhyl webserver is available for public use at: http://www.cs.nuim.ie/distributed/multiphyl.php. PMID:17553837

Phylogenomics and barcoding of Panax: toward the identification of ginseng species.

PubMed

Manzanilla, V; Kool, A; Nguyen Nhat, L; Nong Van, H; Le Thi Thu, H; de Boer, H J

2018-04-03

The economic value of ginseng in the global medicinal plant trade is estimated to be in excess of US$2.1 billion. At the same time, the evolutionary placement of ginseng (Panax ginseng) and the complex evolutionary history of the genus is poorly understood despite several molecular phylogenetic studies. In this study, we use a full plastome phylogenomic framework to resolve relationships in Panax and to identify molecular markers for species discrimination. We used high-throughput sequencing of MBD2-Fc fractionated Panax DNA to supplement publicly available plastid genomes to create a phylogeny based on fully assembled and annotated plastid genomes from 60 accessions of 8 species. The plastome phylogeny based on a 163 kbp matrix resolves the sister relationship of Panax ginseng with P. quinquefolius. The closely related species P. vietnamensis is supported as sister of P. japonicus. The plastome matrix also shows that the markers trnC-rps16, trnS-trnG, and trnE-trnM could be used for unambiguous molecular identification of all the represented species in the genus. MBD2 depletion reduces the cost of plastome sequencing, which makes it a cost-effective alternative to Sanger sequencing based DNA barcoding for molecular identification. The plastome phylogeny provides a robust framework that can be used to study the evolution of morphological characters and biosynthesis pathways of ginsengosides for phylogenetic bioprospecting. Molecular identification of ginseng species is essential for authenticating ginseng in international trade and it provides an incentive for manufacturers to create authentic products with verified ingredients.

Protein 3D Structure Computed from Evolutionary Sequence Variation

PubMed Central

Sheridan, Robert; Hopf, Thomas A.; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

2011-01-01

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing. In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy. We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues., including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7–4.8 Å Cα-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes. PMID:22163331

Detecting Signatures of Positive Selection along Defined Branches of a Population Tree Using LSD.

PubMed

Librado, Pablo; Orlando, Ludovic

2018-06-01

Identifying the genomic basis underlying local adaptation is paramount to evolutionary biology, and bears many applications in the fields of conservation biology, crop, and animal breeding, as well as personalized medicine. Although many approaches have been developed to detect signatures of positive selection within single populations and population pairs, the increasing wealth of high-throughput sequencing data requires improved methods capable of handling multiple, and ideally large number of, populations in a single analysis. In this study, we introduce LSD (levels of exclusively shared differences), a fast and flexible framework to perform genome-wide selection scans, along the internal and external branches of a given population tree. We use forward simulations to demonstrate that LSD can identify branches targeted by positive selection with remarkable sensitivity and specificity. We illustrate a range of potential applications by analyzing data from the 1000 Genomes Project and uncover a list of adaptive candidates accompanying the expansion of anatomically modern humans out of Africa and their spread to Europe.

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution

PubMed Central

Yu, Yanbao; Leng, Taohua; Yun, Dong; Liu, Na; Yao, Jun; Dai, Ying; Yang, Pengyuan; Chen, Xian

2013-01-01

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomics technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing ‘core proteome’, and the ‘evolutionary proteome’ is actually a relatively static proteome. PMID:20443191

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

Diversity, evolution and medical applications of insect antimicrobial peptides

PubMed Central

Mylonakis, Eleftherios; Podsiadlowski, Lars; Muhammed, Maged

2016-01-01

Antimicrobial peptides (AMPs) are short proteins with antimicrobial activity. A large portion of known AMPs originate from insects, and the number and diversity of these molecules in different species varies considerably. Insect AMPs represent a potential source of alternative antibiotics to address the limitation of current antibiotics, which has been caused by the emergence and spread of multidrug-resistant pathogens. To get more insight into AMPs, we investigated the diversity and evolution of insect AMPs by mapping their phylogenetic distribution, allowing us to predict the evolutionary origins of selected AMP families and to identify evolutionarily conserved and taxon-specific families. Furthermore, we highlight the use of the nematode Caenorhabditis elegans as a whole-animal model in high-throughput screening methods to identify AMPs with efficacy against human pathogens, including Acinetobacter baumanii and methicillin-resistant Staphylococcus aureus. We also discuss the potential medical applications of AMPs, including their use as alternatives for conventional antibiotics in ectopic therapies, their combined use with antibiotics to restore the susceptibility of multidrug-resistant pathogens, and their use as templates for the rational design of peptidomimetic drugs that overcome the disadvantages of therapeutic peptides. The article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’. PMID:27160593

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Comparative qualitative phosphoproteomics analysis identifies shared phosphorylation motifs and associated biological processes in evolutionary divergent plants.

PubMed

Al-Momani, Shireen; Qi, Da; Ren, Zhe; Jones, Andrew R

2018-06-15

Phosphorylation is one of the most prevalent post-translational modifications and plays a key role in regulating cellular processes. We carried out a bioinformatics analysis of pre-existing phosphoproteomics data, to profile two model species representing the largest subclasses in flowering plants the dicot Arabidopsis thaliana and the monocot Oryza sativa, to understand the extent to which phosphorylation signaling and function is conserved across evolutionary divergent plants. We identified 6537 phosphopeptides from 3189 phosphoproteins in Arabidopsis and 2307 phosphopeptides from 1613 phosphoproteins in rice. We identified phosphorylation motifs, finding nineteen pS motifs and two pT motifs shared in rice and Arabidopsis. The majority of shared motif-containing proteins were mapped to the same biological processes with similar patterns of fold enrichment, indicating high functional conservation. We also identified shared patterns of crosstalk between phosphoserines with enrichment for motifs pSXpS, pSXXpS and pSXXXpS, where X is any amino acid. Lastly, our results identified several pairs of motifs that are significantly enriched to co-occur in Arabidopsis proteins, indicating cross-talk between different sites, but this was not observed in rice. Our results demonstrate that there are evolutionary conserved mechanisms of phosphorylation-mediated signaling in plants, via analysis of high-throughput phosphorylation proteomics data from key monocot and dicot species: rice and Arabidposis thaliana. The results also suggest that there is increased crosstalk between phosphorylation sites in A. thaliana compared with rice. The results are important for our general understanding of cell signaling in plants, and the ability to use A. thaliana as a general model for plant biology. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Automated Antenna Design with Evolutionary Algorithms

NASA Technical Reports Server (NTRS)

Hornby, Gregory S.; Globus, Al; Linden, Derek S.; Lohn, Jason D.

2006-01-01

Current methods of designing and optimizing antennas by hand are time and labor intensive, and limit complexity. Evolutionary design techniques can overcome these limitations by searching the design space and automatically finding effective solutions. In recent years, evolutionary algorithms have shown great promise in finding practical solutions in large, poorly understood design spaces. In particular, spacecraft antenna design has proven tractable to evolutionary design techniques. Researchers have been investigating evolutionary antenna design and optimization since the early 1990s, and the field has grown in recent years as computer speed has increased and electromagnetic simulators have improved. Two requirements-compliant antennas, one for ST5 and another for TDRS-C, have been automatically designed by evolutionary algorithms. The ST5 antenna is slated to fly this year, and a TDRS-C phased array element has been fabricated and tested. Such automated evolutionary design is enabled by medium-to-high quality simulators and fast modern computers to evaluate computer-generated designs. Evolutionary algorithms automate cut-and-try engineering, substituting automated search though millions of potential designs for intelligent search by engineers through a much smaller number of designs. For evolutionary design, the engineer chooses the evolutionary technique, parameters and the basic form of the antenna, e.g., single wire for ST5 and crossed-element Yagi for TDRS-C. Evolutionary algorithms then search for optimal configurations in the space defined by the engineer. NASA's Space Technology 5 (ST5) mission will launch three small spacecraft to test innovative concepts and technologies. Advanced evolutionary algorithms were used to automatically design antennas for ST5. The combination of wide beamwidth for a circularly-polarized wave and wide impedance bandwidth made for a challenging antenna design problem. From past experience in designing wire antennas, we chose to constrain the evolutionary design to a monopole wire antenna. The results of the runs produced requirements-compliant antennas that were subsequently fabricated and tested. The evolved antenna has a number of advantages with regard to power consumption, fabrication time and complexity, and performance. Lower power requirements result from achieving high gain across a wider range of elevation angles, thus allowing a broader range of angles over which maximum data throughput can be achieved. Since the evolved antenna does not require a phasing circuit, less design and fabrication work is required. In terms of overall work, the evolved antenna required approximately three person-months to design and fabricate whereas the conventional antenna required about five. Furthermore, when the mission was modified and new orbital parameters selected, a redesign of the antenna to new requirements was required. The evolutionary system was rapidly modified and a new antenna evolved in a few weeks. The evolved antenna was shown to be compliant to the ST5 mission requirements. It has an unusual organic looking structure, one that expert antenna designers would not likely produce. This antenna has been tested, baselined and is scheduled to fly this year. In addition to the ST5 antenna, our laboratory has evolved an S-band phased array antenna element design that meets the requirements for NASA's TDRS-C communications satellite scheduled for launch early next decade. A combination of fairly broad bandwidth, high efficiency and circular polarization at high gain made for another challenging design problem. We chose to constrain the evolutionary design to a crossed-element Yagi antenna. The specification called for two types of elements, one for receive only and one for transmit/receive. We were able to evolve a single element design that meets both specifications thereby simplifying the antenna and reducing testing and integration costs. The highest performance antenna found using a getic algorithm and stochastic hill-climbing has been fabricated and tested. Laboratory results correspond well with simulation. Aerospace component design is an expensive and important step in space development. Evolutionary design can make a significant contribution wherever sufficiently fast, accurate and capable software simulators are available. We have demonstrated successful real-world design in the spacecraft antenna domain; and there is good reason to believe that these results could be replicated in other design spaces.

Accelerating Virtual High-Throughput Ligand Docking: current technology and case study on a petascale supercomputer.

PubMed

Ellingson, Sally R; Dakshanamurthy, Sivanesan; Brown, Milton; Smith, Jeremy C; Baudry, Jerome

2014-04-25

In this paper we give the current state of high-throughput virtual screening. We describe a case study of using a task-parallel MPI (Message Passing Interface) version of Autodock4 [1], [2] to run a virtual high-throughput screen of one-million compounds on the Jaguar Cray XK6 Supercomputer at Oak Ridge National Laboratory. We include a description of scripts developed to increase the efficiency of the predocking file preparation and postdocking analysis. A detailed tutorial, scripts, and source code for this MPI version of Autodock4 are available online at http://www.bio.utk.edu/baudrylab/autodockmpi.htm.

Exploiting the ecogenomics toolbox for environmental diagnostics of organohalide-respiring bacteria.

PubMed

Maphosa, Farai; de Vos, Willem M; Smidt, Hauke

2010-06-01

Various 'omics' methods have enabled environmental probing at the molecular level and have created an important new paradigm in bioremediation design and management. Ecogenomics - the application of genomics to ecological and environmental sciences - defines phylogenetic and functional biodiversity at the DNA, RNA and protein levels. It capitalizes on this knowledge to elucidate functions and interactions of organisms at the ecosystem level in relation to ecological and evolutionary processes. Effective bioremediation of widespread halo-organic pollutants in anaerobic environments requires knowledge of catabolic potential and in situ dynamics of organohalide-respiring and co-metabolizing microorganisms. Here, we discuss the potential of ecogenomics approaches in developing high-throughput methods for detecting and monitoring organohalide respirers, and for providing improvements to selection, specificity and sensitivity of target biomarkers and their application to evaluate bioremediation strategies.

SigTree: A Microbial Community Analysis Tool to Identify and Visualize Significantly Responsive Branches in a Phylogenetic Tree.

PubMed

Stevens, John R; Jones, Todd R; Lefevre, Michael; Ganesan, Balasubramanian; Weimer, Bart C

2017-01-01

Microbial community analysis experiments to assess the effect of a treatment intervention (or environmental change) on the relative abundance levels of multiple related microbial species (or operational taxonomic units) simultaneously using high throughput genomics are becoming increasingly common. Within the framework of the evolutionary phylogeny of all species considered in the experiment, this translates to a statistical need to identify the phylogenetic branches that exhibit a significant consensus response (in terms of operational taxonomic unit abundance) to the intervention. We present the R software package SigTree , a collection of flexible tools that make use of meta-analysis methods and regular expressions to identify and visualize significantly responsive branches in a phylogenetic tree, while appropriately adjusting for multiple comparisons.

Coalescent Inference Using Serially Sampled, High-Throughput Sequencing Data from Intrahost HIV Infection

PubMed Central

Dialdestoro, Kevin; Sibbesen, Jonas Andreas; Maretty, Lasse; Raghwani, Jayna; Gall, Astrid; Kellam, Paul; Pybus, Oliver G.; Hein, Jotun; Jenkins, Paul A.

2016-01-01

Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, so genetic diversity within a single infection can be very high. High-throughput “deep” sequencing can now measure this diversity in unprecedented detail, particularly since it can be performed at different time points during an infection, and this offers a potentially powerful way to infer the evolutionary dynamics of the intrahost viral population. However, population genomic inference from HIV sequence data is challenging because of high rates of mutation and recombination, rapid demographic changes, and ongoing selective pressures. In this article we develop a new method for inference using HIV deep sequencing data, using an approach based on importance sampling of ancestral recombination graphs under a multilocus coalescent model. The approach further extends recent progress in the approximation of so-called conditional sampling distributions, a quantity of key interest when approximating coalescent likelihoods. The chief novelties of our method are that it is able to infer rates of recombination and mutation, as well as the effective population size, while handling sampling over different time points and missing data without extra computational difficulty. We apply our method to a data set of HIV-1, in which several hundred sequences were obtained from an infected individual at seven time points over 2 years. We find mutation rate and effective population size estimates to be comparable to those produced by the software BEAST. Additionally, our method is able to produce local recombination rate estimates. The software underlying our method, Coalescenator, is freely available. PMID:26857628

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

EPA Science Inventory

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

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.

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.

PubMed

Galan, Maxime; Guivier, Emmanuel; Caraux, Gilles; Charbonnel, Nathalie; Cosson, Jean-François

2010-05-11

High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.

Evolution versus "intelligent design": comparing the topology of protein-protein interaction networks to the Internet.

PubMed

Yang, Q; Siganos, G; Faloutsos, M; Lonardi, S

2006-01-01

Recent research efforts have made available genome-wide, high-throughput protein-protein interaction (PPI) maps for several model organisms. This has enabled the systematic analysis of PPI networks, which has become one of the primary challenges for the system biology community. In this study, we attempt to understand better the topological structure of PPI networks by comparing them against man-made communication networks, and more specifically, the Internet. Our comparative study is based on a comprehensive set of graph metrics. Our results exhibit an interesting dichotomy. On the one hand, both networks share several macroscopic properties such as scale-free and small-world properties. On the other hand, the two networks exhibit significant topological differences, such as the cliqueishness of the highest degree nodes. We attribute these differences to the distinct design principles and constraints that both networks are assumed to satisfy. We speculate that the evolutionary constraints that favor the survivability and diversification are behind the building process of PPI networks, whereas the leading force in shaping the Internet topology is a decentralized optimization process geared towards efficient node communication.

Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. Results We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. Conclusions This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1]. PMID:23163954

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

High-throughput screening, predictive modeling and computational embryology

EPA Science Inventory

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

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

The application of the high throughput sequencing technology in the transposable elements.

PubMed

Liu, Zhen; Xu, Jian-hong

2015-09-01

High throughput sequencing technology has dramatically improved the efficiency of DNA sequencing, and decreased the costs to a great extent. Meanwhile, this technology usually has advantages of better specificity, higher sensitivity and accuracy. Therefore, it has been applied to the research on genetic variations, transcriptomics and epigenomics. Recently, this technology has been widely employed in the studies of transposable elements and has achieved fruitful results. In this review, we summarize the application of high throughput sequencing technology in the fields of transposable elements, including the estimation of transposon content, preference of target sites and distribution, insertion polymorphism and population frequency, identification of rare copies, transposon horizontal transfers as well as transposon tagging. We also briefly introduce the major common sequencing strategies and algorithms, their advantages and disadvantages, and the corresponding solutions. Finally, we envision the developing trends of high throughput sequencing technology, especially the third generation sequencing technology, and its application in transposon studies in the future, hopefully providing a comprehensive understanding and reference for related scientific researchers.

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

Acceleration performance of individual European sea bass Dicentrarchus labrax measured with a sprint performance chamber: comparison with high-speed cinematography and correlates with ecological performance.

PubMed

Vandamm, Joshua P; Marras, Stefano; Claireaux, Guy; Handelsman, Corey A; Nelson, Jay A

2012-01-01

Locomotor performance can influence the ecological and evolutionary success of a species. For fish, favorable outcomes of predator-prey encounters are often presumably due to robust acceleration ability. Although escape-response or "fast-start" studies utilizing high-speed cinematography are prevalent, little is known about the contribution of relative acceleration performance to ecological or evolutionary success in a species. This dearth of knowledge may be due to the time-consuming nature of analyzing film, which imposes a practical limit on sample sizes. Herein, we present a high-throughput potential alternative for measuring fish acceleration performance using a sprint performance chamber (SPC). The acceleration performance of a large number of juvenile European sea bass (Dicentrarchus labrax) from two populations was analyzed. Animals from both hatchery and natural ontogenies were assessed, and animals of known acceleration ability had their ecological performance measured in a mesocosm environment. Individuals from one population also had their acceleration performance assessed by both high-speed cinematography and an SPC. Acceleration performance measured in an SPC was lower than that measured by classical high-speed video techniques. However, short-term repeatability and interindividual variation of acceleration performance were similar between the two techniques, and the SPC recorded higher sprint swimming velocities. Wild fish were quicker to accelerate in an SPC and had significantly greater accelerations than all groups of hatchery-raised fish. Acceleration performance had no significant effect on ecological performance (as assessed through animal growth and survival in the mesocosms). However, it is worth noting that wild animals did survive predation in the mesocosm better than farmed ones. Moreover, the hatchery-originated fish that survived the mesocosm experiment, when no predators were present, displayed significantly increased acceleration performance during their 6 mo in the mesocosm; this performance was found to be inversely proportional to growth rate.

Biophysical Fitness Landscapes for Transcription Factor Binding Sites

PubMed Central

Haldane, Allan; Manhart, Michael; Morozov, Alexandre V.

2014-01-01

Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs) and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions. PMID:25010228

The Impact of Data Fragmentation on High-Throughput Clinical Phenotyping

ERIC Educational Resources Information Center

Wei, Weiqi

2012-01-01

Subject selection is essential and has become the rate-limiting step for harvesting knowledge to advance healthcare through clinical research. Present manual approaches inhibit researchers from conducting deep and broad studies and drawing confident conclusions. High-throughput clinical phenotyping (HTCP), a recently proposed approach, leverages…

Population genomics and the causes of local differentiation.

PubMed

Tonsor, Stephen J

2012-11-01

Exactly 50 years ago, a revolution in empirical population genetics began with the introduction of methods for detecting allelic variation using protein electrophoresis (Throckmorton 1962; Hubby 1963; Lewontin & Hubby 1966). These pioneering scientists showed that populations are chock-full of genetic variation. This variation was a surprise that required a re-thinking of evolutionary genetic heuristics. Understanding the causes for the maintenance of this variation became and remains a major area of research. In the process of addressing the causes, this same group of scientists documented geographical genetic structure (Prakash et al. 1969), spawning the continued accumulation of what is now a huge case study catalogue of geographical differentiation (e.g. Loveless & Hamrick 1984; Linhart & Grant 1996). Geographical differentiation is clearly quite common. Yet, a truly general understanding of the patterns in and causes of spatial genetic structure across the genome remains elusive. To what extent is spatial structure driven by drift and phylogeography vs. geographical differences in environmental sources of selection? What proportion of the genome participates? A general understanding requires range-wide data on spatial patterning of variation across the entire genome. In this issue of Molecular Ecology, Lasky et al. (2012) make important strides towards addressing these issues, taking advantage of three contemporary revolutions in evolutionary biology. Two are technological: high-throughput sequencing and burgeoning computational power. One is cultural: open access to data from the community of scientists and especially data sets that result from large collaborative efforts. Together, these developments may at last put answers within reach.

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.

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.

Bayesian estimation and use of high-throughput remote sensing indices for quantitative genetic analyses of leaf growth.

PubMed

Baker, Robert L; Leong, Wen Fung; An, Nan; Brock, Marcus T; Rubin, Matthew J; Welch, Stephen; Weinig, Cynthia

2018-02-01

We develop Bayesian function-valued trait models that mathematically isolate genetic mechanisms underlying leaf growth trajectories by factoring out genotype-specific differences in photosynthesis. Remote sensing data can be used instead of leaf-level physiological measurements. Characterizing the genetic basis of traits that vary during ontogeny and affect plant performance is a major goal in evolutionary biology and agronomy. Describing genetic programs that specifically regulate morphological traits can be complicated by genotypic differences in physiological traits. We describe the growth trajectories of leaves using novel Bayesian function-valued trait (FVT) modeling approaches in Brassica rapa recombinant inbred lines raised in heterogeneous field settings. While frequentist approaches estimate parameter values by treating each experimental replicate discretely, Bayesian models can utilize information in the global dataset, potentially leading to more robust trait estimation. We illustrate this principle by estimating growth asymptotes in the face of missing data and comparing heritabilities of growth trajectory parameters estimated by Bayesian and frequentist approaches. Using pseudo-Bayes factors, we compare the performance of an initial Bayesian logistic growth model and a model that incorporates carbon assimilation (A max ) as a cofactor, thus statistically accounting for genotypic differences in carbon resources. We further evaluate two remotely sensed spectroradiometric indices, photochemical reflectance (pri2) and MERIS Terrestrial Chlorophyll Index (mtci) as covariates in lieu of A max , because these two indices were genetically correlated with A max across years and treatments yet allow much higher throughput compared to direct leaf-level gas-exchange measurements. For leaf lengths in uncrowded settings, including A max improves model fit over the initial model. The mtci and pri2 indices also outperform direct A max measurements. Of particular importance for evolutionary biologists and plant breeders, hierarchical Bayesian models estimating FVT parameters improve heritabilities compared to frequentist approaches.

20180312 - Applying a High-Throughput PBTK Model for IVIVE (SOT)

EPA Science Inventory

The ability to link in vitro and in vivo toxicity enables the use of high-throughput in vitro assays as an alternative to resource intensive animal studies. Toxicokinetics (TK) should help describe this link, but prior work found weak correlation when using a TK model for in vitr...

Applying a High-Throughput PBTK Model for IVIVE

EPA Science Inventory

The ability to link in vitro and in vivo toxicity enables the use of high-throughput in vitro assays as an alternative to resource intensive animal studies. Toxicokinetics (TK) should help describe this link, but prior work found weak correlation when using a TK model for in vitr...

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

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

PubMed

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

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

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

BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

PubMed Central

Lutsik, Pavlo; Feuerbach, Lars; Arand, Julia; Lengauer, Thomas; Walter, Jörn; Bock, Christoph

2011-01-01

Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data. PMID:21565797

Tackling Drug Resistant Infection Outbreaks of Global Pandemic Escherichia coli ST131 Using Evolutionary and Epidemiological Genomics

PubMed Central

Downing, Tim

2015-01-01

High-throughput molecular screening is required to investigate the origin and diffusion of antimicrobial resistance in pathogen outbreaks. The most frequent cause of human infection is Escherichia coli, which is dominated by sequence type 131 (ST131)—a set of rapidly radiating pandemic clones. The highly infectious clades of ST131 originated firstly by a mutation enhancing conjugation and adhesion. Secondly, single-nucleotide polymorphisms occurred enabling fluoroquinolone-resistance, which is near-fixed in all ST131. Thirdly, broader resistance through beta-lactamases has been gained and lost frequently, symptomatic of conflicting environmental selective effects. This flexible approach to gene exchange is worrying and supports the proposition that ST131 will develop an even wider range of plasmid and chromosomal elements promoting antimicrobial resistance. To stop ST131, deep genome sequencing is required to understand the origin, evolution and spread of antimicrobial resistance genes. Phylogenetic methods that decipher past events can predict future patterns of virulence and transmission based on genetic signatures of adaptation and gene exchange. Both the effect of partial antimicrobial exposure and cell dormancy caused by variation in gene expression may accelerate the development of resistance. High-throughput sequencing can decode measurable evolution of cell populations within patients associated with systems-wide changes in gene expression during treatments. A multi-faceted approach can enhance assessment of antimicrobial resistance in E. coli ST131 by examining transmission dynamics between hosts to achieve a goal of pre-empting resistance before it emerges by optimising antimicrobial treatment protocols. PMID:27682088

A deep sequencing reveals significant diversity among dominant variants and evolutionary dynamics of avian leukosis viruses in two infectious ecosystems.

PubMed

Meng, Fanfeng; Dong, Xuan; Hu, Tao; Chang, Shuang; Fan, Jianhua; Zhao, Peng; Cui, Zhizhong

2016-12-19

As a typical retrovirus, the evolution of Avian leukosis virus subgroup J (ALV-J) in different infectious ecosystems is not characterized, what we know is there are a cloud of diverse variants, namely quasispecies with considerable genetic diversity. This study is to explore the selection of infectious ecosystems on dominant variants and their evolutionary dynamics of ALV-J between DF1 cells and specific-pathogen-free (SPF) chickens. High-throughput sequencing platforms provide an approach for detecting quasispecies diversity more fully. An average of about 20,000 valid reads were obtained from two variable regions of gp85 gene and LTR-U3 region from each sample in different infectious ecosystems. The top 10 dominant variants among ALV-J from chicken plasmas, DF1 cells and liver tumor were completely different from each other. Also there was a difference of shannon entropy and global selection pressure values (ω) in different infectious ecosystems. In the plasmas of two chickens, a large portion of quasispecies contained a 3-peptides "LSD" repeat insertion that was only less than 0.01% in DF1 cell culture supernatants. In parallel studies, the LTR-U3 region of ALV-J from the chicken plasmas demonstrated more variants with mutations in their transcription regulatory elements than those from DF1 cells. Our data taken together suggest that the molecular epidemiology based on isolated ALV-J in cell culture may not represent the true evolution of virus in chicken flocks in the field. The biological significance of the "LSD" insert and mutations in LTR-U3 needs to be further studied.

From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

PubMed Central

2014-01-01

Background Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources. PMID:24460871

MosaicSolver: a tool for determining recombinants of viral genomes from pileup data

PubMed Central

Wood, Graham R.; Ryabov, Eugene V.; Fannon, Jessica M.; Moore, Jonathan D.; Evans, David J.; Burroughs, Nigel

2014-01-01

Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, contributing to changes in tropism and possibly assisting transmission across species barriers. The ability to determine whether recombination has occurred and to locate associated specific recombination junctions is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method from both siRNAs and reads sampling the viral genome population (cDNA library); our results are confirmed experimentally. Matlab software (MosaicSolver) is available. PMID:25120266

Satellite DNA: An Evolving Topic

PubMed Central

Garrido-Ramos, Manuel A.

2017-01-01

Satellite DNA represents one of the most fascinating parts of the repetitive fraction of the eukaryotic genome. Since the discovery of highly repetitive tandem DNA in the 1960s, a lot of literature has extensively covered various topics related to the structure, organization, function, and evolution of such sequences. Today, with the advent of genomic tools, the study of satellite DNA has regained a great interest. Thus, Next-Generation Sequencing (NGS), together with high-throughput in silico analysis of the information contained in NGS reads, has revolutionized the analysis of the repetitive fraction of the eukaryotic genomes. The whole of the historical and current approaches to the topic gives us a broad view of the function and evolution of satellite DNA and its role in chromosomal evolution. Currently, we have extensive information on the molecular, chromosomal, biological, and population factors that affect the evolutionary fate of satellite DNA, knowledge that gives rise to a series of hypotheses that get on well with each other about the origin, spreading, and evolution of satellite DNA. In this paper, I review these hypotheses from a methodological, conceptual, and historical perspective and frame them in the context of chromosomal organization and evolution. PMID:28926993

Interspecific Plastome Recombination Reflects Ancient Reticulate Evolution in Picea (Pinaceae).

PubMed

Sullivan, Alexis R; Schiffthaler, Bastian; Thompson, Stacey Lee; Street, Nathaniel R; Wang, Xiao-Ru

2017-07-01

Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of ∼35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce (P. abies) and 10 other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing

USDA-ARS?s Scientific Manuscript database

The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High quality samples of raw buffalo milk were obtained from five dairy farms in the Guangxi province of China. A sample of each milk was pasteurized, and both r...

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

PubMed

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Genome-Wide Characterization and Expression Profiling of the AUXIN RESPONSE FACTOR (ARF) Gene Family in Eucalyptus grandis

PubMed Central

Yu, Hong; Soler, Marçal; Mila, Isabelle; San Clemente, Hélène; Savelli, Bruno; Dunand, Christophe; Paiva, Jorge A. P.; Myburg, Alexander A.; Bouzayen, Mondher; Grima-Pettenati, Jacqueline; Cassan-Wang, Hua

2014-01-01

Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF) are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation. PMID:25269088

Proteome-wide Prediction of Self-interacting Proteins Based on Multiple Properties*

PubMed Central

Liu, Zhongyang; Guo, Feifei; Zhang, Jiyang; Wang, Jian; Lu, Liang; Li, Dong; He, Fuchu

2013-01-01

Self-interacting proteins, whose two or more copies can interact with each other, play important roles in cellular functions and the evolution of protein interaction networks (PINs). Knowing whether a protein can self-interact can contribute to and sometimes is crucial for the elucidation of its functions. Previous related research has mainly focused on the structures and functions of specific self-interacting proteins, whereas knowledge on their overall properties is limited. Meanwhile, the two current most common high throughput protein interaction assays have limited ability to detect self-interactions because of biological artifacts and design limitations, whereas the bioinformatic prediction method of self-interacting proteins is lacking. This study aims to systematically study and predict self-interacting proteins from an overall perspective. We find that compared with other proteins the self-interacting proteins in the structural aspect contain more domains; in the evolutionary aspect they tend to be conserved and ancient; in the functional aspect they are significantly enriched with enzyme genes, housekeeping genes, and drug targets, and in the topological aspect tend to occupy important positions in PINs. Furthermore, based on these features, after feature selection, we use logistic regression to integrate six representative features, including Gene Ontology term, domain, paralogous interactor, enzyme, model organism self-interacting protein, and betweenness centrality in the PIN, to develop a proteome-wide prediction model of self-interacting proteins. Using 5-fold cross-validation and an independent test, this model shows good performance. Finally, the prediction model is developed into a user-friendly web service SLIPPER (SeLf-Interacting Protein PrEdictoR). Users may submit a list of proteins, and then SLIPPER will return the probability_scores measuring their possibility to be self-interacting proteins and various related annotation information. This work helps us understand the role self-interacting proteins play in cellular functions from an overall perspective, and the constructed prediction model may contribute to the high throughput finding of self-interacting proteins and provide clues for elucidating their functions. PMID:23422585

RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes

PubMed Central

Singh, Guramrit; Ricci, Emiliano P.; Moore, Melissa J.

2013-01-01

Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. PMID:24096052

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

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution.

PubMed

Renner, Daniel W; Szpara, Moriah L

2018-01-01

Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Copyright © 2017 Renner and Szpara.

A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase.

PubMed

Bond, Thomas E H; Sorenson, Alanna E; Schaeffer, Patrick M

2017-12-01

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔT m =+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (K obs =7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns. Copyright © 2017 Elsevier GmbH. All rights reserved.

Covariance Matrix Adaptation Evolutionary Strategy for Drift Correction of Electronic Nose Data

NASA Astrophysics Data System (ADS)

Di Carlo, S.; Falasconi, M.; Sanchez, E.; Sberveglieri, G.; Scionti, A.; Squillero, G.; Tonda, A.

2011-09-01

Electronic Noses (ENs) might represent a simple, fast, high sample throughput and economic alternative to conventional analytical instruments [1]. However, gas sensors drift still limits the EN adoption in real industrial setups due to high recalibration effort and cost [2]. In fact, pattern recognition (PaRC) models built in the training phase become useless after a period of time, in some cases a few weeks. Although algorithms to mitigate the drift date back to the early 90 this is still a challenging issue for the chemical sensor community [3]. Among other approaches, adaptive drift correction methods adjust the PaRC model in parallel with data acquisition without need of periodic calibration. Self-Organizing Maps (SOMs) [4] and Adaptive Resonance Theory (ART) networks [5] have been already tested in the past with fair success. This paper presents and discusses an original methodology based on a Covariance Matrix Adaptation Evolution Strategy (CMA-ES) [6], suited for stochastic optimization of complex problems.

Fluorescence-based high-throughput screening of dicer cleavage activity.

PubMed

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

PubMed

Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

2017-04-01

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved.

Comparative aerial- and ground-based high-throughput phenotyping for the genetic dissection of NDVI as a proxy for drought-adaptive traits in durum wheat

USDA-ARS?s Scientific Manuscript database

High-throughput phenotyping platforms (HTPPs) provide novel opportunities to more effectively dissect the genetic basis of drought-adaptive traits. This genome-wide association study (GWAS) compares the results obtained with two Unmanned Aerial Vehicles (UAVs) and a ground-based platform used to mea...

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

EPA Science Inventory

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

Moving Toward Integrating Gene Expression Profiling into High-throughput Testing:A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium

EPA Science Inventory

Microarray profiling of chemical-induced effects is being increasingly used in medium and high-throughput formats. In this study, we describe computational methods to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), ...

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

Microfluidics in microbiology: putting a magnifying glass on microbes.

PubMed

Siddiqui, Sanya; Tufenkji, Nathalie; Moraes, Christopher

2016-09-12

Microfluidic technologies enable unique studies in the field of microbiology to facilitate our understanding of microorganisms. Using miniaturized and high-throughput experimental capabilities in microfluidics, devices with controlled microenvironments can be created for microbial studies in research fields such as healthcare and green energy. In this research highlight, we describe recently developed tools for diagnostic assays, high-throughput mutant screening, and the study of human disease development as well as a future outlook on microbes for renewable energy.

Kleptoplast Regulation by an Antarctic Dinoflagellate

NASA Astrophysics Data System (ADS)

Gast, R. J.; Hehenberger, E.; Keeling, P.

2016-02-01

We are studying the evolutionary history and expression of plastid- targeted genes in an Antarctic dinoflagellate that steals chloroplasts from the haptophyte, Phaeocystis. Our project seeks to determine whether the kleptoplastidic dinoflagellate utilizes ancestral plastid proteins to regulate its stolen plastid, and how their transcription is related to environmental factors that are relevant to the Southern Ocean environment (temperature and light). To accomplish our goals, we have utilized high throughput transciptome analysis and RNA-Seq experiments of the dinoflagellate and Phaeocystis. Analysis of the dinoflagellate transcriptome has revealed complete mevalonic acid-independent and heme plastid-associated pathways as well as petF and petH transcripts with peridinin-plastid targeting sequences. In contrast, the proteins psaE, petJ, petC show similarity to non-Phaeocystis haptophyte homologs in their respective trees, and potentially carry haptophyte transit peptides. Anaylsis of RNA-Seq temperature and light experiments for the dinoflagellate indicate that there are significant differences in gene expression under the different environmental conditions, and we are in the process of identifying the genes associated with these changes. This work will help us to understand the environmental success of this alternative nutritional strategy.

Contrasting Microbial Community Assembly Hypotheses: A Reconciling Tale from the Río Tinto

PubMed Central

Palacios, Carmen; Zettler, Erik; Amils, Ricardo; Amaral-Zettler, Linda

2008-01-01

Background The Río Tinto (RT) is distinguished from other acid mine drainage systems by its natural and ancient origins. Microbial life from all three domains flourishes in this ecosystem, but bacteria dominate metabolic processes that perpetuate environmental extremes. While the patchy geochemistry of the RT likely influences the dynamics of bacterial populations, demonstrating which environmental variables shape microbial diversity and unveiling the mechanisms underlying observed patterns, remain major challenges in microbial ecology whose answers rely upon detailed assessments of community structures coupled with fine-scale measurements of physico-chemical parameters. Methodology/Principal Findings By using high-throughput environmental tag sequencing we achieved saturation of richness estimators for the first time in the RT. We found that environmental factors dictate the distribution of the most abundant taxa in this system, but stochastic niche differentiation processes, such as mutation and dispersal, also contribute to observed diversity patterns. Conclusions/Significance We predict that studies providing clues to the evolutionary and ecological processes underlying microbial distributions will reconcile the ongoing debate between the Baas Becking vs. Hubbell community assembly hypotheses. PMID:19052647

Contrasting microbial community assembly hypotheses: a reconciling tale from the Río Tinto.

PubMed

Palacios, Carmen; Zettler, Erik; Amils, Ricardo; Amaral-Zettler, Linda

2008-01-01

The Río Tinto (RT) is distinguished from other acid mine drainage systems by its natural and ancient origins. Microbial life from all three domains flourishes in this ecosystem, but bacteria dominate metabolic processes that perpetuate environmental extremes. While the patchy geochemistry of the RT likely influences the dynamics of bacterial populations, demonstrating which environmental variables shape microbial diversity and unveiling the mechanisms underlying observed patterns, remain major challenges in microbial ecology whose answers rely upon detailed assessments of community structures coupled with fine-scale measurements of physico-chemical parameters. By using high-throughput environmental tag sequencing we achieved saturation of richness estimators for the first time in the RT. We found that environmental factors dictate the distribution of the most abundant taxa in this system, but stochastic niche differentiation processes, such as mutation and dispersal, also contribute to observed diversity patterns. We predict that studies providing clues to the evolutionary and ecological processes underlying microbial distributions will reconcile the ongoing debate between the Baas Becking vs. Hubbell community assembly hypotheses.

Diversity and evolution of the primate skin microbiome

PubMed Central

Council, Sarah E.; Savage, Amy M.; Urban, Julie M.; Ehlers, Megan E.; Skene, J. H. Pate; Platt, Michael L.; Dunn, Robert R.; Horvath, Julie E.

2016-01-01

Skin microbes play a role in human body odour, health and disease. Compared with gut microbes, we know little about the changes in the composition of skin microbes in response to evolutionary changes in hosts, or more recent behavioural and cultural changes in humans. No studies have used sequence-based approaches to consider the skin microbe communities of gorillas and chimpanzees, for example. Comparison of the microbial associates of non-human primates with those of humans offers unique insights into both the ancient and modern features of our skin-associated microbes. Here we describe the microbes found on the skin of humans, chimpanzees, gorillas, rhesus macaques and baboons. We focus on the bacterial and archaeal residents in the axilla using high-throughput sequencing of the 16S rRNA gene. We find that human skin microbial communities are unique relative to those of other primates, in terms of both their diversity and their composition. These differences appear to reflect both ancient shifts during millions of years of primate evolution and more recent changes due to modern hygiene. PMID:26763711

The challenges of modelling antibody repertoire dynamics in HIV infection

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

Luo, Shishi; Perelson, Alan S.

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

The challenges of modelling antibody repertoire dynamics in HIV infection

DOE PAGES

Luo, Shishi; Perelson, Alan S.

2015-07-20

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

'Enzyme Test Bench': A biochemical application of the multi-rate modeling

NASA Astrophysics Data System (ADS)

Rachinskiy, K.; Schultze, H.; Boy, M.; Büchs, J.

2008-11-01

In the expanding field of 'white biotechnology' enzymes are frequently applied to catalyze the biochemical reaction from a resource material to a valuable product. Evolutionary designed to catalyze the metabolism in any life form, they selectively accelerate complex reactions under physiological conditions. Modern techniques, such as directed evolution, have been developed to satisfy the increasing demand on enzymes. Applying these techniques together with rational protein design, we aim at improving of enzymes' activity, selectivity and stability. To tap the full potential of these techniques, it is essential to combine them with adequate screening methods. Nowadays a great number of high throughput colorimetric and fluorescent enzyme assays are applied to measure the initial enzyme activity with high throughput. However, the prediction of enzyme long term stability within short experiments is still a challenge. A new high throughput technique for enzyme characterization with specific attention to the long term stability, called 'Enzyme Test Bench', is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests conducted under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimum non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The results of the characterization, based on micro liter format experiments of hours, are in good agreement with the results of long term experiments in 1L format. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature. The presented article gives a successful example for the application of multi-rate modeling, experimental design and parameter estimation within biochemical engineering. At the same time, it shows the limitations of the methods at the state of the art and addresses the current problems to the applied mathematics community.

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.

A High-Throughput Biological Calorimetry Core: Steps to Startup, Run, and Maintain a Multiuser Facility.

PubMed

Yennawar, Neela H; Fecko, Julia A; Showalter, Scott A; Bevilacqua, Philip C

2016-01-01

Many labs have conventional calorimeters where denaturation and binding experiments are setup and run one at a time. While these systems are highly informative to biopolymer folding and ligand interaction, they require considerable manual intervention for cleaning and setup. As such, the throughput for such setups is limited typically to a few runs a day. With a large number of experimental parameters to explore including different buffers, macromolecule concentrations, temperatures, ligands, mutants, controls, replicates, and instrument tests, the need for high-throughput automated calorimeters is on the rise. Lower sample volume requirements and reduced user intervention time compared to the manual instruments have improved turnover of calorimetry experiments in a high-throughput format where 25 or more runs can be conducted per day. The cost and efforts to maintain high-throughput equipment typically demands that these instruments be housed in a multiuser core facility. We describe here the steps taken to successfully start and run an automated biological calorimetry facility at Pennsylvania State University. Scientists from various departments at Penn State including Chemistry, Biochemistry and Molecular Biology, Bioengineering, Biology, Food Science, and Chemical Engineering are benefiting from this core facility. Samples studied include proteins, nucleic acids, sugars, lipids, synthetic polymers, small molecules, natural products, and virus capsids. This facility has led to higher throughput of data, which has been leveraged into grant support, attracting new faculty hire and has led to some exciting publications. © 2016 Elsevier Inc. All rights reserved.

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...

Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data

PubMed Central

Fujimori, Shigeo; Hirai, Naoya; Ohashi, Hiroyuki; Masuoka, Kazuyo; Nishikimi, Akihiko; Fukui, Yoshinori; Washio, Takanori; Oshikubo, Tomohiro; Yamashita, Tatsuhiro; Miyamoto-Sato, Etsuko

2012-01-01

Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem. Here, we describe a cell-free display technology combined with NGS that can improve both the coverage and reliability of interactome datasets. The completely cell-free method gives a high-throughput and a large detection space, testing the interactions without using clones. The quantitative information provided by NGS reduces the number of false positives. The method is suitable for the in vitro detection of proteins that interact not only with the bait protein, but also with DNA, RNA and chemical compounds. Thus, it could become a universal approach for exploring the large space of protein sequences and interactome networks. PMID:23056904

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis.

PubMed

Morrison, Cheryl L; Iwanowicz, Luke; Work, Thierry M; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deb; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

USGS Publications Warehouse

Morrison, Cheryl L.; Iwanowicz, Luke R.; Work, Thierry M.; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deborah; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S.

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

PubMed Central

Iwanowicz, Luke; Work, Thierry M.; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deb; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S.

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants. PMID:29479497

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

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Perspective: Composition–structure–property mapping in high-throughput experiments: Turning data into knowledge

DOE PAGES

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

2016-05-26

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

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...

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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.

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

PubMed

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

2017-01-01

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.

Combining high-throughput sequencing with fruit body surveys reveals contrasting life-history strategies in fungi

PubMed Central

Ovaskainen, Otso; Schigel, Dmitry; Ali-Kovero, Heini; Auvinen, Petri; Paulin, Lars; Nordén, Björn; Nordén, Jenni

2013-01-01

Before the recent revolution in molecular biology, field studies on fungal communities were mostly confined to fruit bodies, whereas mycelial interactions were studied in the laboratory. Here we combine high-throughput sequencing with a fruit body inventory to study simultaneously mycelial and fruit body occurrences in a community of fungi inhabiting dead wood of Norway spruce. We studied mycelial occurrence by extracting DNA from wood samples followed by 454-sequencing of the ITS1 and ITS2 regions and an automated procedure for species identification. In total, we detected 198 species as mycelia and 137 species as fruit bodies. The correlation between mycelial and fruit body occurrences was high for the majority of the species, suggesting that high-throughput sequencing can successfully characterize the dominating fungal communities, despite possible biases related to sampling, PCR, sequencing and molecular identification. We used the fruit body and molecular data to test hypothesized links between life history and population dynamic parameters. We show that the species that have on average a high mycelial abundance also have a high fruiting rate and produce large fruit bodies, leading to a positive feedback loop in their population dynamics. Earlier studies have shown that species with specialized resource requirements are rarely seen fruiting, for which reason they are often classified as red-listed. We show with the help of high-throughput sequencing that some of these species are more abundant as mycelium in wood than what could be expected from their occurrence as fruit bodies. PMID:23575372

Extending Ion Engine Technology to NEXT and Beyond

NASA Technical Reports Server (NTRS)

Domonkos, Matthew T.; Patterson, Michael J.; Foster, John E.; Rawlin, Vince K.; Soulas, George C.; Sovey, James S.; Kovaleski, Scott D.; Roman, Robert F.; Williams, George J., Jr.; Lyons, Valerie J. (Technical Monitor)

2002-01-01

Extending ion engine technology beyond the current state-of-the art primary interplanetary electric propulsion system, the 2.3-kW NASA Solar Electric Propulsion Technology and Applications Readiness (NSTAR) system, will require thrusters with improved propellant throughput and total impulse capability. Many of the design choices that culminated in the NSTAR thrusters must be revisited, and their application to next generation ion engine technology must be evaluated. The concept of derating, which was successfully employed in NSTAR, has been applied to the 40 cm NASA Evolutionary Xenon Thruster (NEXT) currently under development at NASA Glenn Research Center (GRC). At 5-kW, NEXT operates with the same average beam current density as NSTAR, and at 10-kW, the peak beam current density is only ten percent greater than NSTAR. The result is that similar Ion optics technology is expected to yield comparable lifetime. Thick-accelerator- grid ion optics are also being tested to realize additional lifetime benefits. A 40-A discharge cathode is being developed for NEXT based on scaling the NSTAR design. Nevertheless, the experiences of the NSTAR ground tests and the thruster on the Deep Space One spacecraft indicate that the discharge cathode wear must be studied experimentally and theoretically to ensure that it meets the lifetime requirements. Although NEXT is in its infancy, investigations have already begun to examine possible modifications to engine design for even higher-power and higher-specific impulse engines. Ion optics using alternate materials such as titanium, graphite, or carbon-carbon composite are currently being investigated due to their low sputter yields at high voltage. To avoid the difficulties encountered using electrodes at high-currents, the use of a microwave-based ion thruster is under investigation for potential high-power ion thruster systems requiring long lifetimes. Additionally, alternative propellants are being considered for applications requiring high-specific impulse (>> 5000 s) and extremely long-life (>> 15,000 hr). Testing requirements make condensable propellants attractive for high-power engines. Although the NSTAR ion engine demonstrated the flight maturity of ion thruster technology, many challenges remain for the development of thrusters with improved propellant throughput and power handling capabilities.

Assessing the genetic diversity of Cu resistance in mine tailings through high-throughput recovery of full-length copA genes

PubMed Central

Li, Xiaofang; Zhu, Yong-Guan; Shaban, Babak; Bruxner, Timothy J. C.; Bond, Philip L.; Huang, Longbin

2015-01-01

Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses. PMID:26286020

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).

Automated mini-column solid-phase extraction cleanup for high-throughput analysis of chemical contaminants in foods by low-pressure gas chromatography – tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

This study demonstrated the application of an automated high-throughput mini-cartridge solid-phase extraction (mini-SPE) cleanup for the rapid low-pressure gas chromatography – tandem mass spectrometry (LPGC-MS/MS) analysis of pesticides and environmental contaminants in QuEChERS extracts of foods. ...

From drug to protein: using yeast genetics for high-throughput target discovery.

PubMed

Armour, Christopher D; Lum, Pek Yee

2005-02-01

The budding yeast Saccharomyces cerevisiae has long been an effective eukaryotic model system for understanding basic cellular processes. The genetic tractability and ease of manipulation in the laboratory make yeast well suited for large-scale chemical and genetic screens. Several recent studies describing the use of yeast genetics for high-throughput drug target identification are discussed in this review.

Relative Impact of Incorporating Pharmacokinetics on Predicting In Vivo Hazard and Mode of Action from High-Throughput In Vitro Toxicity Assays

EPA Science Inventory

The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo e...

Detecting adulterants in milk powder using high-throughput Raman chemical imaging

USDA-ARS?s Scientific Manuscript database

This study used a line-scan high-throughput Raman imaging system to authenticate milk powder. A 5 W 785 nm line laser (240 mm long and 1 mm wide) was used as a Raman excitation source. The system was used to acquire hyperspectral Raman images in a wavenumber range of 103–2881 cm-1 from the skim milk...

Detection and quantification of adulterants in milk powder using high-throughput Raman chemical imaging technique

USDA-ARS?s Scientific Manuscript database

Milk is a vulnerable target for economically motivated adulteration. In this study, a line-scan high-throughput Raman imaging system was used to authenticate milk powder. A 5 W 785 nm line laser (240 mm long and 1 mm wide) was used as a Raman excitation source. The system was used to acquire hypersp...

Characterizing visible and invisible cell wall mutant phenotypes

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

Carpita, Nicholas C.; McCann, Maureen C.

2015-04-06

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basismore » of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.« less

Chromosome-scale selective sweeps shape Caenorhabditis elegans genomic diversity

PubMed Central

Andersen, Erik C.; Gerke, Justin P.; Shapiro, Joshua A.; Crissman, Jonathan R.; Ghosh, Rajarshi; Bloom, Joshua S.; Félix, Marie-Anne; Kruglyak, Leonid

2011-01-01

The nematode Caenorhabditis elegans is central to research in molecular, cell, and developmental biology, but nearly all of this research has been conducted on a single strain. Comparatively little is known about the population genomic and evolutionary history of this species. We characterized C. elegans genetic variation by high-throughput selective sequencing of a worldwide collection of 200 wild strains, identifying 41,188 single nucleotide polymorphisms. Unexpectedly, C. elegans genome variation is dominated by a set of commonly shared haplotypes on four of the six chromosomes, each spanning many megabases. Population-genetic modeling shows that this pattern was generated by chromosome-scale selective sweeps that have reduced variation worldwide; at least one of these sweeps likely occurred in the past few hundred years. These sweeps, which we hypothesize to be a result of human activity, have dramatically reshaped the global C. elegans population in the recent past. PMID:22286215

Motifs, modules and games in bacteria.

PubMed

Wolf, Denise M; Arkin, Adam P

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment. Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.

Motifs, modules and games in bacteria

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

Wolf, Denise M.; Arkin, Adam P.

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment.more » Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.« less

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...

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...

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

PubMed

Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

2016-10-07

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

Turbulent flow chromatography TFC-tandem mass spectrometry supporting in vitro/vivo studies of NCEs in high throughput fashion.

PubMed

Verdirame, Maria; Veneziano, Maria; Alfieri, Anna; Di Marco, Annalise; Monteagudo, Edith; Bonelli, Fabio

2010-03-11

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput. Copyright 2009 Elsevier B.V. All rights reserved.

Antibiotics as selectors and accelerators of diversity in the mechanisms of resistance: from the resistome to genetic plasticity in the β-lactamases world.

PubMed

Galán, Juan-Carlos; González-Candelas, Fernando; Rolain, Jean-Marc; Cantón, Rafael

2013-01-01

Antibiotics and antibiotic resistance determinants, natural molecules closely related to bacterial physiology and consistent with an ancient origin, are not only present in antibiotic-producing bacteria. Throughput sequencing technologies have revealed an unexpected reservoir of antibiotic resistance in the environment. These data suggest that co-evolution between antibiotic and antibiotic resistance genes has occurred since the beginning of time. This evolutionary race has probably been slow because of highly regulated processes and low antibiotic concentrations. Therefore to understand this global problem, a new variable must be introduced, that the antibiotic resistance is a natural event, inherent to life. However, the industrial production of natural and synthetic antibiotics has dramatically accelerated this race, selecting some of the many resistance genes present in nature and contributing to their diversification. One of the best models available to understand the biological impact of selection and diversification are β-lactamases. They constitute the most widespread mechanism of resistance, at least among pathogenic bacteria, with more than 1000 enzymes identified in the literature. In the last years, there has been growing concern about the description, spread, and diversification of β-lactamases with carbapenemase activity and AmpC-type in plasmids. Phylogenies of these enzymes help the understanding of the evolutionary forces driving their selection. Moreover, understanding the adaptive potential of β-lactamases contribute to exploration the evolutionary antagonists trajectories through the design of more efficient synthetic molecules. In this review, we attempt to analyze the antibiotic resistance problem from intrinsic and environmental resistomes to the adaptive potential of resistance genes and the driving forces involved in their diversification, in order to provide a global perspective of the resistance problem.

Antibiotics as selectors and accelerators of diversity in the mechanisms of resistance: from the resistome to genetic plasticity in the β-lactamases world

PubMed Central

Galán, Juan-Carlos; González-Candelas, Fernando; Rolain, Jean-Marc; Cantón, Rafael

2013-01-01

Antibiotics and antibiotic resistance determinants, natural molecules closely related to bacterial physiology and consistent with an ancient origin, are not only present in antibiotic-producing bacteria. Throughput sequencing technologies have revealed an unexpected reservoir of antibiotic resistance in the environment. These data suggest that co-evolution between antibiotic and antibiotic resistance genes has occurred since the beginning of time. This evolutionary race has probably been slow because of highly regulated processes and low antibiotic concentrations. Therefore to understand this global problem, a new variable must be introduced, that the antibiotic resistance is a natural event, inherent to life. However, the industrial production of natural and synthetic antibiotics has dramatically accelerated this race, selecting some of the many resistance genes present in nature and contributing to their diversification. One of the best models available to understand the biological impact of selection and diversification are β-lactamases. They constitute the most widespread mechanism of resistance, at least among pathogenic bacteria, with more than 1000 enzymes identified in the literature. In the last years, there has been growing concern about the description, spread, and diversification of β-lactamases with carbapenemase activity and AmpC-type in plasmids. Phylogenies of these enzymes help the understanding of the evolutionary forces driving their selection. Moreover, understanding the adaptive potential of β-lactamases contribute to exploration the evolutionary antagonists trajectories through the design of more efficient synthetic molecules. In this review, we attempt to analyze the antibiotic resistance problem from intrinsic and environmental resistomes to the adaptive potential of resistance genes and the driving forces involved in their diversification, in order to provide a global perspective of the resistance problem. PMID:23404545

Remodeling Cildb, a popular database for cilia and links for ciliopathies

PubMed Central

2014-01-01

Background New generation technologies in cell and molecular biology generate large amounts of data hard to exploit for individual proteins. This is particularly true for ciliary and centrosomal research. Cildb is a multi–species knowledgebase gathering high throughput studies, which allows advanced searches to identify proteins involved in centrosome, basal body or cilia biogenesis, composition and function. Combined to localization of genetic diseases on human chromosomes given by OMIM links, candidate ciliopathy proteins can be compiled through Cildb searches. Methods Othology between recent versions of the whole proteomes was computed using Inparanoid and ciliary high throughput studies were remapped on these recent versions. Results Due to constant evolution of the ciliary and centrosomal field, Cildb has been recently upgraded twice, with new species whole proteomes and new ciliary studies, and the latter version displays a novel BioMart interface, much more intuitive than the previous ones. Conclusions This already popular database is designed now for easier use and is up to date in regard to high throughput ciliary studies. PMID:25422781

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

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

Break-up of droplets in a concentrated emulsion flowing through a narrow constriction

NASA Astrophysics Data System (ADS)

Kim, Minkyu; Rosenfeld, Liat; Tang, Sindy; Tang Lab Team

2014-11-01

Droplet microfluidics has enabled a wide range of high throughput screening applications. Compared with other technologies such as robotic screening technology, droplet microfluidics has 1000 times higher throughput, which makes the technology one of the most promising platforms for the ultrahigh throughput screening applications. Few studies have considered the throughput of the droplet interrogation process, however. In this research, we show that the probability of break-up increases with increasing flow rate, entrance angle to the constriction, and size of the drops. Since single drops do not break at the highest flow rate used in the system, break-ups occur primarily from the interactions between highly packed droplets close to each other. Moreover, the probabilistic nature of the break-up process arises from the stochastic variations in the packing configuration. Our results can be used to calculate the maximum throughput of the serial interrogation process. For 40 pL-drops, the highest throughput with less than 1% droplet break-up was measured to be approximately 7,000 drops per second. In addition, the results are useful for understanding the behavior of concentrated emulsions in applications such as mobility control in enhanced oil recovery.

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.

Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates.

PubMed

Andris, Sebastian; Wendeler, Michaela; Wang, Xiangyang; Hubbuch, Jürgen

2018-07-20

Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Developing a gene biomarker at the tipping point of adaptive and adverse responses in human bronchial epithelial cells

EPA Science Inventory

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk...

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

PubMed

Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

2018-01-01

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018. © 2017 American Institute of Chemical Engineers.

Selecting the most appropriate time points to profile in high-throughput studies

PubMed Central

Kleyman, Michael; Sefer, Emre; Nicola, Teodora; Espinoza, Celia; Chhabra, Divya; Hagood, James S; Kaminski, Naftali; Ambalavanan, Namasivayam; Bar-Joseph, Ziv

2017-01-01

Biological systems are increasingly being studied by high throughput profiling of molecular data over time. Determining the set of time points to sample in studies that profile several different types of molecular data is still challenging. Here we present the Time Point Selection (TPS) method that solves this combinatorial problem in a principled and practical way. TPS utilizes expression data from a small set of genes sampled at a high rate. As we show by applying TPS to study mouse lung development, the points selected by TPS can be used to reconstruct an accurate representation for the expression values of the non selected points. Further, even though the selection is only based on gene expression, these points are also appropriate for representing a much larger set of protein, miRNA and DNA methylation changes over time. TPS can thus serve as a key design strategy for high throughput time series experiments. Supporting Website: www.sb.cs.cmu.edu/TPS DOI: http://dx.doi.org/10.7554/eLife.18541.001 PMID:28124972

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.

Establishment of integrated protocols for automated high throughput kinetic chlorophyll fluorescence analyses.

PubMed

Tschiersch, Henning; Junker, Astrid; Meyer, Rhonda C; Altmann, Thomas

2017-01-01

Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge. We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (Φ PSII ). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of Φ PSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency. The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

PubMed Central

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-01-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

PubMed

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-10-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

A Study of the IEEE 802.16 MAC Layer and its Utility in Augmenting the ADNS Architecture to Provide Adaptable Intra-Strike Group High-Speed Packet Switched Data, Imagery, and Voice Communications

DTIC Science & Technology

2005-09-01

This research explores the need for a high throughput, high speed network for use in a network centric wartime environment and how commercial...Automated Digital Network System (ADNS). This research explores the need for a high-throughput, high-speed network for use in a network centric ...1 C. DEPARTMENT OF DEFENSE (DOD) DESIRED END STATE ..............2 1. DOD Transformation to Network Centric Warfare (NCW) Operations

microRNAs Databases: Developmental Methodologies, Structural and Functional Annotations.

PubMed

Singh, Nagendra Kumar

2017-09-01

microRNA (miRNA) is an endogenous and evolutionary conserved non-coding RNA, involved in post-transcriptional process as gene repressor and mRNA cleavage through RNA-induced silencing complex (RISC) formation. In RISC, miRNA binds in complementary base pair with targeted mRNA along with Argonaut proteins complex, causes gene repression or endonucleolytic cleavage of mRNAs and results in many diseases and syndromes. After the discovery of miRNA lin-4 and let-7, subsequently large numbers of miRNAs were discovered by low-throughput and high-throughput experimental techniques along with computational process in various biological and metabolic processes. The miRNAs are important non-coding RNA for understanding the complex biological phenomena of organism because it controls the gene regulation. This paper reviews miRNA databases with structural and functional annotations developed by various researchers. These databases contain structural and functional information of animal, plant and virus miRNAs including miRNAs-associated diseases, stress resistance in plant, miRNAs take part in various biological processes, effect of miRNAs interaction on drugs and environment, effect of variance on miRNAs, miRNAs gene expression analysis, sequence of miRNAs, structure of miRNAs. This review focuses on the developmental methodology of miRNA databases such as computational tools and methods used for extraction of miRNAs annotation from different resources or through experiment. This study also discusses the efficiency of user interface design of every database along with current entry and annotations of miRNA (pathways, gene ontology, disease ontology, etc.). Here, an integrated schematic diagram of construction process for databases is also drawn along with tabular and graphical comparison of various types of entries in different databases. Aim of this paper is to present the importance of miRNAs-related resources at a single place.

High-Throughput, Motility-Based Sorter for Microswimmers such as C. elegans

PubMed Central

Yuan, Jinzhou; Zhou, Jessie; Raizen, David M.; Bau, Haim H.

2015-01-01

Animal motility varies with genotype, disease, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method is implemented in a simple microfluidic device capable of sorting thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriches for known C. elegans motility mutants. Furthermore, using this device, we isolate low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates C. elegans sleep. By performing genetic complementation tests, we demonstrate that our motility-based sorting device efficiently isolates mutants for the same gene identified by tedious visual inspection of behavior on an agar surface. Therefore, our motility-based sorter is capable of performing high throughput gene discovery approaches to investigate fundamental biological processes. PMID:26008643

A Phylogenomic Approach Based on PCR Target Enrichment and High Throughput Sequencing: Resolving the Diversity within the South American Species of Bartsia L. (Orobanchaceae)

PubMed Central

Tank, David C.

2016-01-01

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples. PMID:26828929

WormQTLHD—a web database for linking human disease to natural variation data in C. elegans

PubMed Central

van der Velde, K. Joeri; de Haan, Mark; Zych, Konrad; Arends, Danny; Snoek, L. Basten; Kammenga, Jan E.; Jansen, Ritsert C.; Swertz, Morris A.; Li, Yang

2014-01-01

Interactions between proteins are highly conserved across species. As a result, the molecular basis of multiple diseases affecting humans can be studied in model organisms that offer many alternative experimental opportunities. One such organism—Caenorhabditis elegans—has been used to produce much molecular quantitative genetics and systems biology data over the past decade. We present WormQTLHD (Human Disease), a database that quantitatively and systematically links expression Quantitative Trait Loci (eQTL) findings in C. elegans to gene–disease associations in man. WormQTLHD, available online at http://www.wormqtl-hd.org, is a user-friendly set of tools to reveal functionally coherent, evolutionary conserved gene networks. These can be used to predict novel gene-to-gene associations and the functions of genes underlying the disease of interest. We created a new database that links C. elegans eQTL data sets to human diseases (34 337 gene–disease associations from OMIM, DGA, GWAS Central and NHGRI GWAS Catalogue) based on overlapping sets of orthologous genes associated to phenotypes in these two species. We utilized QTL results, high-throughput molecular phenotypes, classical phenotypes and genotype data covering different developmental stages and environments from WormQTL database. All software is available as open source, built on MOLGENIS and xQTL workbench. PMID:24217915

WormQTLHD--a web database for linking human disease to natural variation data in C. elegans.

PubMed

van der Velde, K Joeri; de Haan, Mark; Zych, Konrad; Arends, Danny; Snoek, L Basten; Kammenga, Jan E; Jansen, Ritsert C; Swertz, Morris A; Li, Yang

2014-01-01

Interactions between proteins are highly conserved across species. As a result, the molecular basis of multiple diseases affecting humans can be studied in model organisms that offer many alternative experimental opportunities. One such organism-Caenorhabditis elegans-has been used to produce much molecular quantitative genetics and systems biology data over the past decade. We present WormQTL(HD) (Human Disease), a database that quantitatively and systematically links expression Quantitative Trait Loci (eQTL) findings in C. elegans to gene-disease associations in man. WormQTL(HD), available online at http://www.wormqtl-hd.org, is a user-friendly set of tools to reveal functionally coherent, evolutionary conserved gene networks. These can be used to predict novel gene-to-gene associations and the functions of genes underlying the disease of interest. We created a new database that links C. elegans eQTL data sets to human diseases (34 337 gene-disease associations from OMIM, DGA, GWAS Central and NHGRI GWAS Catalogue) based on overlapping sets of orthologous genes associated to phenotypes in these two species. We utilized QTL results, high-throughput molecular phenotypes, classical phenotypes and genotype data covering different developmental stages and environments from WormQTL database. All software is available as open source, built on MOLGENIS and xQTL workbench.

Detecting gene subnetworks under selection in biological pathways.

PubMed

Gouy, Alexandre; Daub, Joséphine T; Excoffier, Laurent

2017-09-19

Advances in high throughput sequencing technologies have created a gap between data production and functional data analysis. Indeed, phenotypes result from interactions between numerous genes, but traditional methods treat loci independently, missing important knowledge brought by network-level emerging properties. Therefore, detecting selection acting on multiple genes affecting the evolution of complex traits remains challenging. In this context, gene network analysis provides a powerful framework to study the evolution of adaptive traits and facilitates the interpretation of genome-wide data. We developed a method to analyse gene networks that is suitable to evidence polygenic selection. The general idea is to search biological pathways for subnetworks of genes that directly interact with each other and that present unusual evolutionary features. Subnetwork search is a typical combinatorial optimization problem that we solve using a simulated annealing approach. We have applied our methodology to find signals of adaptation to high-altitude in human populations. We show that this adaptation has a clear polygenic basis and is influenced by many genetic components. Our approach, implemented in the R package signet, improves on gene-level classical tests for selection by identifying both new candidate genes and new biological processes involved in adaptation to altitude. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription.

PubMed

Kayal, Ehsan; Bentlage, Bastian; Cartwright, Paulyn; Yanagihara, Angel A; Lindsay, Dhugal J; Hopcroft, Russell R; Collins, Allen G

2015-01-01

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms.

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription

PubMed Central

Bentlage, Bastian; Cartwright, Paulyn; Yanagihara, Angel A.; Lindsay, Dhugal J.; Hopcroft, Russell R.; Collins, Allen G.

2015-01-01

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III–IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I–II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms. PMID:26618080

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

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.

Complementing in vitro hazard assessment with exposure and pharmacokinetics considerations for chemical prioritization

EPA Science Inventory

Traditional toxicity testing involves a large investment in resources, often using low-throughput in vivo animal studies for limited numbers of chemicals. An alternative strategy is the emergence of high-throughput (HT) in vitro assays as a rapid, cost-efficient means to screen t...

Comparative study on gastrointestinal microbiota of eight fish species with different feeding habits.

PubMed

Li, J; Ni, J; Li, J; Wang, C; Li, X; Wu, S; Zhang, T; Yu, Y; Yan, Q

2014-12-01

To reveal the effects of fish genotype, feeding habits and serum physiological index on the composition of gastrointestinal microbiota, eight fish species with four different feeding habits were investigated. The V1 to V3 regions of 16S rRNA gene were analysed by high-throughput sequencing (454 platform) to compare the gut microbiota of different fish species. A total of 551 995 high-quality sequences with an average length of 463 bp were obtained from the 48 samples. No significant difference was observed among the detected sequences obtained from fishes with different feeding habits (One-way anova, F = 1·003, P = 0·400), but the number of OTUs among different feeding habits was significantly different (One-way anova, F = 7·564, P < 0·001). Additionally, significant correlations were detected between the fish genotype and microbial composition (partial Mantel test, all P values = 0·001) in the stomach, foregut and hindgut. Moreover, different core intestinal microbiota was also noticed in the eight fish species with different feeding habits. Feeding habits and genotype clearly affected the gastrointestinal microbiota of fish. Moreover, the evolutionary process shaped the serum physiological indexes of fish. This study provided much important information for developing commercial fish feeds. © 2014 The Society for Applied Microbiology.

Integrative data mining of high-throughput in vitro screens, in vivo data, and disease information to identify Adverse Outcome Pathway (AOP) signatures:ToxCast high-throughput screening data and Comparative Toxicogenomics Database (CTD) as a case study.

EPA Science Inventory

The Adverse Outcome Pathway (AOP) framework provides a systematic way to describe linkages between molecular and cellular processes and organism or population level effects. The current AOP assembly methods however, are inefficient. Our goal is to generate computationally-pr...

[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.

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 on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

NASA Astrophysics Data System (ADS)

Rohde, Christopher B.; Zeng, Fei; Gilleland, Cody; Samara, Chrysanthi; Yanik, Mehmet F.

2009-02-01

In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.

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

PubMed

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

2015-08-06

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

Nanosphere Templating Through Controlled Evaporation: A High Throughput Method For Building SERS Substrates

NASA Astrophysics Data System (ADS)

Alexander, Kristen; Hampton, Meredith; Lopez, Rene; Desimone, Joseph

2009-03-01

When a pair of noble metal nanoparticles are brought close together, the plasmonic properties of the pair (known as a ``dimer'') give rise to intense electric field enhancements in the interstitial gap. These fields present a simple yet exquisitely sensitive system for performing single molecule surface-enhanced Raman spectroscopy (SM-SERS). Problems associated with current fabrication methods of SERS-active substrates include reproducibility issues, high cost of production and low throughput. In this study, we present a novel method for the high throughput fabrication of high quality SERS substrates. Using a polymer templating technique followed by the placement of thiolated nanoparticles through meniscus force deposition, we are able to fabricate large arrays of identical, uniformly spaced dimers in a quick, reproducible manner. Subsequent theoretical and experimental studies have confirmed the strong dependence of the SERS enhancement on both substrate geometry (e.g. dimer size, shape and gap size) and the polarization of the excitation source.

Nanosphere Templating Through Controlled Evaporation: A High Throughput Method For Building SERS Substrates

NASA Astrophysics Data System (ADS)

Alexander, Kristen; Lopez, Rene; Hampton, Meredith; Desimone, Joseph

2008-10-01

When a pair of noble metal nanoparticles are brought close together, the plasmonic properties of the pair (known as a ``dimer'') give rise to intense electric field enhancements in the interstitial gap. These fields present a simple yet exquisitely sensitive system for performing single molecule surface-enhanced Raman spectroscopy (SM-SERS). Problems associated with current fabrication methods of SERS-active substrates include reproducibility issues, high cost of production and low throughput. In this study, we present a novel method for the high throughput fabrication of high quality SERS substrates. Using a polymer templating technique followed by the placement of thiolated nanoparticles through meniscus force deposition, we are able to fabricate large arrays of identical, uniformly spaced dimers in a quick, reproducible manner. Subsequent theoretical and experimental studies have confirmed the strong dependence of the SERS enhancement on both substrate geometry (e.g. dimer size, shape and gap size) and the polarization of the excitation source.

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

PubMed

Kizaki, Seiichiro; Chandran, Anandhakumar; Sugiyama, Hiroshi

2016-03-02

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biodiversity comparison among phylogenetic diversity metrics and between three North American prairies1

PubMed Central

Kellar, P. Roxanne (Steele); Ahrendsen, Dakota L.; Aust, Shelly K.; Jones, Amanda R.; Pires, J. Chris

2015-01-01

Protection of Earth’s ecosystems requires identification of geographical areas of greatest biodiversity. Assessment of biodiversity begins with knowledge of the evolutionary histories of species in a geographic area. Multiple phylogenetic diversity (PD) metrics have been developed to describe biodiversity beyond species counts, but sufficient empirical studies, particularly at fine phylogenetic scales, have not been conducted to provide conservation planners with evidence for incorporating PD metrics into selection of priority regions. We review notable studies that are contributing to a growing database of empirical results, we report on the effect of using high-throughput sequencing to estimate the phylogenies used to calculate PD metrics, and we discuss difficulties in selecting appropriate diversity indices. We focused on two of the most speciose angiosperm families in prairies—Asteraceae and Fabaceae—and compared 12 PD metrics and four traditional measures of biodiversity between three North American prairie sites. The varying results from the literature and from the current data reveal the wide range of applications of PD metrics and the necessity for many more empirical studies. The accumulation of results from further investigations will eventually lead to a scientific understanding upon which conservation planners can make informed decisions about where to apply limited preservation funds. PMID:26191461

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

PubMed Central

Engel, Krysta L.; Mackiewicz, Mark; Hardigan, Andrew A.; Myers, Richard M.; Savic, Daniel

2016-01-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. PMID:27224938

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation.

PubMed

Engel, Krysta L; Mackiewicz, Mark; Hardigan, Andrew A; Myers, Richard M; Savic, Daniel

2016-09-01

Deciphering the intricate molecular processes that orchestrate the spatial and temporal regulation of genes has become an increasingly major focus of biological research. The differential expression of genes by diverse cell types with a common genome is a hallmark of complex cellular functions, as well as the basis for multicellular life. Importantly, a more coherent understanding of gene regulation is critical for defining developmental processes, evolutionary principles and disease etiologies. Here we present our current understanding of gene regulation by focusing on the role of enhancer elements in these complex processes. Although functional genomic methods have provided considerable advances to our understanding of gene regulation, these assays, which are usually performed on a genome-wide scale, typically provide correlative observations that lack functional interpretation. Recent innovations in genome editing technologies have placed gene regulatory studies at an exciting crossroads, as systematic, functional evaluation of enhancers and other transcriptional regulatory elements can now be performed in a coordinated, high-throughput manner across the entire genome. This review provides insights on transcriptional enhancer function, their role in development and disease, and catalogues experimental tools commonly used to study these elements. Additionally, we discuss the crucial role of novel techniques in deciphering the complex gene regulatory landscape and how these studies will shape future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

Pinzon, NM; Aukema, KG; Gralnick, JA

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

Pervasive Effects of Aging on Gene Expression in Wild Wolves

PubMed Central

Charruau, Pauline; Johnston, Rachel A.; Stahler, Daniel R.; Lea, Amanda; Snyder-Mackler, Noah; Smith, Douglas W.; vonHoldt, Bridgett M.; Cole, Steven W.; Tung, Jenny; Wayne, Robert K.

2016-01-01

Abstract Gene expression levels change as an individual ages and responds to environmental conditions. With the exception of humans, such patterns have principally been studied under controlled conditions, overlooking the array of developmental and environmental influences that organisms encounter under conditions in which natural selection operates. We used high-throughput RNA sequencing (RNA-Seq) of whole blood to assess the relative impacts of social status, age, disease, and sex on gene expression levels in a natural population of gray wolves (Canis lupus). Our findings suggest that age is broadly associated with gene expression levels, whereas other examined factors have minimal effects on gene expression patterns. Further, our results reveal evolutionarily conserved signatures of senescence, such as immunosenescence and metabolic aging, between wolves and humans despite major differences in life history and environment. The effects of aging on gene expression levels in wolves exhibit conservation with humans, but the more rapid expression differences observed in aging wolves is evolutionarily appropriate given the species’ high level of extrinsic mortality due to intraspecific aggression. Some expression changes that occur with age can facilitate physical age-related changes that may enhance fitness in older wolves. However, the expression of these ancestral patterns of aging in descendant modern dogs living in highly modified domestic environments may be maladaptive and cause disease. This work provides evolutionary insight into aging patterns observed in domestic dogs and demonstrates the applicability of studying natural populations to investigate the mechanisms of aging. PMID:27189566

High-throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps

PubMed Central

Hoedjes, K M; Steidle, J L M; Werren, J H; Vet, L E M; Smid, H M

2012-01-01

Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high-throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T-maze olfactometer facilitates high-throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species-specific differences in ecological factors, for example, host preference. The high-throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species. PMID:22804968

Lessons from high-throughput protein crystallization screening: 10 years of practical experience

PubMed Central

JR, Luft; EH, Snell; GT, DeTitta

2011-01-01

Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

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.

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions.

PubMed

De Diego, Nuria; Fürst, Tomáš; Humplík, Jan F; Ugena, Lydia; Podlešáková, Kateřina; Spíchal, Lukáš

2017-01-01

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidops is as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance.

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past.

PubMed

Der Sarkissian, Clio; Pichereau, Vianney; Dupont, Catherine; Ilsøe, Peter C; Perrigault, Mickael; Butler, Paul; Chauvaud, Laurent; Eiríksson, Jón; Scourse, James; Paillard, Christine; Orlando, Ludovic

2017-09-01

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo-) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro-) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high-throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome-scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management. © 2017 John Wiley & Sons Ltd.

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

NASA Astrophysics Data System (ADS)

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

2013-06-01

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

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

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

PubMed

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

2016-02-01

Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. © 2015 Society for Laboratory Automation and Screening.

Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology.

PubMed

Kokel, David; Rennekamp, Andrew J; Shah, Asmi H; Liebel, Urban; Peterson, Randall T

2012-08-01

For decades, studying the behavioral effects of individual drugs and genetic mutations has been at the heart of efforts to understand and treat nervous system disorders. High-throughput technologies adapted from other disciplines (e.g., high-throughput chemical screening, genomics) are changing the scale of data acquisition in behavioral neuroscience. Massive behavioral datasets are beginning to emerge, particularly from zebrafish labs, where behavioral assays can be performed rapidly and reproducibly in 96-well, high-throughput format. Mining these datasets and making comparisons across different assays are major challenges for the field. Here, we review behavioral barcoding, a process by which complex behavioral assays are reduced to a string of numeric features, facilitating analysis and comparison within and across datasets. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Experimental and Study Design Considerations for Uncovering Oncometabolites.

PubMed

Haznadar, Majda; Mathé, Ewy A

2017-01-01

Metabolomics as a field has gained attention due to its potential for biomarker discovery, namely because it directly reflects disease phenotype and is the downstream effect of posttranslational modifications. The field provides a "top-down," integrated view of biochemistry in complex organisms, as opposed to the traditional "bottom-up" approach that aims to analyze networks of interactions between genes, proteins and metabolites. It also allows for the detection of thousands of endogenous metabolites in various clinical biospecimens in a high-throughput manner, including tissue and biofluids such as blood and urine. Of note, because biological fluid samples can be collected relatively easily, the time-dependent fluctuations of metabolites can be readily studied in detail.In this chapter, we aim to provide an overview of (1) analytical methods that are currently employed in the field, and (2) study design concepts that should be considered prior to conducting high-throughput metabolomics studies. While widely applicable, the concepts presented here are namely applicable to high-throughput untargeted studies that aim to search for metabolite biomarkers that are associated with a particular human disease.

Effectiveness of a high-throughput genetic analysis in the identification of responders/non-responders to CYP2D6-metabolized drugs.

PubMed

Savino, Maria; Seripa, Davide; Gallo, Antonietta P; Garrubba, Maria; D'Onofrio, Grazia; Bizzarro, Alessandra; Paroni, Giulia; Paris, Francesco; Mecocci, Patrizia; Masullo, Carlo; Pilotto, Alberto; Santini, Stefano A

2011-01-01

Recent studies investigating the single cytochrome P450 (CYP) 2D6 allele *2A reported an association with the response to drug treatments. More genetic data can be obtained, however, by high-throughput based-technologies. Aim of this study is the high-throughput analysis of the CYP2D6 polymorphisms to evaluate its effectiveness in the identification of patient responders/non-responders to CYP2D6-metabolized drugs. An attempt to compare our results with those previously obtained with the standard analysis of CYP2D6 allele *2A was also made. Sixty blood samples from patients treated with CYP2D6-metabolized drugs previously genotyped for the allele CYP2D6*2A, were analyzed for the CYP2D6 polymorphisms with the AutoGenomics INFINITI CYP4502D6-I assay on the AutoGenomics INFINITI analyzer. A higher frequency of mutated alleles in responder than in non-responder patients (75.38 % vs 43.48 %; p = 0.015) was observed. Thus, the presence of a mutated allele of CYP2D6 was associated with a response to CYP2D6-metabolized drugs (OR = 4.044 (1.348 - 12.154). No difference was observed in the distribution of allele *2A (p = 0.320). The high-throughput genetic analysis of the CYP2D6 polymorphisms better discriminate responders/non-responders with respect to the standard analysis of the CYP2D6 allele *2A. A high-throughput genetic assay of the CYP2D6 may be useful to identify patients with different clinical responses to CYP2D6-metabolized drugs.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes

PubMed Central

Na, Hong; Laver, John D.; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J. Timothy; Kim, Philip M.; Smibert, Craig A.; Lipshitz, Howard D.; Sidhu, Sachdev S.

2016-01-01

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins. PMID:26847261

A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform

PubMed Central

Rizvi, Imran; Moon, Sangjun; Hasan, Tayyaba; Demirci, Utkan

2013-01-01

In vitro 3D cancer models that provide a more accurate representation of disease in vivo are urgently needed to improve our understanding of cancer pathology and to develop better cancer therapies. However, development of 3D models that are based on manual ejection of cells from micropipettes suffer from inherent limitations such as poor control over cell density, limited repeatability, low throughput, and, in the case of coculture models, lack of reproducible control over spatial distance between cell types (e.g., cancer and stromal cells). In this study, we build on a recently introduced 3D model in which human ovarian cancer (OVCAR-5) cells overlaid on Matrigel™ spontaneously form multicellular acini. We introduce a high-throughput automated cell printing system to bioprint a 3D coculture model using cancer cells and normal fibroblasts micropatterned on Matrigel™. Two cell types were patterned within a spatially controlled microenvironment (e.g., cell density, cell-cell distance) in a high-throughput and reproducible manner; both cell types remained viable during printing and continued to proliferate following patterning. This approach enables the miniaturization of an established macro-scale 3D culture model and would allow systematic investigation into the multiple unknown regulatory feedback mechanisms between tumor and stromal cells and provide a tool for high-throughput drug screening. PMID:21298805

Evaluating the Value of Augmenting In Vitro Hazard Assessment with Exposure and Pharmacokinetics Considerations for Chemical Prioritization

EPA Science Inventory

Over time, toxicity-testing paradigms have progressed from low-throughput in vivo animal studies for limited numbers of chemicals to high-throughput (HT) in vitro screening assays for thousands of chemicals. Such HT in vitro methods, along with HT in silico predictions of popula...

Next Generation Sequencing Technologies: The Doorway to the Unexplored Genomics of Non-Model Plants

PubMed Central

Unamba, Chibuikem I. N.; Nag, Akshay; Sharma, Ram K.

2015-01-01

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping. PMID:26734016

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...

Recent Development Activities and Future Mission Applications of NASA's Evolutionary Xenon Thruster (NEXT)

NASA Technical Reports Server (NTRS)

Patterson, Michael J.; Pencil, Eric J.

2014-01-01

NASAs Evolutionary Xenon Thruster (NEXT) project is developing next generation ion propulsion technologies to enhance the performance and lower the costs of future NASA space science missions. This is being accomplished by producing Engineering Model (EM) and Prototype Model (PM) components, validating these via qualification-level and integrated system testing, and preparing the transition of NEXT technologies to flight system development. This presentation is a follow-up to the NEXT project overviews presented in 2009-2010. It reviews the status of the NEXT project, presents the current system performance characteristics, and describes planned activities in continuing the transition of NEXT technology to a first flight. In 2013 a voluntary decision was made to terminate the long duration test of the NEXT thruster, given the thruster design has exceeded all expectations by accumulating over 50,000 hours of operation to demonstrate around 900 kg of xenon throughput. Besides its promise for upcoming NASA science missions, NEXT has excellent potential for future commercial and international spacecraft applications.

Genomics of coloration in natural animal populations.

PubMed

San-Jose, Luis M; Roulin, Alexandre

2017-07-05

Animal coloration has traditionally been the target of genetic and evolutionary studies. However, until very recently, the study of the genetic basis of animal coloration has been mainly restricted to model species, whereas research on non-model species has been either neglected or mainly based on candidate approaches, and thereby limited by the knowledge obtained in model species. Recent high-throughput sequencing technologies allow us to overcome previous limitations, and open new avenues to study the genetic basis of animal coloration in a broader number of species and colour traits, and to address the general relevance of different genetic structures and their implications for the evolution of colour. In this review, we highlight aspects where genome-wide studies could be of major utility to fill in the gaps in our understanding of the biology and evolution of animal coloration. The new genomic approaches have been promptly adopted to study animal coloration although substantial work is still needed to consider a larger range of species and colour traits, such as those exhibiting continuous variation or based on reflective structures. We argue that a robust advancement in the study of animal coloration will also require large efforts to validate the functional role of the genes and variants discovered using genome-wide tools.This article is part of the themed issue 'Animal coloration: production, perception, function and application'. © 2017 The Author(s).

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.

Accelerating the design of solar thermal fuel materials through high throughput simulations.

PubMed

Liu, Yun; Grossman, Jeffrey C

2014-12-10

Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastable structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions.

High-throughput automatic defect review for 300mm blank wafers with atomic force microscope

NASA Astrophysics Data System (ADS)

Zandiatashbar, Ardavan; Kim, Byong; Yoo, Young-kook; Lee, Keibock; Jo, Ahjin; Lee, Ju Suk; Cho, Sang-Joon; Park, Sang-il

2015-03-01

While feature size in lithography process continuously becomes smaller, defect sizes on blank wafers become more comparable to device sizes. Defects with nm-scale characteristic size could be misclassified by automated optical inspection (AOI) and require post-processing for proper classification. Atomic force microscope (AFM) is known to provide high lateral and the highest vertical resolution by mechanical probing among all techniques. However, its low throughput and tip life in addition to the laborious efforts for finding the defects have been the major limitations of this technique. In this paper we introduce automatic defect review (ADR) AFM as a post-inspection metrology tool for defect study and classification for 300 mm blank wafers and to overcome the limitations stated above. The ADR AFM provides high throughput, high resolution, and non-destructive means for obtaining 3D information for nm-scale defect review and classification.

[Weighted gene co-expression network analysis in biomedicine research].

PubMed

Liu, Wei; Li, Li; Ye, Hua; Tu, Wei

2017-11-25

High-throughput biological technologies are now widely applied in biology and medicine, allowing scientists to monitor thousands of parameters simultaneously in a specific sample. However, it is still an enormous challenge to mine useful information from high-throughput data. The emergence of network biology provides deeper insights into complex bio-system and reveals the modularity in tissue/cellular networks. Correlation networks are increasingly used in bioinformatics applications. Weighted gene co-expression network analysis (WGCNA) tool can detect clusters of highly correlated genes. Therefore, we systematically reviewed the application of WGCNA in the study of disease diagnosis, pathogenesis and other related fields. First, we introduced principle, workflow, advantages and disadvantages of WGCNA. Second, we presented the application of WGCNA in disease, physiology, drug, evolution and genome annotation. Then, we indicated the application of WGCNA in newly developed high-throughput methods. We hope this review will help to promote the application of WGCNA in biomedicine research.

High-throughput method to predict extrusion pressure of ceramic pastes.

PubMed

Cao, Kevin; Liu, Yang; Tucker, Christopher; Baumann, Michael; Grit, Grote; Lakso, Steven

2014-04-14

A new method was developed to measure the rheology of extrudable ceramic pastes using a Hamilton MicroLab Star liquid handler. The Hamilton instrument, normally used for high throughput liquid processing, was expanded to function as a low pressure capillary rheometer. Diluted ceramic pastes were forced through the modified pipettes, which produced pressure drop data that was converted to standard rheology data. A known ceramic paste containing cellulose ether was made and diluted to various concentrations in water. The most dilute paste samples were tested in the Hamilton instrument and the more typical, highly concentrated, ceramic paste were tested with a hydraulic ram extruder fitted with a capillary die and pressure measurement system. The rheology data from this study indicates that the dilute high throughput method using the Hamilton instrument correlates to, and can predict, the rheology of concentrated ceramic pastes normally used in ceramic extrusion production processes.

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

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.

pH measurement and a rational and practical pH control strategy for high throughput cell culture system.

PubMed

Zhou, Haiying; Purdie, Jennifer; Wang, Tongtong; Ouyang, Anli

2010-01-01

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large-scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF-4F 5-(-and 6)-carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO(2), HCO(3)(-), and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO(2) from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96-well plates and shake flasks was also demonstrated in this study. This work shed light on mini-bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. Copyright 2009 American Institute of Chemical Engineers

Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing.

PubMed

Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun

2018-02-27

Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation.

PubMed

Chatterjee, Anirban; Mirer, Paul L; Zaldivar Santamaria, Elvira; Klapperich, Catherine; Sharon, Andre; Sauer-Budge, Alexis F

2010-06-01

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format.

PubMed

Huang, Dejian; Ou, Boxin; Hampsch-Woodill, Maureen; Flanagan, Judith A; Prior, Ronald L

2002-07-31

The oxygen radical absorbance capacity (ORAC) assay has been widely accepted as a standard tool to measure the antioxidant activity in the nutraceutical, pharmaceutical, and food industries. However, the ORAC assay has been criticized for a lack of accessibility due to the unavailability of the COBAS FARA II analyzer, an instrument discontinued by the manufacturer. In addition, the manual sample preparation is time-consuming and labor-intensive. The objective of this study was to develop a high-throughput instrument platform that can fully automate the ORAC assay procedure. The new instrument platform consists of a robotic eight-channel liquid handling system and a microplate fluorescence reader. By using the high-throughput platform, the efficiency of the assay is improved with at least a 10-fold increase in sample throughput over the current procedure. The mean of intra- and interday CVs was

An introduction to microbiome analysis for human biology applications.

PubMed

Amato, Katherine R

2017-01-01

Research examining the gut microbiota is currently exploding, and results are providing new perspectives on human biology. Factors such as host diet and physiology influence the composition and function of the gut microbiota, which in turn affects human nutrition, health, and behavior via interactions with metabolism, the immune system, and the brain. These findings represent an exciting new twist on familiar topics, and as a result, gut microbiome research is likely to provide insight into unresolved biological mechanisms driving human health. However, much remains to be learned about the broader ecological and evolutionary contexts within which gut microbes and humans are affecting each other. Here, I outline the procedures for generating data describing the gut microbiota with the goal of facilitating the wider integration of microbiome analyses into studies of human biology. I describe the steps involved in sample collection, DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics. While this review serves only as an introduction to these topics, it provides sufficient resources for researchers interested in launching new microbiome initiatives. As knowledge of these methods spreads, microbiome analysis should become a standard tool in the arsenal of human biology research. © 2016 Wiley Periodicals, Inc.

Toward systems biology in brown algae to explore acclimation and adaptation to the shore environment.

PubMed

Tonon, Thierry; Eveillard, Damien; Prigent, Sylvain; Bourdon, Jérémie; Potin, Philippe; Boyen, Catherine; Siegel, Anne

2011-12-01

Brown algae belong to a phylogenetic lineage distantly related to land plants and animals. They are almost exclusively found in the intertidal zone, a harsh and frequently changing environment where organisms are submitted to marine and terrestrial constraints. In relation with their unique evolutionary history and their habitat, they feature several peculiarities, including at the level of their primary and secondary metabolism. The establishment of Ectocarpus siliculosus as a model organism for brown algae has represented a framework in which several omics techniques have been developed, in particular, to study the response of these organisms to abiotic stresses. With the recent publication of medium to high throughput profiling data, it is now possible to envision integrating observations at the cellular scale to apply systems biology approaches. As a first step, we propose a protocol focusing on integrating heterogeneous knowledge gained on brown algal metabolism. The resulting abstraction of the system will then help understanding how brown algae cope with changes in abiotic parameters within their unique habitat, and to decipher some of the mechanisms underlying their (1) acclimation and (2) adaptation, respectively consequences of (1) the behavior or (2) the topology of the system resulting from the integrative approach.

Pathogen-driven selection in the human genome.

PubMed

Cagliani, Rachele; Sironi, Manuela

2013-01-01

Infectious diseases and epidemics have always accompanied and characterized human history, representing one of the main causes of death. Even today, despite progress in sanitation and medical research, infections are estimated to account for about 15% of deaths. The hypothesis whereby infectious diseases have been acting as a powerful selective pressure was formulated long ago, but it was not until the availability of large-scale genetic data and the development of novel methods to study molecular evolution that we could assess how pervasively infectious agents have shaped human genetic diversity. Indeed, recent evidences indicated that among the diverse environmental factors that acted as selective pressures during the evolution of our species, pathogen load had the strongest influence. Beside the textbook example of the major histocompatibility complex, selection signatures left by pathogen-exerted pressure can be identified at several human loci, including genes not directly involved in immune response. In the future, high-throughput technologies and the availability of genetic data from different populations are likely to provide novel insights into the evolutionary relationships between the human host and its pathogens. Hopefully, this will help identify the genetic determinants modulating the susceptibility to infectious diseases and will translate into new treatment strategies.

The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa

PubMed Central

Wagner, Andreas; MacLean, R. Craig

2016-01-01

Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs. PMID:27149698

Marine genomics: News and views.

PubMed

Ribeiro, Ângela M; Foote, Andrew D; Kupczok, Anne; Frazão, Bárbara; Limborg, Morten T; Piñeiro, Rosalía; Abalde, Samuel; Rocha, Sara; da Fonseca, Rute R

2017-02-01

Marine ecosystems occupy 71% of the surface of our planet, yet we know little about their diversity. Although the inventory of species is continually increasing, as registered by the Census of Marine Life program, only about 10% of the estimated two million marine species are known. This lag between observed and estimated diversity is in part due to the elusiveness of most aquatic species and the technical difficulties of exploring extreme environments, as for instance the abyssal plains and polar waters. In the last decade, the rapid development of affordable and flexible high-throughput sequencing approaches have been helping to improve our knowledge of marine biodiversity, from the rich microbial biota that forms the base of the tree of life to a wealth of plant and animal species. In this review, we present an overview of the applications of genomics to the study of marine life, from evolutionary biology of non-model organisms to species of commercial relevance for fishing, aquaculture and biomedicine. Instead of providing an exhaustive list of available genomic data, we rather set to present contextualized examples that best represent the current status of the field of marine genomics. Copyright © 2016 Elsevier B.V. All rights reserved.

The spatial architecture of protein function and adaptation

PubMed Central

McLaughlin, Richard N.; Poelwijk, Frank J.; Raman, Arjun; Gosal, Walraj S.; Ranganathan, Rama

2014-01-01

Statistical analysis of protein evolution suggests a design for natural proteins in which sparse networks of coevolving amino acids (termed sectors) comprise the essence of three-dimensional structure and function1, 2, 3, 4, 5. However, proteins are also subject to pressures deriving from the dynamics of the evolutionary process itself—the ability to tolerate mutation and to be adaptive to changing selection pressures6, 7, 8, 9, 10. To understand the relationship of the sector architecture to these properties, we developed a high-throughput quantitative method for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid. Using a PDZ domain (PSD95pdz3) model system, we show that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution. In addition, we find that adaptation to a new binding specificity initiates exclusively through variation within sector residues. A combination of just two sector mutations located near and away from the ligand-binding site suffices to switch the binding specificity of PSD95pdz3 quantitatively towards a class-switching ligand. The localization of functional constraint and adaptive variation within the sector has important implications for understanding and engineering proteins. PMID:23041932

A high throughput array microscope for the mechanical characterization of biomaterials

NASA Astrophysics Data System (ADS)

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

2015-02-01

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

Continuous inertial microparticle and blood cell separation in straight channels with local microstructures.

PubMed

Wu, Zhenlong; Chen, Yu; Wang, Moran; Chung, Aram J

2016-02-07

Fluid inertia which has conventionally been neglected in microfluidics has been gaining much attention for particle and cell manipulation because inertia-based methods inherently provide simple, passive, precise and high-throughput characteristics. Particularly, the inertial approach has been applied to blood separation for various biomedical research studies mainly using spiral microchannels. For higher throughput, parallelization is essential; however, it is difficult to realize using spiral channels because of their large two dimensional layouts. In this work, we present a novel inertial platform for continuous sheathless particle and blood cell separation in straight microchannels containing microstructures. Microstructures within straight channels exert secondary flows to manipulate particle positions similar to Dean flow in curved channels but with higher controllability. Through a balance between inertial lift force and microstructure-induced secondary flow, we deterministically position microspheres and cells based on their sizes to be separated downstream. Using our inertial platform, we successfully sorted microparticles and fractionized blood cells with high separation efficiencies, high purities and high throughputs. The inertial separation platform developed here can be operated to process diluted blood with a throughput of 10.8 mL min(-1)via radially arrayed single channels with one inlet and two rings of outlets.

Lens-free shadow image based high-throughput continuous cell monitoring technique.

PubMed

Jin, Geonsoo; Yoo, In-Hwa; Pack, Seung Pil; Yang, Ji-Woon; Ha, Un-Hwan; Paek, Se-Hwan; Seo, Sungkyu

2012-01-01

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72 h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing. Copyright © 2012 Elsevier B.V. All rights reserved.

High-throughput tetrad analysis.

PubMed

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

2013-07-01

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

Performance comparison of SNP detection tools with illumina exome sequencing data—an assessment using both family pedigree information and sample-matched SNP array data

PubMed Central

Yi, Ming; Zhao, Yongmei; Jia, Li; He, Mei; Kebebew, Electron; Stephens, Robert M.

2014-01-01

To apply exome-seq-derived variants in the clinical setting, there is an urgent need to identify the best variant caller(s) from a large collection of available options. We have used an Illumina exome-seq dataset as a benchmark, with two validation scenarios—family pedigree information and SNP array data for the same samples, permitting global high-throughput cross-validation, to evaluate the quality of SNP calls derived from several popular variant discovery tools from both the open-source and commercial communities using a set of designated quality metrics. To the best of our knowledge, this is the first large-scale performance comparison of exome-seq variant discovery tools using high-throughput validation with both Mendelian inheritance checking and SNP array data, which allows us to gain insights into the accuracy of SNP calling through such high-throughput validation in an unprecedented way, whereas the previously reported comparison studies have only assessed concordance of these tools without directly assessing the quality of the derived SNPs. More importantly, the main purpose of our study was to establish a reusable procedure that applies high-throughput validation to compare the quality of SNP discovery tools with a focus on exome-seq, which can be used to compare any forthcoming tool(s) of interest. PMID:24831545

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

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

Su, Hui

2001-01-01

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

Mapping the miRNA interactome by crosslinking ligation and sequencing of hybrids (CLASH)

PubMed Central

Helwak, Aleksandra; Tollervey, David

2014-01-01

RNA-RNA interactions play critical roles in many cellular processes but studying them is difficult and laborious. Here, we describe an experimental procedure, termed crosslinking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein is UV crosslinked in vivo to stabilise RNA interactions and purified under denaturing conditions. RNAs associated with the bait protein are partially truncated, and the ends of RNA-duplexes are ligated together. Following linker addition, cDNA library preparation and high-throughput sequencing, the ligated duplexes give rise to chimeric cDNAs, which unambiguously identify RNA-RNA interaction sites independent of bioinformatic predictions. This protocol is optimized for studying miRNA targets bound by Argonaute proteins, but should be easily adapted for other RNA-binding proteins and classes of RNA. The protocol requires around 5 days to complete, excluding the time required for high-throughput sequencing and bioinformatic analyses. PMID:24577361

A Method of High Throughput Monitoring Crop Physiology Using Chlorophyll Fluorescence and Multispectral Imaging.

PubMed

Wang, Heng; Qian, Xiangjie; Zhang, Lan; Xu, Sailong; Li, Haifeng; Xia, Xiaojian; Dai, Liankui; Xu, Liang; Yu, Jingquan; Liu, Xu

2018-01-01

We present a high throughput crop physiology condition monitoring system and corresponding monitoring method. The monitoring system can perform large-area chlorophyll fluorescence imaging and multispectral imaging. The monitoring method can determine the crop current condition continuously and non-destructively. We choose chlorophyll fluorescence parameters and relative reflectance of multispectral as the indicators of crop physiological status. Using tomato as experiment subject, the typical crop physiological stress, such as drought, nutrition deficiency and plant disease can be distinguished by the monitoring method. Furthermore, we have studied the correlation between the physiological indicators and the degree of stress. Besides realizing the continuous monitoring of crop physiology, the monitoring system and method provide the possibility of machine automatic diagnosis of the plant physiology. Highlights: A newly designed high throughput crop physiology monitoring system and the corresponding monitoring method are described in this study. Different types of stress can induce distinct fluorescence and spectral characteristics, which can be used to evaluate the physiological status of plants.

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

Application of High-Throughput In Vitro Assays for Risk-Based ...

EPA Pesticide Factsheets

Multiple drivers shape the types of human-health assessments performed on chemicals by U.S. EPA resulting in chemical assessments are “fit-for-purpose” ranging from prioritization for further testing to full risk assessments. Layered on top of the diverse assessment needs are the resource intensive nature of traditional toxicological studies used to test chemicals and the lack of toxicity information on many chemicals. To address these challenges, the Agency initiated the ToxCast program to screen thousands of chemicals across hundreds of high-throughput screening assays in concentrations-response format. One of the findings of the project has been that the majority of chemicals interact with multiple biological targets within a narrow concentration range and the extent of interactions increases rapidly near the concentration causing cytotoxicity. This means that application of high-throughput in vitro assays to chemical assessments will need to identify both the relative selectivity at chemicals interact with biological targets and the concentration at which these interactions perturb signaling pathways. The integrated analyses will be used to both define a point-of-departure for comparison with human exposure estimates and identify which chemicals may benefit from further studies in a mode-of-action or adverse outcome pathway framework. The application of new technologies in a risk-based, tiered manner provides flexibility in matching throughput and cos

20150325 - Application of High-Throughput In Vitro Assays for ...

EPA Pesticide Factsheets

Multiple drivers shape the types of human-health assessments performed on chemicals by U.S. EPA resulting in chemical assessments are “fit-for-purpose” ranging from prioritization for further testing to full risk assessments. Layered on top of the diverse assessment needs are the resource intensive nature of traditional toxicological studies used to test chemicals and the lack of toxicity information on many chemicals. To address these challenges, the Agency initiated the ToxCast program to screen thousands of chemicals across hundreds of high-throughput screening assays in concentrations-response format. One of the findings of the project has been that the majority of chemicals interact with multiple biological targets within a narrow concentration range and the extent of interactions increases rapidly near the concentration causing cytotoxicity. This means that application of high-throughput in vitro assays to chemical assessments will need to identify both the relative selectivity at chemicals interact with biological targets and the concentration at which these interactions perturb signaling pathways. The integrated analyses will be used to both define a point-of-departure for comparison with human exposure estimates and identify which chemicals may benefit from further studies in a mode-of-action or adverse outcome pathway framework. The application of new technologies in a risk-based, tiered manner provides flexibility in matching throughput and cos

PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants.

PubMed

Jin, Jinpu; Tian, Feng; Yang, De-Chang; Meng, Yu-Qi; Kong, Lei; Luo, Jingchu; Gao, Ge

2017-01-04

With the goal of providing a comprehensive, high-quality resource for both plant transcription factors (TFs) and their regulatory interactions with target genes, we upgraded plant TF database PlantTFDB to version 4.0 (http://planttfdb.cbi.pku.edu.cn/). In the new version, we identified 320 370 TFs from 165 species, presenting a more comprehensive genomic TF repertoires of green plants. Besides updating the pre-existing abundant functional and evolutionary annotation for identified TFs, we generated three new types of annotation which provide more directly clues to investigate functional mechanisms underlying: (i) a set of high-quality, non-redundant TF binding motifs derived from experiments; (ii) multiple types of regulatory elements identified from high-throughput sequencing data; (iii) regulatory interactions curated from literature and inferred by combining TF binding motifs and regulatory elements. In addition, we upgraded previous TF prediction server, and set up four novel tools for regulation prediction and functional enrichment analyses. Finally, we set up a novel companion portal PlantRegMap (http://plantregmap.cbi.pku.edu.cn) for users to access the regulation resource and analysis tools conveniently. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

PubMed

D'Antonio, Matteo; Woodruff, Grace; Nathanson, Jason L; D'Antonio-Chronowska, Agnieszka; Arias, Angelo; Matsui, Hiroko; Williams, Roy; Herrera, Cheryl; Reyna, Sol M; Yeo, Gene W; Goldstein, Lawrence S B; Panopoulos, Athanasia D; Frazer, Kelly A

2017-04-11

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost. Published by Elsevier Inc.

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.

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.

Photon-Counting H33D Detector for Biological Fluorescence Imaging

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2010-01-01

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms

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

Yang, Shihui; Franden, Mary A; Yang, Qing

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strainsmore » as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. Here, this study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.« less

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms

DOE PAGES

Yang, Shihui; Franden, Mary A; Yang, Qing; ...

2018-04-04

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strainsmore » as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. Here, this study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.« less

PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

PubMed

Vierna, J; Doña, J; Vizcaíno, A; Serrano, D; Jovani, R

2017-10-01

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

High-throughput and selective solid-phase extraction of urinary catecholamines by crown ether-modified resin composite fiber.

PubMed

Chen, LiQin; Wang, Hui; Xu, Zhen; Zhang, QiuYue; Liu, Jia; Shen, Jun; Zhang, WanQi

2018-08-03

In the present study, we developed a simple and high-throughput solid phase extraction (SPE) procedure for selective extraction of catecholamines (CAs) in urine samples. The SPE adsorbents were electrospun composite fibers functionalized with 4-carboxybenzo-18-crown-6 ether modified XAD resin and polystyrene, which were packed into 96-well columns and used for high-throughput selective extraction of CAs in healthy human urine samples. Moreover, the extraction efficiency of packed-fiber SPE (PFSPE) was examined by high performance liquid chromatography coupled with fluorescence detector. The parameters affecting the extraction efficiency and impurity removal efficiency were optimized, and good linearity ranging from 0.5 to 400 ng/mL was obtained with a low limit of detection (LOD, 0.2-0.5 ng/mL) and a good repeatability (2.7%-3.7%, n = 6). The extraction recoveries of three CAs ranged from 70.5% to 119.5%. Furthermore, stable and reliable results obtained by the fluorescence detector were superior to those obtained by the electrochemical detector. Collectively, PFSPE coupled with 96-well columns was a simple, rapid, selective, high-throughput and cost-efficient method, and the proposed method could be applied in clinical chemistry. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Carbohydrate Microarray Technology Applied to High-Throughput Mapping of Plant Cell Wall Glycans Using Comprehensive Microarray Polymer Profiling (CoMPP).

PubMed

Kračun, Stjepan Krešimir; Fangel, Jonatan Ulrik; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Vidal-Melgosa, Silvia; Willats, William George Tycho

2017-01-01

Cell walls are an important feature of plant cells and a major component of the plant glycome. They have both structural and physiological functions and are critical for plant growth and development. The diversity and complexity of these structures demand advanced high-throughput techniques to answer questions about their structure, functions and roles in both fundamental and applied scientific fields. Microarray technology provides both the high-throughput and the feasibility aspects required to meet that demand. In this chapter, some of the most recent microarray-based techniques relating to plant cell walls are described together with an overview of related contemporary techniques applied to carbohydrate microarrays and their general potential in glycoscience. A detailed experimental procedure for high-throughput mapping of plant cell wall glycans using the comprehensive microarray polymer profiling (CoMPP) technique is included in the chapter and provides a good example of both the robust and high-throughput nature of microarrays as well as their applicability to plant glycomics.

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.

A high-throughput semi-automated preparation for filtered synaptoneurosomes.

PubMed

Murphy, Kathryn M; Balsor, Justin; Beshara, Simon; Siu, Caitlin; Pinto, Joshua G A

2014-09-30

Synaptoneurosomes have become an important tool for studying synaptic proteins. The filtered synaptoneurosomes preparation originally developed by Hollingsworth et al. (1985) is widely used and is an easy method to prepare synaptoneurosomes. The hand processing steps in that preparation, however, are labor intensive and have become a bottleneck for current proteomic studies using synaptoneurosomes. For this reason, we developed new steps for tissue homogenization and filtration that transform the preparation of synaptoneurosomes to a high-throughput, semi-automated process. We implemented a standardized protocol with easy to follow steps for homogenizing multiple samples simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, LLC) and then filtering all of the samples in centrifugal filter units (EMD Millipore, Corp). The new steps dramatically reduce the time to prepare synaptoneurosomes from hours to minutes, increase sample recovery, and nearly double enrichment for synaptic proteins. These steps are also compatible with biosafety requirements for working with pathogen infected brain tissue. The new high-throughput semi-automated steps to prepare synaptoneurosomes are timely technical advances for studies of low abundance synaptic proteins in valuable tissue samples. Copyright © 2014 Elsevier B.V. All rights reserved.

MS-REDUCE: an ultrafast technique for reduction of big mass spectrometry data for high-throughput processing.

PubMed

Awan, Muaaz Gul; Saeed, Fahad

2016-05-15

Modern proteomics studies utilize high-throughput mass spectrometers which can produce data at an astonishing rate. These big mass spectrometry (MS) datasets can easily reach peta-scale level creating storage and analytic problems for large-scale systems biology studies. Each spectrum consists of thousands of peaks which have to be processed to deduce the peptide. However, only a small percentage of peaks in a spectrum are useful for peptide deduction as most of the peaks are either noise or not useful for a given spectrum. This redundant processing of non-useful peaks is a bottleneck for streaming high-throughput processing of big MS data. One way to reduce the amount of computation required in a high-throughput environment is to eliminate non-useful peaks. Existing noise removing algorithms are limited in their data-reduction capability and are compute intensive making them unsuitable for big data and high-throughput environments. In this paper we introduce a novel low-complexity technique based on classification, quantization and sampling of MS peaks. We present a novel data-reductive strategy for analysis of Big MS data. Our algorithm, called MS-REDUCE, is capable of eliminating noisy peaks as well as peaks that do not contribute to peptide deduction before any peptide deduction is attempted. Our experiments have shown up to 100× speed up over existing state of the art noise elimination algorithms while maintaining comparable high quality matches. Using our approach we were able to process a million spectra in just under an hour on a moderate server. The developed tool and strategy has been made available to wider proteomics and parallel computing community and the code can be found at https://github.com/pcdslab/MSREDUCE CONTACT: : fahad.saeed@wmich.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

History, applications, and challenges of immune repertoire research.

PubMed

Liu, Xiao; Wu, Jinghua

2018-02-27

The diversity of T and B cells in terms of their receptor sequences is huge in the vertebrate's immune system and provides broad protection against the vast diversity of pathogens. Immune repertoire is defined as the sum of T cell receptors and B cell receptors (also named immunoglobulin) that makes the organism's adaptive immune system. Before the emergence of high-throughput sequencing, the studies on immune repertoire were limited by the underdeveloped methodologies, since it was impossible to capture the whole picture by the low-throughput tools. The massive paralleled sequencing technology suits perfectly the researches on immune repertoire. In this article, we review the history of immune repertoire studies, in terms of technologies and research applications. Particularly, we discuss several aspects of challenges in this field and highlight the efforts to develop potential solutions, in the era of high-throughput sequencing of the immune repertoire.

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.

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

PubMed

Li, Tianbo; Lu, Gang; Chiang, Eugene Y; Chernov-Rogan, Tania; Grogan, Jane L; Chen, Jun

2017-01-01

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Accelerating the Design of Solar Thermal Fuel Materials through High Throughput Simulations

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

Liu, Y; Grossman, JC

2014-12-01

Solar thermal fuels (STF) store the energy of sunlight, which can then be released later in the form of heat, offering an emission-free and renewable solution for both solar energy conversion and storage. However, this approach is currently limited by the lack of low-cost materials with high energy density and high stability. In this Letter, we present an ab initio high-throughput computational approach to accelerate the design process and allow for searches over a broad class of materials. The high-throughput screening platform we have developed can run through large numbers of molecules composed of earth-abundant elements and identifies possible metastablemore » structures of a given material. Corresponding isomerization enthalpies associated with the metastable structures are then computed. Using this high-throughput simulation approach, we have discovered molecular structures with high isomerization enthalpies that have the potential to be new candidates for high-energy density STF. We have also discovered physical principles to guide further STF materials design through structural analysis. More broadly, our results illustrate the potential of using high-throughput ab initio simulations to design materials that undergo targeted structural transitions.« less

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

PubMed Central

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

2015-01-01

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

PubMed

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

2015-03-30

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

Genomics pipelines and data integration: challenges and opportunities in the research setting

PubMed Central

Davis-Turak, Jeremy; Courtney, Sean M.; Hazard, E. Starr; Glen, W. Bailey; da Silveira, Willian; Wesselman, Timothy; Harbin, Larry P.; Wolf, Bethany J.; Chung, Dongjun; Hardiman, Gary

2017-01-01

Introduction The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert Commentary The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings. PMID:28092471

OnTheFly: a database of Drosophila melanogaster transcription factors and their binding sites.

PubMed

Shazman, Shula; Lee, Hunjoong; Socol, Yakov; Mann, Richard S; Honig, Barry

2014-01-01

We present OnTheFly (http://bhapp.c2b2.columbia.edu/OnTheFly/index.php), a database comprising a systematic collection of transcription factors (TFs) of Drosophila melanogaster and their DNA-binding sites. TFs predicted in the Drosophila melanogaster genome are annotated and classified and their structures, obtained via experiment or homology models, are provided. All known preferred TF DNA-binding sites obtained from the B1H, DNase I and SELEX methodologies are presented. DNA shape parameters predicted for these sites are obtained from a high throughput server or from crystal structures of protein-DNA complexes where available. An important feature of the database is that all DNA-binding domains and their binding sites are fully annotated in a eukaryote using structural criteria and evolutionary homology. OnTheFly thus provides a comprehensive view of TFs and their binding sites that will be a valuable resource for deciphering non-coding regulatory DNA.

Perspectives on the mechanism of transcriptional regulation by long non-coding RNAs.

PubMed

Roberts, Thomas C; Morris, Kevin V; Weinberg, Marc S

2014-01-01

Long non-coding RNAs (lncRNAs) are increasingly being recognized as epigenetic regulators of gene transcription. The diversity and complexity of lncRNA genes means that they exert their regulatory effects by a variety of mechanisms. Although there is still much to be learned about the mechanism of lncRNA function, general principles are starting to emerge. In particular, the application of high throughput (deep) sequencing methodologies has greatly advanced our understanding of lncRNA gene function. lncRNAs function as adaptors that link specific chromatin loci with chromatin-remodeling complexes and transcription factors. lncRNAs can act in cis or trans to guide epigenetic-modifier complexes to distinct genomic sites, or act as scaffolds which recruit multiple proteins simultaneously, thereby coordinating their activities. In this review we discuss the genomic organization of lncRNAs, the importance of RNA secondary structure to lncRNA functionality, the multitude of ways in which they interact with the genome, and what evolutionary conservation tells us about their function.

Genomics pipelines and data integration: challenges and opportunities in the research setting.

PubMed

Davis-Turak, Jeremy; Courtney, Sean M; Hazard, E Starr; Glen, W Bailey; da Silveira, Willian A; Wesselman, Timothy; Harbin, Larry P; Wolf, Bethany J; Chung, Dongjun; Hardiman, Gary

2017-03-01

The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered: The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert commentary: The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings.

Characterizing visible and invisible cell wall mutant phenotypes.

PubMed

Carpita, Nicholas C; McCann, Maureen C

2015-07-01

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Accelerating Yeast Prion Biology using Droplet Microfluidics

NASA Astrophysics Data System (ADS)

Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

2012-02-01

Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

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...

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.

High-throughput telomere length quantification by FISH and its application to human population studies.

PubMed

Canela, Andrés; Vera, Elsa; Klatt, Peter; Blasco, María A

2007-03-27

A major limitation of studies of the relevance of telomere length to cancer and age-related diseases in human populations and to the development of telomere-based therapies has been the lack of suitable high-throughput (HT) assays to measure telomere length. We have developed an automated HT quantitative telomere FISH platform, HT quantitative FISH (Q-FISH), which allows the quantification of telomere length as well as percentage of short telomeres in large human sample sets. We show here that this technique provides the accuracy and sensitivity to uncover associations between telomere length and human disease.

Conceptual dissonance: evaluating the efficacy of natural language processing techniques for validating translational knowledge constructs.

PubMed

Payne, Philip R O; Kwok, Alan; Dhaval, Rakesh; Borlawsky, Tara B

2009-03-01

The conduct of large-scale translational studies presents significant challenges related to the storage, management and analysis of integrative data sets. Ideally, the application of methodologies such as conceptual knowledge discovery in databases (CKDD) provides a means for moving beyond intuitive hypothesis discovery and testing in such data sets, and towards the high-throughput generation and evaluation of knowledge-anchored relationships between complex bio-molecular and phenotypic variables. However, the induction of such high-throughput hypotheses is non-trivial, and requires correspondingly high-throughput validation methodologies. In this manuscript, we describe an evaluation of the efficacy of a natural language processing-based approach to validating such hypotheses. As part of this evaluation, we will examine a phenomenon that we have labeled as "Conceptual Dissonance" in which conceptual knowledge derived from two or more sources of comparable scope and granularity cannot be readily integrated or compared using conventional methods and automated tools.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

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

2016-01-01

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

Spotsizer: High-throughput quantitative analysis of microbial growth.

PubMed

Bischof, Leanne; Převorovský, Martin; Rallis, Charalampos; Jeffares, Daniel C; Arzhaeva, Yulia; Bähler, Jürg

2016-10-01

Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license.

From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

PubMed

Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

2016-01-01

Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

Selection and optimization of hits from a high-throughput phenotypic screen against Trypanosoma cruzi.

PubMed

Keenan, Martine; Alexander, Paul W; Chaplin, Jason H; Abbott, Michael J; Diao, Hugo; Wang, Zhisen; Best, Wayne M; Perez, Catherine J; Cornwall, Scott M J; Keatley, Sarah K; Thompson, R C Andrew; Charman, Susan A; White, Karen L; Ryan, Eileen; Chen, Gong; Ioset, Jean-Robert; von Geldern, Thomas W; Chatelain, Eric

2013-10-01

Inhibitors of Trypanosoma cruzi with novel mechanisms of action are urgently required to diversify the current clinical and preclinical pipelines. Increasing the number and diversity of hits available for assessment at the beginning of the discovery process will help to achieve this aim. We report the evaluation of multiple hits generated from a high-throughput screen to identify inhibitors of T. cruzi and from these studies the discovery of two novel series currently in lead optimization. Lead compounds from these series potently and selectively inhibit growth of T. cruzi in vitro and the most advanced compound is orally active in a subchronic mouse model of T. cruzi infection. High-throughput screening of novel compound collections has an important role to play in diversifying the trypanosomatid drug discovery portfolio. A new T. cruzi inhibitor series with good drug-like properties and promising in vivo efficacy has been identified through this process.

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

Improving protein–protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model

PubMed Central

An, Ji‐Yong; Meng, Fan‐Rong; Chen, Xing; Yan, Gui‐Ying; Hu, Ji‐Pu

2016-01-01

Abstract Predicting protein–protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high‐throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM‐BiGP that combines the relevance vector machine (RVM) model and Bi‐gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi‐gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five‐fold cross‐validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state‐of‐the‐art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM‐BiGP method is significantly better than the SVM‐based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic decision support tool for future proteomics research. For facilitating extensive studies for future proteomics research, we developed a freely available web server called RVM‐BiGP‐PPIs in Hypertext Preprocessor (PHP) for predicting PPIs. The web server including source code and the datasets are available at http://219.219.62.123:8888/BiGP/. PMID:27452983

Characterisation of the Manduca sexta sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.

PubMed

Whittington, Emma; Zhao, Qian; Borziak, Kirill; Walters, James R; Dorus, Steve

2015-07-01

The application of mass spectrometry based proteomics to sperm biology has greatly accelerated progress in understanding the molecular composition and function of spermatozoa. To date, these approaches have been largely restricted to model organisms, all of which produce a single sperm morph capable of oocyte fertilisation. Here we apply high-throughput mass spectrometry proteomic analysis to characterise sperm composition in Manduca sexta, the tobacco hornworm moth, which produce heteromorphic sperm, including one fertilisation competent (eupyrene) and one incompetent (apyrene) sperm type. This resulted in the high confidence identification of 896 proteins from a co-mixed sample of both sperm types, of which 167 are encoded by genes with strict one-to-one orthology in Drosophila melanogaster. Importantly, over half (55.1%) of these orthologous proteins have previously been identified in the D. melanogaster sperm proteome and exhibit significant conservation in quantitative protein abundance in sperm between the two species. Despite the complex nature of gene expression across spermatogenic stages, a significant correlation was also observed between sperm protein abundance and testis gene expression. Lepidopteran-specific sperm proteins (e.g., proteins with no homology to proteins in non-Lepidopteran taxa) were present in significantly greater abundance on average than those with homology outside the Lepidoptera. Given the disproportionate production of apyrene sperm (96% of all mature sperm in Manduca) relative to eupyrene sperm, these evolutionarily novel and highly abundant proteins are candidates for possessing apyrene-specific functions. Lastly, comparative genomic analyses of testis-expressed, ovary-expressed and sperm genes identified a concentration of novel sperm proteins shared amongst Lepidoptera of potential relevance to the evolutionary origin of heteromorphic spermatogenesis. As the first published Lepidopteran sperm proteome, this whole-cell proteomic characterisation will facilitate future evolutionary genetic and developmental studies of heteromorphic sperm production and parasperm function. Furthermore, the analyses presented here provide useful annotation information regarding sex-biased gene expression, novel Lepidopteran genes and gene function in the male gamete to complement the newly sequenced and annotated Manduca genome. Copyright © 2015 Elsevier Ltd. All rights reserved.

Measuring the gut microbiome in birds: Comparison of faecal and cloacal sampling.

PubMed

Videvall, Elin; Strandh, Maria; Engelbrecht, Anel; Cloete, Schalk; Cornwallis, Charlie K

2018-05-01

The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling; however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches, Struthio camelus, and high-throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of each sampling method in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas Actinobacteria, for example, was only strongly correlated between faecal and colon samples. Based on our results, we recommend sampling faeces, whenever possible, as this sample type provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique accurately portrayed the bacterial community of the ileum nor the caecum illustrates the difficulty in noninvasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies. © 2017 John Wiley & Sons Ltd.

High-throughput two-dimensional root system phenotyping platform facilitates genetic analysis of root growth and development.

PubMed

Clark, Randy T; Famoso, Adam N; Zhao, Keyan; Shaff, Jon E; Craft, Eric J; Bustamante, Carlos D; McCouch, Susan R; Aneshansley, Daniel J; Kochian, Leon V

2013-02-01

High-throughput phenotyping of root systems requires a combination of specialized techniques and adaptable plant growth, root imaging and software tools. A custom phenotyping platform was designed to capture images of whole root systems, and novel software tools were developed to process and analyse these images. The platform and its components are adaptable to a wide range root phenotyping studies using diverse growth systems (hydroponics, paper pouches, gel and soil) involving several plant species, including, but not limited to, rice, maize, sorghum, tomato and Arabidopsis. The RootReader2D software tool is free and publicly available and was designed with both user-guided and automated features that increase flexibility and enhance efficiency when measuring root growth traits from specific roots or entire root systems during large-scale phenotyping studies. To demonstrate the unique capabilities and high-throughput capacity of this phenotyping platform for studying root systems, genome-wide association studies on rice (Oryza sativa) and maize (Zea mays) root growth were performed and root traits related to aluminium (Al) tolerance were analysed on the parents of the maize nested association mapping (NAM) population. © 2012 Blackwell Publishing Ltd.

Bibliometrics for Social Validation.

PubMed

Hicks, Daniel J

2016-01-01

This paper introduces a bibliometric, citation network-based method for assessing the social validation of novel research, and applies this method to the development of high-throughput toxicology research at the US Environmental Protection Agency. Social validation refers to the acceptance of novel research methods by a relevant scientific community; it is formally independent of the technical validation of methods, and is frequently studied in history, philosophy, and social studies of science using qualitative methods. The quantitative methods introduced here find that high-throughput toxicology methods are spread throughout a large and well-connected research community, which suggests high social validation. Further assessment of social validation involving mixed qualitative and quantitative methods are discussed in the conclusion.

Bibliometrics for Social Validation

PubMed Central

2016-01-01

This paper introduces a bibliometric, citation network-based method for assessing the social validation of novel research, and applies this method to the development of high-throughput toxicology research at the US Environmental Protection Agency. Social validation refers to the acceptance of novel research methods by a relevant scientific community; it is formally independent of the technical validation of methods, and is frequently studied in history, philosophy, and social studies of science using qualitative methods. The quantitative methods introduced here find that high-throughput toxicology methods are spread throughout a large and well-connected research community, which suggests high social validation. Further assessment of social validation involving mixed qualitative and quantitative methods are discussed in the conclusion. PMID:28005974

Transcriptome Analysis of the Octopus vulgaris Central Nervous System

PubMed Central

Zhang, Xiang; Mao, Yong; Huang, Zixia; Qu, Meng; Chen, Jun; Ding, Shaoxiong; Hong, Jingni; Sun, Tiantian

2012-01-01

Background Cephalopoda are a class of Mollusca species found in all the world's oceans. They are an important model organism in neurobiology. Unfortunately, the lack of neuronal molecular sequences, such as ESTs, transcriptomic or genomic information, has limited the development of molecular neurobiology research in this unique model organism. Results With high-throughput Illumina Solexa sequencing technology, we have generated 59,859 high quality sequences from 12,918,391 paired-end reads. Using BLASTx/BLASTn, 12,227 contigs have blast hits in the Swissprot, NR protein database and NT nucleotide database with E-value cutoff 1e−5. The comparison between the Octopus vulgaris central nervous system (CNS) library and the Aplysia californica/Lymnaea stagnalis CNS ESTs library yielded 5.93%/13.45% of O. vulgaris sequences with significant matches (1e−5) using BLASTn/tBLASTx. Meanwhile the hit percentage of the recently published Schistocerca gregaria, Tilapia or Hirudo medicinalis CNS library to the O. vulgaris CNS library is 21.03%–46.19%. We constructed the Phylogenetic tree using two genes related to CNS function, Synaptotagmin-7 and Synaptophysin. Lastly, we demonstrated that O. vulgaris may have a vertebrate-like Blood-Brain Barrier based on bioinformatic analysis. Conclusion This study provides a mass of molecular information that will contribute to further molecular biology research on O. vulgaris. In our presentation of the first CNS transcriptome analysis of O. vulgaris, we hope to accelerate the study of functional molecular neurobiology and comparative evolutionary biology. PMID:22768275

High-Throughput rRNA Gene Sequencing Reveals High
and Complex Bacterial Diversity Associated with
Brazilian Coffee Bean Fermentation

PubMed Central

Vinícius de Melo, Gilberto

2018-01-01

Summary Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

PubMed

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Efficient high-throughput sequencing of a laser microdissected chromosome arm

PubMed Central

2013-01-01

Background Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. Results We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. Conclusion We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds. PMID:23714049

Key Ecological Roles for Zoosporic True Fungi in Aquatic Habitats.

PubMed

Gleason, Frank H; Scholz, Bettina; Jephcott, Thomas G; van Ogtrop, Floris F; Henderson, Linda; Lilje, Osu; Kittelmann, Sandra; Macarthur, Deborah J

2017-03-01

The diversity and abundance of zoosporic true fungi have been analyzed recently using fungal sequence libraries and advances in molecular methods, such as high-throughput sequencing. This review focuses on four evolutionary primitive true fungal phyla: the Aphelidea, Chytridiomycota, Neocallimastigomycota, and Rosellida (Cryptomycota), most species of which are not polycentric or mycelial (filamentous), rather they tend to be primarily monocentric (unicellular). Zoosporic fungi appear to be both abundant and diverse in many aquatic habitats around the world, with abundance often exceeding other fungal phyla in these habitats, and numerous novel genetic sequences identified. Zoosporic fungi are able to survive extreme conditions, such as high and extremely low pH; however, more work remains to be done. They appear to have important ecological roles as saprobes in decomposition of particulate organic substrates, pollen, plant litter, and dead animals; as parasites of zooplankton and algae; as parasites of vertebrate animals (such as frogs); and as symbionts in the digestive tracts of mammals. Some chytrids cause economically important diseases of plants and animals. They regulate sizes of phytoplankton populations. Further metagenomics surveys of aquatic ecosystems are expected to enlarge our knowledge of the diversity of true zoosporic fungi. Coupled with studies on their functional ecology, we are moving closer to unraveling the role of zoosporic fungi in carbon cycling and the impact of climate change on zoosporic fungal populations.

Luminex and other multiplex high throughput technologies for the identification of, and host response to, environmental triggers of type 1 diabetes.

PubMed

Purohit, Sharad; Sharma, Ashok; She, Jin-Xiong

2015-01-01

Complex interactions between a series of environmental factors and genes result in progression to clinical type 1 diabetes in genetically susceptible individuals. Despite several decades of research in the area, these interactions remain poorly understood. Several studies have yielded associations of certain foods, infections, and immunizations with the onset and progression of diabetes autoimmunity, but most findings are still inconclusive. Environmental triggers are difficult to identify mainly due to (i) large number and complex nature of environmental exposures, including bacteria, viruses, dietary factors, and environmental pollutants, (ii) reliance on low throughput technology, (iii) less efforts in quantifying host response, (iv) long silent period between the exposure and clinical onset of T1D which may lead to loss of the exposure fingerprints, and (v) limited sample sets. Recent development in multiplex technologies has enabled systematic evaluation of different classes of molecules or macroparticles in a high throughput manner. However, the use of multiplex assays in type 1 diabetes research is limited to cytokine assays. In this review, we will discuss the potential use of multiplex high throughput technologies in identification of environmental triggers and host response in type 1 diabetes.

High Throughput, Polymeric Aqueous Two-Phase Printing of Tumor Spheroids

PubMed Central

Atefi, Ehsan; Lemmo, Stephanie; Fyffe, Darcy; Luker, Gary D.; Tavana, Hossein

2014-01-01

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the “patterning” phase containing cells into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models. PMID:25411577

Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells.

PubMed

Mock, Andreas; Chiblak, Sara; Herold-Mende, Christel

2014-01-01

A growing body of evidence suggests that glioma stem cells (GSCs) account for tumor initiation, therapy resistance, and the subsequent regrowth of gliomas. Thus, continuous efforts have been undertaken to further characterize this subpopulation of less differentiated tumor cells. Although we are able to enrich GSCs, we still lack a comprehensive understanding of GSC phenotypes and behavior. The advent of high-throughput technologies raised hope that incorporation of these newly developed platforms would help to tackle such questions. Since then a couple of comparative genome-, transcriptome- and proteome-wide studies on GSCs have been conducted giving new insights in GSC biology. However, lessons had to be learned in designing high-throughput experiments and some of the resulting conclusions fell short of expectations because they were performed on only a few GSC lines or at one molecular level instead of an integrative poly-omics approach. Despite these shortcomings, our knowledge of GSC biology has markedly expanded due to a number of survival-associated biomarkers as well as glioma-relevant signaling pathways and therapeutic targets being identified. In this article we review recent findings obtained by comparative high-throughput analyses of GSCs. We further summarize fundamental concepts of systems biology as well as its applications for glioma stem cell research.

Throughput, latency and cost comparisons of microcontroller-based implementations of wireless sensor network (WSN) in high jump sports

NASA Astrophysics Data System (ADS)

Ahmad, Afandi; Roslan, Muhammad Faris; Amira, Abbes

2017-09-01

In high jump sports, approach take-off speed and force during the take-off are two (2) main important parts to gain maximum jump. To measure both parameters, wireless sensor network (WSN) that contains microcontroller and sensor are needed to describe the results of speed and force for jumpers. Most of the microcontroller exhibit transmission issues in terms of throughput, latency and cost. Thus, this study presents the comparison of wireless microcontrollers in terms of throughput, latency and cost, and the microcontroller that have best performances and cost will be implemented in high jump wearable device. In the experiments, three (3) parts have been integrated - input, process and output. Force (for ankle) and global positioning system (GPS) sensor (for body waist) acts as an input for data transmission. These data were then being processed by both microcontrollers, ESP8266 and Arduino Yun Mini to transmit the data from sensors to the server (host-PC) via message queuing telemetry transport (MQTT) protocol. The server acts as receiver and the results was calculated from the MQTT log files. At the end, results obtained have shown ESP8266 microcontroller had been chosen since it achieved high throughput, low latency and 11 times cheaper in term of prices compared to Arduino Yun Mini microcontroller.

A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

PubMed Central

Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

2014-01-01

The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

The evolution of vancomycin intermediate Staphylococcus aureus (VISA) and heterogenous-VISA.

PubMed

Howden, Benjamin P; Peleg, Anton Y; Stinear, Timothy P

2014-01-01

Resistance to new antimicrobials is generally recognized in Staphylococcus aureus soon after they are released for clinical use. In the case of vancomycin, which was first released in the 1950s, resistance was not reported until the mid 1990s, with the description of vancomycin-intermediate S. aureus (VISA), and heterogenous-VISA (hVISA). Unraveling the complex genetic and cell wall structural changes conferring low-level vancomycin resistance in S. aureus has proved challenging. However the recent advances in high throughput whole-genome sequencing has played a key role in determining the breadth of bacterial chromosomal changes linked with resistance. Diverse mutations in a small number of staphylococcal regulatory genes, in particular walKR, graRS, vraSR and rpoB, have been associated with hVISA and VISA. Only a small number of these mutations have been experimentally proven to confer the resistance phenotype and some of these only partially contribute to resistance. It also appears that the evolution of VISA from VSSA is a step-wise process. Transcriptomics studies, and analysis of host pathogen interactions, indicate that the evolution of vancomycin-susceptible S. aureus to VISA is associated not only with antibiotic resistance, but with other changes likely to promote persistent infection. These include predicted alterations in central metabolism, altered expression of virulence associated factors, attenuated virulence in vivo, and alterations in susceptibility to host innate immune responses, together with reduced susceptibility to other antibiotics. In fact, current data suggests that hVISA and VISA represent a bacterial evolutionary state favoring persistence in the face of not only antibiotics, but also the host environment. The additional knowledge of staphylococcal biology that has been uncovered during the study of hVISA and VISA is significant. The present review will detail the current understanding of the evolutionary process in the generation of hVISA and VISA, and explore the diverse additional changes that occur in these strains. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-04-01

We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Robot-Based High-Throughput Engineering of Alcoholic Polymer: Fullerene Nanoparticle Inks for an Eco-Friendly Processing of Organic Solar Cells.

PubMed

Xie, Chen; Tang, Xiaofeng; Berlinghof, Marvin; Langner, Stefan; Chen, Shi; Späth, Andreas; Li, Ning; Fink, Rainer H; Unruh, Tobias; Brabec, Christoph J

2018-06-27

Development of high-quality organic nanoparticle inks is a significant scientific challenge for the industrial production of solution-processed organic photovoltaics (OPVs) with eco-friendly processing methods. In this work, we demonstrate a novel, robot-based, high-throughput procedure performing automatic poly(3-hexylthio-phene-2,5-diyl) and indene-C 60 bisadduct nanoparticle ink synthesis in nontoxic alcohols. A novel methodology to prepare particle dispersions for fully functional OPVs by manipulating the particle size and solvent system was studied in detail. The ethanol dispersion with a particle diameter of around 80-100 nm exhibits reduced degradation, yielding a power conversion efficiency of 4.52%, which is the highest performance reported so far for water/alcohol-processed OPV devices. By successfully deploying the high-throughput robot-based approach for an organic nanoparticle ink preparation, we believe that the findings demonstrated in this work will trigger more research interest and effort on eco-friendly industrial production of OPVs.

Novel method for high-throughput colony PCR screening in nanoliter-reactors

PubMed Central

Walser, Marcel; Pellaux, Rene; Meyer, Andreas; Bechtold, Matthias; Vanderschuren, Herve; Reinhardt, Richard; Magyar, Joseph; Panke, Sven; Held, Martin

2009-01-01

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. PMID:19282448

New data from basal Australian songbird lineages show that complex structure of MHC class II β genes has early evolutionary origins within passerines.

PubMed

Balasubramaniam, Shandiya; Bray, Rebecca D; Mulder, Raoul A; Sunnucks, Paul; Pavlova, Alexandra; Melville, Jane

2016-05-21

The major histocompatibility complex (MHC) plays a crucial role in the adaptive immune system and has been extensively studied across vertebrate taxa. Although the function of MHC genes appears to be conserved across taxa, there is great variation in the number and organisation of these genes. Among avian species, for instance, there are notable differences in MHC structure between passerine and non-passerine lineages: passerines typically have a high number of highly polymorphic MHC paralogs whereas non-passerines have fewer loci and lower levels of polymorphism. Although the occurrence of highly polymorphic MHC paralogs in passerines is well documented, their evolutionary origins are relatively unexplored. The majority of studies have focussed on the more derived passerine lineages and there is very little empirical information on the diversity of the MHC in basal passerine lineages. We undertook a study of MHC diversity and evolutionary relationships across seven species from four families (Climacteridae, Maluridae, Pardalotidae, Meliphagidae) that comprise a prominent component of the basal passerine lineages. We aimed to determine if highly polymorphic MHC paralogs have an early evolutionary origin within passerines or are a more derived feature of the infraorder Passerida. We identified 177 alleles of the MHC class II β exon 2 in seven basal passerine species, with variation in numbers of alleles across individuals and species. Overall, we found evidence of multiple gene loci, pseudoalleles, trans-species polymorphism and high allelic diversity in these basal lineages. Phylogenetic reconstruction of avian lineages based on MHC class II β exon 2 sequences strongly supported the monophyletic grouping of basal and derived passerine species. Our study provides evidence of a large number of highly polymorphic MHC paralogs in seven basal passerine species, with strong similarities to the MHC described in more derived passerine lineages rather than the simpler MHC in non-passerine lineages. These findings indicate an early evolutionary origin of highly polymorphic MHC paralogs in passerines and shed light on the evolutionary forces shaping the avian MHC.

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

ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration.

PubMed

Zhao, Siwei; Zhu, Kan; Zhang, Yan; Zhu, Zijie; Xu, Zhengping; Zhao, Min; Pan, Tingrui

2014-11-21

Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R-2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing.

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.

Evolutionary dynamism in bryophytes: Phylogenomic inferences confirm rapid radiation in the moss family Funariaceae.

PubMed

Medina, Rafael; Johnson, Matthew; Liu, Yang; Wilding, Nicholas; Hedderson, Terry A; Wickett, Norman; Goffinet, Bernard

2018-03-01

Rapid diversifications of plants are primarily documented and studied in angiosperms, which are perceived as evolutionarily dynamic. Recent studies have, however, revealed that bryophytes have also undergone periods of rapid radiation. The speciose family Funariaceae, including the model taxon Physcomitrella patens, is one such lineage. Here, we infer relationships among major lineages within the Entosthodon-Physcomitrium complex from virtually complete organellar exomes (i.e., 123 genes) obtained through high throughput sequencing of genomic libraries enriched in these loci via targeted locus capture. Based on these extensive exonic data we (1) reconstructed a robust backbone topology of the Funariaceae, (2) confirmed the monophyly of Funaria and the polyphyly of Entosthodon, Physcomitrella, and Physcomitrium, and (3) argue for the occurrence of a rapid radiation within the Entosthodon-Physcomitrium complex that began 28 mya and gave rise more than half of the species diversity of the family. This diversification may have been triggered by a whole genome duplication and coincides with global Eocene cooling that continued through the Oligocene and Miocene. The Funariaceae join a growing list of bryophyte lineages whose history is marked by at least one burst of diversification, and our study thereby strengthens the view that bryophytes are evolutionarily dynamic lineages and that patterns and processes characterizing the evolution of angiosperms may be universal among land plants. Copyright © 2017 Elsevier Inc. All rights reserved.

Post-Test Inspection of NASA's Evolutionary Xenon Thruster Long-Duration Test Hardware: Discharge Chamber

NASA Technical Reports Server (NTRS)

Shastry, Rohit; Soulas, George C.

2016-01-01

The NEXT Long-Duration Test is part of a comprehensive thruster service life assessment intended to demonstrate overall throughput capability, validate service life models, quantify wear rates as a function of time and operating condition, and identify any unknown life-limiting mechanisms. The test was voluntarily terminated in February 2014 after demonstrating 51,184 hours of high-voltage operation, 918 kg of propellant throughput, and 35.5 MN-s of total impulse. The post-test inspection of the thruster hardware began shortly afterwards with a combination of non-destructive and destructive analysis techniques, and is presently nearing completion. This paper presents relevant results of the post-test inspection for the discharge chamber as well as other miscellaneous components such as the high-voltage propellant isolators and electrical cabling. Comparison of magnetic field measurements taken during pretest and post-test inspections indicate that the field strength did not degrade, consistent with performance data obtained during the test. Inspection of discharge chamber mesh samples show a deposition coating primarily composed of grid material that is approximately 15 micrometers in thickness. This thickness is well within the retention capability of the mesh and is therefore not expected to present any issues. Approximately 3.1 grams of deposition flakes were found at the bottom of the discharge chamber, composed primarily of grid material and carbon. Calculated size histograms of these flakes indicate that 99% have a maximum dimension of 200 micrometers or smaller, which is significantly less than the ion optics grid gap. Larger flakes that are capable of causing a grid-to-grid short will be analyzed to determine if their formation will occur in flight or is a facility effect. The high-voltage propellant isolators as well as numerous other electrical insulators were inspected and no evidence of arcing or any other issues were found.

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...

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

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.

Evolutionary rescue in vertebrates: evidence, applications and uncertainty

PubMed Central

Vander Wal, E.; Garant, D.; Festa-Bianchet, M.; Pelletier, F.

2013-01-01

The current rapid rate of human-driven environmental change presents wild populations with novel conditions and stresses. Theory and experimental evidence for evolutionary rescue present a promising case for species facing environmental change persisting via adaptation. Here, we assess the potential for evolutionary rescue in wild vertebrates. Available information on evolutionary rescue was rare and restricted to abundant and highly fecund species that faced severe intentional anthropogenic selective pressures. However, examples from adaptive tracking in common species and genetic rescues in species of conservation concern provide convincing evidence in favour of the mechanisms of evolutionary rescue. We conclude that low population size, long generation times and limited genetic variability will result in evolutionary rescue occurring rarely for endangered species without intervention. Owing to the risks presented by current environmental change and the possibility of evolutionary rescue in nature, we suggest means to study evolutionary rescue by mapping genotype → phenotype → demography → fitness relationships, and priorities for applying evolutionary rescue to wild populations. PMID:23209171

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

PubMed

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

The evolutionary origins of Syngnathidae: pipefishes and seahorses.

PubMed

Wilson, A B; Orr, J W

2011-06-01

Despite their importance as evolutionary and ecological model systems, the phylogenetic relationships among gasterosteiforms remain poorly understood, complicating efforts to understand the evolutionary origins of the exceptional morphological and behavioural diversity of this group. The present review summarizes current knowledge on the origin and evolution of syngnathids, a gasterosteiform family with a highly developed form of male parental care, combining inferences based on morphological and molecular data with paleontological evidence documenting the evolutionary history of the group. Molecular methods have provided new tools for the study of syngnathid relationships and have played an important role in recent conservation efforts. Despite recent insights into syngnathid evolution, however, a survey of the literature reveals a strong taxonomic bias towards studies on the species-rich genera Hippocampus and Syngnathus, with a lack of data for many morphologically unique members of the family. The study of the evolutionary pressures responsible for generating the high diversity of syngnathids would benefit from a wider perspective, providing a comparative framework in which to investigate the evolution of the genetic, morphological and behavioural traits of the group as a whole. © 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.

Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries.

PubMed

Jiang, Rongzhong

2007-07-01

An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10 to 20 mAcm(2). The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150 mAcm(2), respectively.

Research progress of plant population genomics based on high-throughput sequencing.

PubMed

Wang, Yun-sheng

2016-08-01

Population genomics, a new paradigm for population genetics, combine the concepts and techniques of genomics with the theoretical system of population genetics and improve our understanding of microevolution through identification of site-specific effect and genome-wide effects using genome-wide polymorphic sites genotypeing. With the appearance and improvement of the next generation high-throughput sequencing technology, the numbers of plant species with complete genome sequences increased rapidly and large scale resequencing has also been carried out in recent years. Parallel sequencing has also been done in some plant species without complete genome sequences. These studies have greatly promoted the development of population genomics and deepened our understanding of the genetic diversity, level of linking disequilibium, selection effect, demographical history and molecular mechanism of complex traits of relevant plant population at a genomic level. In this review, I briely introduced the concept and research methods of population genomics and summarized the research progress of plant population genomics based on high-throughput sequencing. I also discussed the prospect as well as existing problems of plant population genomics in order to provide references for related studies.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

PubMed Central

Zielonka, Jacek; Zielonka, Monika; Sikora, Adam; Adamus, Jan; Joseph, Joy; Hardy, Micael; Ouari, Olivier; Dranka, Brian P.; Kalyanaraman, Balaraman

2012-01-01

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants. PMID:22139901

HTS-Net: An integrated regulome-interactome approach for establishing network regulation models in high-throughput screenings

PubMed Central

Rioualen, Claire; Da Costa, Quentin; Chetrit, Bernard; Charafe-Jauffret, Emmanuelle; Ginestier, Christophe

2017-01-01

High-throughput RNAi screenings (HTS) allow quantifying the impact of the deletion of each gene in any particular function, from virus-host interactions to cell differentiation. However, there has been less development for functional analysis tools dedicated to RNAi analyses. HTS-Net, a network-based analysis program, was developed to identify gene regulatory modules impacted in high-throughput screenings, by integrating transcription factors-target genes interaction data (regulome) and protein-protein interaction networks (interactome) on top of screening z-scores. HTS-Net produces exhaustive HTML reports for results navigation and exploration. HTS-Net is a new pipeline for RNA interference screening analyses that proves better performance than simple gene rankings by z-scores, by re-prioritizing genes and replacing them in their biological context, as shown by the three studies that we reanalyzed. Formatted input data for the three studied datasets, source code and web site for testing the system are available from the companion web site at http://htsnet.marseille.inserm.fr/. We also compared our program with existing algorithms (CARD and hotnet2). PMID:28949986

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.

Robo-Lector - a novel platform for automated high-throughput cultivations in microtiter plates with high information content.

PubMed

Huber, Robert; Ritter, Daniel; Hering, Till; Hillmer, Anne-Kathrin; Kensy, Frank; Müller, Carsten; Wang, Le; Büchs, Jochen

2009-08-01

In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays. To meet this demand, a novel automated microfermentation platform consisting of a BioLector and a liquid-handling robot (Robo-Lector) was sucessfully built and tested. The BioLector provides a cultivation system that is able to permanently monitor microbial growth and the fluorescence of reporter proteins under defined conditions in microtiter plates. Three examplary methods were programed on the Robo-Lector platform to study in detail high-throughput cultivation processes and especially recombinant protein expression. The host/vector system E. coli BL21(DE3) pRhotHi-2-EcFbFP, expressing the fluorescence protein EcFbFP, was hereby investigated. With the method 'induction profiling' it was possible to conduct 96 different induction experiments (varying inducer concentrations from 0 to 1.5 mM IPTG at 8 different induction times) simultaneously in an automated way. The method 'biomass-specific induction' allowed to automatically induce cultures with different growth kinetics in a microtiter plate at the same biomass concentration, which resulted in a relative standard deviation of the EcFbFP production of only +/- 7%. The third method 'biomass-specific replication' enabled to generate equal initial biomass concentrations in main cultures from precultures with different growth kinetics. This was realized by automatically transferring an appropiate inoculum volume from the different preculture microtiter wells to respective wells of the main culture plate, where subsequently similar growth kinetics could be obtained. The Robo-Lector generates extensive kinetic data in high-throughput cultivations, particularly for biomass and fluorescence protein formation. Based on the non-invasive on-line-monitoring signals, actions of the liquid-handling robot can easily be triggered. This interaction between the robot and the BioLector (Robo-Lector) combines high-content data generation with systematic high-throughput experimentation in an automated fashion, offering new possibilities to study biological production systems. The presented platform uses a standard liquid-handling workstation with widespread automation possibilities. Thus, high-throughput cultivations can now be combined with small-scale downstream processing techniques and analytical assays. Ultimately, this novel versatile platform can accelerate and intensify research and development in the field of systems biology as well as modelling and bioprocess optimization.

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 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

Using concepts from biology to improve problem-solving methods

NASA Astrophysics Data System (ADS)

Goodman, Erik D.; Rothwell, Edward J.; Averill, Ronald C.

2011-06-01

Observing nature has been a cornerstone of engineering design. Today, engineers look not only at finished products, but imitate the evolutionary process by which highly optimized artifacts have appeared in nature. Evolutionary computation began by capturing only the simplest ideas of evolution, but today, researchers study natural evolution and incorporate an increasing number of concepts in order to evolve solutions to complex engineering problems. At the new BEACON Center for the Study of Evolution in Action, studies in the lab and field and in silico are laying the groundwork for new tools for evolutionary engineering design. This paper, which accompanies a keynote address, describes various steps in development and application of evolutionary computation, particularly as regards sensor design, and sets the stage for future advances.

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

PubMed

Carvalho, Rimenys J; Cruz, Thayana A

2018-01-01

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

Tackling the widespread and critical impact of batch effects in high-throughput data.

PubMed

Leek, Jeffrey T; Scharpf, Robert B; Bravo, Héctor Corrada; Simcha, David; Langmead, Benjamin; Johnson, W Evan; Geman, Donald; Baggerly, Keith; Irizarry, Rafael A

2010-10-01

High-throughput technologies are widely used, for example to assay genetic variants, gene and protein expression, and epigenetic modifications. One often overlooked complication with such studies is batch effects, which occur because measurements are affected by laboratory conditions, reagent lots and personnel differences. This becomes a major problem when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. Using both published studies and our own analyses, we argue that batch effects (as well as other technical and biological artefacts) are widespread and critical to address. We review experimental and computational approaches for doing so.

Assessment of DNA damage in car spray painters exposed to organic solvents by the high-throughput comet assay.

PubMed

Londoño-Velasco, Elizabeth; Martínez-Perafán, Fabián; Carvajal-Varona, Silvio; García-Vallejo, Felipe; Hoyos-Giraldo, Luz Stella

2016-05-01

Occupational exposure as a painter is associated with DNA damage and development of cancer. Comet assay has been widely adopted as a sensitive and quantitative tool for DNA damage assessment at the individual cell level in populations exposed to genotoxics. The aim of this study was to assess the application of the high-throughput comet assay, to determine the DNA damage in car spray painters. The study population included 52 car spray painters and 52 unexposed subjects. A significant increase in the %TDNA median (p <  0.001) was observed in the exposed group in comparison to the unexposed group. Neither age (%TDNA: p =  0.913) nor time of exposure (%TDNA: p = 0.398) were significantly correlated with DNA damage. The car spray painters who consumed alcohol did not show a significant increase in DNA damage compared to nonalcohol consumers (p  > 0.05). The results showed an increase in DNA breaks in car spray painters exposed to organic solvents and paints; furthermore, they demonstrated the application of high-throughput comet assay in an occupational exposure study to genotoxic agents.

Study of data I/O performance on distributed disk system in mask data preparation

NASA Astrophysics Data System (ADS)

Ohara, Shuichiro; Odaira, Hiroyuki; Chikanaga, Tomoyuki; Hamaji, Masakazu; Yoshioka, Yasuharu

2010-09-01

Data volume is getting larger every day in Mask Data Preparation (MDP). In the meantime, faster data handling is always required. MDP flow typically introduces Distributed Processing (DP) system to realize the demand because using hundreds of CPU is a reasonable solution. However, even if the number of CPU were increased, the throughput might be saturated because hard disk I/O and network speeds could be bottlenecks. So, MDP needs to invest a lot of money to not only hundreds of CPU but also storage and a network device which make the throughput faster. NCS would like to introduce new distributed processing system which is called "NDE". NDE could be a distributed disk system which makes the throughput faster without investing a lot of money because it is designed to use multiple conventional hard drives appropriately over network. NCS studies I/O performance with OASIS® data format on NDE which contributes to realize the high throughput in this paper.

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...

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.

Evolutionary responses to climate change in parasitic systems.

PubMed

Chaianunporn, Thotsapol; Hovestadt, Thomas

2015-08-01

Species may respond to climate change in many ecological and evolutionary ways. In this simulation study, we focus on the concurrent evolution of three traits in response to climate change, namely dispersal probability, temperature tolerance (or niche width), and temperature preference (optimal habitat). More specifically, we consider evolutionary responses in host species involved in different types of interaction, that is parasitism or commensalism, and for low or high costs of a temperature tolerance-fertility trade-off (cost of generalization). We find that host species potentially evolve all three traits simultaneously in response to increasing temperature but that the evolutionary response interacts and may be compensatory depending on the conditions. The evolutionary adjustment of temperature preference is slower in the parasitism than in commensalism scenario. Parasitism, in turn, selects for higher temperature tolerance and increased dispersal. High costs for temperature tolerance (i.e. generalization) restrict evolution of tolerance and thus lead to a faster response in temperature preference than that observed under low costs. These results emphasize the possible role of biotic interactions and the importance of 'multidimensional' evolutionary responses to climate change. © 2015 John Wiley & Sons Ltd.

Evolutionary engineering of industrial microorganisms-strategies and applications.

PubMed

Zhu, Zhengming; Zhang, Juan; Ji, Xiaomei; Fang, Zhen; Wu, Zhimeng; Chen, Jian; Du, Guocheng

2018-06-01

Microbial cells have been widely used in the industry to obtain various biochemical products, and evolutionary engineering is a common method in biological research to improve their traits, such as high environmental tolerance and improvement of product yield. To obtain better integrate functions of microbial cells, evolutionary engineering combined with other biotechnologies have attracted more attention in recent years. Classical laboratory evolution has been proven effective to letting more beneficial mutations occur in different genes but also has some inherent limitations such as a long evolutionary period and uncontrolled mutation frequencies. However, recent studies showed that some new strategies may gradually overcome these limitations. In this review, we summarize the evolutionary strategies commonly used in industrial microorganisms and discuss the combination of evolutionary engineering with other biotechnologies such as systems biology and inverse metabolic engineering. Finally, we prospect the importance and application prospect of evolutionary engineering as a powerful tool especially in optimization of industrial microbial cell factories.

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

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

Su, Hui

2001-01-01

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

Digital imaging of root traits (DIRT): a high-throughput computing and collaboration platform for field-based root phenomics.

PubMed

Das, Abhiram; Schneider, Hannah; Burridge, James; Ascanio, Ana Karine Martinez; Wojciechowski, Tobias; Topp, Christopher N; Lynch, Jonathan P; Weitz, Joshua S; Bucksch, Alexander

2015-01-01

Plant root systems are key drivers of plant function and yield. They are also under-explored targets to meet global food and energy demands. Many new technologies have been developed to characterize crop root system architecture (CRSA). These technologies have the potential to accelerate the progress in understanding the genetic control and environmental response of CRSA. Putting this potential into practice requires new methods and algorithms to analyze CRSA in digital images. Most prior approaches have solely focused on the estimation of root traits from images, yet no integrated platform exists that allows easy and intuitive access to trait extraction and analysis methods from images combined with storage solutions linked to metadata. Automated high-throughput phenotyping methods are increasingly used in laboratory-based efforts to link plant genotype with phenotype, whereas similar field-based studies remain predominantly manual low-throughput. Here, we present an open-source phenomics platform "DIRT", as a means to integrate scalable supercomputing architectures into field experiments and analysis pipelines. DIRT is an online platform that enables researchers to store images of plant roots, measure dicot and monocot root traits under field conditions, and share data and results within collaborative teams and the broader community. The DIRT platform seamlessly connects end-users with large-scale compute "commons" enabling the estimation and analysis of root phenotypes from field experiments of unprecedented size. DIRT is an automated high-throughput computing and collaboration platform for field based crop root phenomics. The platform is accessible at http://www.dirt.iplantcollaborative.org/ and hosted on the iPlant cyber-infrastructure using high-throughput grid computing resources of the Texas Advanced Computing Center (TACC). DIRT is a high volume central depository and high-throughput RSA trait computation platform for plant scientists working on crop roots. It enables scientists to store, manage and share crop root images with metadata and compute RSA traits from thousands of images in parallel. It makes high-throughput RSA trait computation available to the community with just a few button clicks. As such it enables plant scientists to spend more time on science rather than on technology. All stored and computed data is easily accessible to the public and broader scientific community. We hope that easy data accessibility will attract new tool developers and spur creative data usage that may even be applied to other fields of science.

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...

Mitochondrial DNA haplotype distribution patterns in Pinus ponderosa (pinaceae): range-wide evolutionary history and implications for conservation

Treesearch

Kevin M. Potter; Valerie D. Hipkins; Mary F. Mahalovich; Robert E. Means

2013-01-01

Premise of the study: Ponderosa pine ( Pinus ponderosa Douglas ex P. Lawson & C. Lawson) exhibits complicated patterns of morphological and genetic variation across its range in western North America. This study aims to clarify P. ponderosa evolutionary history and phylogeography using a highly polymorphic...

Novel throughput phenotyping platforms in plant genetic studies.

PubMed

Montes, Juan M; Melchinger, Albrecht E; Reif, Jochen C

2007-10-01

Unraveling the genetic basis of complex traits in plants is limited by the lack of appropriate phenotyping platforms that enable high-throughput screening of many genotypes in multilocation field trials. Near-infrared spectroscopy on agricultural harvesters and spectral reflectance of plant canopies have recently been reported as promising components of novel phenotyping platforms. Understanding the genetic basis of complex traits is now within reach with the use of these new techniques.

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

PubMed Central

Borges, Chad R.; Jarvis, Jason W.; Oran, Paul E.; Rogers, Stephen P.; Nelson, Randall W.

2008-01-01

Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID:19137103

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

PubMed

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

A Customizable Flow Injection System for Automated, High Throughput, and Time Sensitive Ion Mobility Spectrometry and Mass Spectrometry Measurements

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

Orton, Daniel J.; Tfaily, Malak M.; Moore, Ronald J.

To better understand disease conditions and environmental perturbations, multi-omic studies (i.e. proteomic, lipidomic, metabolomic, etc. analyses) are vastly increasing in popularity. In a multi-omic study, a single sample is typically extracted in multiple ways and numerous analyses are performed using different instruments. Thus, one sample becomes many analyses, making high throughput and reproducible evaluations a necessity. One way to address the numerous samples and varying instrumental conditions is to utilize a flow injection analysis (FIA) system for rapid sample injection. While some FIA systems have been created to address these challenges, many have limitations such as high consumable costs, lowmore » pressure capabilities, limited pressure monitoring and fixed flow rates. To address these limitations, we created an automated, customizable FIA system capable of operating at diverse flow rates (~50 nL/min to 500 µL/min) to accommodate low- and high-flow instrument sources. This system can also operate at varying analytical throughputs from 24 to 1200 samples per day to enable different MS analysis approaches. Applications ranging from native protein analyses to molecular library construction were performed using the FIA system. The results from these studies showed a highly robust platform, providing consistent performance over many days without carryover as long as washing buffers specific to each molecular analysis were utilized.« less

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.

NEXT Long-Duration Test Neutralizer Performance and Erosion Characteristics

NASA Technical Reports Server (NTRS)

Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2009-01-01

The NASA's Evolutionary Xenon Thruster (NEXT) program is developing the next-generation ion propulsion system with significant enhancements beyond the state-of-the-art to provide future NASA science missions with enhanced capabilities at a low total development cost. A Long-Duration Test (LDT) was initiated in June 2005, to verify the NEXT propellant throughput capability to a qualification-level of 450 kg, 1.5 times the anticipated throughput requirement of 300 kg per thruster based on mission analyses. As of September 2, 2009, the thruster has accumulated 24,400 hr of operation with extensive durations at the following input powers: 6.9, 4.7, 1.1, and 0.5 kW. The thruster has processed 434 kg of xenon, surpassing the NASA Solar Technology Application Readiness (NSTAR) program thruster propellant throughput demonstrated during the extended life testing of the Deep Space 1 flight spare ion thruster and approaching the NEXT development qualification throughput goal of 450 kg. The NEXT LDT has demonstrated a total impulse of 16.1 10(exp 6zzz0 N s; the highest total impulse ever demonstrated by an ion thruster. A reduction in neutralizer flow margin has been the only appreciable source of thruster performance degradation. The behavior of the neutralizer is not easily predicted due to both erosion and deposition observed in previous wear tests. Spot-to-plume mode transition flow data and in-situ erosion results for the LDT neutralizer are discussed. This loss of flow margin has been addressed through a combination of a design change in the prototype-model neutralizer to increase flow margin at low emission current and to update the NEXT throttle table to ensure adequate flow margin as a function of propellant throughput processed. The new throttle table will be used for future LDT operations. The performance of the NEXT LDT neutralizer is consistent with that observed for long-life hollow cathodes. The neutralizer life-limiting failure modes are progressing as expected and the neutralizer data indicate none of the neutralizer failures are imminent.

Status of the NEXT Long-Duration Test After 23,300 Hours of Operation

NASA Technical Reports Server (NTRS)

Herman, Daniel A.; Soulas, George C.; Patterson, Michael J.

2009-01-01

The NASA s Evolutionary Xenon Thruster (NEXT) program is developing the next-generation ion propulsion system with significant enhancements beyond the state-of-the-art in ion propulsion to provide future NASA science missions with enhanced mission capabilities at a low total development cost. As part of a comprehensive thruster service life assessment utilizing both testing and analyses, a Long-Duration Test (LDT) was initiated in June 2005, to verify the NEXT propellant throughput capability to a qualification-level of 450 kg, 1.5 times the anticipated throughput requirement of 300 kg per thruster from mission analyses. The LDT is being conducted with a modified, flight-representative NEXT engineering model ion thruster, designated EM3. As of July 2009, the thruster has accumulated 23,300 h of operation with extensive durations at the following input powers: 6.9, 4.7, 1.1, and 0.5 kW. The thruster has processed 427 kg of xenon surpassing the NSTAR propellant throughput demonstrated during the extended life testing of the Deep Space 1 flight spare ion thruster and approaching the NEXT development qualification throughput goal. The NEXT LDT has demonstrated a total impulse of 16.0 10(exp 6) N/s; the highest total impulse ever demonstrated by an ion thruster. Thruster performance tests are conducted periodically over the entire NEXT throttle table with input power ranging 0.5 to 6.9 kW. Thruster performance parameters including thrust, input power, specific impulse, and thruster efficiency have been nominal with little variation to date. The NSTAR first-failure mode, accelerator aperture erosion leading to electron backstreaming, has been mitigated in the NEXT design. The severe NSTAR discharge cathode assembly erosion has been mitigated by a graphite keeper in the NEXT thruster. Tracking of the NEXT first failure mode, charge-exchange ion impingement on the accelerator grid causing hexagonal groove erosion, is consistent with model predictions and indicates thruster life greater than or equal to 750 kg throughput. This paper presents the status, performance data, and wear characteristics of the NEXT LDT to date.

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, ...

Assessing Morphological and Physiological Properties of Forest Species Using High Throughput Plant Phenotyping and Imaging Techniques

NASA Astrophysics Data System (ADS)

Mazis, A.; Hiller, J.; Morgan, P.; Awada, T.; Stoerger, V.

2017-12-01

High throughput plant phenotyping is increasingly being used to assess morphological and biophysical traits of economically important crops in agriculture. In this study, the potential application of this technique in natural resources management, through the characterization of woody plants regeneration, establishment, growth, and responses to water and nutrient manipulations was assessed. Two woody species were selected for this study, Quercus prinoides and Quercus bicolor. Seeds were collected from trees growing at the edge of their natural distribution in Nebraska and Missouri, USA. Seeds were germinated in the greenhouse and transferred to the Nebraska Innovation Campus Lemnatec3D High Throughput facility at the University of Nebraska-Lincoln. Seedlings subjected to water and N manipulations, were imaged twice or three times a week using four cameras (Visible, Fluorescence, Infrared and Hyperspectral), throughout the growing season. Traditional leaf to plant levels ecophysiological measurements were concurrently acquired to assess the relationship between these two techniques. These include gas exchange (LI 6400 and LI 6800, LICOR Inc., Lincoln NE), chlorophyll content, optical characteristics (Ocean Optics USB200), water and osmotic potentials, leaf area and weight and carbon isotope ratio. In the presentation, we highlight results on the potential use of high throughput plant phenotyping techniques to assess the morphology and physiology of woody species including responses to water availability and nutrient manipulation, and its broader application under field conditions and natural resources management. Also, we explore the different capabilities imaging provides us for modeling the plant physiological and morphological growth and how it can complement the current techniques

Arabidopsis Seed Content QTL Mapping Using High-Throughput Phenotyping: The Assets of Near Infrared Spectroscopy

PubMed Central

Jasinski, Sophie; Lécureuil, Alain; Durandet, Monique; Bernard-Moulin, Patrick; Guerche, Philippe

2016-01-01

Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well-known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of 100s of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon, and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analyzed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 × Col-0 recombinant inbred line population. Some QTL co-localized with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and genome wide association studies. PMID:27891138

Experimental Study of an Advanced Concept of Moderate-resolution Holographic Spectrographs

NASA Astrophysics Data System (ADS)

Muslimov, Eduard; Valyavin, Gennady; Fabrika, Sergei; Musaev, Faig; Galazutdinov, Gazinur; Pavlycheva, Nadezhda; Emelianov, Eduard

2018-07-01

We present the results of an experimental study of an advanced moderate-resolution spectrograph based on a cascade of narrow-band holographic gratings. The main goal of the project is to achieve a moderately high spectral resolution with R up to 5000 simultaneously in the 4300–6800 Å visible spectral range on a single standard CCD, together with an increased throughput. The experimental study consisted of (1) resolution and image quality tests performed using the solar spectrum, and (2) a total throughput test performed for a number of wavelengths using a calibrated lab monochromator. The measured spectral resolving power reaches values over R > 4000 while the experimental throughput is as high as 55%, which agrees well with the modeling results. Comparing the obtained characteristics of the spectrograph under consideration with the best existing spectrographs, we conclude that the used concept can be considered as a very competitive and cheap alternative to the existing spectrographs of the given class. We propose several astrophysical applications for the instrument and discuss the prospect of creating its full-scale version.

High-throughput analysis using non-depletive SPME: challenges and applications to the determination of free and total concentrations in small sample volumes.

PubMed

Boyacı, Ezel; Bojko, Barbara; Reyes-Garcés, Nathaly; Poole, Justen J; Gómez-Ríos, Germán Augusto; Teixeira, Alexandre; Nicol, Beate; Pawliszyn, Janusz

2018-01-18

In vitro high-throughput non-depletive quantitation of chemicals in biofluids is of growing interest in many areas. Some of the challenges facing researchers include the limited volume of biofluids, rapid and high-throughput sampling requirements, and the lack of reliable methods. Coupled to the above, growing interest in the monitoring of kinetics and dynamics of miniaturized biosystems has spurred the demand for development of novel and revolutionary methodologies for analysis of biofluids. The applicability of solid-phase microextraction (SPME) is investigated as a potential technology to fulfill the aforementioned requirements. As analytes with sufficient diversity in their physicochemical features, nicotine, N,N-Diethyl-meta-toluamide, and diclofenac were selected as test compounds for the study. The objective was to develop methodologies that would allow repeated non-depletive sampling from 96-well plates, using 100 µL of sample. Initially, thin film-SPME was investigated. Results revealed substantial depletion and consequent disruption in the system. Therefore, new ultra-thin coated fibers were developed. The applicability of this device to the described sampling scenario was tested by determining the protein binding of the analytes. Results showed good agreement with rapid equilibrium dialysis. The presented method allows high-throughput analysis using small volumes, enabling fast reliable free and total concentration determinations without disruption of system equilibrium.

Study on high throughput nanomanufacturing of photopatternable nanofibers using tube nozzle electrospinning with multi-tubes and multi-nozzles

NASA Astrophysics Data System (ADS)

Fang, Sheng-Po; Jao, PitFee; Senior, David E.; Kim, Kyoung-Tae; Yoon, Yong-Kyu

2017-12-01

High throughput nanomanufacturing of photopatternable nanofibers and subsequent photopatterning is reported. For the production of high density nanofibers, the tube nozzle electrospinning (TNE) process has been used, where an array of micronozzles on the sidewall of a plastic tube are used as spinnerets. By increasing the density of nozzles, the electric fields of adjacent nozzles confine the cone of electrospinning and give a higher density of nanofibers. With TNE, higher density nozzles are easily achievable compared to metallic nozzles, e.g. an inter-nozzle distance as small as 0.5 cm and an average semi-vertical repulsion angle of 12.28° for 8-nozzles were achieved. Nanofiber diameter distribution, mass throughput rate, and growth rate of nanofiber stacks in different operating conditions and with different numbers of nozzles, such as 2, 4 and 8 nozzles, and scalability with single and double tube configurations are discussed. Nanofibers made of SU-8, photopatternable epoxy, have been collected to a thickness of over 80 μm in 240 s of electrospinning and the production rate of 0.75 g/h is achieved using the 2 tube 8 nozzle systems, followed by photolithographic micropatterning. TNE is scalable to a large number of nozzles, and offers high throughput production, plug and play capability with standard electrospinning equipment, and little waste of polymer.

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

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

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

DOE PAGES

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

2015-12-09

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

High-Throughput, Motility-Based Sorter for Microswimmers and Gene Discovery Platform

NASA Astrophysics Data System (ADS)

Yuan, Jinzhou; Raizen, David; Bau, Haim

2015-11-01

Animal motility varies with genotype, disease progression, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method was implemented in a simple microfluidic device capable of sorting many thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriched for known C. elegans motility mutants. Furthermore, using this device, we isolated low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates sleep-like quiescence in C. elegans. Subsequent genomic sequencing led to the identification of a flp-13-suppressor gene. This research was supported, in part, by NIH NIA Grant 5R03AG042690-02.

Mathematical and Computational Modeling in Complex Biological Systems

PubMed Central

Li, Wenyang; Zhu, Xiaoliang

2017-01-01

The biological process and molecular functions involved in the cancer progression remain difficult to understand for biologists and clinical doctors. Recent developments in high-throughput technologies urge the systems biology to achieve more precise models for complex diseases. Computational and mathematical models are gradually being used to help us understand the omics data produced by high-throughput experimental techniques. The use of computational models in systems biology allows us to explore the pathogenesis of complex diseases, improve our understanding of the latent molecular mechanisms, and promote treatment strategy optimization and new drug discovery. Currently, it is urgent to bridge the gap between the developments of high-throughput technologies and systemic modeling of the biological process in cancer research. In this review, we firstly studied several typical mathematical modeling approaches of biological systems in different scales and deeply analyzed their characteristics, advantages, applications, and limitations. Next, three potential research directions in systems modeling were summarized. To conclude, this review provides an update of important solutions using computational modeling approaches in systems biology. PMID:28386558

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

PubMed Central

Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain. PMID:28472077

Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening.

PubMed

Fan, Xiaoguang; Wu, Heyun; Li, Guoliang; Yuan, Hui; Zhang, Hongchao; Li, Yanjun; Xie, Xixian; Chen, Ning

2017-01-01

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5'-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.

A gas trapping method for high-throughput metabolic experiments.

PubMed

Krycer, James R; Diskin, Ciana; Nelson, Marin E; Zeng, Xiao-Yi; Fazakerley, Daniel J; James, David E

2018-01-01

Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. We used this trap design to measure CO2 production from glucose and fatty acid metabolism, as well as hydrogen sulfide production from cysteine-treated cells. Our data clearly show that this gas trap can be applied to liquid and solid gas-collection media and can be used to study gaseous product generation by both adherent cells and cells in suspension. Since our gas traps can be adapted to multiwell plates of various sizes, they present a convenient, cost-effective solution that can accommodate the trend toward high-throughput measurements in metabolic research.

Mathematical and Computational Modeling in Complex Biological Systems.

PubMed

Ji, Zhiwei; Yan, Ke; Li, Wenyang; Hu, Haigen; Zhu, Xiaoliang

2017-01-01

The biological process and molecular functions involved in the cancer progression remain difficult to understand for biologists and clinical doctors. Recent developments in high-throughput technologies urge the systems biology to achieve more precise models for complex diseases. Computational and mathematical models are gradually being used to help us understand the omics data produced by high-throughput experimental techniques. The use of computational models in systems biology allows us to explore the pathogenesis of complex diseases, improve our understanding of the latent molecular mechanisms, and promote treatment strategy optimization and new drug discovery. Currently, it is urgent to bridge the gap between the developments of high-throughput technologies and systemic modeling of the biological process in cancer research. In this review, we firstly studied several typical mathematical modeling approaches of biological systems in different scales and deeply analyzed their characteristics, advantages, applications, and limitations. Next, three potential research directions in systems modeling were summarized. To conclude, this review provides an update of important solutions using computational modeling approaches in systems biology.

High-throughput technology for novel SO2 oxidation catalysts

PubMed Central

Loskyll, Jonas; Stoewe, Klaus; Maier, Wilhelm F

2011-01-01

We review the state of the art and explain the need for better SO2 oxidation catalysts for the production of sulfuric acid. A high-throughput technology has been developed for the study of potential catalysts in the oxidation of SO2 to SO3. High-throughput methods are reviewed and the problems encountered with their adaptation to the corrosive conditions of SO2 oxidation are described. We show that while emissivity-corrected infrared thermography (ecIRT) can be used for primary screening, it is prone to errors because of the large variations in the emissivity of the catalyst surface. UV-visible (UV-Vis) spectrometry was selected instead as a reliable analysis method of monitoring the SO2 conversion. Installing plain sugar absorbents at reactor outlets proved valuable for the detection and quantitative removal of SO3 from the product gas before the UV-Vis analysis. We also overview some elements used for prescreening and those remaining after the screening of the first catalyst generations. PMID:27877427

Synthesis and evaluation of chromogenic and fluorogenic substrates for high-throughput detection of enzymes that hydrolyze inorganic polyphosphate.

PubMed

Hebbard, Carleigh F F; Wang, Yan; Baker, Catherine J; Morrissey, James H

2014-08-11

Inorganic polyphosphates, linear polymers of orthophosphate, occur naturally throughout biology and have many industrial applications. Their biodegradable nature makes them attractive for a multitude of uses, and it would be important to understand how polyphosphates are turned over enzymatically. Studies of inorganic polyphosphatases are, however, hampered by the lack of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. We now report chromogenic and fluorogenic polyphosphate substrates that permit spectrophotometric monitoring of polyphosphate hydrolysis and allow for high-throughput analyses of both endopolyphosphatase and exopolyphosphatase activities, depending on assay configuration. These substrates contain 4-nitrophenol or 4-methylumbelliferone moieties that are covalently attached to the terminal phosphates of polyphosphate via phosphoester linkages formed during reactions mediated by EDAC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide). This report identifies Nudt2 as an inorganic polyphosphatase and also adds to the known coupling chemistry for polyphosphates, permitting facile covalent linkage of alcohols with the terminal phosphates of inorganic polyphosphate.

An automated high throughput tribometer for adhesion, wear, and friction measurements

NASA Astrophysics Data System (ADS)

Kalihari, Vivek; Timpe, Shannon J.; McCarty, Lyle; Ninke, Matthew; Whitehead, Jim

2013-03-01

Understanding the origin and correlation of different surface properties under a multitude of operating conditions is critical in tribology. Diverse tribological properties and a lack of a single instrument to measure all make it difficult to compare and correlate properties, particularly in light of the wide range of interfaces commonly investigated. In the current work, a novel automated tribometer has been designed and validated, providing a unique experimental platform capable of high throughput adhesion, wear, kinetic friction, and static friction measurements. The innovative design aspects are discussed that allow for a variety of probes, sample surfaces, and testing conditions. Critical components of the instrument and their design criteria are described along with examples of data collection schemes. A case study is presented with multiple surface measurements performed on a set of characteristic substrates. Adhesion, wear, kinetic friction, and static friction are analyzed and compared across surfaces, highlighting the comprehensive nature of the surface data that can be generated using the automated high throughput tribometer.

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,...

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.

HTP-NLP: A New NLP System for High Throughput Phenotyping.

PubMed

Schlegel, Daniel R; Crowner, Chris; Lehoullier, Frank; Elkin, Peter L

2017-01-01

Secondary use of clinical data for research requires a method to quickly process the data so that researchers can quickly extract cohorts. We present two advances in the High Throughput Phenotyping NLP system which support the aim of truly high throughput processing of clinical data, inspired by a characterization of the linguistic properties of such data. Semantic indexing to store and generalize partially-processed results and the use of compositional expressions for ungrammatical text are discussed, along with a set of initial timing results for the system.

High count-rate study of two TES x-ray microcalorimeters with different transition temperatures

NASA Astrophysics Data System (ADS)

Lee, Sang-Jun; Adams, Joseph S.; Bandler, Simon R.; Betancourt-Martinez, Gabriele L.; Chervenak, James A.; Eckart, Megan E.; Finkbeiner, Fred M.; Kelley, Richard L.; Kilbourne, Caroline A.; Porter, Frederick S.; Sadleir, John E.; Smith, Stephen J.; Wassell, Edward J.

2017-10-01

We have developed transition-edge sensor (TES) microcalorimeter arrays with high count-rate capability and high energy resolution to carry out x-ray imaging spectroscopy observations of various astronomical sources and the Sun. We have studied the dependence of the energy resolution and throughput (fraction of processed pulses) on the count rate for such microcalorimeters with two different transition temperatures (T c). Devices with both transition temperatures were fabricated within a single microcalorimeter array directly on top of a solid substrate where the thermal conductance of the microcalorimeter is dependent upon the thermal boundary resistance between the TES sensor and the dielectric substrate beneath. Because the thermal boundary resistance is highly temperature dependent, the two types of device with different T cs had very different thermal decay times, approximately one order of magnitude different. In our earlier report, we achieved energy resolutions of 1.6 and 2.3 eV at 6 keV from lower and higher T c devices, respectively, using a standard analysis method based on optimal filtering in the low flux limit. We have now measured the same devices at elevated x-ray fluxes ranging from 50 Hz to 1000 Hz per pixel. In the high flux limit, however, the standard optimal filtering scheme nearly breaks down because of x-ray pile-up. To achieve the highest possible energy resolution for a fixed throughput, we have developed an analysis scheme based on the so-called event grade method. Using the new analysis scheme, we achieved 5.0 eV FWHM with 96% throughput for 6 keV x-rays of 1025 Hz per pixel with the higher T c (faster) device, and 5.8 eV FWHM with 97% throughput with the lower T c (slower) device at 722 Hz.

Precise, High-throughput Analysis of Bacterial Growth.

PubMed

Kurokawa, Masaomi; Ying, Bei-Wen

2017-09-19

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

High throughput screening of CO2-tolerating microalgae using GasPak bags

PubMed Central

2013-01-01

Background Microalgae are diverse in terms of their speciation and function. More than 35,000 algal strains have been described, and thousands of algal cultures are maintained in different culture collection centers. The ability of CO2 uptake by microalgae varies dramatically among algal species. It becomes challenging to select suitable algal candidates that can proliferate under high CO2 concentration from a large collection of algal cultures. Results Here, we described a high throughput screening method to rapidly identify high CO2 affinity microalgae. The system integrates a CO2 mixer, GasPak bags and microplates. Microalgae on the microplates will be cultivated in GasPak bags charged with different CO2 concentrations. Using this method, we identified 17 algal strains whose growth rates were not influenced when the concentration of CO2 was increased from 2 to 20% (v/v). Most CO2 tolerant strains identified in this study were closely related to the species Scenedesmus and Chlorococcum. One of Scenedesmus strains (E7A) has been successfully tested in in the scale up photo bioreactors (500 L) bubbled with flue gas which contains 10-12% CO2. Conclusion Our high throughput CO2 testing system provides a rapid and reliable way for identifying microalgal candidate strains that can grow under high CO2 condition from a large pool of culture collection species. This high throughput system can also be modified for selecting algal strains that can tolerate other gases, such as NOx, SOx, or flue gas. PMID:24341988

In-Space Propulsion Technology Program Solar Electric Propulsion Technologies

NASA Technical Reports Server (NTRS)

Dankanich, John W.

2006-01-01

NASA's In-space Propulsion (ISP) Technology Project is developing new propulsion technologies that can enable or enhance near and mid-term NASA science missions. The Solar Electric Propulsion (SEP) technology area has been investing in NASA s Evolutionary Xenon Thruster (NEXT), the High Voltage Hall Accelerator (HiVHAC), lightweight reliable feed systems, wear testing, and thruster modeling. These investments are specifically targeted to increase planetary science payload capability, expand the envelope of planetary science destinations, and significantly reduce the travel times, risk, and cost of NASA planetary science missions. Status and expected capabilities of the SEP technologies are reviewed in this presentation. The SEP technology area supports numerous mission studies and architecture analyses to determine which investments will give the greatest benefit to science missions. Both the NEXT and HiVHAC thrusters have modified their nominal throttle tables to better utilize diminished solar array power on outbound missions. A new life extension mechanism has been implemented on HiVHAC to increase the throughput capability on low-power systems to meet the needs of cost-capped missions. Lower complexity, more reliable feed system components common to all electric propulsion (EP) systems are being developed. ISP has also leveraged commercial investments to further validate new ion and hall thruster technologies and to potentially lower EP mission costs.

Setting the stage - building and working in an ancient DNA laboratory.

PubMed

Knapp, Michael; Clarke, Andrew C; Horsburgh, K Ann; Matisoo-Smith, Elizabeth A

2012-01-20

With the introduction of next generation high throughput sequencing in 2005 and the resulting revolution in genetics, ancient DNA research has rapidly developed from an interesting but marginal field within evolutionary biology into one that can contribute significantly to our understanding of evolution in general and the development of our own species in particular. While the amount of sequence data available from ancient human, other animal and plant remains has increased dramatically over the past five years, some key limitations of ancient DNA research remain. Most notably, reduction of contamination and the authentication of results are of utmost importance. A number of studies have addressed different aspects of sampling, DNA extraction and DNA manipulation in order to establish protocols that most efficiently generate reproducible and authentic results. As increasing numbers of researchers from different backgrounds become interested in using ancient DNA technology to address key questions, the need for practical guidelines on how to construct and use an ancient DNA facility arises. The aim of this article is therefore to provide practical tips for building a state-of-the-art ancient DNA facility. It is intended to help researchers new to the field of ancient DNA research generally, and those considering the application of next generation sequencing, in their planning process. Copyright © 2011 Elsevier GmbH. All rights reserved.

Combined Effect of Random Transmit Power Control and Inter-Path Interference Cancellation on DS-CDMA Packet Mobile Communications

NASA Astrophysics Data System (ADS)

Kudoh, Eisuke; Ito, Haruki; Wang, Zhisen; Adachi, Fumiyuki

In mobile communication systems, high speed packet data services are demanded. In the high speed data transmission, throughput degrades severely due to severe inter-path interference (IPI). Recently, we proposed a random transmit power control (TPC) to increase the uplink throughput of DS-CDMA packet mobile communications. In this paper, we apply IPI cancellation in addition to the random TPC. We derive the numerical expression of the received signal-to-interference plus noise power ratio (SINR) and introduce IPI cancellation factor. We also derive the numerical expression of system throughput when IPI is cancelled ideally to compare with the Monte Carlo numerically evaluated system throughput. Then we evaluate, by Monte-Carlo numerical computation method, the combined effect of random TPC and IPI cancellation on the uplink throughput of DS-CDMA packet mobile communications.

The tale of the shrinking weapon: seasonal changes in nutrition affect weapon size and sexual dimorphism, but not contemporary evolution.

PubMed

Miller, C W; McDonald, G C; Moore, A J

2016-11-01

Sexually selected traits are often highly variable in size within populations due to their close link with the physical condition of individuals. Nutrition has a large impact on physical condition, and thus, any seasonal changes in nutritional quality are predicted to alter the average size of sexually selected traits as well as the degree of sexual dimorphism in populations. However, although traits affected by mate choice are well studied, we have a surprising lack of knowledge of how natural variation in nutrition affects the expression of sexually selected weapons and sexual dimorphism. Further, few studies explicitly test for differences in the heritability and mean-scaled evolvability of sexually selected traits across conditions. We studied Narnia femorata (Hemiptera: Coreidae), an insect where males use their hind legs as weapons and the femurs are enlarged, to understand the extent to which weapon expression, sexual dimorphism and evolvability change across the actual range of nutrition available in the wild. We found that insects raised on a poor diet (cactus without fruit) are nearly monomorphic, whereas those raised on a high-quality diet (cactus with ripe fruit) are distinctly sexually dimorphic via the expression of large hind leg weapons in males. Contrary to our expectations, we found little evidence of a potential for evolutionary change for any trait measured. Thus, although we show weapons are highly condition dependent, and changes in weapon expression and dimorphism could alter evolutionary dynamics, our populations are unlikely to experience further evolutionary changes under current conditions. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

High-throughput quantum cascade laser (QCL) spectral histopathology: a practical approach towards clinical translation.

PubMed

Pilling, Michael J; Henderson, Alex; Bird, Benjamin; Brown, Mick D; Clarke, Noel W; Gardner, Peter

2016-06-23

Infrared microscopy has become one of the key techniques in the biomedical research field for interrogating tissue. In partnership with multivariate analysis and machine learning techniques, it has become widely accepted as a method that can distinguish between normal and cancerous tissue with both high sensitivity and high specificity. While spectral histopathology (SHP) is highly promising for improved clinical diagnosis, several practical barriers currently exist, which need to be addressed before successful implementation in the clinic. Sample throughput and speed of acquisition are key barriers and have been driven by the high volume of samples awaiting histopathological examination. FTIR chemical imaging utilising FPA technology is currently state-of-the-art for infrared chemical imaging, and recent advances in its technology have dramatically reduced acquisition times. Despite this, infrared microscopy measurements on a tissue microarray (TMA), often encompassing several million spectra, takes several hours to acquire. The problem lies with the vast quantities of data that FTIR collects; each pixel in a chemical image is derived from a full infrared spectrum, itself composed of thousands of individual data points. Furthermore, data management is quickly becoming a barrier to clinical translation and poses the question of how to store these incessantly growing data sets. Recently, doubts have been raised as to whether the full spectral range is actually required for accurate disease diagnosis using SHP. These studies suggest that once spectral biomarkers have been predetermined it may be possible to diagnose disease based on a limited number of discrete spectral features. In this current study, we explore the possibility of utilising discrete frequency chemical imaging for acquiring high-throughput, high-resolution chemical images. Utilising a quantum cascade laser imaging microscope with discrete frequency collection at key diagnostic wavelengths, we demonstrate that we can diagnose prostate cancer with high sensitivity and specificity. Finally we extend the study to a large patient dataset utilising tissue microarrays, and show that high sensitivity and specificity can be achieved using high-throughput, rapid data collection, thereby paving the way for practical implementation in the clinic.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

PubMed

Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

2017-12-01

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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

EPA Science Inventory

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

Fulfilling the promise of the materials genome initiative with high-throughput experimental methodologies

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

Green, Martin L.; Choi, C. L.; Hattrick-Simpers, J. R.

The Materials Genome Initiative, a national effort to introduce new materials into the market faster and at lower cost, has made significant progress in computational simulation and modeling of materials. To build on this progress, a large amount of experimental data for validating these models, and informing more sophisticated ones, will be required. High-throughput experimentation generates large volumes of experimental data using combinatorial materials synthesis and rapid measurement techniques, making it an ideal experimental complement to bring the Materials Genome Initiative vision to fruition. This paper reviews the state-of-the-art results, opportunities, and challenges in high-throughput experimentation for materials design. Asmore » a result, a major conclusion is that an effort to deploy a federated network of high-throughput experimental (synthesis and characterization) tools, which are integrated with a modern materials data infrastructure, is needed.« less

Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

PubMed Central

Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

2012-01-01

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

A Self-Reporting Photocatalyst for Online Fluorescence Monitoring of High Throughput RAFT Polymerization.

PubMed

Yeow, Jonathan; Joshi, Sanket; Chapman, Robert; Boyer, Cyrille Andre Jean Marie

2018-04-25

Translating controlled/living radical polymerization (CLRP) from batch to the high throughput production of polymer libraries presents several challenges in terms of both polymer synthesis and characterization. Although recently there have been significant advances in the field of low volume, high throughput CLRP, techniques able to simultaneously monitor multiple polymerizations in an "online" manner have not yet been developed. Here, we report our discovery that 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) is a self-reporting photocatalyst that can mediate PET-RAFT polymerization as well as report on monomer conversion via changes in its fluorescence properties. This enables the use of a microplate reader to conduct high throughput "online" monitoring of PET-RAFT polymerizations performed directly in 384-well, low volume microtiter plates. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fulfilling the promise of the materials genome initiative with high-throughput experimental methodologies

DOE PAGES

Green, Martin L.; Choi, C. L.; Hattrick-Simpers, J. R.; ...

2017-03-28

The Materials Genome Initiative, a national effort to introduce new materials into the market faster and at lower cost, has made significant progress in computational simulation and modeling of materials. To build on this progress, a large amount of experimental data for validating these models, and informing more sophisticated ones, will be required. High-throughput experimentation generates large volumes of experimental data using combinatorial materials synthesis and rapid measurement techniques, making it an ideal experimental complement to bring the Materials Genome Initiative vision to fruition. This paper reviews the state-of-the-art results, opportunities, and challenges in high-throughput experimentation for materials design. Asmore » a result, a major conclusion is that an effort to deploy a federated network of high-throughput experimental (synthesis and characterization) tools, which are integrated with a modern materials data infrastructure, is needed.« less

Germination Potential of Dormant and Nondormant Arabidopsis Seeds Is Driven by Distinct Recruitment of Messenger RNAs to Polysomes

PubMed Central

Basbouss-Serhal, Isabelle; Soubigou-Taconnat, Ludivine; Bailly, Christophe; Leymarie, Juliette

2015-01-01

Dormancy is a complex evolutionary trait that temporally prevents seed germination, thus allowing seedling growth at a favorable season. High-throughput analyses of transcriptomes have led to significant progress in understanding the molecular regulation of this process, but the role of posttranscriptional mechanisms has received little attention. In this work, we have studied the dynamics of messenger RNA association with polysomes and compared the transcriptome with the translatome in dormant and nondormant seeds of Arabidopsis (Arabidopsis thaliana) during their imbibition at 25°C in darkness, a temperature preventing germination of dormant seeds only. DNA microarray analysis revealed that 4,670 and 7,028 transcripts were differentially abundant in dormant and nondormant seeds in the transcriptome and the translatome, respectively. We show that there is no correlation between transcriptome and translatome and that germination regulation is also largely translational, implying a selective and dynamic recruitment of messenger RNAs to polysomes in both dormant and nondormant seeds. The study of 5′ untranslated region features revealed that GC content and the number of upstream open reading frames could play a role in selective translation occurring during germination. Gene Ontology clustering showed that the functions of polysome-associated transcripts differed between dormant and nondormant seeds and revealed actors in seed dormancy and germination. In conclusion, our results demonstrate the essential role of selective polysome loading in this biological process. PMID:26019300

Peroxisystem: harnessing systems cell biology to study peroxisomes.

PubMed

Schuldiner, Maya; Zalckvar, Einat

2015-04-01

In recent years, high-throughput experimentation with quantitative analysis and modelling of cells, recently dubbed systems cell biology, has been harnessed to study the organisation and dynamics of simple biological systems. Here, we suggest that the peroxisome, a fascinating dynamic organelle, can be used as a good candidate for studying a complete biological system. We discuss several aspects of peroxisomes that can be studied using high-throughput systematic approaches and be integrated into a predictive model. Such approaches can be used in the future to study and understand how a more complex biological system, like a cell and maybe even ultimately a whole organism, works. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

QPatch: the missing link between HTS and ion channel drug discovery.

PubMed

Mathes, Chris; Friis, Søren; Finley, Michael; Liu, Yi

2009-01-01

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

High Throughput Genotoxicity Profiling of the US EPA ToxCast Chemical Library

EPA Science Inventory

A key aim of the ToxCast project is to investigate modern molecular and genetic high content and high throughput screening (HTS) assays, along with various computational tools to supplement and perhaps replace traditional assays for evaluating chemical toxicity. Genotoxicity is a...

Improving protein-protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model.

PubMed

An, Ji-Yong; Meng, Fan-Rong; You, Zhu-Hong; Chen, Xing; Yan, Gui-Ying; Hu, Ji-Pu

2016-10-01

Predicting protein-protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high-throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM-BiGP that combines the relevance vector machine (RVM) model and Bi-gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi-gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five-fold cross-validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-BiGP method is significantly better than the SVM-based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic decision support tool for future proteomics research. For facilitating extensive studies for future proteomics research, we developed a freely available web server called RVM-BiGP-PPIs in Hypertext Preprocessor (PHP) for predicting PPIs. The web server including source code and the datasets are available at http://219.219.62.123:8888/BiGP/. © 2016 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Microfluidic-SANS: insitu molecular insight into complex fluid processing and high throughput characterisation

NASA Astrophysics Data System (ADS)

Lopez, Carlos; Watanabe, Takaichi; Cabral, Joao; Graham, Peter; Porcar, Lionel; Martel, Anne

2014-03-01

The coupling of microfluidics and small angle neutron scattering (SANS) is successfully demonstrated for the first time. We have developed novel microdevices with suitably low SANS background and high pressure compatibility for the investigation of flow-induced phenomena and high throughput phase mapping of complex fluids. We successfully obtained scattering profiles from 50 micron channels, in 10s - 100s second acquisition times. The microfluidic geometry enables the variation of both flow type and magnitude, beyond traditional rheo-SANS setups, and is exceptionally well-suited for complex fluids due to the commensurability of relevant time and lengthscales. We demonstrate our approach by studying model flow responsive systems, including surfactant/co-surfactant/water mixtures, with well-known equilibrium phase behaviour,: sodium dodecyl sulfate (SDS)/octanol/brine, cetyltrimethyl ammonium chloride (C16TAC)/pentanol/water and a model microemulsion system (C10E4 /decane/ D20), as well as polyelectrolyte solutions. Finally, using an online micromixer we are able to implement a high throughput approach, scanning in excess of 10 scattering profiles/min for a continuous aqueous surfactant dilution over two decades in concentration.

A compact imaging spectroscopic system for biomolecular detections on plasmonic chips.

PubMed

Lo, Shu-Cheng; Lin, En-Hung; Wei, Pei-Kuen; Tsai, Wan-Shao

2016-10-17

In this study, we demonstrate a compact imaging spectroscopic system for high-throughput detection of biomolecular interactions on plasmonic chips, based on a curved grating as the key element of light diffraction and light focusing. Both the curved grating and the plasmonic chips are fabricated on flexible plastic substrates using a gas-assisted thermal-embossing method. A fiber-coupled broadband light source and a camera are included in the system. Spectral resolution within 1 nm is achieved in sensing environmental index solutions and protein bindings. The detected sensitivities of the plasmonic chip are comparable with a commercial spectrometer. An extra one-dimensional scanning stage enables high-throughput detection of protein binding on a designed plasmonic chip consisting of several nanoslit arrays with different periods. The detected resonance wavelengths match well with the grating equation under an air environment. Wavelength shifts between 1 and 9 nm are detected for antigens of various concentrations binding with antibodies. A simple, mass-productive and cost-effective method has been demonstrated on the imaging spectroscopic system for real-time, label-free, highly sensitive and high-throughput screening of biomolecular interactions.

Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

PubMed Central

Lu, Rong; Neff, Norma F.; Quake, Stephen R.; Weissman, Irving L.

2011-01-01

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here, we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single cell transplantation studies, but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggests that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse post irradiation. This technique can be applied to any viral accessible cell type for both in vitro and in vivo processes. PMID:21964413

Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system.

PubMed

Sugano, Shigeo S; Suzuki, Hiroko; Shimokita, Eisuke; Chiba, Hirofumi; Noji, Sumihare; Osakabe, Yuriko; Osakabe, Keishi

2017-04-28

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

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.

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering☆

PubMed Central

Rabitz, Herschel; Welsh, William J.; Kohn, Joachim; de Boer, Jan

2016-01-01

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. PMID:26876875

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

PubMed Central

2013-01-01

Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described. PMID:23475615

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

Heusinkveld, Harm J.; Westerink, Remco H.S., E-mail: R.Westerink@uu.nl

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

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

PubMed

Jung, Paul P; Christian, Nils; Kay, Daniel P; Skupin, Alexander; Linster, Carole L

2015-01-01

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.