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Sample records for pyrosequencing reveals contrasting

  1. Pyrosequencing Reveals Fungal Communities in the Rhizosphere of Xinjiang Jujube

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    Peng Liu

    2015-01-01

    Full Text Available Fungi are important soil components as both decomposers and plant symbionts and play a major role in ecological and biogeochemical processes. However, little is known about the richness and structure of fungal communities. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. We therefore used 454 pyrosequencing to investigate the fungal communities in the rhizosphere of Xinjiang jujube. We obtained no less than 40,488 internal transcribed spacer (ITS rDNA reads, the number of each sample was 6943, 6647, 6584, 6550, 6860, and 6904, and we used bioinformatics and multivariate statistics to analyze the results. The index of diversity showed greater richness in the rhizosphere fungal community of a 3-year-old jujube than in that of an 8-year-old jujube. Most operational taxonomic units belonged to Ascomycota, and taxonomic analyses identified Hypocreales as the dominant fungal order. Our results demonstrated that the fungal orders are present in different proportions in different sampling areas. Redundancy analysis (RDA revealed a significant correlation between soil properties and the abundance of fungal phyla. Our results indicated lower fungal diversity in the rhizosphere of Xinjiang jujube than that reported in other studies, and we hope our findings provide a reference for future research.

  2. Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome.

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    Melody S Clark

    Full Text Available BACKGROUND: The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. METHODOLOGY/PRINCIPAL FINDINGS: The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp. In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS, providing a resource for population and also gene function studies. CONCLUSIONS: This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding

  3. Spatial and Species Variations in Bacterial Communities Associated with Corals from the Red Sea as Revealed by Pyrosequencing

    KAUST Repository

    Lee, O. O.; Yang, J.; Bougouffa, S.; Wang, Y.; Batang, Zenon B.; Tian, R.; Al-Suwailem, A.; Qian, P.-Y.

    2012-01-01

    -pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than

  4. Sputum microbiota in tuberculosis as revealed by 16S rRNA pyrosequencing.

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    Man Kit Cheung

    Full Text Available BACKGROUND: Tuberculosis (TB remains a global threat in the 21st century. Traditional studies of the disease are focused on the single pathogen Mycobacterium tuberculosis. Recent studies have revealed associations of some diseases with an imbalance in the microbial community. Characterization of the TB microbiota could allow a better understanding of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Here, the sputum microbiota in TB infection was examined by using 16S rRNA pyrosequencing. A total of 829,873 high-quality sequencing reads were generated from 22 TB and 14 control sputum samples. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were the five major bacterial phyla recovered, which together composed over 98% of the microbial community. Proteobacteria and Bacteroidetes were more represented in the TB samples and Firmicutes was more predominant in the controls. Sixteen major bacterial genera were recovered. Streptococcus, Neisseria and Prevotella were the most predominant genera, which were dominated by several operational taxonomic units grouped at a 97% similarity level. Actinomyces, Fusobacterium, Leptotrichia, Prevotella, Streptococcus, and Veillonella were found in all TB samples, possibly representing the core genera in TB sputum microbiota. The less represented genera Mogibacterium, Moryella and Oribacterium were enriched statistically in the TB samples, while a genus belonging to the unclassified Lactobacillales was enriched in the controls. The diversity of microbiota was similar in the TB and control samples. CONCLUSIONS/SIGNIFICANCE: The composition and diversity of sputum microbiota in TB infection was characterized for the first time by using high-throughput pyrosequencing. It lays the framework for examination of potential roles played by the diverse microbiota in TB pathogenesis and progression, and could ultimately facilitate advances in TB treatment.

  5. Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

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    Lee, Onon; Wang, Yong; Yang, Jiangke; Lafi, Feras Fawzi; Al-Suwailem, Abdulaziz M.; Qian, Peiyuan

    2010-01-01

    Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus

  6. The diversity and structure of marine protists in the coastal waters of China revealed by morphological observation and 454 pyrosequencing

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    Liu, Yun; Song, Shuqun; Chen, Tiantian; Li, Caiwen

    2017-04-01

    Pyrosequencing of the 18S rRNA gene has been widely adopted to study the eukaryotic diversity in various types of environments, and has an advantage over traditional morphology methods in exploring unknown microbial communities. To comprehensively assess the diversity and community composition of marine protists in the coastal waters of China, we applied both morphological observations and high-throughput sequencing of the V2 and V3 regions of 18S rDNA simultaneously to analyze samples collected from the surface layer of the Yellow and East China Seas. Dinoflagellates, diatoms and ciliates were the three dominant protistan groups as revealed by the two methods. Diatoms were the first dominant protistan group in the microscopic observations, with Skeletonema mainly distributed in the nearshore eutrophic waters and Chaetoceros in higher temperature and higher pH waters. The mixotrophic dinoflagellates, Gymnodinium and Gyrodinium, were more competitive in the oligotrophic waters. The pyrosequencing method revealed an extensive diversity of dinoflagellates. Chaetoceros was the only dominant diatom group in the pyrosequencing dataset. Gyrodinium represented the most abundant reads and dominated the offshore oligotrophic protistan community as they were in the microscopic observations. The dominance of parasitic dinoflagellates in the pyrosequencing dataset, which were overlooked in the morphological observations, indicates more attention should be paid to explore the potential role of this group. Both methods provide coherent clustering of samples. Nutrient levels, salinity and pH were the main factors influencing the distribution of protists. This study demonstrates that different primer pairs used in the pyrosequencing will indicate different protistan community structures. A suitable marker may reveal more comprehensive composition of protists and provide valuable information on environmental drivers.

  7. Rhizosphere bacteriome of the medicinal plant Sapindus saponaria L. revealed by pyrosequencing.

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    Garcia, A; Polonio, J C; Polli, A D; Santos, C M; Rhoden, S A; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-11-03

    Sapindus saponaria L. of Sapindaceae family is popularly known as soldier soap and is found in Central and South America. A study of such medicinal plants might reveal a more complex diversity of microorganisms as compared to non-medicinal plants, considering their metabolic potential and the chemical communication between their natural microbiota. Rhizosphere is a highly diverse microbial habitat with respect to both the diversity of species and the size of the community. Rhizosphere bacteriome associated with medicinal plant S. saponaria is still poorly known. The objective of this study was to assess the rhizosphere microbiome of the medicinal plant S. saponaria using pyrosequencing, a culture-independent approach that is increasingly being used to estimate the number of bacterial species present in different environments. In their rhizosphere microbiome, 26 phyla were identified from 5089 sequences of 16S rRNA gene, with a predominance of Actinobacteria (33.54%), Acidobacteria (22.62%), and Proteobacteria (24.72%). The rarefaction curve showed a linear increase, with 2660 operational taxonomic units at 3% distance sequence dissimilarity, indicating that the rhizosphere microbiome associated with S. saponaria was highly diverse with groups of bacteria important for soil management, which could be further exploited for agricultural and biotechnological purposes.

  8. Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing.

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    Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin; Zhou, Zhi; Wu, Chen; Nagarajan, Sanjay

    2015-01-01

    Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere.

  9. Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

    KAUST Repository

    Lee, Onon

    2010-11-18

    Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored. © 2011 International Society for Microbial Ecology All rights reserved.

  10. Pyrosequencing of environmental soil samples reveals biodiversity of the Phytophthora resident community in chestnut forests.

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    Vannini, Andrea; Bruni, Natalia; Tomassini, Alessia; Franceschini, Selma; Vettraino, Anna Maria

    2013-09-01

    Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. Pyrosequencing Reveals the Predominance of Pseudomonadaceae in Gut Microbiome of a Gall Midge

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    Raman Bansal

    2014-06-01

    Full Text Available Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs were obtained at the >97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall

  12. Potential human pathogenic bacteria in a mixed urban watershed as revealed by pyrosequencing.

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    A Mark Ibekwe

    Full Text Available Current microbial source tracking (MST methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs, recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP, Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%, agricultural runoff sediment (6.52%, and Prado Park sediment (6.00%, respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78-4.08%. Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.

  13. Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing.

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    Ye, Lin; Zhang, Tong

    2011-09-01

    This study applied 454 high-throughput pyrosequencing to analyze potentially pathogenic bacteria in activated sludge from 14 municipal wastewater treatment plants (WWTPs) across four countries (China, U.S., Canada, and Singapore), plus the influent and effluent of one of the 14 WWTPs. A total of 370,870 16S rRNA gene sequences with average length of 207 bps were obtained and all of them were assigned to corresponding taxonomic ranks by using RDP classifier and MEGAN. It was found that the most abundant potentially pathogenic bacteria in the WWTPs were affiliated with the genera of Aeromonas and Clostridium. Aeromonas veronii, Aeromonas hydrophila, and Clostridium perfringens were species most similar to the potentially pathogenic bacteria found in this study. Some sequences highly similar (>99%) to Corynebacterium diphtheriae were found in the influent and activated sludge samples from a saline WWTP. Overall, the percentage of the sequences closely related (>99%) to known pathogenic bacteria sequences was about 0.16% of the total sequences. Additionally, a platform-independent Java application (BAND) was developed for graphical visualization of the data of microbial abundance generated by high-throughput pyrosequencing. The approach demonstrated in this study could examine most of the potentially pathogenic bacteria simultaneously instead of one-by-one detection by other methods.

  14. Pyrosequencing reveals diverse microbial community associated with the zoanthid Palythoa australiae from the South China Sea.

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    Sun, Wei; Zhang, Fengli; He, Liming; Li, Zhiyong

    2014-05-01

    Diverse sessile organisms inhabit the coral reef ecosystems, including corals, sponges, and sea anemones. In the past decades, scleractinian corals (Cnidaria, Anthozoa, Scleractinia) and their associated microorganisms have attracted much attention. Zoanthids (Cnidaria, Anthozoa, Zoanthidea) are commonly found in coral reefs. However, little is known about the community structure of zoanthid-associated microbiota. In this study, the microbial community associated with the zoanthid Palythoa australiae in the South China Sea was investigated by 454 pyrosequencing. As a result, 2,353 bacterial, 583 archaeal, and 36 eukaryotic microbial ribotypes were detected, respectively. A total of 22 bacterial phyla (16 formally described phyla and six candidate phyla) were recovered. Proteobacteria was the most abundant group, followed by Chloroflexi and Actinobacteria. High-abundance Rhizobiales and diverse Chloroflexi were observed in the bacterial community. The archaeal population was composed of Crenarchaeota and Euryarchaeota, with Marine Group I as the dominant lineage. In particular, Candidatus Nitrosopumilus dominated the archaeal community. Besides bacteria and archaea, the zoanthid harbored eukaryotic microorganisms including fungi and algae though their diversity was very low. This study provided the first insights into the microbial community associated with P. australiae by 454 pyrosequencing, consequently laid a basis for the understanding of the association of P. australiae-microbes symbioses.

  15. Pyrosequencing of microbial community of typical chernozem in contrast land use conditions

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    Ivanova, Ekaterina; Olga, Kutovaya; Azida, Tkhakakhova

    2015-04-01

    Chernozems are the principal soil resourse of Russia, so the sustainable use of these fertile soils in the intensive agriculturural production is of great importance, especially in terms of agro-ecological security of the country. The increase in agricultural inputs - intensive cropping, soil fallowing application accompanied with high frequency of mechanical treatment, result in decrease in soil organic matter content as well as soil structure degradation and, finally, lead to the loss of soil fertility. Soil microorganisms can serve as bioindicators of anthropogenic stress experienced by the soil during its agricultural use, so they may be universal indicators of soil quality (soil health) used for optimization and biologization of agricultural systems. The way to study the relationship between the structural status of the soil, its microbial communities and the organic matter content is the comparative analysis of soil aggregates in conditions of different land use practices. The objects of our research were soil samples of soil with permanent wheat cropping (50 years), continuous dead fallow (50 years) soil, and recovering soil (for 18 years under native steppe vegetation, fallowed in previous). The analysis of 16 S rRNA gene amplicon libraries of typical chernozem in conditions of different land use systems revealed that the way of agricultural use is a strong determinant of soil microbiome taxonomic composition. It was shown that the continuous «dead fallowing» application (for 50 years) lead to the establishment of olygothrophic components of microbial community, including spore-forming members of phylum Firmicutes. The increase of Acidobacteria lineages in this variant may be an indicator of some acidification of soil during long-time fallowing application. The variant of continuous wheat cropping lead to increasing in Proteobacteria lineages. The variant of soil under native steppe vegetation was characterized by the highest values of biodiversity

  16. Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

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    Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

    2012-08-01

    Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

  17. Diversity and structure of soil bacterial communities in the Fildes Region (maritime Antarctica as revealed by 454 pyrosequencing

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    Neng Fei eWang

    2015-10-01

    Full Text Available This study assessed the diversity and composition of bacterial communities in four different soils (human-, penguin-, seal-colony impacted soils and pristine soil in the Fildes Region (King George Island, Antarctica using 454 pyrosequencing with bacterial-specific primers targeting the 16S rRNA gene. Proteobacteria, Actinobacteria, Acidobacteria, and Verrucomicrobia were abundant phyla in almost all the soil samples. The four types of soils were significantly different in geochemical properties and bacterial community structure. Thermotogae, Cyanobacteria, Fibrobacteres, Deinococcus-Thermus, and Chlorobi obviously varied in their abundance among the 4 soil types. Considering all the samples together, members of the genera Gaiella, Chloracidobacterium, Nitrospira, Polaromonas, Gemmatimonas, Sphingomonas and Chthoniobacter were found to predominate, whereas members of the genera Chamaesiphon, Herbaspirillum, Hirschia, Nevskia, Nitrosococcus, Rhodococcus, Rhodomicrobium, and Xanthomonas varied obviously in their abundance among the four soil types. Distance-based redundancy analysis revealed that pH (p < 0.01, phosphate phosphorus (p < 0.01, organic carbon (p < 0.05, and organic nitrogen (p < 0.05 were the most significant factors that correlated with the community distribution of soil bacteria. To our knowledge, this is the first study to explore the soil bacterial communities in human-, penguin-, and seal- colony impacted soils from ice-free areas in maritime Antarctica using high-throughput pyrosequencing.

  18. Arbuscular mycorrhizal fungi assemblages in Chernozem great groups revealed by massively parallel pyrosequencing.

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    Dai, Mulan; Hamel, Chantal; St Arnaud, Marc; He, Yong; Grant, Cynthia; Lupwayi, Newton; Janzen, Henry; Malhi, Sukhdev S; Yang, Xiaohong; Zhou, Zhiqin

    2012-01-01

    The arbuscular mycorrhizal (AM) fungal resources present in wheat fields of the Canadian Prairie were explored using 454 pyrosequencing. Of the 33 dominant AM fungal operational taxonomic units (OTUs) found in the 76 wheat fields surveyed at anthesis in 2009, 14 clustered as Funneliformis - Rhizophagus, 16 as Claroideoglomus, and 3 as Diversisporales. An OTU of Funneliformis mosseae and one OTU of Diversisporales each accounted for approximately 16% of all AM fungal OTUs. The former was ubiquitous, and the latter was mainly restricted to the Black and Dark Brown Chernozems. AM fungal OTU community composition was better explained by the Chernozem great groups (P = 0.044) than by measured soil properties. Fifty-two percent of the AM fungal OTUs were unrelated to measured soil properties. Black Chernozems hosted the largest AM fungal OTU diversity and almost twice the number of AM fungal sequences seen in Dark Brown Chernozems, the great group ranking second for AM fungal sequence abundance. Brown Chernozems hosted the lowest AM fungal abundance and an AM fungal diversity as low as that seen in Gray soils. We concluded that Black Chernozems are most conducive to AM fungal proliferation. AM fungi are generally distributed according to Chernozem great groups in the Canadian Prairie, although some taxa are evenly distributed in all soil groups.

  19. Pyrosequencing reveals changes in soil bacterial communities after conversion of Yungas forests to agriculture.

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    Marcela S Montecchia

    Full Text Available The Southern Andean Yungas in Northwest Argentina constitute one of the main biodiversity hotspots in the world. Considerable changes in land use have taken place in this ecoregion, predominantly related to forest conversion to croplands, inducing losses in above-ground biodiversity and with potential impact on soil microbial communities. In this study, we used high-throughput pyrosequencing of the 16S ribosomal RNA gene to assess whether land-use change and time under agriculture affect the composition and diversity of soil bacterial communities. We selected two areas dedicated to sugarcane and soybean production, comprising both short- and long-term agricultural sites, and used the adjacent native forest soils as a reference. Land-use change altered the composition of bacterial communities, with differences between productive areas despite the similarities between both forests. At the phylum level, only Verrucomicrobia and Firmicutes changed in abundance after deforestation for sugarcane and soybean cropping, respectively. In cultivated soils, Verrucomicrobia decreased sharply (~80%, while Firmicutes were more abundant. Despite the fact that local diversity was increased in sugarcane systems and was not altered by soybean cropping, phylogenetic beta diversity declined along both chronosequences, evidencing a homogenization of soil bacterial communities over time. In spite of the detected alteration in composition and diversity, we found a core microbiome resistant to the disturbances caused by the conversion of forests to cultivated lands and few or none exclusive OTUs for each land-use type. The overall changes in the relative abundance of copiotrophic and oligotrophic taxa may have an impact in soil ecosystem functionality. However, communities with many taxa in common may also share many functional attributes, allowing to maintain at least some soil ecosystem services after forest conversion to croplands.

  20. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

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    Jan S Suchodolski

    Full Text Available BACKGROUND: Canine idiopathic inflammatory bowel disease (IBD is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6 and dogs with moderate IBD (n = 7 or severe IBD (n = 7 as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001. Proportions of Fusobacteria (p = 0.010, Bacteroidaceae (p = 0.015, Prevotellaceae (p = 0.022, and Clostridiales (p = 0.019 were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044 and Acinetobacter (p = 0.040, were either more abundant or more frequently identified in IBD dogs. CONCLUSIONS/SIGNIFICANCE: In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

  1. Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance.

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    Wen Xie

    Full Text Available BACKGROUND: Bemisia tabaci (Gennadius is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. METHODOLOGY AND PRINCIPAL FINDINGS: Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45% unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10-5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. CONCLUSIONS: This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the

  2. Pyrosequencing Reveals Soil Enzyme Activities and Bacterial Communities Impacted by Graphene and Its Oxides.

    Science.gov (United States)

    Rong, Yan; Wang, Yi; Guan, Yina; Ma, Jiangtao; Cai, Zhiqiang; Yang, Guanghua; Zhao, Xiyue

    2017-10-25

    Graphene (GN) and graphene oxides (GOs) are novel carbon nanomaterial; they have been attracting much attention because of their excellent properties and are widely applied in many areas, including energy, electronics, biomedicine, environmental science, etc. With industrial production and consumption of GN/GO, they will inevitably enter the soil and water environments. GN/GO may directly cause certain harm to microorganisms and lead to ecological and environmental risks. GOs are GN derivatives with abundant oxygen-containing functional groups in their graphitic backbone. The structure and chemistry of GN show obvious differences compared to those of GO, which lead to the different environmental behaviors. In this study, four different types of soil (S1-S4) were employed to investigate the effect of GN and GO on soil enzymatic activity, microbial population, and bacterial community through pyrosequencing of 16S rRNA gene amplicons. The results showed that soil enzyme activity (invertase, protease, catalase, and urease) and microbial population (bacteria, actinomycetes, and fungi) changed after GN/GO release into soils. Soil microbial community species are more rich, and the diversity also increases after GO/GN application. The phylum of Proteobacteria increased at 90 days after treatment (DAT) after GN/GO application. The phylum of Chloroflexi occurred after GN application at 90 DAT in S1 soil and reached 4.6%. Proteobacteria was the most abundant phylum in S2, S3, and S4 soils; it ranged from 43.6 to 71.4% in S2 soil, from 45.6 to 73.7% in S3 soil, and from 38.1 to 56.7% in S4 soil. The most abundant genera were Bacillus (37.5-47.0%) and Lactococcus (28.0-39.0%) in S1 soil, Lysobacter and Flavobacterium in S2 soil, Pedobacter in S3 soil, and Massilia in S4 soil. The effect of GN and GO on the soil microbial community is time-dependent, and there are no significant differences between the samples at 10 and 90 DAT.

  3. Spatial and Species Variations in Bacterial Communities Associated with Corals from the Red Sea as Revealed by Pyrosequencing

    KAUST Repository

    Lee, O. O.

    2012-08-03

    Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi, Chlamydiae, and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio, Pseudoalteromonas, Serratia, Stenotrophomonas, Pseudomonas, and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.

  4. Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing.

    Science.gov (United States)

    Milanović, Vesna; Osimani, Andrea; Taccari, Manuela; Garofalo, Cristiana; Butta, Alessandro; Clementi, Francesca; Aquilanti, Lucia

    2017-07-01

    The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

  5. Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

    Science.gov (United States)

    Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

    2016-04-01

    Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

  6. Different continuous cropping spans significantly affect microbial community membership and structure in a vanilla-grown soil as revealed by deep pyrosequencing.

    Science.gov (United States)

    Xiong, Wu; Zhao, Qingyun; Zhao, Jun; Xun, Weibing; Li, Rong; Zhang, Ruifu; Wu, Huasong; Shen, Qirong

    2015-07-01

    In the present study, soil bacterial and fungal communities across vanilla continuous cropping time-series fields were assessed through deep pyrosequencing of 16S ribosomal RNA (rRNA) genes and internal transcribed spacer (ITS) regions. The results demonstrated that the long-term monoculture of vanilla significantly altered soil microbial communities. Soil fungal diversity index increased with consecutive cropping years, whereas soil bacterial diversity was relatively stable. Bray-Curtis dissimilarity cluster and UniFrac-weighted principal coordinate analysis (PCoA) revealed that monoculture time was the major determinant for fungal community structure, but not for bacterial community structure. The relative abundances (RAs) of the Firmicutes, Actinobacteria, Bacteroidetes, and Basidiomycota phyla were depleted along the years of vanilla monoculture. Pearson correlations at the phyla level demonstrated that Actinobacteria, Armatimonadetes, Bacteroidetes, Verrucomicrobia, and Firmicutes had significant negative correlations with vanilla disease index (DI), while no significant correlation for fungal phyla was observed. In addition, the amount of the pathogen Fusarium oxysporum accumulated with increasing years and was significantly positively correlated with vanilla DI. By contrast, the abundance of beneficial bacteria, including Bradyrhizobium and Bacillus, significantly decreased over time. In sum, soil weakness and vanilla stem wilt disease after long-term continuous cropping can be attributed to the alteration of the soil microbial community membership and structure, i.e., the reduction of the beneficial microbes and the accumulation of the fungal pathogen.

  7. Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans.

    Directory of Open Access Journals (Sweden)

    Lauren M G Davis

    Full Text Available Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by

  8. Bacterial communities potentially involved in iron-cycling in Baltic Sea and North Sea sediments revealed by pyrosequencing

    DEFF Research Database (Denmark)

    Reyes, Carlen; Dellwig, Olaf; Dähnke, K.

    2016-01-01

    To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5-1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing....

  9. Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil

    Energy Technology Data Exchange (ETDEWEB)

    Lladó, S., E-mail: llado@biomed.cas.cz [Department of Microbiology, University of Barcelona, Diagonal 645, E-08028 Barcelona (Spain); Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4 (Czech Republic); Covino, S., E-mail: covino@biomed.cas.cz [Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4 (Czech Republic); Solanas, A.M., E-mail: asolanas@ub.edu [Department of Microbiology, University of Barcelona, Diagonal 645, E-08028 Barcelona (Spain); Petruccioli, M., E-mail: petrucci@unitus.it [Department for Innovation in Biological, Agro-Food and Forest Systems [DIBAF], University of Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo (Italy); D’annibale, A., E-mail: dannib@unitus.it [Department for Innovation in Biological, Agro-Food and Forest Systems [DIBAF], University of Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo (Italy); Viñas, M., E-mail: marc.vinas@irta.cat [GIRO Joint Research Unit IRTA-UPC, Institute of Research and Technology Food and Agriculture [IRTA], Torre Marimon, E-08140 Caldes de Montbui (Spain)

    2015-02-11

    Highlights: • Soil microbial community assessment through classical (MPN) and molecular tools (DGGE and pyrosequencing) is provided. • A failure of exogenous white rot fungi to colonize the polluted soil is shown by DGGE and pyrosequencing. • Surfactant Brij 30 hampers 4-ring PAHs degradation due to toxicity over Actinobacteria and Bacteroidetes populations. • A high prevalence of Fusarium and Scedosporium populations is revealed during soil bioremediation. • Cupriavidus, Mycobacterium and Chithinophagaceae are potential HMW–PAH degraders in the soil. - Abstract: Bacterial and fungal biodiversity throughout different biostimulation and bioaugmentation treatments applied to an industrial creosote-polluted soil were analyzed by means of polyphasic approach in order to gain insight into the microbial community structure and dynamics. Pyrosequencing data obtained from initial creosote polluted soil (after a biopiling step) revealed that Alpha and Gammaproteobacteria were the most abundant bacterial groups, whereas Fusarium and Scedosporium were the main fungal genera in the contaminated soil. At the end of 60-days laboratory scale bioremediation assays, pyrosequencing and DGGE data showed that (i) major bacterial community shifts were caused by the type of mobilizing agent added to the soil and, to a lesser extent, by the addition of lignocellulosic substrate; and (ii) the presence of the non-ionic surfactant (Brij 30) hampered the proliferation of Actinobacteria (Mycobacteriaceae) and Bacteroidetes (Chitinophagaceae) and, in the absence of lignocellulosic substrate, also impeded polycyclic aromatic hydrocarbons (PAHs) degradation. The results show the importance of implementing bioremediation experiments combined with microbiome assessment to gain insight on the effect of crucial parameters (e.g. use of additives) over the potential functions of complex microbial communities harbored in polluted soils, essential for bioremediation success.

  10. Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil

    International Nuclear Information System (INIS)

    Lladó, S.; Covino, S.; Solanas, A.M.; Petruccioli, M.; D’annibale, A.; Viñas, M.

    2015-01-01

    Highlights: • Soil microbial community assessment through classical (MPN) and molecular tools (DGGE and pyrosequencing) is provided. • A failure of exogenous white rot fungi to colonize the polluted soil is shown by DGGE and pyrosequencing. • Surfactant Brij 30 hampers 4-ring PAHs degradation due to toxicity over Actinobacteria and Bacteroidetes populations. • A high prevalence of Fusarium and Scedosporium populations is revealed during soil bioremediation. • Cupriavidus, Mycobacterium and Chithinophagaceae are potential HMW–PAH degraders in the soil. - Abstract: Bacterial and fungal biodiversity throughout different biostimulation and bioaugmentation treatments applied to an industrial creosote-polluted soil were analyzed by means of polyphasic approach in order to gain insight into the microbial community structure and dynamics. Pyrosequencing data obtained from initial creosote polluted soil (after a biopiling step) revealed that Alpha and Gammaproteobacteria were the most abundant bacterial groups, whereas Fusarium and Scedosporium were the main fungal genera in the contaminated soil. At the end of 60-days laboratory scale bioremediation assays, pyrosequencing and DGGE data showed that (i) major bacterial community shifts were caused by the type of mobilizing agent added to the soil and, to a lesser extent, by the addition of lignocellulosic substrate; and (ii) the presence of the non-ionic surfactant (Brij 30) hampered the proliferation of Actinobacteria (Mycobacteriaceae) and Bacteroidetes (Chitinophagaceae) and, in the absence of lignocellulosic substrate, also impeded polycyclic aromatic hydrocarbons (PAHs) degradation. The results show the importance of implementing bioremediation experiments combined with microbiome assessment to gain insight on the effect of crucial parameters (e.g. use of additives) over the potential functions of complex microbial communities harbored in polluted soils, essential for bioremediation success

  11. Fungal and bacterial successions in the process of co-composting of organic wastes as revealed by 454 pyrosequencing.

    Science.gov (United States)

    Galitskaya, Polina; Biktasheva, Liliya; Saveliev, Anatoly; Grigoryeva, Tatiana; Boulygina, Eugenia; Selivanovskaya, Svetlana

    2017-01-01

    Composting is viewed as one of the primary methods to treat organic wastes. Co-composting may improve the efficiency of this treatment by establishing the most suitable conditions for decomposers than those present in the individual wastes. Given that bacteria and fungi are the driving agents of composting, information about the composition of their communities and dynamics during composting may improve reproducibility, performance and quality of the final compost as well as help to evaluate the potential human health risk and the choice of the most appropriate application procedure. In this study, the co-composting of mixtures containing two similar components (organic fraction of municipal solid waste and sawdust polluted by oil) and one discriminate component (sewage sludges of different origin) were investigated. Bacterial and fungal community successions in the two mixtures were analyzed during the composting process by determining the change in their structural dynamics using qPCR and 454 pyrosequencing methods in a lab experiment for a period of 270 days. During the initial composting stage, the number of 16S bacterial copies was (3.0±0.2) x 106 and (0.4±0.0) x 107 g-1, and the Rhodospiralles and Lactobacialles orders dominated. Fungal communities had (2.9±0.0) x105 and (6.1±0.2) x105 ITS copies g-1, and the Saccharomycetales order dominated. At the end of the thermophilic stage on the 30th day of composting, bacterial and fungal communities underwent significant changes: dominants changed and their relative abundance decreased. Typical compost residents included Flavobacteriales, Chitinophagaceae and Bacterioidetes for bacteria and Microascaceae, Dothideomycetes, Eurotiomycetes, Sordariomycetes, and Agaricomycetes for fungi. During the later composting stages, the dominating taxa of both bacterial and fungal communities remained, while their relative abundance decreased. In accordance with the change in the dominating OTUs, it was concluded that the

  12. Fungal and bacterial successions in the process of co-composting of organic wastes as revealed by 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Polina Galitskaya

    Full Text Available Composting is viewed as one of the primary methods to treat organic wastes. Co-composting may improve the efficiency of this treatment by establishing the most suitable conditions for decomposers than those present in the individual wastes. Given that bacteria and fungi are the driving agents of composting, information about the composition of their communities and dynamics during composting may improve reproducibility, performance and quality of the final compost as well as help to evaluate the potential human health risk and the choice of the most appropriate application procedure. In this study, the co-composting of mixtures containing two similar components (organic fraction of municipal solid waste and sawdust polluted by oil and one discriminate component (sewage sludges of different origin were investigated. Bacterial and fungal community successions in the two mixtures were analyzed during the composting process by determining the change in their structural dynamics using qPCR and 454 pyrosequencing methods in a lab experiment for a period of 270 days. During the initial composting stage, the number of 16S bacterial copies was (3.0±0.2 x 106 and (0.4±0.0 x 107 g-1, and the Rhodospiralles and Lactobacialles orders dominated. Fungal communities had (2.9±0.0 x105 and (6.1±0.2 x105 ITS copies g-1, and the Saccharomycetales order dominated. At the end of the thermophilic stage on the 30th day of composting, bacterial and fungal communities underwent significant changes: dominants changed and their relative abundance decreased. Typical compost residents included Flavobacteriales, Chitinophagaceae and Bacterioidetes for bacteria and Microascaceae, Dothideomycetes, Eurotiomycetes, Sordariomycetes, and Agaricomycetes for fungi. During the later composting stages, the dominating taxa of both bacterial and fungal communities remained, while their relative abundance decreased. In accordance with the change in the dominating OTUs, it was

  13. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

    Science.gov (United States)

    Suchodolski, Jan S; Dowd, Scot E; Wilke, Vicky; Steiner, Jörg M; Jergens, Albert E

    2012-01-01

    Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, pmicrobial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

  14. Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes

    Directory of Open Access Journals (Sweden)

    Gorman Kevin

    2011-01-01

    Full Text Available Abstract Background The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. Results Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication. BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. Conclusion Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.

  15. Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454-pyrosequencing.

    Science.gov (United States)

    Lopes, Ana R; Manaia, Célia M; Nunes, Olga C

    2014-03-01

    Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  16. Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar (Coleoptera: Curculionidae Reveals Multiple Protease-Like Transcripts.

    Directory of Open Access Journals (Sweden)

    Arnubio Valencia

    Full Text Available The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

  17. Pyrosequencing revealed shifts of prokaryotic communities between healthy and disease-like tissues of the Red Sea sponge Crella cyathophora

    KAUST Repository

    Gao, Zhao-Ming

    2015-06-11

    Sponge diseases have been widely reported, yet the causal factors and major pathogenic microbes remain elusive. In this study, two individuals of the sponge Crella cyathophora in total that showed similar disease-like characteristics were collected from two different locations along the Red Sea coast separated by more than 30 kilometers. The disease-like parts of the two individuals were both covered by green surfaces, and the body size was much smaller compared with adjacent healthy regions. Here, using high-throughput pyrosequencing technology, we investigated the prokaryotic communities in healthy and disease-like sponge tissues as well as adjacent seawater. Microbes in healthy tissues belonged mainly to the Proteobacteria, Cyanobacteria and Bacteroidetes, and were much more diverse at the phylum level than reported previously. Interestingly, the disease-like tissues from the two sponge individuals underwent shifts of prokaryotic communities and were both enriched with a novel clade affiliated with the phylum Verrucomicrobia, implying its intimate connection with the disease-like Red Sea sponge C. cyathophora. Enrichment of the phylum Verrucomicrobia was also considered to be correlated with the presence of algae assemblages forming the green surface of the disease-like sponge tissues. This finding represents an interesting case of sponge disease and is valuable for further study.

  18. Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

    Science.gov (United States)

    Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair

    2016-01-01

    The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

  19. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    KAUST Repository

    Qian, Peiyuan

    2010-07-29

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea. © 2011 International Society for Microbial Ecology All rights reserved.

  20. Bar-coded pyrosequencing reveals the responses of PBDE-degrading microbial communities to electron donor amendments.

    Directory of Open Access Journals (Sweden)

    Meiying Xu

    Full Text Available Polybrominated diphenyl ethers (PBDEs can be reductively degraded by microorganisms under anaerobic conditions. However, little is known about the effect of electron donors on microbial communities involved in PBDEs degradation. Here we employed 454 Titanium pyrosequencing to examine the phylogenetic diversity, composition, structure and dynamics of microbial communities from microcosms under the conditions of different electron donor amendments. The community structures in each of the five alternate electron donor enrichments were significantly shifted in comparison with those of the control microcosm. Commonly existing OTUs between the treatment and control consortia increased from 5 to 17 and more than 50% of OTUs increased around 13.7 to 186 times at least in one of the microcosms after 90-days enrichment. Although the microbial communities at different taxonomic levels were significantly changed by different environmental variable groups in redundancy analysis, significant correlations were observed between the microbial communities and PBDE congener profiles. The lesser-brominated PBDE congeners, tri-BDE congener (BDE-32 and hexa-BDE, were identified as the key factors shaping the microbial community structures at OTU level. Some rare populations, including the known dechlorinating bacterium, Dehalobacter, showed significant positive-correlation with the amounts of PBDE congeners in the consortia. The same results were also observed on some unclassified bacteria. These results suggest that PBDEs-degrading microbial communities can be successfully enriched, and their structures and compositions can be manipulated through adjusting the environmental parameters.

  1. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    KAUST Repository

    Qian, Peiyuan; Wang, Yong; Lee, Onon; Lau, Chunkwan; Yang, Jiangke; Lafi, Feras Fawzi; Al-Suwailem, Abdulaziz M.; Wong, Tim

    2010-01-01

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea. © 2011 International Society for Microbial Ecology All rights reserved.

  2. Bacterial diversity and community structure of supragingival plaques in adults with dental health or caries revealed by 16S pyrosequencing

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    Cuicui Xiao

    2016-07-01

    Full Text Available Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700 clustered into 10,365 operational taxonomic units (OTUs; 97% identity, representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. Smooth rarefaction curves were suggestive of plaque microbial diversity. α diversity analysis showed that healthy plaque microbial diversity exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups and that group members were relatively constant, only showing differences in abundance. Analysis of composition differences identified 10 health-related and 21 caries-related genera. Key genera (27 that potentially contributed to plaque microbiota distributions between groups were identified. Finally, co-occurrence network analysis and function prediction were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed.

  3. Pyrosequencing reveals bacterial communities in unchlorinated drinking water distribution system: an integral study of bulk water, suspended solids, loose deposits, and pipe wall biofilm.

    Science.gov (United States)

    Liu, G; Bakker, G L; Li, S; Vreeburg, J H G; Verberk, J Q J C; Medema, G J; Liu, W T; Van Dijk, J C

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  4. Pyrosequencing Reveals Bacterial Communities in Unchlorinated Drinking Water Distribution System: An Integral Study of Bulk Water, Suspended Solids, Loose Deposits, and Pipe Wall Biofilm

    KAUST Repository

    Liu, G.

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  5. Pyrosequencing of the bacteria associated with Platygyra carnosus corals with skeletal growth anomalies reveals differences in bacterial community composition in apparently healthy and diseased tissues

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    Jenny Chun-Yee Ng

    2015-10-01

    Full Text Available Corals are rapidly declining globally due to coral diseases. Skeletal growth anomalies (SGA or coral tumors are a group of coral diseases that affect coral reefs worldwide, including Hong Kong waters in the Indo-Pacific region. To better understand how bacterial communities may vary in corals with SGA, for the first time, we examined the bacterial composition associated with the apparently healthy and the diseased tissues of SGA-affected Platgyra carnosus using 16S ribosomal rRNA gene pyrosequencing. Taxonomic analysis revealed Proteobacteria, Bacteroidetes, Cyanobacteria, and Actinobacteria as the main phyla in both the apparently healthy and the diseased tissues. A significant difference in the bacterial community composition was observed between the two conditions at the OTU level. Diseased tissues were associated with higher abundances of Acidobacteria and Gemmatimonadetes, and a lower abundance of Spirochaetes. Several OTUs belonging to Rhodobacteraceae, Rhizobiales, Gammaproteobacteria, and Cytophaga-Flavobacterium-Bacteroidetes (CFB were strongly associated with the diseased tissues. These groups of bacteria may contain potential pathogens involved with the development of SGA or opportunistic secondary or tertiary colonizers that proliferated upon the health-compromised coral host. We suggest that these bacterial groups to be further studied based on inoculation experiments and testing of Koch’s postulates in efforts to understand the etiology and progression of SGA.

  6. Pyrosequencing reveals the microbial communities in the Red Sea sponge Carteriospongia foliascens and their impressive shifts in abnormal tissues.

    Science.gov (United States)

    Gao, Zhao-Ming; Wang, Yong; Lee, On On; Tian, Ren-Mao; Wong, Yue Him; Bougouffa, Salim; Batang, Zenon; Al-Suwailem, Abdulaziz; Lafi, Feras F; Bajic, Vladimir B; Qian, Pei-Yuan

    2014-10-01

    Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species "Candidatus Synechococcus spongiarum," were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

  7. Pyrosequencing Reveals the Microbial Communities in the Red Sea Sponge Carteriospongia foliascens and Their Impressive Shifts in Abnormal Tissues

    KAUST Repository

    Gao, Zhaoming

    2014-04-24

    Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species “Candidatus Synechococcus spongiarum,” were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

  8. Pyrosequencing Analysis Reveals Changes in Intestinal Microbiota of Healthy Adults Who Received a Daily Dose of Immunomodulatory Probiotic Strains

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    Julio Plaza-Díaz

    2015-05-01

    Full Text Available The colon microbiota plays a crucial role in human gastrointestinal health. Current attempts to manipulate the colon microbiota composition are aimed at finding remedies for various diseases. We have recently described the immunomodulatory effects of three probiotic strains (Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034, and Bifidobacterium breve CNCM I-4035. The goal of the present study was to analyze the compositions of the fecal microbiota of healthy adults who received one of these strains using high-throughput 16S ribosomal RNA gene sequencing. Bacteroides was the most abundant genus in the groups that received L. rhamnosus CNCM I-4036 or L. paracasei CNCM I-4034. The Shannon indices were significantly increased in these two groups. Our results also revealed a significant increase in the Lactobacillus genus after the intervention with L. rhamnosus CNCM I-4036. The initially different colon microbiota became homogeneous in the subjects who received L. rhamnosus CNCM I-4036. While some orders that were initially present disappeared after the administration of L. rhamnosus CNCM I-4036, other orders, such as Sphingobacteriales, Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes, were detected after the intervention. In summary, our results show that the intake of these three bacterial strains induced changes in the colon microbiota.

  9. Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Zuo, Yu; Xie, Wenfang; Pang, Yue; Li, Tiesong; Li, Qingwei; Li, Yingying

    2017-01-01

    The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.

  10. Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing.

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    Yu Zuo

    Full Text Available The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.

  11. Pyrosequencing reveals high-temperature cellulolytic microbial consortia in Great Boiling Spring after in situ lignocellulose enrichment.

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    Joseph P Peacock

    Full Text Available To characterize high-temperature cellulolytic microbial communities, two lignocellulosic substrates, ammonia fiber-explosion-treated corn stover and aspen shavings, were incubated at average temperatures of 77 and 85°C in the sediment and water column of Great Boiling Spring, Nevada. Comparison of 109,941 quality-filtered 16S rRNA gene pyrosequences (pyrotags from eight enrichments to 37,057 quality-filtered pyrotags from corresponding natural samples revealed distinct enriched communities dominated by phylotypes related to cellulolytic and hemicellulolytic Thermotoga and Dictyoglomus, cellulolytic and sugar-fermenting Desulfurococcales, and sugar-fermenting and hydrogenotrophic Archaeoglobales. Minor enriched populations included close relatives of hydrogenotrophic Thermodesulfobacteria, the candidate bacterial phylum OP9, and candidate archaeal groups C2 and DHVE3. Enrichment temperature was the major factor influencing community composition, with a negative correlation between temperature and richness, followed by lignocellulosic substrate composition. This study establishes the importance of these groups in the natural degradation of lignocellulose at high temperatures and suggests that a substantial portion of the diversity of thermophiles contributing to consortial cellulolysis may be contained within lineages that have representatives in pure culture.

  12. Deep 16S rRNA Pyrosequencing Reveals a Bacterial Community Associated with Banana Fusarium Wilt Disease Suppression Induced by Bio-Organic Fertilizer Application

    Science.gov (United States)

    Ruan, Yunze; Xue, Chao; Zhang, Jian; Li, Rong; Shen, Qirong

    2014-01-01

    Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas. PMID:24871319

  13. Bacterial community composition in the gut content and ambient sediment of sea cucumber Apostichopus japonicus revealed by 16S rRNA gene pyrosequencing.

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    Fei Gao

    Full Text Available The composition of the bacterial communities in the contents of the foregut and hindgut of the sea cucumber Apostichopus japonicus and in the ambient surface sediment was surveyed by 16S rRNA gene 454-pyrosequencing. A total of 188,623 optimized reads and 15,527 operational taxonomic units (OTUs were obtained from the ten gut contents samples and four surface sediment samples. The sequences in the sediments, foregut contents, and hindgut contents were assigned to 38.0±4.7, 31.2±6.2 and 27.8±6.5 phyla, respectively. The bacterial richness and Shannon diversity index were both higher in the ambient sediments than in the gut contents. Proteobacteria was the predominant phylum in both the gut contents and sediment samples. The predominant classes in the foregut, hindgut, and ambient sediment were Holophagae and Gammaproteobacteria, Deltaproteobacteria and Gammaproteobacteria, and Gammaproteobacteria and Deltaproteobacteria, respectively. The potential probiotics, including sequences related to Bacillus, lactic acid bacteria (Lactobacillus, Lactococcus, and Streptococcus and Pseudomonas were detected in the gut of A. japonicus. Principle component analysis and heatmap figure showed that the foregut, hindgut, and ambient sediment respectively harbored different characteristic bacterial communities. Selective feeding of A. japonicus may be the primary source of the different bacterial communities between the foregut contents and ambient sediments.

  14. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

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    Annette K. Møller

    2013-04-01

    Full Text Available The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition of the microbial assemblages was different within the snow layers and between snow and freshwater. The highest diversity was seen in snow. In the middle and top snow layers, Proteobacteria, Bacteroidetes and Cyanobacteria dominated, although Actinobacteria and Firmicutes were relatively abundant also. High numbers of chloroplasts were also observed. In the deepest snow layer, large percentages of Firmicutes and Fusobacteria were seen. In freshwater, Bacteroidetes, Actinobacteria and Verrucomicrobia were the most abundant phyla while relatively few Proteobacteria and Cyanobacteria were present. Possibly, light intensity controlled the distribution of the Cyanobacteria and algae in the snow while carbon and nitrogen fixed by these autotrophs in turn fed the heterotrophic bacteria. In the lake, a probable lower light input relative to snow resulted in low numbers of Cyanobacteria and chloroplasts and, hence, limited input of organic carbon and nitrogen to the heterotrophic bacteria. Thus, differences in the physicochemical conditions may play an important role in the processes leading to distinctive bacterial community structures in High-Arctic snow and freshwater.

  15. Diversity and distribution of lichen-associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by 454 pyrosequencing

    Science.gov (United States)

    Zhang, Tao; Wei, Xin-Li; Zhang, Yu-Qin; Liu, Hong-Yu; Yu, Li-Yan

    2015-01-01

    This study assessed the diversity and distribution of fungal communities associated with seven lichen species in the Ny-Ålesund Region (Svalbard, High Arctic) using Roche 454 pyrosequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Lichen-associated fungal communities showed high diversity, with a total of 42,259 reads belonging to 370 operational taxonomic units (OTUs) being found. Of these OTUs, 294 belonged to Ascomycota, 54 to Basidiomycota, 2 to Zygomycota, and 20 to unknown fungi. Leotiomycetes, Dothideomycetes, and Eurotiomycetes were the major classes, whereas the dominant orders were Helotiales, Capnodiales, and Chaetothyriales. Interestingly, most fungal OTUs were closely related to fungi from various habitats (e.g., soil, rock, plant tissues) in the Arctic, Antarctic and alpine regions, which suggests that living in association with lichen thalli may be a transient stage of life cycle for these fungi and that long-distance dispersal may be important to the fungi in the Arctic. In addition, host-related factors shaped the lichen-associated fungal communities in this region. Taken together, these results suggest that lichens thalli act as reservoirs of diverse fungi from various niches, which may improve our understanding of fungal evolution and ecology in the Arctic. PMID:26463847

  16. Diversity and distribution of lichen-associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by 454 pyrosequencing.

    Science.gov (United States)

    Zhang, Tao; Wei, Xin-Li; Zhang, Yu-Qin; Liu, Hong-Yu; Yu, Li-Yan

    2015-10-14

    This study assessed the diversity and distribution of fungal communities associated with seven lichen species in the Ny-Ålesund Region (Svalbard, High Arctic) using Roche 454 pyrosequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Lichen-associated fungal communities showed high diversity, with a total of 42,259 reads belonging to 370 operational taxonomic units (OTUs) being found. Of these OTUs, 294 belonged to Ascomycota, 54 to Basidiomycota, 2 to Zygomycota, and 20 to unknown fungi. Leotiomycetes, Dothideomycetes, and Eurotiomycetes were the major classes, whereas the dominant orders were Helotiales, Capnodiales, and Chaetothyriales. Interestingly, most fungal OTUs were closely related to fungi from various habitats (e.g., soil, rock, plant tissues) in the Arctic, Antarctic and alpine regions, which suggests that living in association with lichen thalli may be a transient stage of life cycle for these fungi and that long-distance dispersal may be important to the fungi in the Arctic. In addition, host-related factors shaped the lichen-associated fungal communities in this region. Taken together, these results suggest that lichens thalli act as reservoirs of diverse fungi from various niches, which may improve our understanding of fungal evolution and ecology in the Arctic.

  17. Pyrosequencing reveals correlations between extremely acidophilic bacterial communities with hydrogen sulphide concentrations, pH and inert polymer coatings at concrete sewer crown surfaces.

    Science.gov (United States)

    Pagaling, E; Yang, K; Yan, T

    2014-07-01

    To investigate the acidophilic bacterial communities involved in microbially induced concrete corrosion (MICC). Our study sites located downstream from a forced main provided a unique opportunity to study the microbial communities involved in MICC under different environmental conditions (gradients of atmospheric H(2)S, sulphate concentration and pH) and under pipe modifications (coated vs uncoated). Bacterial cell density estimated by both cultivation- and DNA-based methods was low in the corroded sewer samples. Pyrosequencing and cloning showed that Mycobacterium and Acidithiobacillus dominated the acidophilic microbial communities. Methylacidiphilum was also dominant in samples where methane was detected. Correlation analysis indicated that Mycobacterium and Acidithiobacillus were significantly affected by pH and that Mycobacterium could better withstand highly acidic conditions compared to Acidithiobacillus. Communities dominated by Mycobacterium favoured conditions in the lined sewer pipes, while communities with a higher relative abundance of Acidithiobacillus favoured the unlined sewer pipes. Identifying the key micro-organisms involved in MICC and knowing how they interact with their environment are essential aspects for identifying steps towards concrete corrosion management. © 2014 The Society for Applied Microbiology.

  18. Pyrosequencing assessment of rhizosphere fungal communities from a soybean field.

    Science.gov (United States)

    Sugiyama, Akifumi; Ueda, Yoshikatsu; Takase, Hisabumi; Yazaki, Kazufumi

    2014-10-01

    Soil fungal communities play essential roles in soil ecosystems, affecting plant growth and health. Rhizosphere bacterial communities have been shown to undergo dynamic changes during plant growth. This study utilized 454 pyrosequencing to analyze rhizosphere fungal communities during soybean growth. Members of the Ascomycota and Basiodiomycota dominated in all soils. There were no statistically significant changes at the phylum level among growth stages or between bulk and rhizosphere soils. In contrast, the relative abundance of small numbers of operational taxonomic units, 4 during growth and 28 between bulk and rhizosphere soils, differed significantly. Clustering analysis revealed that rhizosphere fungal communities were different from bulk fungal communities during growth stages of soybeans. Taken together, these results suggest that in contrast to rhizosphere bacterial communities, most constituents of rhizosphere fungal communities remained stable during soybean growth.

  19. Microbial diversity in hummock and hollow soils of three wetlands on the Qinghai-Tibetan Plateau revealed by 16S rRNA pyrosequencing.

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    Yongcui Deng

    Full Text Available The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo on the Qinghai-Tibetan Plateau, the world's highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance, Actinobacteria (17.3% and Bacteroidetes (11% had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1-10%. In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales. Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta and methanotrophs (mainly Methylocystis compared with the other two wetlands. Principal coordinate analysis (PCoA indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of

  20. Microbial diversity in hummock and hollow soils of three wetlands on the Qinghai-Tibetan Plateau revealed by 16S rRNA pyrosequencing.

    Science.gov (United States)

    Deng, Yongcui; Cui, Xiaoyong; Hernández, Marcela; Dumont, Marc G

    2014-01-01

    The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world's highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1-10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai

  1. Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

    Science.gov (United States)

    Lenchi, Nesrine; İnceoğlu, Özgül; Kebbouche-Gana, Salima; Gana, Mohamed Lamine; Llirós, Marc; Servais, Pierre; García-Armisen, Tamara

    2013-01-01

    The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already

  2. Functional bacterial and archaeal diversity revealed by 16S rRNA gene pyrosequencing during potato starch processing wastewater treatment in an UASB.

    Science.gov (United States)

    Antwi, Philip; Li, Jianzheng; Opoku Boadi, Portia; Meng, Jia; Shi, En; Xue, Chi; Zhang, Yupeng; Ayivi, Frederick

    2017-07-01

    Microbial community structure of sludge sampled from an UASB treating potato starch processing wastewater (PSPW) was investigated. Operational taxonomic units revealed at 97% sequence identity tolerance was 2922, 2869 and 3919 for bottom, middle and top sections of the reactor, respectively. Overall abundant phylum observed within the UASB was low-G+C-Gram-positive bacteria affiliated to Firmicutes (26.01%) followed by Chloroflexi (16.70%), Proteobacteria (12.71%), Cloacimonetes (10.72%), Bacteroidetes (7.87%), Synergistetes (9.02%) and Euryarchaeota (8.82%). Whiles Firmicutes had dominated the bottom and top section by 34.01% and 28.64%, respectively, middle section was predominantly Euryarchaeota (24.32%) with major dominance in methanogens affiliated to genus Methanosaeta. The results demonstrated substantial stratification of the microbial community structure along the reactor height with various functional bacterial groups which subsequently allowed degradation of organics in PSPW in sequential mode. The findings herein would provide guidance for optimizing the anaerobic process and operation of the UASB. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Evaluation of Automated Ribosomal Intergenic Spacer Analysis for Bacterial Fingerprinting of Rumen Microbiome Compared to Pyrosequencing Technology

    Directory of Open Access Journals (Sweden)

    Elie Jami

    2014-01-01

    Full Text Available The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA, an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data.

  4. Growth and carbon isotopes of Mediterranean trees reveal contrasting responses to increased carbon dioxide and drought.

    Science.gov (United States)

    Granda, Elena; Rossatto, Davi Rodrigo; Camarero, J Julio; Voltas, Jordi; Valladares, Fernando

    2014-01-01

    Forest dynamics will depend upon the physiological performance of individual tree species under more stressful conditions caused by climate change. In order to compare the idiosyncratic responses of Mediterranean tree species (Quercus faginea, Pinus nigra, Juniperus thurifera) coexisting in forests of central Spain, we evaluated the temporal changes in secondary growth (basal area increment; BAI) and intrinsic water-use efficiency (iWUE) during the last four decades, determined how coexisting species are responding to increases in atmospheric CO2 concentrations (C(a)) and drought stress, and assessed the relationship among iWUE and growth during climatically contrasting years. All species increased their iWUE (ca. +15 to +21%) between the 1970s and the 2000s. This increase was positively related to C(a) for J. thurifera and to higher C(a) and drought for Q. faginea and P. nigra. During climatically favourable years the study species either increased or maintained their growth at rising iWUE, suggesting a higher CO2 uptake. However, during unfavourable climatic years Q. faginea and especially P. nigra showed sharp declines in growth at enhanced iWUE, likely caused by a reduced stomatal conductance to save water under stressful dry conditions. In contrast, J. thurifera showed enhanced growth also during unfavourable years at increased iWUE, denoting a beneficial effect of C(a) even under climatically harsh conditions. Our results reveal significant inter-specific differences in growth driven by alternative physiological responses to increasing drought stress. Thus, forest composition in the Mediterranean region might be altered due to contrasting capacities of coexisting tree species to withstand increasingly stressful conditions.

  5. Polarization-dependent Imaging Contrast (PIC) mapping reveals nanocrystal orientation patterns in carbonate biominerals

    Energy Technology Data Exchange (ETDEWEB)

    Gilbert, Pupa U.P.A., E-mail: pupa@physics.wisc.edu [University of Wisconsin-Madison, Departments of Physics and Chemistry, Madison, WI 53706 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer Nanocrystal orientation shown by Polarization-dependent Imaging Contrast (PIC) maps. Black-Right-Pointing-Pointer PIC-mapping of carbonate biominerals reveals their ultrastructure at the nanoscale. Black-Right-Pointing-Pointer The formation mechanisms of biominerals is discovered by PIC-mapping using PEEM. -- Abstract: Carbonate biominerals are one of the most interesting systems a physicist can study. They play a major role in the CO{sub 2} cycle, they master templation, self-assembly, nanofabrication, phase transitions, space filling, crystal nucleation and growth mechanisms. A new imaging modality was introduced in the last 5 years that enables direct observation of the orientation of carbonate single crystals, at the nano- and micro-scale. This is Polarization-dependent Imaging Contrast (PIC) mapping, which is based on X-ray linear dichroism, and uses PhotoElectron Emission spectroMicroscopy (PEEM). Here we present PIC-mapping results from biominerals, including the nacre and prismatic layers of mollusk shells, and sea urchin teeth. We describe various PIC-mapping approaches, and show that these lead to fundamental discoveries on the formation mechanisms of biominerals.

  6. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

    Science.gov (United States)

    Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

  7. Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

    Science.gov (United States)

    Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

  8. Characterization of the avian Trojan gene family reveals contrasting evolutionary constraints.

    Directory of Open Access Journals (Sweden)

    Petar Petrov

    Full Text Available "Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules.

  9. Characterization of the avian Trojan gene family reveals contrasting evolutionary constraints.

    Science.gov (United States)

    Petrov, Petar; Syrjänen, Riikka; Smith, Jacqueline; Gutowska, Maria Weronika; Uchida, Tatsuya; Vainio, Olli; Burt, David W

    2015-01-01

    "Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules.

  10. Removing Noise From Pyrosequenced Amplicons

    Directory of Open Access Journals (Sweden)

    Davenport Russell J

    2011-01-01

    Full Text Available Abstract Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high

  11. Comparative transcriptomic analysis of roots of contrasting Gossypium herbaceum genotypes revealing adaptation to drought

    Directory of Open Access Journals (Sweden)

    Ranjan Alok

    2012-11-01

    Full Text Available Abstract Background Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of Gossypium herbaceum that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation. Results The variations in root length and growth were found among four genotypes of G.herbaceum when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols were also highly expressed in Vagad and Gujcot-21. Conclusion Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these

  12. Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.

    Science.gov (United States)

    Whittington, Emma; Forsythe, Desiree; Borziak, Kirill; Karr, Timothy L; Walters, James R; Dorus, Steve

    2017-12-02

    Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Our results identify a burst of genetic novelty

  13. Exploitation of Contrasts in Low Energy SEM to Reveal True Microstructure

    Czech Academy of Sciences Publication Activity Database

    Müllerová, Ilona; Mikmeková, Šárka; Mikmeková, Eliška; Pokorná, Zuzana; Frank, Luděk

    2014-01-01

    Roč. 20, S3 (2014), s. 858-859 ISSN 1431-9276 R&D Projects: GA TA ČR TE01020118; GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : scanning low energy electron microscopy * contrast * cathode lens Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.877, year: 2014

  14. Revealing life-history traits by contrasting genetic estimations with predictions of effective population size.

    Science.gov (United States)

    Greenbaum, Gili; Renan, Sharon; Templeton, Alan R; Bouskila, Amos; Saltz, David; Rubenstein, Daniel I; Bar-David, Shirli

    2017-12-22

    Effective population size, a central concept in conservation biology, is now routinely estimated from genetic surveys and can also be theoretically predicted from demographic, life-history, and mating-system data. By evaluating the consistency of theoretical predictions with empirically estimated effective size, insights can be gained regarding life-history characteristics and the relative impact of different life-history traits on genetic drift. These insights can be used to design and inform management strategies aimed at increasing effective population size. We demonstrated this approach by addressing the conservation of a reintroduced population of Asiatic wild ass (Equus hemionus). We estimated the variance effective size (N ev ) from genetic data (N ev =24.3) and formulated predictions for the impacts on N ev of demography, polygyny, female variance in lifetime reproductive success (RS), and heritability of female RS. By contrasting the genetic estimation with theoretical predictions, we found that polygyny was the strongest factor affecting genetic drift because only when accounting for polygyny were predictions consistent with the genetically measured N ev . The comparison of effective-size estimation and predictions indicated that 10.6% of the males mated per generation when heritability of female RS was unaccounted for (polygyny responsible for 81% decrease in N ev ) and 19.5% mated when female RS was accounted for (polygyny responsible for 67% decrease in N ev ). Heritability of female RS also affected N ev ; hf2=0.91 (heritability responsible for 41% decrease in N ev ). The low effective size is of concern, and we suggest that management actions focus on factors identified as strongly affecting Nev, namely, increasing the availability of artificial water sources to increase number of dominant males contributing to the gene pool. This approach, evaluating life-history hypotheses in light of their impact on effective population size, and contrasting

  15. Spatially Explicit Modeling Reveals Cephalopod Distributions Match Contrasting Trophic Pathways in the Western Mediterranean Sea.

    Directory of Open Access Journals (Sweden)

    Patricia Puerta

    Full Text Available Populations of the same species can experience different responses to the environment throughout their distributional range as a result of spatial and temporal heterogeneity in habitat conditions. This highlights the importance of understanding the processes governing species distribution at local scales. However, research on species distribution often averages environmental covariates across large geographic areas, missing variability in population-environment interactions within geographically distinct regions. We used spatially explicit models to identify interactions between species and environmental, including chlorophyll a (Chla and sea surface temperature (SST, and trophic (prey density conditions, along with processes governing the distribution of two cephalopods with contrasting life-histories (octopus and squid across the western Mediterranean Sea. This approach is relevant for cephalopods, since their population dynamics are especially sensitive to variations in habitat conditions and rarely stable in abundance and location. The regional distributions of the two cephalopod species matched two different trophic pathways present in the western Mediterranean Sea, associated with the Gulf of Lion upwelling and the Ebro river discharges respectively. The effects of the studied environmental and trophic conditions were spatially variant in both species, with usually stronger effects along their distributional boundaries. We identify areas where prey availability limited the abundance of cephalopod populations as well as contrasting effects of temperature in the warmest regions. Despite distributional patterns matching productive areas, a general negative effect of Chla on cephalopod densities suggests that competition pressure is common in the study area. Additionally, results highlight the importance of trophic interactions, beyond other common environmental factors, in shaping the distribution of cephalopod populations. Our study presents

  16. Active methanotrophs in two contrasting North American peatland ecosystems revealed using DNA-SIP.

    Science.gov (United States)

    Gupta, Varun; Smemo, Kurt A; Yavitt, Joseph B; Basiliko, Nathan

    2012-02-01

    The active methanotroph community was investigated in two contrasting North American peatlands, a nutrient-rich sedge fen and nutrient-poor Sphagnum bog using in vitro incubations and (13)C-DNA stable-isotope probing (SIP) to measure methane (CH(4)) oxidation rates and label active microbes followed by fingerprinting and sequencing of bacterial and archaeal 16S rDNA and methane monooxygenase (pmoA and mmoX) genes. Rates of CH(4) oxidation were slightly, but significantly, faster in the bog and methanotrophs belonged to the class Alphaproteobacteria and were similar to other methanotrophs of the genera Methylocystis, Methylosinus, and Methylocapsa or Methylocella detected in, or isolated from, European bogs. The fen had a greater phylogenetic diversity of organisms that had assimilated (13)C, including methanotrophs from both the Alpha- and Gammaproteobacteria classes and other potentially non-methanotrophic organisms that were similar to bacteria detected in a UK and Finnish fen. Based on similarities between bacteria in our sites and those in Europe, including Russia, we conclude that site physicochemical characteristics rather than biogeography controlled the phylogenetic diversity of active methanotrophs and that differences in phylogenetic diversity between the bog and fen did not relate to measured CH(4) oxidation rates. A single crenarchaeon in the bog site appeared to be assimilating (13)C in 16S rDNA; however, its phylogenetic similarity to other CO(2)-utilizing archaea probably indicates that this organism is not directly involved in CH(4) oxidation in peat.

  17. Evapotranspiration patterns in complex upland forests reveal contrasting topographic thresholds of non-linearity

    Science.gov (United States)

    Metzen, D.; Sheridan, G. J.; Benyon, R. G.; Bolstad, P. V.; Nyman, P.; Lane, P. N. J.

    2017-12-01

    Large areas of forest are often treated as being homogeneous just because they fall in a single climate category. However, we observe strong vegetation patterns in relation to topography in SE Australian forests and thus hypothesise that ET will vary spatially as well. Spatial heterogeneity evolves over different temporal scales in response to climatic forcing with increasing time lag from soil moisture (sub-yearly), to vegetation (10s -100s of years) to soil properties and topography (>100s of years). Most importantly, these processes and time scales are not independent, creating feedbacks that result in "co-evolved stable states" which yield the current spatial terrain, vegetation and ET patterns. We used up-scaled sap flux and understory ET measurements from water-balance plots, as well as LiDAR derived terrain and vegetation information, to infer links between spatio-temporal energy and water fluxes, topography and vegetation patterns at small catchment scale. Topography caused variations in aridity index between polar and equatorial-facing slopes (1.3 vs 1.8), which in turn manifested in significant differences in sapwood area index (6.9 vs 5.8), overstory LAI (3.0 vs 2.3), understory LAI (0.5 vs 0.4), sub-canopy radiation load (4.6 vs 6.8 MJ m-2 d-1), overstory transpiration (501 vs 347 mm a-1) and understory ET (79 vs 155 mm a-1). Large spatial variation in overstory transpiration (195 to 891 mm a-1) was observed over very short distances (100s m); a range representative of diverse forests such as arid open woodlands and wet mountain ash forests. Contrasting, non-linear overstory and understory ET patterns were unveiled between aspects, and topographic thresholds were lower for overstory than understory ET. While ET partitioning remained stable on polar-facing slopes regardless of slope position, overstory contribution gradually decreased with increasing slope inclination on equatorial aspects. Further, we show that ET patterns and controls underlie strong

  18. Going Begging:Casino Culture and its Contrasts as Revealed in the New Macao Poetry

    Directory of Open Access Journals (Sweden)

    Christopher Kelen

    2009-12-01

    Full Text Available Among the key themes of contemporary Macao poetry, chance and luck loom large, along with their figuration in Macao life through sites such as casinos and temples, through personae such as those of the gambler, the beggar, the prostitute. Macao as dot-on-the-map is likewise conceived as a site for all kinds of portal semiotics, as paradigm for cultural crossing and cultural shift. Macao may be re-garded as a work enduring (in Brechtian terms because it is unfinished. While this is a formula that could be notionally applied to any city, this view seems par-ticularly apt given both the extraordinary pace of change in post-handover Macao (i.e. since 1999, and the present bubble-bursting effect of the 2008 "financial tsunami". Relating Augé's conception of "non-places" to Eco's notion of open (as op-posed to closed text, this paper observes that consciousness of place in contem-porary Macao poetry appears to be dominated by two kinds of space, glossed here as "Macao space" and "anywhere space". Macao space is uniquely of an historical moment and place, something culturally positioned; in anywhere space (e.g. in-side of a casino or an airport subjects are hailed by consumption-oriented reifica-tions of putative universal value. The contemporary Macao poetry typically values Macao space and sees it as under threat from the "non-negotiable" space of cul-ture that could be anywhere. Interested in the paradoxes, ironies and hypocrisies inherent in the present-day culture, politics and international position of Macao, the new Macao poetry re-veals a place-based poetics deeply concerned with Macao identity, its evolution and potentials.

  19. Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site.

    Science.gov (United States)

    Durand, Alexis; Maillard, François; Foulon, Julie; Gweon, Hyun S; Valot, Benoit; Chalot, Michel

    2017-11-01

    Characterization of microbial communities in stressful conditions at a field level is rather scarce, especially when considering fungal communities from aboveground habitats. We aimed at characterizing fungal communities from different poplar habitats at a Hg-contaminated phytomanagement site by using Illumina-based sequencing, network analysis approach, and direct isolation of Hg-resistant fungal strains. The highest diversity estimated by the Shannon index was found for soil communities, which was negatively affected by soil Hg concentration. Among the significant correlations between soil operational taxonomic units (OTUs) in the co-occurrence network, 80% were negatively correlated revealing dominance of a pattern of mutual exclusion. The fungal communities associated with Populus roots mostly consisted of OTUs from the symbiotic guild, such as members of the Thelephoraceae, thus explaining the lowest diversity found for root communities. Additionally, root communities showed the highest network connectivity index, while rarely detected OTUs from the Glomeromycetes may have a central role in the root network. Unexpectedly high richness and diversity were found for aboveground habitats, compared to the root habitat. The aboveground habitats were dominated by yeasts from the Lalaria, Davidiella, and Bensingtonia genera, not detected in belowground habitats. Leaf and stem habitats were characterized by few dominant OTUs such as those from the Dothideomycete class producing mutual exclusion with other OTUs. Aureobasidium pullulans, one of the dominating OTUs, was further isolated from the leaf habitat, in addition to Nakazawaea populi species, which were found to be Hg resistant. Altogether, these findings will provide an improved point of reference for microbial research on inoculation-based programs of tailings dumps.

  20. Pyrosequencing and genetic diversity of microeukaryotes

    DEFF Research Database (Denmark)

    Harder, Christoffer Bugge

    carefully selected waterworks (Article IV), where the bacterial metabolic diversity and its important for water purification was described. Building on this, the most important part of the thesis consists of two pyrosequencing analyses of protozoa with newly developed 18S primers. One specifically targets...... Cercozoa, a particularly abundant phylum of protozoa (Article III), on heath land that had been subjected to prolonged artificially induced drought in a Danish free-air climate-manipulation experiment (CLIMAITE). Article III showed that the testate cercozoan forms responded negatively to prolonged drought...

  1. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  2. "Contrasting patterns of selection at Pinus pinaster Ait. Drought stress candidate genes as revealed by genetic differentiation analyses".

    Science.gov (United States)

    Eveno, Emmanuelle; Collada, Carmen; Guevara, M Angeles; Léger, Valérie; Soto, Alvaro; Díaz, Luis; Léger, Patrick; González-Martínez, Santiago C; Cervera, M Teresa; Plomion, Christophe; Garnier-Géré, Pauline H

    2008-02-01

    The importance of natural selection for shaping adaptive trait differentiation among natural populations of allogamous tree species has long been recognized. Determining the molecular basis of local adaptation remains largely unresolved, and the respective roles of selection and demography in shaping population structure are actively debated. Using a multilocus scan that aims to detect outliers from simulated neutral expectations, we analyzed patterns of nucleotide diversity and genetic differentiation at 11 polymorphic candidate genes for drought stress tolerance in phenotypically contrasted Pinus pinaster Ait. populations across its geographical range. We compared 3 coalescent-based methods: 2 frequentist-like, including 1 approach specifically developed for biallelic single nucleotide polymorphisms (SNPs) here and 1 Bayesian. Five genes showed outlier patterns that were robust across methods at the haplotype level for 2 of them. Two genes presented higher F(ST) values than expected (PR-AGP4 and erd3), suggesting that they could have been affected by the action of diversifying selection among populations. In contrast, 3 genes presented lower F(ST) values than expected (dhn-1, dhn2, and lp3-1), which could represent signatures of homogenizing selection among populations. A smaller proportion of outliers were detected at the SNP level suggesting the potential functional significance of particular combinations of sites in drought-response candidate genes. The Bayesian method appeared robust to low sample sizes, flexible to assumptions regarding migration rates, and powerful for detecting selection at the haplotype level, but the frequentist-like method adapted to SNPs was more efficient for the identification of outlier SNPs showing low differentiation. Population-specific effects estimated in the Bayesian method also revealed populations with lower immigration rates, which could have led to favorable situations for local adaptation. Outlier patterns are discussed

  3. Local-scale models reveal ecological niche variability in amphibian and reptile communities from two contrasting biogeographic regions

    Directory of Open Access Journals (Sweden)

    Alberto Muñoz

    2016-10-01

    Full Text Available Ecological Niche Models (ENMs are widely used to describe how environmental factors influence species distribution. Modelling at a local scale, compared to a large scale within a high environmental gradient, can improve our understanding of ecological species niches. The main goal of this study is to assess and compare the contribution of environmental variables to amphibian and reptile ENMs in two Spanish national parks located in contrasting biogeographic regions, i.e., the Mediterranean and the Atlantic area. The ENMs were built with maximum entropy modelling using 11 environmental variables in each territory. The contributions of these variables to the models were analysed and classified using various statistical procedures (Mann–Whitney U tests, Principal Components Analysis and General Linear Models. Distance to the hydrological network was consistently the most relevant variable for both parks and taxonomic classes. Topographic variables (i.e., slope and altitude were the second most predictive variables, followed by climatic variables. Differences in variable contribution were observed between parks and taxonomic classes. Variables related to water availability had the larger contribution to the models in the Mediterranean park, while topography variables were decisive in the Atlantic park. Specific response curves to environmental variables were in accordance with the biogeographic affinity of species (Mediterranean and non-Mediterranean species and taxonomy (amphibians and reptiles. Interestingly, these results were observed for species located in both parks, particularly those situated at their range limits. Our findings show that ecological niche models built at local scale reveal differences in habitat preferences within a wide environmental gradient. Therefore, modelling at local scales rather than assuming large-scale models could be preferable for the establishment of conservation strategies for herptile species in natural

  4. Local-scale models reveal ecological niche variability in amphibian and reptile communities from two contrasting biogeographic regions

    Science.gov (United States)

    Santos, Xavier; Felicísimo, Ángel M.

    2016-01-01

    Ecological Niche Models (ENMs) are widely used to describe how environmental factors influence species distribution. Modelling at a local scale, compared to a large scale within a high environmental gradient, can improve our understanding of ecological species niches. The main goal of this study is to assess and compare the contribution of environmental variables to amphibian and reptile ENMs in two Spanish national parks located in contrasting biogeographic regions, i.e., the Mediterranean and the Atlantic area. The ENMs were built with maximum entropy modelling using 11 environmental variables in each territory. The contributions of these variables to the models were analysed and classified using various statistical procedures (Mann–Whitney U tests, Principal Components Analysis and General Linear Models). Distance to the hydrological network was consistently the most relevant variable for both parks and taxonomic classes. Topographic variables (i.e., slope and altitude) were the second most predictive variables, followed by climatic variables. Differences in variable contribution were observed between parks and taxonomic classes. Variables related to water availability had the larger contribution to the models in the Mediterranean park, while topography variables were decisive in the Atlantic park. Specific response curves to environmental variables were in accordance with the biogeographic affinity of species (Mediterranean and non-Mediterranean species) and taxonomy (amphibians and reptiles). Interestingly, these results were observed for species located in both parks, particularly those situated at their range limits. Our findings show that ecological niche models built at local scale reveal differences in habitat preferences within a wide environmental gradient. Therefore, modelling at local scales rather than assuming large-scale models could be preferable for the establishment of conservation strategies for herptile species in natural parks. PMID

  5. First- and second-order contrast sensitivity functions reveal disrupted visual processing following mild traumatic brain injury.

    Science.gov (United States)

    Spiegel, Daniel P; Reynaud, Alexandre; Ruiz, Tatiana; Laguë-Beauvais, Maude; Hess, Robert; Farivar, Reza

    2016-05-01

    Vision is disrupted by traumatic brain injury (TBI), with vision-related complaints being amongst the most common in this population. Based on the neural responses of early visual cortical areas, injury to the visual cortex would be predicted to affect both 1(st) order and 2(nd) order contrast sensitivity functions (CSFs)-the height and/or the cut-off of the CSF are expected to be affected by TBI. Previous studies have reported disruptions only in 2(nd) order contrast sensitivity, but using a narrow range of parameters and divergent methodologies-no study has characterized the effect of TBI on the full CSF for both 1(st) and 2(nd) order stimuli. Such information is needed to properly understand the effect of TBI on contrast perception, which underlies all visual processing. Using a unified framework based on the quick contrast sensitivity function, we measured full CSFs for static and dynamic 1(st) and 2(nd) order stimuli. Our results provide a unique dataset showing alterations in sensitivity for both 1(st) and 2(nd) order visual stimuli. In particular, we show that TBI patients have increased sensitivity for 1(st) order motion stimuli and decreased sensitivity to orientation-defined and contrast-defined 2(nd) order stimuli. In addition, our data suggest that TBI patients' sensitivity for both 1(st) order stimuli and 2(nd) order contrast-defined stimuli is shifted towards higher spatial frequencies. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Strategien zur HLA-Typisierung mit PyrosequencingTM

    OpenAIRE

    Entz, Patricia

    2006-01-01

    Der Haupthistokompatibilitätskomplex ist durch seine biologische Funktion eine für die Diagnostik und Forschung äußerst wichtige Region im humanen Genom. Die Untersuchung von HLA-Genorten stellt ein wichtiges Instrument in der molekulargenetischen Praxis dar. Die Pyrosequencing-Technik ist gut geeignet, um kurze DNA-Abschnitte mit weitgehend bekannter Sequenz schnell und effizient zu untersuchen. Ziel dieser Arbeit war die Entwicklung von Pyrosequencing-basierten Methoden zur HLA-Typisierung....

  7. Pyrosequencing the Canine Faecal Microbiota: Breadth and Depth of Biodiversity

    Science.gov (United States)

    Hand, Daniel; Wallis, Corrin; Colyer, Alison; Penn, Charles W.

    2013-01-01

    Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5′ region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a “core microbiota”. Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs) at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs. PMID:23382835

  8. Pyrosequencing the canine faecal microbiota: breadth and depth of biodiversity.

    Directory of Open Access Journals (Sweden)

    Daniel Hand

    Full Text Available Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5' region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a "core microbiota". Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs.

  9. Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions.

    Science.gov (United States)

    Park, O-J; Yi, H; Jeon, J H; Kang, S-S; Koo, K-T; Kum, K-Y; Chun, J; Yun, C-H; Han, S H

    2015-07-01

    Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases. © International & American Associations for Dental Research 2015.

  10. Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

    KAUST Repository

    Prest, Emmanuelle I E C

    2014-10-01

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.

  11. Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

    Science.gov (United States)

    Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

    2014-10-15

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Quantitative Molecular Imaging with a Single Gd-Based Contrast Agent Reveals Specific Tumor Binding and Retention in Vivo.

    Science.gov (United States)

    Johansen, Mette L; Gao, Ying; Hutnick, Melanie A; Craig, Sonya E L; Pokorski, Jonathan K; Flask, Chris A; Brady-Kalnay, Susann M

    2017-06-06

    Magnetic resonance imaging (MRI) has become an indispensable tool in the diagnosis and treatment of many diseases, especially cancer. However, the poor sensitivity of MRI relative to other imaging modalities, such as PET, has hindered the development and clinical use of molecular MRI contrast agents that could provide vital diagnostic information by specifically locating a molecular target altered in the disease process. This work describes the specific and sustained in vivo binding and retention of a protein tyrosine phosphatase mu (PTPμ)-targeted, molecular magnetic resonance (MR) contrast agent with a single gadolinium (Gd) chelate using a quantitative MRI T 1 mapping technique in glioma xenografts. Quantitative T 1 mapping is an imaging method used to measure the longitudinal relaxation time, the T 1 relaxation time, of protons in a magnetic field after excitation by a radiofrequency pulse. T 1 relaxation times can in turn be used to calculate the concentration of a gadolinium-containing contrast agent in a region of interest, thereby allowing the retention or clearance of an agent to be quantified. In this context, retention is a measure of molecular contrast agent binding. Using conventional peptide chemistry, a PTPμ-targeted peptide was linked to a chelator that had been conjugated to a lysine residue. Following complexation with Gd, this PTPμ-targeted molecular contrast agent containing a single Gd ion showed significant tumor enhancement and a sustained increase in Gd concentration in both heterotopic and orthotopic tumors using dynamic quantitative MRI. This single Gd-containing PTPμ agent was more effective than our previous version with three Gd ions. Differences between nonspecific and specific agents, due to specific tumor binding, can be determined within the first 30 min after agent administration by examining clearance rates. This more facile chemistry, when combined with quantitative MR techniques, allows for widespread adoption by academic

  13. Pyrosequencing Analysis of Cryogenically Ground Samples from Primary and Secondary/Persistent Endodontic Infections.

    Science.gov (United States)

    Keskin, Cangül; Demiryürek, Ebru Özsezer; Onuk, Ertan Emek

    2017-08-01

    This study aimed to characterize the microbial communities of primary and secondary/persistent endodontic infections using high-throughput pyrosequencing from the pulverized samples. The roots of 20 extracted human teeth with primary endodontic infection and 20 teeth with secondary/persistent endodontic infection were collected. The outer surfaces of the roots were disinfected, and whole roots were cryopulverized. 16S amplicon pyrosequencing data from the DNA extracted from the pulverized root powders were obtained, and microorganism abundance and diversity were calculated. Data were analyzed using statistical and bioinformatic methods. Pyrosequencing analysis resulted a total of 2,606,128 sequences from 40 samples. A total of 15 phyla, 160 genera, and 368 species were detected. No significant difference between primary and secondary/persistent endodontic infections was found regarding the diversity and richness of operational taxonomic units at the phyla, genera, and species levels (P > .005). The present study revealed that the microbial diversity of secondary/persistent endodontic infections did not differ than those of primary endodontic infections. A new archaeal species, Candidatus Nitrosoarchaeum limnia, was detected in root canals of 1 patient with primary endodontic infection for the first time. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. Wrinkles in the rare biosphere: Pyrosequencing errors can lead to artificial inflation of diversity estimates

    Energy Technology Data Exchange (ETDEWEB)

    Kunin, Victor; Engelbrektson, Anna; Ochman, Howard; Hugenholtz, Philip

    2009-08-01

    Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the 'rare biosphere', is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere.

  15. Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual- and Combined- Biotic and Abiotic Stress Tolerance Mechanisms.

    Directory of Open Access Journals (Sweden)

    Vemanna S Ramu

    Full Text Available In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses.

  16. 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

    Science.gov (United States)

    Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

    2017-06-16

    COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

  17. Protein Profiles Reveal Diverse Responsive Signaling Pathways in Kernels of Two Maize Inbred Lines with Contrasting Drought Sensitivity

    Directory of Open Access Journals (Sweden)

    Liming Yang

    2014-10-01

    Full Text Available Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964 with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP, and protein profiles were investigated in developing kernels (35 DAP using iTRAQ (isobaric tags for relative and absolute quantitation. Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels.

  18. Distribution of unresolvable anisotropic microstructures revealed in visibility-contrast images using x-ray Talbot interferometry

    International Nuclear Information System (INIS)

    Yashiro, Wataru; Harasse, Sebastien; Kawabata, Katsuyuki; Kuwabara, Hiroaki; Yamazaki, Takashi; Momose, Atsushi

    2011-01-01

    X-ray Talbot interferometry has been widely used as a technique for x-ray phase imaging and tomography. We propose a method using this interferometry for mapping distribution of parameters characterizing anisotropic microstructures, which are typically of the order of μm in size and cannot be resolved by the imaging system, in a sample. The method uses reduction in fringe visibility, which is caused by such unresolvable microstructures, in moire images obtained using an interferometer. We applied the method to a chloroprene rubber sponge sample, which exhibited uniaxial anisotropy of reduced visibility. We measured the dependencies of reduced visibility on both the Talbot order and the orientation of the sample and obtained maps of three parameters and their anisotropies that characterize the unresolvable anisotropic microstructures in the sample. The maps indicated that the anisotropy of the sample's visibility contrast mainly originated from the anisotropy of the microstructure elements' average size. Our method directly provides structural information on unresolvable microstructures in real space, which is only accessible through the ultra-small-angle x-ray scattering measurements in reciprocal space, and is expected to be broadly applied to material, biological, and medical sciences.

  19. Contrasting patterns of survival and dispersal in multiple habitats reveal an ecological trap in a food-caching bird.

    Science.gov (United States)

    Norris, D Ryan; Flockhart, D T Tyler; Strickland, Dan

    2013-11-01

    A comprehensive understanding of how natural and anthropogenic variation in habitat influences populations requires long-term information on how such variation affects survival and dispersal throughout the annual cycle. Gray jays Perisoreus canadensis are widespread boreal resident passerines that use cached food to survive over the winter and to begin breeding during the late winter. Using multistate capture-recapture analysis, we examined apparent survival and dispersal in relation to habitat quality in a gray jay population over 34 years (1977-2010). Prior evidence suggests that natural variation in habitat quality is driven by the proportion of conifers on territories because of their superior ability to preserve cached food. Although neither adults (>1 year) nor juveniles (conifer territories, both age classes were less likely to leave high-conifer territories and, when they did move, were more likely to disperse to high-conifer territories. In contrast, survival rates were lower on territories that were adjacent to a major highway compared to territories that did not border the highway but there was no evidence for directional dispersal towards or away from highway territories. Our results support the notion that natural variation in habitat quality is driven by the proportion of coniferous trees on territories and provide the first evidence that high-mortality highway habitats can act as an equal-preference ecological trap for birds. Reproductive success, as shown in a previous study, but not survival, is sensitive to natural variation in habitat quality, suggesting that gray jays, despite living in harsh winter conditions, likely favor the allocation of limited resources towards self-maintenance over reproduction.

  20. Contrasting Networks for Recognition Memory and Recency Memory Revealed by Immediate-Early Gene Imaging in the Rat

    Science.gov (United States)

    2014-01-01

    The expression of the immediate-early gene c-fos was used to compare networks of activity associated with recency memory (temporal order memory) and recognition memory. In Experiment 1, rats were first familiarized with sets of objects and then given pairs of different, familiar objects to explore. For the recency test group, each object in a pair was separated by 110 min in the time between their previous presentations. For the recency control test, each object in a pair was separated by less than a 1 min between their prior presentations. Temporal discrimination of the objects correlated with c-fos activity in the recency test group in several sites, including area Te2, the perirhinal cortex, lateral entorhinal cortex, as well as the dentate gyrus, hippocampal fields CA3 and CA1. For both the test and control conditions, network models were derived using structural equation modeling. The recency test model emphasized serial connections from the perirhinal cortex to lateral entorhinal cortex and then to the CA1 subfield. The recency control condition involved more parallel pathways, but again highlighted CA1 within the hippocampus. Both models contrasted with those derived from tests of object recognition (Experiment 2), because stimulus novelty was associated with pathways from the perirhinal cortex to lateral entorhinal cortex that then involved both the dentate gyrus (and CA3) and CA1 in parallel. The present findings implicate CA1 for the processing of familiar stimuli, including recency discriminations, while the dentate gyrus and CA3 pathways are recruited when the perirhinal cortex signals novel stimuli. PMID:24933661

  1. Laminar microvascular transit time distribution in the mouse somatosensory cortex revealed by Dynamic Contrast Optical Coherence Tomography.

    Science.gov (United States)

    Merkle, Conrad W; Srinivasan, Vivek J

    2016-01-15

    The transit time distribution of blood through the cerebral microvasculature both constrains oxygen delivery and governs the kinetics of neuroimaging signals such as blood-oxygen-level-dependent functional Magnetic Resonance Imaging (BOLD fMRI). However, in spite of its importance, capillary transit time distribution has been challenging to quantify comprehensively and efficiently at the microscopic level. Here, we introduce a method, called Dynamic Contrast Optical Coherence Tomography (DyC-OCT), based on dynamic cross-sectional OCT imaging of an intravascular tracer as it passes through the field-of-view. Quantitative transit time metrics are derived from temporal analysis of the dynamic scattering signal, closely related to tracer concentration. Since DyC-OCT does not require calibration of the optical focus, quantitative accuracy is achieved even deep in highly scattering brain tissue where the focal spot degrades. After direct validation of DyC-OCT against dilution curves measured using a fluorescent plasma label in surface pial vessels, we used DyC-OCT to investigate the transit time distribution in microvasculature across the entire depth of the mouse somatosensory cortex. Laminar trends were identified, with earlier transit times and less heterogeneity in the middle cortical layers. The early transit times in the middle cortical layers may explain, at least in part, the early BOLD fMRI onset times observed in these layers. The layer-dependencies in heterogeneity may help explain how a single vascular supply manages to deliver oxygen to individual cortical layers with diverse metabolic needs. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Contrasting Responses of the Humboldt Current Ecosystem between the Holocene and MIS5e Interglacials Revealed from Multiple Sediment Records

    Science.gov (United States)

    Salvatteci, R.; Schneider, R. R.; Blanz, T.; Martinez, P.; Crosta, X.

    2016-12-01

    The Humboldt Current Ecosystem (HCE) off Peru yields about 10% of the global fish catch, producing more fish per unit area than any other region in the world. The high productivity is maintained by the upwelling of cold, nutrient-rich water from the oxygen minimum zone (OMZ), driven by strong trade winds. However, the potential impacts of climate change on upwelling dynamics and oceanographic conditions in the near future are uncertain, threatening local and global economies. Here, we unravel the response of the HCE to contrasting climatic conditions during the last two interglacials (i.e. Holocene and MIS5e) providing an independent insight about the relation between climatic factors and upwelling and productivity dynamics. For this purpose, we used multiple cores to reconstruct past changes in OMZ and upwelling intensity, productivity and fish biomass variability. Chronologies for the Holocene were obtained by multiple 14C ages and laminae correlations among cores, while for the MIS5e they were mainly done by correlation of prominent features in several proxies with other published records. We used a multiproxy approach including alkenones to reconstruct sea surface temperatures, δ15N as a proxy for water column denitrification, redox sensitive metals as proxies for sediment redox conditions, and diatom and fish debris assemblages to reconstruct ecological changes. The results show a very different response of the HCE to climate conditions during the last 2 interglacials, likely driven by changes in Tropical Pacific dynamics. During the Holocene we find that 1) the Late Holocene exhibits higher multi-centennial scale variability compared to the Early Holocene, 2) increased upwelling and a weak OMZ during the mid-Holocene, and 3) long term increase in productivity (diatoms and fishes) from the Early to the Late Holocene. During the MIS5e we find an 1) intense OMZ, 2) strong water column stratification, 3) high siliceous biomass, and 4) low fish biomass compared

  3. Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes communities in the gut microbiota of obese humans

    Directory of Open Access Journals (Sweden)

    Raoult Didier

    2008-12-01

    Full Text Available Abstract Background Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans. Results The main result was the identification of DNA barcodes targeting the Firmicutes and Bacteroidetes phyla. These barcodes were validated using previously published 16S rRNA gut microbiota clone libraries. In addition, an accurate F/B ratio was found when the DNA barcodes were applied to short pyrosequencing reads of published gut metagenomes. Finally, the barcodes were utilized to define the F/B ratio of 16S rRNA pyrosequencing data generated from brain abscess pus and cystic fibrosis sputum. Conclusion Using DNA barcodes of Bacteroidetes and Firmicutes 16S rRNA genes combined with pyrosequencing is a cost-effective method for monitoring relevant changes in the relative abundance of Firmicutes and Bacteroidetes bacterial communities in microbial ecosystems.

  4. Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

    Science.gov (United States)

    Boissy, Robert J; Romberger, Debra J; Roughead, William A; Weissenburger-Moser, Lisa; Poole, Jill A; LeVan, Tricia D

    2014-01-01

    Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses

  5. Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

    Directory of Open Access Journals (Sweden)

    Robert J Boissy

    Full Text Available Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance. The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70% at the phylum level, Clostridia (44% at the Class level, and Clostridiales at the Order level (41%. In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the

  6. Ancient DNA analyses reveal contrasting phylogeographic patterns amongst kiwi (Apteryx spp. and a recently extinct lineage of spotted kiwi.

    Directory of Open Access Journals (Sweden)

    Lara D Shepherd

    Full Text Available The little spotted kiwi (Apteryx owenii is a flightless ratite formerly found throughout New Zealand but now greatly reduced in distribution. Previous phylogeographic studies of the related brown kiwi (A. mantelli, A. rowi and A. australis, with which little spotted kiwi was once sympatric, revealed extremely high levels of genetic structuring, with mitochondrial DNA haplotypes often restricted to populations. We surveyed genetic variation throughout the present and pre-human range of little spotted kiwi by obtaining mitochondrial DNA sequences from contemporary and ancient samples. Little spotted kiwi and great spotted kiwi (A. haastii formed a monophyletic clade sister to brown kiwi. Ancient samples of little spotted kiwi from the northern North Island, where it is now extinct, formed a lineage that was distinct from remaining little spotted kiwi and great spotted kiwi lineages, potentially indicating unrecognized taxonomic diversity. Overall, little spotted kiwi exhibited much lower levels of genetic diversity and structuring than brown kiwi, particularly through the South Island. Our results also indicate that little spotted kiwi (or at least hybrids involving this species survived on the South Island mainland until more recently than previously thought.

  7. Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium

    Energy Technology Data Exchange (ETDEWEB)

    Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar, E-mail: aptesk@barc.gov.in

    2017-01-15

    Highlights: • Response of two native cyanobacterial strains to uranium exposure was studied. • Anabaena L-31 exhibited higher tolerance to uranium as compared to Anabaena 7120. • Uranium exposure differentially affected the proteome profiles of the two strains. • Anabaena L-31 showed better sustenance of photosynthesis and carbon metabolism. • Anabaena L-31 displayed superior oxidative stress defense than Anabaena 7120. - Abstract: Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD{sub 50} dose), following 3 h exposure to 75 μM and 200 μM uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120. Significance: Uranium responsive proteome modulations in two nitrogen-fixing strains of Anabaena, native to Indian paddy fields, revealed that rapid adaptation to better oxidative stress management, and maintenance of metabolic and energy homeostasis underlies superior uranium tolerance of Anabaena sp. strain L-31 compared to Anabaena sp. strain PCC 7120.

  8. A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

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    Binhua Liang

    Full Text Available BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

  9. Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

    Science.gov (United States)

    How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

    2015-07-01

    Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

  10. Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

    Energy Technology Data Exchange (ETDEWEB)

    Bomberg, M.; Nyyssoenen, M.; Itaevaara, M. [VTT Technical Research Centre of Finland, Espoo (Finland)

    2012-06-15

    Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.

  11. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

  12. Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

    Energy Technology Data Exchange (ETDEWEB)

    Bomberg, M; Nyyssoenen, M; Itaevaara, M [VTT Technical Research Centre of Finland, Espoo (Finland)

    2012-06-15

    Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.

  13. Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

    International Nuclear Information System (INIS)

    Bomberg, M.; Nyyssoenen, M.; Itaevaara, M.

    2012-06-01

    Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected

  14. 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters.

    Science.gov (United States)

    Sundberg, Carina; Al-Soud, Waleed A; Larsson, Madeleine; Alm, Erik; Yekta, Sepehr S; Svensson, Bo H; Sørensen, Søren J; Karlsson, Anna

    2013-09-01

    The microbial community of 21 full-scale biogas reactors was examined using 454 pyrosequencing of 16S rRNA gene sequences. These reactors included seven (six mesophilic and one thermophilic) digesting sewage sludge (SS) and 14 (ten mesophilic and four thermophilic) codigesting (CD) various combinations of wastes from slaughterhouses, restaurants, households, etc. The pyrosequencing generated more than 160,000 sequences representing 11 phyla, 23 classes, and 95 genera of Bacteria and Archaea. The bacterial community was always both more abundant and more diverse than the archaeal community. At the phylum level, the foremost populations in the SS reactors included Actinobacteria, Proteobacteria, Chloroflexi, Spirochetes, and Euryarchaeota, while Firmicutes was the most prevalent in the CD reactors. The main bacterial class in all reactors was Clostridia. Acetoclastic methanogens were detected in the SS, but not in the CD reactors. Their absence suggests that methane formation from acetate takes place mainly via syntrophic acetate oxidation in the CD reactors. A principal component analysis of the communities at genus level revealed three clusters: SS reactors, mesophilic CD reactors (including one thermophilic CD and one SS), and thermophilic CD reactors. Thus, the microbial composition was mainly governed by the substrate differences and the process temperature. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  15. Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing.

    Science.gov (United States)

    Barker, Christopher J; Gillett, Amber; Polkinghorne, Adam; Timms, Peter

    2013-12-27

    As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study

    Directory of Open Access Journals (Sweden)

    Guillaume eMinard

    2014-05-01

    Full Text Available The Asian tiger mosquito Aedes (Stegomya albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated however. Some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimise the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 98,520 sequences obtained. Ae. albopictus is known to harbour two Wolbachia strains, wAlbA and wAlbB, and quantitative PCR was used to estimate the relative densities, i.e. the bacteria-to-host gene ratios, of the strains in individual mosquitoes. Relative densities were between 6.25 × 100.01 and 5.47 × 100.1 for wAlbA and between 2.03 × 100.1 and 1.4 × 101 for wAlbB. Apart from Wolbachia, a total of 32 bacterial taxa were identified at the genus level using the different in method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from

  17. MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

    Science.gov (United States)

    Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

    2016-01-01

    The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

  18. Transcriptome Analysis of Two Rice Varieties Contrasting for Nitrogen Use Efficiency under Chronic N Starvation Reveals Differences in Chloroplast and Starch Metabolism-Related Genes

    Directory of Open Access Journals (Sweden)

    Subodh Kumar Sinha

    2018-04-01

    Full Text Available The nitrogen use efficiency (NUE of crop plants is limited and enhancing it in rice, a major cereal crop, would be beneficial for farmers and the environment alike. Here we report the genome-wide transcriptome analysis of two rice genotypes, IR 64 (IR64 and Nagina 22 (N22 under optimal (+N and chronic starvation (-N of nitrogen (N from 15-day-old root and shoot tissues. The two genotypes were found to be contrasting in their response to -N; IR64 root architecture and root dry weight remained almost equivalent to that under +N conditions, while N22 showed high foraging ability but a substantial reduction in biomass under -N. Similarly, the photosynthetic pigments showed a drastic reduction in N22 under low N, while IR64 was more resilient. Nitrate reductase showed significantly low specific activity under -N in both genotypes. Glutamate synthase (GOGAT and citrate synthase CS activity were highly reduced in N22 but not in IR64. Transcriptome analysis of these genotypes revealed nearly double the number of genes to be differentially expressed (DEGs in roots (1016 compared to shoots (571. The response of the two genotypes to N starvation was distinctly different reflecting their morphological/biochemical response with just two and eight common DEGs in the root and shoot tissues. There were a total of 385 nitrogen-responsive DEGs (106 in shoots and 279 in roots between the two genotypes. Fifty-two of the 89 DEGs identified as specific to N22 root tissues were also found to be differentially expressed between the two genotypes under -N. Most of these DEGs belonged to starch and chloroplast metabolism, followed by membrane and signaling proteins. Physical mapping of DEGs revealed 95 DEGs in roots and 76 in shoots to be present in quantitative trait loci (QTL known for NUE.

  19. Efficient alignment of pyrosequencing reads for re-sequencing applications

    Directory of Open Access Journals (Sweden)

    Russo Luis MS

    2011-05-01

    Full Text Available Abstract Background Over the past few years, new massively parallel DNA sequencing technologies have emerged. These platforms generate massive amounts of data per run, greatly reducing the cost of DNA sequencing. However, these techniques also raise important computational difficulties mostly due to the huge volume of data produced, but also because of some of their specific characteristics such as read length and sequencing errors. Among the most critical problems is that of efficiently and accurately mapping reads to a reference genome in the context of re-sequencing projects. Results We present an efficient method for the local alignment of pyrosequencing reads produced by the GS FLX (454 system against a reference sequence. Our approach explores the characteristics of the data in these re-sequencing applications and uses state of the art indexing techniques combined with a flexible seed-based approach, leading to a fast and accurate algorithm which needs very little user parameterization. An evaluation performed using real and simulated data shows that our proposed method outperforms a number of mainstream tools on the quantity and quality of successful alignments, as well as on the execution time. Conclusions The proposed methodology was implemented in a software tool called TAPyR--Tool for the Alignment of Pyrosequencing Reads--which is publicly available from http://www.tapyr.net.

  20. Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  1. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

    Directory of Open Access Journals (Sweden)

    Tongjit Thanchomnang

    Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  2. Evaluation of culture-based techniques and 454 pyrosequencing for the analysis of fungal diversity in potting media and organic fertilizers.

    Science.gov (United States)

    Al-Sadi, A M; Al-Mazroui, S S; Phillips, A J L

    2015-08-01

    Potting media and organic fertilizers (OFs) are commonly used in agricultural systems. However, there is a lack of studies on the efficiency of culture-based techniques in assessing the level of fungal diversity in these products. A study was conducted to investigate the efficiency of seven culture-based techniques and pyrosequencing for characterizing fungal diversity in potting media and OFs. Fungal diversity was evaluated using serial dilution, direct plating and baiting with carrot slices, potato slices, radish seeds, cucumber seeds and cucumber cotyledons. Identity of all the isolates was confirmed on the basis of the internal transcribed spacer region of the ribosomal RNA (ITS rRNA) sequence data. The direct plating technique was found to be superior over other culture-based techniques in the number of fungal species detected. It was also found to be simple and the least time consuming technique. Comparing the efficiency of direct plating with 454 pyrosequencing revealed that pyrosequencing detected 12 and 15 times more fungal species from potting media and OFs respectively. Analysis revealed that there were differences between potting media and OFs in the dominant phyla, classes, orders, families, genera and species detected. Zygomycota (52%) and Chytridiomycota (60%) were the predominant phyla in potting media and OFs respectively. The superiority of pyrosequencing over cultural methods could be related to the ability to detect obligate fungi, slow growing fungi and fungi that exist at low population densities. The evaluated methods in this study, especially direct plating and pyrosequencing, may be used as tools to help detect and reduce movement of unwanted fungi between countries and regions. © 2015 The Society for Applied Microbiology.

  3. [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

    Science.gov (United States)

    Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

    2012-05-01

    To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, PNested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, Pnested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

  4. Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses.

    Directory of Open Access Journals (Sweden)

    Patricia Moya

    Full Text Available The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones, and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to

  5. Bacterial flora-typing with targeted, chip-based Pyrosequencing

    Directory of Open Access Journals (Sweden)

    El-Sayed Yasser Y

    2007-11-01

    Full Text Available Abstract Background The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the Results We describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques. Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region. Conclusion This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora

  6. Challenges in Whole-Genome Annotation of Pyrosequenced Eukaryotic Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Alan; Grigoriev, Igor

    2009-04-17

    Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentous ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.

  7. Pyrosequencing of Plaque Microflora In Twin Children with Discordant Caries Phenotypes.

    Directory of Open Access Journals (Sweden)

    Meng Zhang

    Full Text Available Despite recent successes in the control of dental caries, the mechanism of caries development remains unclear. To investigate the causes of dental decay, especially in early childhood caries, the supragingival microflora composition of 20 twins with discordant caries phenotypes were analyzed using high-throughput pyrosequencing. In addition, the parents completed a lifestyle questionnaire. A total of 228,789 sequencing reads revealed 10 phyla, 84 genera, and 155 species of microflora, the relative abundances of these strains varied dramatically among the children, Comparative analysis between groups revealed that Veillonella, Corynebacterium and Actinomyces were presumed to be caries-related genera, Fusobacterium, Kingella and Leptotrichia were presumed to be healthy-related genus, yet this six genera were not statistically significant (P>0.05. Moreover, a cluster analysis revealed that the microbial composition of samples in the same group was often dissimilar but that the microbial composition observed in twins was usually similar. Although the genetic and environmental factors that strongly influence the microbial composition of dental caries remains unknown, we speculate that genetic factors primarily influence the individual's susceptibility to dental caries and that environmental factors primarily regulate the microbial composition of the dental plaque and the progression to caries. By using improved twins models and increased sample sizes, our study can be extended to analyze the specific genetic and environmental factors that affect the development of caries.

  8. Phylogenetic characterization of fecal microbial communities of dogs fed diets with or without supplemental dietary fiber using 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Ingmar S Middelbos

    Full Text Available BACKGROUND: Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. PRINCIPAL FINDINGS: Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5% diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23-40% of reads, Firmicutes (14-28% of reads and Bacteroidetes (31-34% of reads being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. CONCLUSIONS: Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases.

  9. Analysis of Bacterial Diversity During Acetic Acid Fermentation of Tianjin Duliu Aged Vinegar by 454 Pyrosequencing.

    Science.gov (United States)

    Peng, Qian; Yang, Yanping; Guo, Yanyun; Han, Ye

    2015-08-01

    The vinegar pei harbors complex bacterial communities. Prior studies revealing the bacterial diversity involved were mainly conducted by culture-dependent methods and PCR-DGGE. In this study, 454 pyrosequencing was used to investigate the bacterial communities in vinegar pei during the acetic acid fermentation (AAF) of Tianjin Duliu aged vinegar (TDAV). The results showed that there were 7 phyla and 24 families existing in the vinegar pei, with 2 phyla (Firmicutes, Protebacteria) and 4 families (Lactobacillaceae, Acetobacteracae, Enterobacteriaceae, Chloroplast) predominating. The genus-level identification revealed that 9 genera were the relatively stable, consistent components in different stages of AAF, including the most abundant genus Lactobacillus followed by Acetobacter and Serratia. Additionally, the bacterial community in the early fermentation stage was more complex than those in the later stages, indicating that the accumulation of organic acids provided an appropriate environment to filter unwanted bacteria and to accelerate the growth of required ones. This study provided basic information of bacterial patterns in vinegar pei and relevant changes during AAF of TDAV, and could be used as references in the following study on the implementation of starter culture as well as the improvement of AAF process.

  10. A polymeric micelle magnetic resonance imaging (MRI) contrast agent reveals blood-brain barrier (BBB) permeability for macromolecules in cerebral ischemia-reperfusion injury.

    Science.gov (United States)

    Shiraishi, Kouichi; Wang, Zuojun; Kokuryo, Daisuke; Aoki, Ichio; Yokoyama, Masayuki

    2017-05-10

    Blood-brain barrier (BBB) opening is a key phenomenon for understanding ischemia-reperfusion injuries that are directly linked to hemorrhagic transformation. The recombinant human tissue-type plasminogen activator (rtPA) increases the risk of symptomatic intracranial hemorrhages. Recent imaging technologies have advanced our understanding of pathological BBB disorders; however, an ongoing challenge in the pre-"rtPA treatment" stage is the task of developing a rigorous method for hemorrhage-risk assessments. Therefore, we examined a novel method for assessment of rtPA-extravasation through a hyper-permeable BBB. To examine the image diagnosis of rtPA-extravasation for a rat transient occlusion-reperfusion model, in this study we used a polymeric micelle MRI contrast-agent (Gd-micelles). Specifically, we used two MRI contrast agents at 1h after reperfusion. Gd-micelles provided very clear contrast images in 15.5±10.3% of the ischemic hemisphere at 30min after i.v. injection, whereas a classic gadolinium chelate MRI contrast agent provided no satisfactorily clear images. The obtained images indicate both the hyper-permeable BBB area for macromolecules and the distribution area of macromolecules in the ischemic hemisphere. Owing to their large molecular weight, Gd-micelles remained in the ischemic hemisphere through the hyper-permeable BBB. Our results indicate the feasibility of a novel clinical diagnosis for evaluating rtPA-related hemorrhage risks. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Magnetic Resonance Imaging with a Weak Albumin Binding Contrast Agent can Reveal Additional Endo leaks in Patients with an Enlarging Aneurysm after EVAR

    NARCIS (Netherlands)

    Habets, J.; Zandvoort, H. J. A.; Moll, F. L.; Bartels, L. W.; Vonken, E. P. A.; van Herwaarden, J. A.; Leiner, T.

    WHAT THIS PAPER ADDS In patients with enlarging aneurysms of unknown origin after endovascular aneurysm repair, magnetic resonance imaging (MRI) with a weak albumin binding contrast agent has additional diagnostic value for both the detection and determination of the origin of the endoleak.

  12. Pyrosequencing Based Microbial Community Analysis of Stabilized Mine Soils

    Science.gov (United States)

    Park, J. E.; Lee, B. T.; Son, A.

    2015-12-01

    Heavy metals leached from exhausted mines have been causing severe environmental problems in nearby soils and groundwater. Environmental mitigation was performed based on the heavy metal stabilization using Calcite and steel slag in Korea. Since the soil stabilization only temporarily immobilizes the contaminants to soil matrix, the potential risk of re-leaching heavy metal still exists. Therefore the follow-up management of stabilized soils and the corresponding evaluation methods are required to avoid the consequent contamination from the stabilized soils. In this study, microbial community analysis using pyrosequencing was performed for assessing the potential leaching of the stabilized soils. As a result of rarefaction curve and Chao1 and Shannon indices, the stabilized soil has shown lower richness and diversity as compared to non-contaminated negative control. At the phyla level, as the degree of contamination increases, most of phyla decreased with only exception of increased proteobacteria. Among proteobacteria, gamma-proteobacteria increased against the heavy metal contamination. At the species level, Methylobacter tundripaludum of gamma-proteobacteria showed the highest relative portion of microbial community, indicating that methanotrophs may play an important role in either solubilization or immobilization of heavy metals in stabilized soils.

  13. 454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

    Directory of Open Access Journals (Sweden)

    David J. Rooks

    2010-07-01

    Full Text Available Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785 were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%. The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases.

  14. The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing

    Czech Academy of Sciences Publication Activity Database

    Kostovčík, Martin; Bateman, C.C.; Kolařík, Miroslav; Stelinski, L.L.; Jordal, B.H.; Hulcr, J.

    2015-01-01

    Roč. 9, č. 1 (2015), s. 126-138 ISSN 1751-7362 Institutional support: RVO:61388971 Keywords : ambrosia symbiosis * pyrosequencing Subject RIV: EE - Microbiology, Virology Impact factor: 9.328, year: 2015

  15. Crossing the front: contrasting storm-forced dispersal dynamics revealed by biological, geological and genetic analysis of beach-cast kelp.

    Science.gov (United States)

    Waters, Jonathan M; King, Tania M; Fraser, Ceridwen I; Craw, Dave

    2018-03-01

    The subtropical front (STF) generally represents a substantial oceanographic barrier to dispersal between cold-sub-Antarctic and warm-temperate water masses. Recent studies have suggested that storm events can drastically influence marine dispersal and patterns. Here we analyse biological and geological dispersal driven by two major, contrasting storm events in southern New Zealand, 2017. We integrate biological and physical data to show that a severe southerly system in July 2017 disrupted this barrier by promoting movement of substantial numbers of southern sub-Antarctic Durvillaea kelp rafts across the STF, to make landfall in mainland NZ. By contrast, a less intense easterly storm (Cyclone Cook, April 2017) resulted in more moderate dispersal distances, with minimal dispersal between the sub-Antarctic and mainland New Zealand. These quantitative analyses of approximately 200 freshly beach-cast kelp specimens indicate that storm intensity and wind direction can strongly influence marine dispersal and landfall outcomes. © 2018 The Author(s).

  16. Pyrosequencing analysis of the microbial diversity of airag, khoormog and tarag, traditional fermented dairy products of mongolia.

    Science.gov (United States)

    Oki, Kaihei; Dugersuren, Jamyan; Demberel, Shirchin; Watanabe, Koichi

    2014-01-01

    Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare's milk), 5 Khoormog (fermented camel's milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 phyla. The genus Lactobacillus is a core bacterial component of Mongolian fermented milks, and Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus delbrueckii were the predominant species of lactic acid bacteria (LAB) in the Airag, Khoormog and Tarag samples, respectively. By using this pyrosequencing approach, we successfully detected most LAB species that have been isolated as well as seven LAB species that have not been found in our previous culture-based study. A subsequent analysis of the principal components of the samples revealed that L. delbrueckii, L. helveticus, L. kefiranofaciens and Streptococcus thermophilus were the main factors influencing the microbial diversity of these Mongolian traditional fermented dairy products and that this diversity correlated with the animal species from which the milk was sourced.

  17. Post-contractile BOLD contrast in skeletal muscle at 7 T reveals inter-individual heterogeneity in the physiological responses to muscle contraction.

    Science.gov (United States)

    Towse, Theodore F; Elder, Christopher P; Bush, Emily C; Klockenkemper, Samuel W; Bullock, Jared T; Dortch, Richard D; Damon, Bruce M

    2016-12-01

    Muscle blood oxygenation-level dependent (BOLD) contrast is greater in magnitude and potentially more influenced by extravascular BOLD mechanisms at 7 T than it is at lower field strengths. Muscle BOLD imaging of muscle contractions at 7 T could, therefore, provide greater or different contrast than at 3 T. The purpose of this study was to evaluate the feasibility of using BOLD imaging at 7 T to assess the physiological responses to in vivo muscle contractions. Thirteen subjects (four females) performed a series of isometric contractions of the calf muscles while being scanned in a Philips Achieva 7 T human imager. Following 2 s maximal isometric plantarflexion contractions, BOLD signal transients ranging from 0.3 to 7.0% of the pre-contraction signal intensity were observed in the soleus muscle. We observed considerable inter-subject variability in both the magnitude and time course of the muscle BOLD signal. A subset of subjects (n = 7) repeated the contraction protocol at two different repetition times (T R : 1000 and 2500 ms) to determine the potential of T 1 -related inflow effects on the magnitude of the post-contractile BOLD response. Consistent with previous reports, there was no difference in the magnitude of the responses for the two T R values (3.8 ± 0.9 versus 4.0 ± 0.6% for T R  = 1000 and 2500 ms, respectively; mean ± standard error). These results demonstrate that studies of the muscle BOLD responses to contractions are feasible at 7 T. Compared with studies at lower field strengths, post-contractile 7 T muscle BOLD contrast may afford greater insight into microvascular function and dysfunction. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Y-chromosome and mtDNA genetics reveal significant contrasts in affinities of modern Middle Eastern populations with European and African populations.

    Science.gov (United States)

    Badro, Danielle A; Douaihy, Bouchra; Haber, Marc; Youhanna, Sonia C; Salloum, Angélique; Ghassibe-Sabbagh, Michella; Johnsrud, Brian; Khazen, Georges; Matisoo-Smith, Elizabeth; Soria-Hernanz, David F; Wells, R Spencer; Tyler-Smith, Chris; Platt, Daniel E; Zalloua, Pierre A

    2013-01-01

    The Middle East was a funnel of human expansion out of Africa, a staging area for the Neolithic Agricultural Revolution, and the home to some of the earliest world empires. Post LGM expansions into the region and subsequent population movements created a striking genetic mosaic with distinct sex-based genetic differentiation. While prior studies have examined the mtDNA and Y-chromosome contrast in focal populations in the Middle East, none have undertaken a broad-spectrum survey including North and sub-Saharan Africa, Europe, and Middle Eastern populations. In this study 5,174 mtDNA and 4,658 Y-chromosome samples were investigated using PCA, MDS, mean-linkage clustering, AMOVA, and Fisher exact tests of F(ST)'s, R(ST)'s, and haplogroup frequencies. Geographic differentiation in affinities of Middle Eastern populations with Africa and Europe showed distinct contrasts between mtDNA and Y-chromosome data. Specifically, Lebanon's mtDNA shows a very strong association to Europe, while Yemen shows very strong affinity with Egypt and North and East Africa. Previous Y-chromosome results showed a Levantine coastal-inland contrast marked by J1 and J2, and a very strong North African component was evident throughout the Middle East. Neither of these patterns were observed in the mtDNA. While J2 has penetrated into Europe, the pattern of Y-chromosome diversity in Lebanon does not show the widespread affinities with Europe indicated by the mtDNA data. Lastly, while each population shows evidence of connections with expansions that now define the Middle East, Africa, and Europe, many of the populations in the Middle East show distinctive mtDNA and Y-haplogroup characteristics that indicate long standing settlement with relatively little impact from and movement into other populations.

  19. Temporal patterns of orchid mycorrhizal fungi in meadows and forests as revealed by 454 pyrosequencing

    Czech Academy of Sciences Publication Activity Database

    Oja, J.; Kohout, Petr; Tedersoo, L.; Kull, T.; Köljalg, U.

    2015-01-01

    Roč. 205, č. 4 (2015), s. 1608-1618 ISSN 0028-646X Institutional support: RVO:67985939 Keywords : mycorrhiza * community ecology * Orchideaceae Subject RIV: EF - Botanics Impact factor: 7.210, year: 2015

  20. Pyrosequencing reveals the predominance of Pseudomonadaceae in gut microbiome of a Gall Midge

    Science.gov (United States)

    Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654...

  1. Pyrosequencing data reveals tissue-specific expression of lineage-specific transcripts in chickpea

    OpenAIRE

    Garg, Rohini; Jain, Mukesh

    2011-01-01

    Chickpea is a very important crop legume plant, which provides a protein-rich supplement to cereal-based diets and has the ability to fix atmospheric nitrogen. Despite its economic importance, the functional genomic resources for chickpea are very limited. Recently, we reported the complete transcriptome of chickpea using next generation sequencing technologies. We analyzed the tissue-specific expression of chickpea transcripts based on RNA-seq data. In addition, we identified two sets of lin...

  2. Protist communities in a marine oxygen minimum zone off Costa Rica by 454 pyrosequencing

    Science.gov (United States)

    Jing, H.; Rocke, E.; Kong, L.; Xia, X.; Liu, H.; Landry, M. R.

    2015-08-01

    Marine planktonic protists, including microalgae and protistan grazers, are an important contributor to global primary production and carbon and mineral cycles, however, little is known about their population shifts along the oxic-anoxic gradient in the water column. We used 454 pyrosequencing of the 18S rRNA gene and gene transcripts to study the community composition of whole and active protists throughout a water column in the Costa Rica Dome, where a stable oxygen minimum zone (OMZ) exists at a depth of 400~700 m. A clear shift of protist composition from photosynthetic Dinoflagellates in the surface to potential parasitic Dinoflagellates and Ciliates in the deeper water was revealed along the vertical profile at both rRNA and rDNA levels. Those protist groups recovered only at the rDNA level represent either lysed aggregates sinking from the upper waters or potential hosts for parasitic groups. UPGMA clustering demonstrated that total and active protists in the anoxic core of OMZ (550 m) were distinct from those in other water depths. The reduced community diversity and presence of a parasitic/symbiotic trophic lifestyle in the OMZ, especially the anoxic core, suggests that OMZs can exert a selective pressure on protist communities. Such changes in community structure and a shift in trophic lifestyle could result in a modulation of the microbial loop and associated biogeochemical cycling.

  3. Contrast Materials

    Science.gov (United States)

    ... is mixed with water before administration liquid paste tablet When iodine-based and barium-sulfate contrast materials ... for patients with kidney failure or allergies to MRI and/or computed tomography (CT) contrast material. Microbubble ...

  4. Contrasting population-level responses to Pleistocene climatic oscillations in an alpine bat revealed by complete mitochondrial genomes and evolutionary history inference

    DEFF Research Database (Denmark)

    Alberdi, Antton; Gilbert, M. Thomas P; Razgour, Orly

    2015-01-01

    Aim: We used an integrative approach to reconstruct the evolutionary history of the alpine long-eared bat, Plecotus macrobullaris, to test whether the variable effects of Pleistocene climatic oscillations across geographical regions led to contrasting population-level demographic histories within...... a single species. Location: The Western Palaearctic. Methods: We sequenced the complete mitochondrial genomes of 57 individuals from across the distribution of the species. The analysis integrated ecological niche modelling (ENM), approximate Bayesian computation (ABC), measures of genetic diversity...... and Bayesian phylogenetic methods. Results: We identified two deep lineages: a western lineage, restricted to the Pyrenees and the Alps, and an eastern lineage, which expanded across the mountain ranges east of the Dinarides (Croatia). ENM projections of past conditions predicted that climatic suitability...

  5. Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection.

    Science.gov (United States)

    Hume, Michael E; Barbosa, Nei A; Dowd, Scot E; Sakomura, Nilva K; Nalian, Armen G; Martynova-Van Kley, Alexandra; Oviedo-Rondón, Edgar O

    2011-11-01

    A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in

  6. Contrasting strategies used by lichen microalgae to cope with desiccation-rehydration stress revealed by metabolite profiling and cell wall analysis.

    Science.gov (United States)

    Centeno, Danilo C; Hell, Aline F; Braga, Marcia R; Del Campo, Eva M; Casano, Leonardo M

    2016-05-01

    Most lichens in general, and their phycobionts in particular, are desiccation tolerant, but their mechanisms of desiccation tolerance (DT) remain obscure. The physiological responses and cell wall features of two putatively contrasting lichen-forming microalgae, Trebouxia sp. TR9 (TR9), isolated from Ramalina farinacea (adapted to frequent desiccation-rehydration cycles), and Coccomyxa solorina-saccatae (Csol), obtained from Solorina saccata (growing in usually humid limestone crevices, subjected to seasonal dry periods) was characterized. Microalgal cultures were desiccated under 25%-30% RH and then rehydrated. Under these conditions, RWC and ψw decreased faster and simultaneously during dehydration in Csol, whereas TR9 maintained its ψw until 70% RWC. The metabolic profile indicated that polyols played a key role in DT of both microalgae. However, TR9 constitutively accumulated higher amounts of polyols, whereas Csol induced the polyol synthesis under desiccation-rehydration. Csol also accumulated ascorbic acid, while TR9 synthesized protective raffinose-family oligosaccharides (RFOs) and increased its content of phenolics. Additionally, TR9 exhibited thicker and qualitatively different cell wall and extracellular polymeric layer compared with Csol, indicating higher water retention capability. The findings were consistent with the notion that lichen microalgae would have evolved distinct strategies to cope with desiccation-rehydration stress in correspondence with the water regime of their respective habitats. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Combined MR direct thrombus imaging and non-contrast magnetic resonance venography reveal the evolution of deep vein thrombosis: a feasibility study

    Energy Technology Data Exchange (ETDEWEB)

    Mendichovszky, I.A.; Lomas, D.J. [Addenbrooke' s Hospital, Department of Radiology, Cambridge (United Kingdom); University of Cambridge, Department of Radiology, Cambridge (United Kingdom); Priest, A.N.; Bowden, D.J.; Hunter, S.; Joubert, I.; Hilborne, S.; Graves, M.J. [Addenbrooke' s Hospital, Department of Radiology, Cambridge (United Kingdom); Baglin, T. [Addenbrooke' s Hospital, Department of Haematology, Cambridge (United Kingdom)

    2017-06-15

    Lower limb deep venous thrombosis (DVT) is a common condition with high morbidity and mortality. The aim of the study was to investigate the temporal evolution of the acute thrombus by magnetic resonance imaging (MRI) and its relationship to venous recanalization in patients with recurrent DVTs. Thirteen patients with newly diagnosed lower limb DVTs underwent MRI with non-contrast MR venography (NC-MRV) and MR direct thrombus imaging (MR-DTI), an inversion-recovery water-selective fast gradient-echo acquisition. Imaging was performed within 7 days of the acute thrombotic event, then at 3 and 6 months. By 3 months from the thrombotic event a third of the thrombi had resolved and by 6 months about half of the cases had resolved on the basis of vein recanalisation using NC-MRV. On the initial MR-DTI acute thrombus was clearly depicted by hyperintense signal, while the remaining thrombi were predominantly low signal at 3 and 6 months. Some residual thrombi contained small and fragmented persisting hyperintense areas at 3 months, clearing almost completely by 6 months. Our study suggests that synergistic venous assessment with combined NC-MRV and MR-DTI is able to distinguish acute venous thrombosis from the established (old) or evolving DVT detected by ultrasound. (orig.)

  8. Combined MR direct thrombus imaging and non-contrast magnetic resonance venography reveal the evolution of deep vein thrombosis: a feasibility study

    International Nuclear Information System (INIS)

    Mendichovszky, I.A.; Lomas, D.J.; Priest, A.N.; Bowden, D.J.; Hunter, S.; Joubert, I.; Hilborne, S.; Graves, M.J.; Baglin, T.

    2017-01-01

    Lower limb deep venous thrombosis (DVT) is a common condition with high morbidity and mortality. The aim of the study was to investigate the temporal evolution of the acute thrombus by magnetic resonance imaging (MRI) and its relationship to venous recanalization in patients with recurrent DVTs. Thirteen patients with newly diagnosed lower limb DVTs underwent MRI with non-contrast MR venography (NC-MRV) and MR direct thrombus imaging (MR-DTI), an inversion-recovery water-selective fast gradient-echo acquisition. Imaging was performed within 7 days of the acute thrombotic event, then at 3 and 6 months. By 3 months from the thrombotic event a third of the thrombi had resolved and by 6 months about half of the cases had resolved on the basis of vein recanalisation using NC-MRV. On the initial MR-DTI acute thrombus was clearly depicted by hyperintense signal, while the remaining thrombi were predominantly low signal at 3 and 6 months. Some residual thrombi contained small and fragmented persisting hyperintense areas at 3 months, clearing almost completely by 6 months. Our study suggests that synergistic venous assessment with combined NC-MRV and MR-DTI is able to distinguish acute venous thrombosis from the established (old) or evolving DVT detected by ultrasound. (orig.)

  9. Rapid and accurate pyrosequencing of angiosperm plastid genomes

    Science.gov (United States)

    Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

    2006-01-01

    Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid

  10. Rapid and accurate pyrosequencing of angiosperm plastid genomes

    Directory of Open Access Journals (Sweden)

    Farmerie William G

    2006-08-01

    Full Text Available Abstract Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20 System (454 Life Sciences Corporation, to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae and Platanus occidentalis (Platanaceae. Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy

  11. Comparative analysis of bacterial communities in a potato field as determined by pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Özgül Inceoğlu

    Full Text Available BACKGROUND: Plants selectively attract particular soil microorganisms, in particular consumers of root-excreted compounds. It is unclear to what extent cultivar type and/or growth stage affect this process. METHODOLOGY/PRINCIPAL FINDINGS: DNA-based pyrosequencing was used to characterize the structure of bacterial communities in a field cropped with potato. The rhizospheres of six cultivars denoted Aveka, Aventra, Karnico, Modena, Premiere and Desiree, at three growth stages (young, flowering and senescence were examined, in addition to corresponding bulk soils. Around 350,000 sequences were obtained (5,700 to 38,000 per sample. Across all samples, rank abundance distributions best fitted the power law model, which indicates a community composed of a few highly dominant species next to numerous rare species. Grouping of the sequences showed that members of the Actinobacteria, Alphaproteobacteria, next to as-yet-unclassified bacteria, dominated. Other groups that were consistently found, albeit at lower abundance, were Beta-, Gamma- and Deltaproteobacteria and Acidobacteria. Principal components analyses revealed that rhizosphere samples were significantly different from corresponding bulk soil in each growth stage. Furthermore, cultivar effects were found in the young plant stage, whereas these became insignificant in the flowering and senescence stages. Besides, an effect of time of season was observed for both rhizosphere and bulk soils. The analyzed rhizosphere samples of the potato cultivars were grouped into two groups, in accordance with the allocation of carbon to starch in their tubers, i.e. Aveka, Aventra and Karnico (high versus Premiere and Desiree (low and thus replicates per group were established. CONCLUSIONS: Across all potato cultivars, the young plant stages revealed cultivar-dependent bacterial community structures, which disappeared in the flowering and senescence stages. Furthermore, Pseudomonas, Beta-, Alpha- and

  12. Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

    Science.gov (United States)

    Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

    2016-03-01

    A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

  13. Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones ▿

    Science.gov (United States)

    Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.

    2009-01-01

    After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642

  14. Combined MR direct thrombus imaging and non-contrast magnetic resonance venography reveal the evolution of deep vein thrombosis: a feasibility study.

    Science.gov (United States)

    Mendichovszky, I A; Priest, A N; Bowden, D J; Hunter, S; Joubert, I; Hilborne, S; Graves, M J; Baglin, T; Lomas, D J

    2017-06-01

    Lower limb deep venous thrombosis (DVT) is a common condition with high morbidity and mortality. The aim of the study was to investigate the temporal evolution of the acute thrombus by magnetic resonance imaging (MRI) and its relationship to venous recanalization in patients with recurrent DVTs. Thirteen patients with newly diagnosed lower limb DVTs underwent MRI with non-contrast MR venography (NC-MRV) and MR direct thrombus imaging (MR-DTI), an inversion-recovery water-selective fast gradient-echo acquisition. Imaging was performed within 7 days of the acute thrombotic event, then at 3 and 6 months. By 3 months from the thrombotic event a third of the thrombi had resolved and by 6 months about half of the cases had resolved on the basis of vein recanalisation using NC-MRV. On the initial MR-DTI acute thrombus was clearly depicted by hyperintense signal, while the remaining thrombi were predominantly low signal at 3 and 6 months. Some residual thrombi contained small and fragmented persisting hyperintense areas at 3 months, clearing almost completely by 6 months. Our study suggests that synergistic venous assessment with combined NC-MRV and MR-DTI is able to distinguish acute venous thrombosis from the established (old) or evolving DVT detected by ultrasound. • MRI can distinguish between acute and evolving or chronic lower limb DVT • Two advanced MRI techniques can follow the evolution of lower limb DVT • MRI could be used to avoid an incorrect diagnosis of recurrent DVT • MRI could help avoid the risks and complications of lifelong anticoagulation therapy.

  15. Developmental morphology of cover crop species exhibit contrasting behaviour to changes in soil bulk density, revealed by X-ray computed tomography.

    Science.gov (United States)

    Burr-Hersey, Jasmine E; Mooney, Sacha J; Bengough, A Glyn; Mairhofer, Stefan; Ritz, Karl

    2017-01-01

    Plant roots growing through soil typically encounter considerable structural heterogeneity, and local variations in soil dry bulk density. The way the in situ architecture of root systems of different species respond to such heterogeneity is poorly understood due to challenges in visualising roots growing in soil. The objective of this study was to visualise and quantify the impact of abrupt changes in soil bulk density on the roots of three cover crop species with contrasting inherent root morphologies, viz. tillage radish (Raphanus sativus), vetch (Vicia sativa) and black oat (Avena strigosa). The species were grown in soil columns containing a two-layer compaction treatment featuring a 1.2 g cm-3 (uncompacted) zone overlaying a 1.4 g cm-3 (compacted) zone. Three-dimensional visualisations of the root architecture were generated via X-ray computed tomography, and an automated root-segmentation imaging algorithm. Three classes of behaviour were manifest as a result of roots encountering the compacted interface, directly related to the species. For radish, there was switch from a single tap-root to multiple perpendicular roots which penetrated the compacted zone, whilst for vetch primary roots were diverted more horizontally with limited lateral growth at less acute angles. Black oat roots penetrated the compacted zone with no apparent deviation. Smaller root volume, surface area and lateral growth were consistently observed in the compacted zone in comparison to the uncompacted zone across all species. The rapid transition in soil bulk density had a large effect on root morphology that differed greatly between species, with major implications for how these cover crops will modify and interact with soil structure.

  16. Contrasting Patterns of Genomic Diversity Reveal Accelerated Genetic Drift but Reduced Directional Selection on X-Chromosome in Wild and Domestic Sheep Species.

    Science.gov (United States)

    Chen, Ze-Hui; Zhang, Min; Lv, Feng-Hua; Ren, Xue; Li, Wen-Rong; Liu, Ming-Jun; Nam, Kiwoong; Bruford, Michael W; Li, Meng-Hua

    2018-04-01

    Analyses of genomic diversity along the X chromosome and of its correlation with autosomal diversity can facilitate understanding of evolutionary forces in shaping sex-linked genomic architecture. Strong selective sweeps and accelerated genetic drift on the X-chromosome have been inferred in primates and other model species, but no such insight has yet been gained in domestic animals compared with their wild relatives. Here, we analyzed X-chromosome variability in a large ovine data set, including a BeadChip array for 943 ewes from the world's sheep populations and 110 whole genomes of wild and domestic sheep. Analyzing whole-genome sequences, we observed a substantially reduced X-to-autosome diversity ratio (∼0.6) compared with the value expected under a neutral model (0.75). In particular, one large X-linked segment (43.05-79.25 Mb) was found to show extremely low diversity, most likely due to a high density of coding genes, featuring highly conserved regions. In general, we observed higher nucleotide diversity on the autosomes, but a flat diversity gradient in X-linked segments, as a function of increasing distance from the nearest genes, leading to a decreased X: autosome (X/A) diversity ratio and contrasting to the positive correlation detected in primates and other model animals. Our evidence suggests that accelerated genetic drift but reduced directional selection on X chromosome, as well as sex-biased demographic events, explain low X-chromosome diversity in sheep species. The distinct patterns of X-linked and X/A diversity we observed between Middle Eastern and non-Middle Eastern sheep populations can be explained by multiple migrations, selection, and admixture during the domestic sheep's recent postdomestication demographic expansion, coupled with natural selection for adaptation to new environments. In addition, we identify important novel genes involved in abnormal behavioral phenotypes, metabolism, and immunity, under selection on the sheep X-chromosome.

  17. Sorghum root-system classification in contrasting P environments reveals three main rooting types and root-architecture-related marker-trait associations.

    Science.gov (United States)

    Parra-Londono, Sebastian; Kavka, Mareike; Samans, Birgit; Snowdon, Rod; Wieckhorst, Silke; Uptmoor, Ralf

    2018-02-12

    Roots facilitate acquisition of macro- and micronutrients, which are crucial for plant productivity and anchorage in the soil. Phosphorus (P) is rapidly immobilized in the soil and hardly available for plants. Adaptation to P scarcity relies on changes in root morphology towards rooting systems well suited for topsoil foraging. Root-system architecture (RSA) defines the spatial organization of the network comprising primary, lateral and stem-derived roots and is important for adaptation to stress conditions. RSA phenotyping is a challenging task and essential for understanding root development. In this study, 19 traits describing RSA were analysed in a diversity panel comprising 194 sorghum genotypes, fingerprinted with a 90-k single-nucleotide polymorphism (SNP) array and grown under low and high P availability. Multivariate analysis was conducted and revealed three different RSA types: (1) a small root system; (2) a compact and bushy rooting type; and (3) an exploratory root system, which might benefit plant growth and development if water, nitrogen (N) or P availability is limited. While several genotypes displayed similar rooting types in different environments, others responded to P scarcity positively by developing more exploratory root systems, or negatively with root growth suppression. Genome-wide association studies revealed significant quantitative trait loci (P root-system development on chromosomes SBI-02 and SBI-03. Sorghum genotypes with a compact, bushy and shallow root system provide potential adaptation to P scarcity in the field by allowing thorough topsoil foraging, while genotypes with an exploratory root system may be advantageous if N or water is the limiting factor, although such genotypes showed highest P uptake levels under the artificial conditions of the present study. © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

    Science.gov (United States)

    Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

    2014-12-01

    Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

  19. ESA-MERIS 10-Year Mission Reveals Contrasting Phytoplankton Bloom Dynamics in Two Tropical Regions of Northern Australia

    Directory of Open Access Journals (Sweden)

    David Blondeau-Patissier

    2014-04-01

    Full Text Available The spatial and temporal variability of phytoplankton blooms was investigated in two tropical coastal regions of northern Australia using the MEdium Resolution Imaging Spectrometer (MERIS full mission (2002–2012 reduced resolution dataset. Satellite-derived proxies for phytoplankton (Chlorophyll-a (Chl, Fluorescence Line Height (FLH, Maximum Chlorophyll Index (MCI and suspended sediment (Total Suspended Matter (TSM were jointly analyzed for two clusters of the Great Barrier Reef Wet tropics (GBRW; 15°–19.5°S; Queensland and the Van Diemen Gulf (VDG; 9°–13°S; Northern Territory. The analysis of time-series and Hovmöller diagrams of the four MERIS products provided a unique perspective on the processes linking phytoplankton blooms and river runoff, or resuspension, across spatio-temporal scales. Both regions are characterized by a complex oceanography and seasonal inflows of sediment, freshwater and nutrients during the tropical wet season months (November to April. The GBRW is characterized by a great variability in water clarity (Secchi depth 0–25 m. A long history of agricultural land use has led to a large increase in the seasonal discharge of sediments and nutrients, triggering seasonal phytoplankton blooms (>0.4 mg∙m−3 between January and April. In contrast, the VDG is a poorly flushed, turbid (Secchi depth <5 m environment with strong tidal-energy (4–8 m and very limited land use. Phytoplankton blooms here were found to have higher Chl concentrations (>1.0 mg∙m−3 than in the GBRW, occurring up to twice a year between January and April. Over the 10-year MERIS mission, a weak decline in Chl and TSM was observed for the VDG (Sen slope: −2.85%/decade, τ = −0.32 and −3.57%/decade, τ = −0.24; p 0.05, while no significant trend in those two satellite products was observed in the GBRW. Cyanobacteria surface algal blooms occur in both regions between August and October. The MCI and FLH products were found to

  20. Characteristics of 454 pyrosequencing data--enabling realistic simulation with flowsim.

    Science.gov (United States)

    Balzer, Susanne; Malde, Ketil; Lanzén, Anders; Sharma, Animesh; Jonassen, Inge

    2010-09-15

    The commercial launch of 454 pyrosequencing in 2005 was a milestone in genome sequencing in terms of performance and cost. Throughout the three available releases, average read lengths have increased to approximately 500 base pairs and are thus approaching read lengths obtained from traditional Sanger sequencing. Study design of sequencing projects would benefit from being able to simulate experiments. We explore 454 raw data to investigate its characteristics and derive empirical distributions for the flow values generated by pyrosequencing. Based on our findings, we implement Flowsim, a simulator that generates realistic pyrosequencing data files of arbitrary size from a given set of input DNA sequences. We finally use our simulator to examine the impact of sequence lengths on the results of concrete whole-genome assemblies, and we suggest its use in planning of sequencing projects, benchmarking of assembly methods and other fields. Flowsim is freely available under the General Public License from http://blog.malde.org/index.php/flowsim/.

  1. Analysis of cagA in Helicobacter pylori strains from Colombian populations with contrasting gastric cancer risk reveals a biomarker for disease severity

    Science.gov (United States)

    Loh, John T.; Shaffer, Carrie L.; Piazuelo, M. Blanca; Bravo, Luis E.; McClain, Mark S.; Correa, Pelayo; Cover, Timothy L.

    2011-01-01

    BACKGROUND Helicobacter pylori infection is a risk factor for the development of gastric cancer, and the bacterial oncoprotein CagA contributes to gastric carcinogenesis. METHODS We analyzed H. pylori isolates from persons in Colombia and observed that there was marked variation among strains in levels of CagA expression. To elucidate the basis for this variation, we analyzed sequences upstream from the CagA translational initiation site in each strain. RESULTS A DNA motif (AATAAGATA) upstream of the translational initiation site of CagA was associated with high levels of CagA expression. Experimental studies showed that this motif was necessary but not sufficient for high-level CagA expression. H. pylori strains from a region of Colombia with high gastric cancer rates expressed higher levels of CagA than did strains from a region with lower gastric cancer rates, and Colombian strains of European phylogeographic origin expressed higher levels of CagA than did strains of African origin. Histopathological analysis of gastric biopsy specimens revealed that strains expressing high levels of CagA or containing the AATAAGATA motif were associated with more advanced precancerous lesions than those found in persons infected with strains expressing low levels of CagA or lacking the AATAAGATA motif. CONCLUSIONS CagA expression varies greatly among H. pylori strains. The DNA motif identified in this study is associated with high levels of CagA expression, and may be a useful biomarker to predict gastric cancer risk. IMPACT These findings help to explain why some persons infected with cagA-positive H. pylori develop gastric cancer and others do not. PMID:21859954

  2. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

    Directory of Open Access Journals (Sweden)

    Raul A Cernadas

    2014-02-01

    Full Text Available Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo, which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

  3. Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone.

    Science.gov (United States)

    Meier, Samuel M; Muqaku, Besnik; Ullmann, Ronald; Bileck, Andrea; Kreutz, Dominique; Mader, Johanna C; Knasmüller, Siegfried; Gerner, Christopher

    2015-01-01

    Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac

  4. Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine.

    Directory of Open Access Journals (Sweden)

    Marcus J Claesson

    Full Text Available BACKGROUND: Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. METHODS AND FINDINGS: Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400-1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. CONCLUSIONS: The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate

  5. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

    Directory of Open Access Journals (Sweden)

    Wolcott Benjamin M

    2008-03-01

    Full Text Available Abstract Background Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D, venous leg ulcers (V, and pressure ulcers (P. Results There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. Conclusion If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in

  6. Prerequisites for Amplicon Pyrosequencing of Microbial Methanol Utilizers in the Environment

    Directory of Open Access Journals (Sweden)

    Steffen eKolb

    2013-09-01

    Full Text Available The commercial availability of next generation sequencing (NGS technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of PQQ-dependent methanol dehydrogenase (mxaF has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2, of the NAD-dependent MDH (mdh, of the methanol oxidoreductase of Actinobacteria (mdo, of the fungal FAD-dependent alcohol oxidase (mod1, mod2, and homologues, and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle.

  7. A sensitive issue: Pyrosequencing as a valuable forensic SNP typing platform

    DEFF Research Database (Denmark)

    Harrison, C.; Musgrave-Brown, E.; Bender, K.

    2006-01-01

    Analysing minute amounts of DNA is a routine challenge in forensics in part due to the poor sensitivity of an instrument and its inability to detect results from forensic samples. In this study, the sensitivity of the Pyrosequencing method is investigated using varying concentrations of DNA and f...

  8. Dialysis and contrast media

    International Nuclear Information System (INIS)

    Morcos, Sameh K.; Thomsen, Henrik S.; Webb, Judith A.W.

    2002-01-01

    In a previous survey we revealed uncertainty among responders about (a) whether or not to perform hemodialysis in patients with severely reduced renal function who had received contrast medium; and (b) when to perform hemodialysis in patients on regular treatment with hemodialysis or continuous ambulatory dialysis who received contrast medium. Therefore, the Contrast Media Safety Committee of The European Society of Urogenital Radiology decided to review the literature and to issue guidelines. The committee performed a Medline search. Based on this, a report and guidelines were prepared. The report was discussed at the Ninth European Symposium on Urogenital Radiology in Genoa, Italy. Hemodialysis and peritoneal dialysis safely remove both iodinated and gadolinium-based contrast media. The effectiveness of hemodialysis depends on many factors including blood and dialysate flow rate, permeability of dialysis membrane, duration of hemodialysis and molecular size, protein binding, hydrophilicity, and electrical charge of the contrast medium. Generally, several hemodialysis sessions are needed to removal all contrast medium, whereas it takes 3 weeks for continuous ambulatory dialysis to remove the agent completely. There is no need to schedule the dialysis in relation to the injection of iodinated or MR contrast media or the injection of contrast agent in relation to the dialysis program. Hemodialysis does not protect poorly functioning kidneys against contrast-medium-induced nephrotoxicity. Simple guidelines are given. (orig.)

  9. Contrast media

    International Nuclear Information System (INIS)

    Decazes, Ph.

    2004-01-01

    The Guerbet firm, which holds 69% of the capital on the contrast media for medical imagery, could sale about 20% of this capital in order to accelerate its development in the United States, one of its next market with the Japan. (O.M.)

  10. The use of genus-specific amplicon pyrosequencing to assess phytophthora species diversity using eDNA from soil and water in Northern Spain.

    Science.gov (United States)

    Català, Santiago; Pérez-Sierra, Ana; Abad-Campos, Paloma

    2015-01-01

    Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

  11. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and and pyrosequencing for the characterisation of the caries-associated microbiome

    Directory of Open Access Journals (Sweden)

    Kathrin eSchulze-Schweifing

    2014-11-01

    Full Text Available Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture.

  12. Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach.

    Science.gov (United States)

    Takeshita, Toru; Yasui, Masaki; Shibata, Yukie; Furuta, Michiko; Saeki, Yoji; Eshima, Nobuoki; Yamashita, Yoshihisa

    2015-01-30

    Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience.

  13. Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing

    Directory of Open Access Journals (Sweden)

    Sanz Libia

    2011-05-01

    Full Text Available Abstract Background A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. Results The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27% were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements and class II (DNA transposons mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large

  14. Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data

    DEFF Research Database (Denmark)

    Rask, Thomas Salhøj; Petersen, Bent; Chen, Donald S.

    2016-01-01

    . The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data....... This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene...... family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon...

  15. Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

    Science.gov (United States)

    Song, Qinxin; Wei, Guijiang; Zhou, Guohua

    2014-07-01

    A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. The microbiome of Brazilian mangrove sediments as revealed by metagenomics

    NARCIS (Netherlands)

    Andreote, Fernando Dini; Jiménez Avella, Diego; Chaves, Diego; Dias, Armando Cavalcante Franco; Luvizotto, Danice Mazzer; Dini-Andreote, Francisco; Fasanella, Cristiane Cipola; Lopez, Maryeimy Varon; Baena, Sandra; Taketani, Rodrigo Gouvêa; de Melo, Itamar Soares

    2012-01-01

    Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct

  17. Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

    Science.gov (United States)

    Shcherbik, Svetlana V; Pearce, Nicholas C; Levine, Marnie L; Klimov, Alexander I; Villanueva, Julie M; Bousse, Tatiana L

    2014-01-01

    Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

  18. Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

    Directory of Open Access Journals (Sweden)

    Svetlana V Shcherbik

    Full Text Available BACKGROUND: Live attenuated influenza vaccine viruses (LAIVs can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV and a seasonal wild-type (wt virus. The vaccine candidates contain hemagglutinin (HA and neuraminidase (NA genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. METHODOLOGY/PRINCIPAL FINDINGS: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2 (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012 or influenza A (H7N9 (A/Anhui/1/2013 wt viruses with the MDV A/Leningrad/134/17/57(H2N2. Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

  19. Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchus labrax) fed functional diets.

    OpenAIRE

    Carda Diéguez, Miguel; Mira, Alex; Fouz Rodríguez, Belén

    2014-01-01

    The routine use of chemotherapy to control bacterial diseases in aquatic populations has resulted in the development and spread of antibiotic resistance. The inclusion of immunostimulants in fish diets (functional diets) is one of the main strategies to solve this threat. This study aimed to analyse the intestinal microbiota of cultured European sea bass (Dicentrarchus labrax) fed two functional diets applying pyrosequencing of PCR-amplified 16S rRNA gene. Quality-filtered reads were assigned...

  20. Development and characterization of microsatellite markers from the humivorous termite Cavitermes tuberosus (Isoptera: Termitinae) using pyrosequencing technology

    Czech Academy of Sciences Publication Activity Database

    Fournier, D.; Hanus, Robert; Roisin, Y.

    2015-01-01

    Roč. 7, č. 2 (2015), s. 521-524 ISSN 1877-7252 Institutional support: RVO:61388963 Keywords : Cavitermes tuberosus * termite * microsatellite * pyrosequencing * population genetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.446, year: 2015

  1. Pyrosequencing analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases.

    Science.gov (United States)

    Hou, X-L; Cao, Q-Y; Jia, H-Y; Chen, Z

    2008-07-01

    Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long ( approximately1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 10(4) CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.

  2. Microbial analysis in primary and persistent endodontic infections by using pyrosequencing.

    Science.gov (United States)

    Hong, Bo-Young; Lee, Tae-Kwon; Lim, Sang-Min; Chang, Seok Woo; Park, Joonhong; Han, Seung Hyun; Zhu, Qiang; Safavi, Kamran E; Fouad, Ashraf F; Kum, Kee Yeon

    2013-09-01

    The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. The Effect of Long-Term Continuous Cropping of Black Pepper on Soil Bacterial Communities as Determined by 454 Pyrosequencing

    Science.gov (United States)

    Xiong, Wu; Li, Zhigang; Liu, Hongjun; Xue, Chao; Zhang, Ruifu; Wu, Huasong; Li, Rong; Shen, Qirong

    2015-01-01

    In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system. PMID:26317364

  4. Bacterial communities in the gut and reproductive organs of Bactrocera minax (Diptera: Tephritidae) based on 454 pyrosequencing.

    Science.gov (United States)

    Wang, Ailin; Yao, Zhichao; Zheng, Weiwei; Zhang, Hongyu

    2014-01-01

    The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%-95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.

  5. Bacterial communities in the gut and reproductive organs of Bactrocera minax (Diptera: Tephritidae based on 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Ailin Wang

    Full Text Available The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs. A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%-95%. Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices and community structure (PCA analysis varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.

  6. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota.

    Directory of Open Access Journals (Sweden)

    Lance B Price

    Full Text Available BACKGROUND: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007 and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. CONCLUSIONS/SIGNIFICANCE: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.

  7. Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing.

    Science.gov (United States)

    Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie

    2010-10-01

    Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

  8. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

    Science.gov (United States)

    Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

    2013-08-01

    The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  9. Characterization of microsatellite loci from two-spotted octopus Octopus bimaculatus Verrill 1883 from pyrosequencing reads

    Science.gov (United States)

    Domínguez-Contreras, J. F.; Munguía-Vega, A.; Ceballos-Vázquez, B. P.; Arellano-Martínez, M.; Culver, Melanie

    2014-01-01

    We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum, respectively.

  10. Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

    Science.gov (United States)

    Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

    2004-03-01

    We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

  11. Diversity of thermophilic bacteria in raw, pasteurized and selectively-cultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing.

    Science.gov (United States)

    Delgado, Susana; Rachid, Caio T C C; Fernández, Elena; Rychlik, Tomasz; Alegría, Angel; Peixoto, Raquel S; Mayo, Baltasar

    2013-10-01

    Thermophilic lactic acid bacteria (LAB) species, such as Streptococcus thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus, enjoy worldwide economic importance as dairy starters. To assess the diversity of thermophilic bacteria in milk, milk samples were enriched in thermophilic organisms through a stepwise procedure which included pasteurization of milk at 63 °C for 30 min (PM samples) and pasteurization followed by incubation at 42 °C for 24 h (IPM samples). The microbial composition of these samples was analyzed by culture-dependent (at 42 °C) and culture-independent (PCR-DGGE and pyrosequencing of 16S rRNA gene amplicons) microbial techniques. The results were then compared to those obtained for their corresponding starting raw milk counterparts (RM samples). Twenty different species were scored by culturing among 352 isolates purified from the counting plates and identified by molecular methods. Mesophilic LAB species (Lactococcus lactis, Lactococcus garvieae) were dominant (87% of the isolates) among the RM samples. However, S. thermophilus and Lb. delbrueckii were found to be the dominant recoverable organisms in both PM and IPM samples. The DGGE profiles of RM and PM samples were found to be very similar; the most prominent bands belonging to Lactococcus, Leuconostoc and Streptococcus species. In contrast, just three DGGE bands were obtained for IPM samples, two of which were assigned to S. thermophilus. The pyrosequencing results scored 95 operational taxonomic units (OTUs) at 3% sequence divergence in an RM sample, while only 13 were encountered in two IPM samples. This technique identified Leuconostoc citreum as the dominant microorganism in the RM sample, while S. thermophilus constituted more than 98% of the reads in the IPM samples. The procedure followed in this study allowed to estimate the bacterial diversity in milk and afford a suitable strategy for the isolation of new thermophilic LAB strains, among which adequate

  12. A sweetpotato gene index established by de novo assembly of pyrosequencing and Sanger sequences and mining for gene-based microsatellite markers

    Directory of Open Access Journals (Sweden)

    Solis Julio

    2010-10-01

    Full Text Available Abstract Background Sweetpotato (Ipomoea batatas (L. Lam., a hexaploid outcrossing crop, is an important staple and food security crop in developing countries in Africa and Asia. The availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. Previously existing sequence data were restricted to around 22,000 expressed sequence tag (EST sequences and ~ 1,500 GenBank sequences. We have used 454 pyrosequencing to augment the available gene sequence information to enhance functional genomics and marker design for this plant species. Results Two quarter 454 pyrosequencing runs used two normalized cDNA collections from stems and leaves from drought-stressed sweetpotato clone Tanzania and yielded 524,209 reads, which were assembled together with 22,094 publically available expressed sequence tags into 31,685 sets of overlapping DNA segments and 34,733 unassembled sequences. Blastx comparisons with the UniRef100 database allowed annotation of 23,957 contigs and 15,342 singletons resulting in 24,657 putatively unique genes. Further, 27,119 sequences had no match to protein sequences of UniRef100database. On the basis of this gene index, we have identified 1,661 gene-based microsatellite sequences, of which 223 were selected for testing and 195 were successfully amplified in a test panel of 6 hexaploid (I. batatas and 2 diploid (I. trifida accessions. Conclusions The sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous EST assemblies. A searchable version of the gene index, including a blastn function, is available at http://www.cipotato.org/sweetpotato_gene_index.

  13. Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms.

    Science.gov (United States)

    Brown, David S; Jarman, Simon N; Symondson, William O C

    2012-03-01

    Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms (N=80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles. © 2011 Blackwell Publishing Ltd.

  14. Comparisons of the fungal and protistan communities among different marine sponge holobionts by pyrosequencing.

    Science.gov (United States)

    He, Liming; Liu, Fang; Karuppiah, Valliappan; Ren, Yi; Li, Zhiyong

    2014-05-01

    To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.

  15. Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Yi-Mo Deng

    Full Text Available BACKGROUND: Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. METHODOLOGY/PRINCIPAL FINDINGS: A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. CONCLUSIONS/SIGNIFICANCE: In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

  16. Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

    Science.gov (United States)

    Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G

    2011-01-01

    Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

  17. Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.

    Science.gov (United States)

    Mark, Kristiina; Cornejo, Carolina; Keller, Christine; Flück, Daniela; Scheidegger, Christoph

    2016-09-01

    Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

  18. Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

    Science.gov (United States)

    Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H

    2010-02-01

    The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

  19. Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing® technology

    Directory of Open Access Journals (Sweden)

    Gallagher Margaret L

    2009-08-01

    Full Text Available Abstract Background Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1–2 are derived from CYP2A7, and exons 3–9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. Methods A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow. Results Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics. Conclusion This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.

  20. Field efficacy of four anthelmintics and confirmation of drug-resistant nematodes by controlled efficacy test and pyrosequencing on a sheep and goat farm in Denmark.

    Science.gov (United States)

    Peña-Espinoza, Miguel; Thamsborg, Stig M; Demeler, Janina; Enemark, Heidi L

    2014-12-15

    We describe a case of anthelmintic resistance on one of the largest organic small ruminant farms in Denmark. The flock was established in 2007 by purchase of animals from other Danish farms and had history of clinical parasitism, high mortality of young stock and anthelmintic treatment failure. In October 2011, 40 lambs and 40 kids were selected for a faecal egg count reduction test (FECRT) with fenbendazole (FBZ), ivermectin (IVM), moxidectin (MOX) and levamisole (LEV). Lambs were treated with the recommended sheep dose of each product while kids received the sheep dose of IVM, 1.5× sheep dose of MOX and 2× sheep dose of FBZ and LEV. Untreated lambs and kids were also included and three methods for calculating faecal egg count (FEC) reduction were compared. In a subsequent investigation, a controlled efficacy test (CET) with FBZ and IVM was performed in lambs infected with Haemonchus contortus and Trichostrongylus colubriformis isolated from adult goats on the farm. Recovered specimens of H. contortus were subjected to pyrosequencing for detection of single nucleotide polymorphisms (SNPs) related to benzimidazole (BZ) resistance. During the FECRT, FECs in untreated lambs dropped significantly by 47%. No FEC reduction was detected in untreated kids. After FBZ treatments, FEC reductions in lambs and kids ranged from 15 to 54% and 49-56%, respectively, according to the different calculation methods. Post IVM treatments, FEC reductions in lambs and kids varied between 71-90% and 81-83%, correspondingly. LEV and MOX reduced FECs by 98-100% in both species. In the CET, FBZ reduced H. contortus worm counts by 52-56% and no reduction in T. colubriformis counts were detected after treatment. IVM eliminated 100% of H. contortus and reduced T. colubriformis counts by 84-92%, according to different calculation methods. Pyrosequencing of isolated H. contortus revealed increased frequencies of the BZ resistance-related SNP in codon 200 of the β-tubulin isotype 1 gene

  1. Pyrosequencing revealed shifts of prokaryotic communities between healthy and disease-like tissues of the Red Sea sponge Crella cyathophora

    KAUST Repository

    Gao, Zhao-Ming; Wang, Yong; Tian, Ren-Mao; Lee, On On; Wong, Yue Him; Batang, Zenon B.; Al-Suwailem, Abdulaziz M.; Lafi, Feras Fawzi; Bajic, Vladimir B.; Qian, Pei-Yuan

    2015-01-01

    Sponge diseases have been widely reported, yet the causal factors and major pathogenic microbes remain elusive. In this study, two individuals of the sponge Crella cyathophora in total that showed similar disease-like characteristics were collected

  2. Pyrosequencing Reveals the Microbial Communities in the Red Sea Sponge Carteriospongia foliascens and Their Impressive Shifts in Abnormal Tissues

    KAUST Repository

    Gao, Zhaoming; Wang, Yong; Lee, Onon; Tian, Renmao; Wong, Yuehim; Bougouffa, Salim; Batang, Zenon B.; Al-Suwailem, Abdulaziz M.; Lafi, Feras Fawzi; Bajic, Vladimir B.; Qian, Peiyuan

    2014-01-01

    Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from

  3. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    Science.gov (United States)

    Daniel L. Lindner; Tor Carlsen; Henrik Nilsson; Marie Davey; Trond Schumacher; Havard. Kauserud

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular...

  4. Fungal root symbionts of the seagrass Posidonia oceanica in the central Adriatic Sea revealed by microscopy, culturing and 454-pyrosequencing

    Czech Academy of Sciences Publication Activity Database

    Vohník, Martin; Borovec, Ondřej; Župan, I.; Kolařík, Miroslav; Sudová, Radka

    2017-01-01

    Roč. 583, November 16 (2017), s. 107-120 ISSN 0171-8630 Institutional support: RVO:67985939 ; RVO:61388971 Keywords : seagrass es * Posidonia oceanica * root mycobionts Subject RIV: EH - Ecology, Behaviour; EE - Microbiology, Virology (MBU-M) OBOR OECD: Ecology; Microbiology (MBU-M) Impact factor: 2.292, year: 2016

  5. New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing

    Czech Academy of Sciences Publication Activity Database

    Helguera, M.; Rivarola, M.; Clavijo, B.; Martis, M.M.; Vanzetti, L.S.; Gonzalez, S.; Garbus, I.; LeRoy, P.; Šimková, Hana; Valárik, Miroslav; Caccamo, M.; Doležel, Jaroslav; Mayer, K. F. X.; Feuillet, C.; Tranquilli, G.; Paniego, N.; Echenique, V.

    2015-01-01

    Roč. 233, APR 2015 (2015), s. 200-212 ISSN 0168-9452 R&D Projects: GA ČR GBP501/12/G090; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Chromosome 4D survey sequence * Gene annotation * Gene content Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.362, year: 2015

  6. Bacterial communities in chitin-amended soil as revealed by 16S rRNA gene based pyrosequencing

    NARCIS (Netherlands)

    Cretoiu, M.S.; Kielak, A.M.; Schluter, A.; van Elsas, J.D.

    2014-01-01

    Chitin and its derivatives are natural biopolymers that are often used as compounds for the control of soil-borne plant pathogens. In spite of recent advances in agricultural practices involving chitin amendments, the microbial communities in chitin-amended soils remain poorly known. The objectives

  7. Bacterial communities in chitin-amended soil as revealed by 16S rRNA gene based pyrosequencing

    NARCIS (Netherlands)

    Cretoiu, Mariana Silvia; Kielak, Anna Maria; Schluter, Andreas; van Elsas, Jan Dirk

    Chitin and its derivatives are natural biopolymers that are often used as compounds for the control of soilborne plant pathogens. In spite of recent advances in agricultural practices involving chitin amendments, the microbial communities in chitin-amended soils remain poorly known. The objectives

  8. Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

    Science.gov (United States)

    Sohn, Sung-Hwa; Kim, Nayoung; Jo, Hyun Jin; Kim, Jaeyeon; Park, Ji Hyun; Nam, Ryoung Hee; Seok, Yeong-Jae; Kim, Yeon-Ran; Lee, Dong Ho

    2017-06-01

    Gastric microbiota along with Helicobacter pylori (HP) plays a key role in gastric disease. The aim of our study is to investigate the difference of human gastric microbiota between antrum and body according to disease (control vs. gastric cancer) and HP status. Each antrum and body biopsy was collected from 12 subjects at Seoul National University Bundang Hospital. Gastric microbiota was analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Twelve subjects consisted of HP-negative control (n = 2), HP-negative cancer (n = 2), HP-positive control (n = 3), and HP-positive cancer (n = 5). The analysis was focused on non-HP urease-producing bacteria (UB) and non-HP nitrosating or nitroreducing bacteria (NB) between antrum and body. Gastric body samples showed higher diversity compared to gastric antrum mucosa samples but there was no significant difference. The mean of operational taxonomic units was higher in HP(-) cancer than HP(+) cancer (antrum, 273.5 vs. 228.2, P = 0.439; body, 585.5 vs. 183.2, P = 0.053). The number of non-HP UB and non-HP NB was higher in HP(-) cancer groups than the others. These differences were more pronounced in the body ( P = 0.051 and P = 0.081, respectively). Analysis of overlap of non-HP UB and non-HP NB revealed the higher composition of Streptococcus pseudopneumoniae, S. parasanguinis , and S. oralis in HP(-) cancer groups than the others, only in the body ( P = 0.030) but not in the antrum ( P = 0.123). Higher diversity and higher composition of S. pseudopneumoniae, S. parasanguinis , and S. oralis in HP(-) cancer group than the other groups in the body suggest that analysis of microbiota from body mucosa could be beneficial to identify a role of non-HP bacteria in the gastric carcinogenesis.

  9. Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

    Directory of Open Access Journals (Sweden)

    Bonten Marc JM

    2010-04-01

    Full Text Available Abstract Background The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. Results We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI, which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. Conclusions Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.

  10. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing

    DEFF Research Database (Denmark)

    Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist

    2011-01-01

    ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus, which were encountered in some cheese samples at low percentages compared with the total bacterial populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient method...

  11. De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing.

    Science.gov (United States)

    Natarajan, Purushothaman; Parani, Madasamy

    2011-04-15

    Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of

  12. Analysis of the scallop microbiota by means of 16S rRNA gene pyrosequencing

    Directory of Open Access Journals (Sweden)

    Alex Mira

    2014-06-01

    Pyrosequencing of the samples resulted in a total of 18520 sequences (3000 per sample, approximately with an average length of 325 bp (base pairs. The taxonomic assignment of sequences allowed the identification to the genus level, being observed a large bacterial diversity with over 110 genera. The most prevalent genera in the samples were Hydrotalea, Acinetobacter, Delftia, Sediminibacter and Pseudomonas, among others. Differences in the microbial communities were observed among the samples, and the PCoA analysis allowed their separation by means on their gender and if they proceed from sampling before or after the spawning. Nevertheless, the rarefaction curves obtained for each sample failed to reach a saturation phase, indicating that more sequencing effort would be necessary.

  13. Development of an ELA-DRA gene typing method based on pyrosequencing technology.

    Science.gov (United States)

    Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G

    2008-11-01

    The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

  14. Analysis of soil fungal communities by amplicon pyrosequencing: current approaches to data analysis and the introduction of the pipeline SEED

    Czech Academy of Sciences Publication Activity Database

    Větrovský, Tomáš; Baldrian, Petr

    2013-01-01

    Roč. 49, č. 8 (2013), s. 1027-1037 ISSN 0178-2762 R&D Projects: GA MŠk LD12050; GA MŠk LD12048; GA ČR GAP504/12/0709 Institutional support: RVO:61388971 Keywords : Fungal community * Internal transcribed spacer * Pyrosequencing pipeline Subject RIV: EE - Microbiology, Virology Impact factor: 3.396, year: 2013

  15. Pyrosequencing Analysis of Norovirus Genogroup II Distribution in Sewage and Oysters: First Detection of GII.17 Kawasaki 2014 in Oysters.

    Science.gov (United States)

    Pu, Jian; Kazama, Shinobu; Miura, Takayuki; Azraini, Nabila Dhyan; Konta, Yoshimitsu; Ito, Hiroaki; Ueki, You; Cahyaningrum, Ermaya Eka; Omura, Tatsuo; Watanabe, Toru

    2016-12-01

    Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.

  16. Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

    Science.gov (United States)

    Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia

    2013-01-01

    There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336

  17. Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units.

    Science.gov (United States)

    Vettraino, A M; Bonants, P; Tomassini, A; Bruni, N; Vannini, A

    2012-11-01

    To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%) Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  18. Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

    Science.gov (United States)

    Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

    2015-11-20

    HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

  19. Pyrosequencing based assessment of bacterial diversity in Turkish Rhipicephalus annulatus and Dermacentor marginatus ticks (Acari: Ixodidae).

    Science.gov (United States)

    Tekin, Saban; Dowd, Scot E; Davinic, Marko; Bursali, Ahmet; Keskin, Adem

    2017-03-01

    Ticks continue to be a threat to human and animal health in Turkey, as they are considered important vectors of human and animal diseases. The objectives of this investigation are to characterize the microbial communities of two tick species, Rhipicephalus annulatus and Dermacenter marginatus, analyze patterns of co-occurrence among microbial taxa, identify and compare pathogens contributing human diseases, and determine whether avirulent symbionts could exclude human pathogens from tick communities. Furthermore, this study explores a microbiome of the R. annulatus and D. marginatus via the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to describe their bacterial diversity. Pyrosequencing was performed on adult males and females isolated from humans from two high-risk Turkish provinces, Sivas and Amasya, during tick outbreaks in 2009. A total of 36,253 sequences were utilized for analyses of the 8 tick samples. Several pathogenic genera such as Francisella, Coxiella, Rickettsia, and Shigella were detected in the ticks tested. The most distinguishable difference between the two species of ticks was the lack of known human pathogen Rickettsia in R. annulatus and in samples 9 and 10 of D. marginatus. These samples had higher relative abundance of Flavobacterium sp., Curvibacter sp., Acidovorax sp., and Bacteroidaceae genera mostly representing symbionts which form a large component of normal tick microbiota. The outcome of this study is consistent with the predictions of the community ecological theory that diversity-rich bacteriomes are more resistant to bacterial invasion (and consequent pathogen dissemination) than diversity-deprived ones.

  20. Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

    Science.gov (United States)

    Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W

    2005-04-01

    Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

  1. The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections.

    Science.gov (United States)

    Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Lieberman, Isador H; Krebs, Viktor; Togawa, Daisuke; Fujishiro, Takaaki; Procop, Gary W

    2006-08-01

    We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

  2. Characterization of the microbial communities along the gastrointestinal tract of sheep by 454 pyrosequencing analysis

    Directory of Open Access Journals (Sweden)

    Jin Wang

    2017-01-01

    Full Text Available Objective The gastrointestinal tract of sheep contain complex microbial communities that influence numerous aspects of the sheep’s health and development. The objective of this study was to analyze the composition and diversity of the microbiota in the gastrointestinal tract sections (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum of sheep. Methods This analysis was performed by 454 pyrosequencing using the V3-V6 region of the 16S rRNA genes. Samples were collected from five healthy, small tailed Han sheep aged 10 months, obtained at market. The bacterial composition of sheep gastrointestinal microbiota was investigated at the phylum, class, order, family, genus, and species levels. Results The dominant bacterial phyla in the entire gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. In the stomach, the three most dominant genera in the sheep were Prevotella, unclassified Lachnospiraceae, and Butyrivibrio. In the small intestine, the three most dominant genera in the sheep were Escherichia, unclassified Lachnospiraceae, and Ruminococcus. In the large intestine, the three most dominant genera in the sheep were Ruminococcus, unclassified Ruminococcaceae, and Prevotella. R. flavefaciens, B. fibrisolvens, and S. ruminantium were three most dominant species in the sheep gastrointestinal tract. Principal Coordinates Analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: stomach (rumen, reticulum, omasum, and abomasum, small intestine (duodenum, jejunum, and ileum, and large intestine (cecum, colon, and rectum. Conclusion This is the first study to characterize the entire gastrointestinal microbiota in sheep by use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the gastrointestinal bacterial community of sheep.

  3. Anisotropic contrast optical microscope.

    Science.gov (United States)

    Peev, D; Hofmann, T; Kananizadeh, N; Beeram, S; Rodriguez, E; Wimer, S; Rodenhausen, K B; Herzinger, C M; Kasputis, T; Pfaunmiller, E; Nguyen, A; Korlacki, R; Pannier, A; Li, Y; Schubert, E; Hage, D; Schubert, M

    2016-11-01

    An optical microscope is described that reveals contrast in the Mueller matrix images of a thin, transparent, or semi-transparent specimen located within an anisotropic object plane (anisotropic filter). The specimen changes the anisotropy of the filter and thereby produces contrast within the Mueller matrix images. Here we use an anisotropic filter composed of a semi-transparent, nanostructured thin film with sub-wavelength thickness placed within the object plane. The sample is illuminated as in common optical microscopy but the light is modulated in its polarization using combinations of linear polarizers and phase plate (compensator) to control and analyze the state of polarization. Direct generalized ellipsometry data analysis approaches permit extraction of fundamental Mueller matrix object plane images dispensing with the need of Fourier expansion methods. Generalized ellipsometry model approaches are used for quantitative image analyses. These images are obtained from sets of multiple images obtained under various polarizer, analyzer, and compensator settings. Up to 16 independent Mueller matrix images can be obtained, while our current setup is limited to 11 images normalized by the unpolarized intensity. We demonstrate the anisotropic contrast optical microscope by measuring lithographically defined micro-patterned anisotropic filters, and we quantify the adsorption of an organic self-assembled monolayer film onto the anisotropic filter. Comparison with an isotropic glass slide demonstrates the image enhancement obtained by our method over microscopy without the use of an anisotropic filter. In our current instrument, we estimate the limit of detection for organic volumetric mass within the object plane of ≈49 fg within ≈7 × 7 μm 2 object surface area. Compared to a quartz crystal microbalance with dissipation instrumentation, where contemporary limits require a total load of ≈500 pg for detection, the instrumentation demonstrated here improves

  4. Development of contrast media

    International Nuclear Information System (INIS)

    Krause, W.

    1993-01-01

    Description of all contrast media (ionic and nonionic monomers, ionic and nonionic dimers) was presented. Chemotoxicity, osmolality and viscosity of some contrast agents were analyzed. The main adverse reactions to ionic and nonionic contrast media were described

  5. Identification and analysis of common bean (Phaseolus vulgaris L. transcriptomes by massively parallel pyrosequencing

    Directory of Open Access Journals (Sweden)

    Thimmapuram Jyothi

    2011-10-01

    Full Text Available Abstract Background Common bean (Phaseolus vulgaris is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. Results We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt. These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC end sequences, and a total of 21% of the unigenes (12,724 including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs and transcription factors were also identified in this study. Conclusions This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and

  6. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus microplus through tag-encoded pyrosequencing

    Directory of Open Access Journals (Sweden)

    Bendele Kylie G

    2011-01-01

    Full Text Available Abstract Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be

  7. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

    Science.gov (United States)

    2011-01-01

    Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts

  8. Radiographic contrast media

    International Nuclear Information System (INIS)

    Golman, K.; Holtz, E.; Almen, T.

    1987-01-01

    Contrast media are used in diagnostic radiology to enhance the X-ray attenuation between a body structure of interest and the surrounding tissue. A detail becomes perceptible on a roentgenogram only when its contrast exceeds a minimum value in relation to the background. Small areas of interest must have higher contrast than the background. The contrast effect depends on concentration of the contrast media with the body. A high contrast media concentration difference thus gives rise to more morphological details in the radiographs. Contrast media can be divided into negative contrast media such as air and gas which attenuate X-rays less than the body tissues, and positive contrast materials which attenuate X-rays more than the body tissues. The positive contrast media all contain either iodine (atomic number 53) or barium (atomic number 56) and can be divided into water-insoluble and water-soluble contrast media

  9. Adriamycin nephrosis and contrast media

    International Nuclear Information System (INIS)

    Thomsen, H.S.; Golman, K.; Hemmingsen, L.; Larsen, S.; Skaarup, P.; Koebenhavns Amts Sygehus, Herlev; Koebenhavns Amts Sygehus, Herlev; Centralsygehuset, Nykoebing Falster; Malmoe Allmaenna Sjukhus

    1990-01-01

    Urine profiles (albumin, glucose, NAG, LDH, GGT and sodium) were followed for 9 days after intravenous injection of either diatrizoate, iohexol, or saline in 27 Wistar rats with nephrosis induced by Adriamycin 42 days before. Another 9 rats exposed to neither Adriamycin nor contrast media served as controls. None of the contrast media caused further increased albuminuria of significance, whereas both induced significantly increased excretion of all 5 tubular components. The excretion of NAG and sodium was significantly higher following diatrizoate than following iohexol. From 24 h post injection there was no significantly greater excretion of any of the components after either diatrizoate or iohexol than after saline among the rats given Adriamycin. At the end of day 9 after contrast medium injection neither serum sodium, potassium, glucose, urea, creatinine, nor albumin revealed any contrast media related changes. Kidney histology showed quantitatively larger lesions in kidneys exposed to Adriamycin and contrast media than in kidneys exposed to Adriamycin and saline. There were no differences between the two contrast media groups. It is thus concluded, that both high osmolar ionic and low osmolar non-ionic contrast media cause temporary tubular dysfunction but no further glomerular dysfunction in rats with nephrosis induced by Adriamycin. The histologic findings indicate that both media may worsen non-reversible renal lesions. (orig.)

  10. Complete genome sequence of a novel Plum pox virus strain W isolate determined by 454 pyrosequencing.

    Science.gov (United States)

    Sheveleva, Anna; Kudryavtseva, Anna; Speranskaya, Anna; Belenikin, Maxim; Melnikova, Natalia; Chirkov, Sergei

    2013-10-01

    The near-complete (99.7 %) genome sequence of a novel Russian Plum pox virus (PPV) isolate Pk, belonging to the strain Winona (W), has been determined by 454 pyrosequencing with the exception of the thirty-one 5'-terminal nucleotides. This region was amplified using 5'RACE kit and sequenced by the Sanger method. Genomic RNA released from immunocaptured PPV particles was employed for generation of cDNA library using TransPlex Whole transcriptome amplification kit (WTA2, Sigma-Aldrich). The entire Pk genome has identity level of 92.8-94.5 % when compared to the complete nucleotide sequences of other PPV-W isolates (W3174, LV-141pl, LV-145bt, and UKR 44189), confirming a high degree of variability within the PPV-W strain. The isolates Pk and LV-141pl are most closely related. The Pk has been found in a wild plum (Prunus domestica) in a new region of Russia indicating widespread dissemination of the PPV-W strain in the European part of the former USSR.

  11. Isolation of 18 Microsatellite Loci in the Desert Mistletoe Phoradendron californicum (Santalaceae Via 454 Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Juan M. Arroyo

    2013-12-01

    Full Text Available Premise of the study: Microsatellite primers were developed for the parasitic mistletoe Phoradendron californicum to investigate to what extent population genetic structure depends on host tree distribution within a highly fragmented landscape. Methods and Results: Fourteen unlinked polymorphic and four monomorphic nuclear microsatellite markers were developed using a genomic shotgun pyrosequencing method. A total of 187 alleles plus four monomorphic loci alleles were found in 98 individuals sampled in three populations from the Sonoran Desert in the Baja California peninsula (Mexico. Loci averaged 13.3 alleles per locus (range 4–28, and observed and expected heterozygosities within populations varied from 0.167–0.879 and 0.364–0.932, respectively. Conclusions: Levels of polymorphism of the reported markers are adequate for studies of diversity and fragmentation in natural populations of this parasitic plant. Cross-species amplifications in P. juniperinum and P. diguetianum only showed four markers that could be useful in P. diguetianum.

  12. Pyrosequencing analysis of oral microbiota in children with severe early childhood dental caries.

    Science.gov (United States)

    Jiang, Wen; Zhang, Jie; Chen, Hui

    2013-11-01

    Severe early childhood caries are a prevalent public health problem among preschool children throughout the world. However, little is known about the microbiota found in association with severe early childhood caries. Our study aimed to explore the bacterial microbiota of dental plaques to study the etiology of severe early childhood caries through pyrosequencing analysis based on 16S rRNA gene V1-V3 hypervariable regions. Forty participants were enrolled in the study, and we obtained twenty samples of supragingival plaque from caries-free subjects and twenty samples from subjects with severe early childhood caries. A total of 175,918 reads met the quality control standards, and the bacteria found belonged to fourteen phyla and sixty-three genera. Our results show the overall structure and microbial composition of oral bacterial communities, and they suggest that these bacteria may present a core microbiome in the dental plaque microbiota. Three genera, Streptococcus, Granulicatella, and Actinomyces, were increased significantly in children with severe dental cavities. These data may facilitate improvements in the prevention and treatment of severe early childhood caries.

  13. Relationship of children's salivary microbiota with their caries status: a pyrosequencing study.

    Science.gov (United States)

    Gomar-Vercher, S; Cabrera-Rubio, R; Mira, A; Montiel-Company, J M; Almerich-Silla, J M

    2014-12-01

    Different dental caries status could be related with alterations in oral microbiota. Previous studies have collected saliva as a representative medium of the oral ecosystem. The purpose of this study was to assess the composition of oral microbiota and its relation to the presence of dental caries at different degrees of severity. One hundred ten saliva samples from 12-year-old children were taken and divided into six groups defined in strict accordance with their dental caries prevalence according to the International Caries Detection and Assessment System II criteria. These samples were studied by pyrosequencing PCR products of the 16S ribosomal RNA gene. The results showed statistically significant intergroup differences at the class and genus taxonomic levels. Streptococcus is the most frequent genus in all groups; although it did not show intergroup statistical differences. In patients with cavities, Porphyromonas and Prevotella showed an increasing percentage compared to healthy individuals. Bacterial diversity diminished as the severity of the disease increased, so those patients with more advanced stages of caries presented less bacterial diversity than healthy subjects. Although microbial composition tended to be different, the intragroup variation is large, as evidenced by the lack of clear intragroup clustering in principal component analyses. Thus, no clear differences were found, indicating that using bacterial composition as the sole source of biomarkers for dental caries may not be reliable in the unstimulated saliva samples used in the current study.

  14. Characterization of the Zoarces viviparus liver transcriptome using massively parallel pyrosequencing

    Directory of Open Access Journals (Sweden)

    Asker Noomi

    2009-07-01

    Full Text Available Abstract Background The teleost Zoarces viviparus (eelpout lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. Results In the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. Conclusion Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.

  15. Evaluation of the bacterial diversity of pressure ulcers using bTEFAP pyrosequencing.

    Science.gov (United States)

    Smith, Drake M; Snow, David E; Rees, Eric; Zischkau, Ann M; Hanson, J Delton; Wolcott, Randall D; Sun, Yan; White, Jennifer; Kumar, Shashi; Dowd, Scot E

    2010-09-21

    Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities. Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics. Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.

  16. Evaluation of the bacterial diversity of Pressure ulcers using bTEFAP pyrosequencing

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    Wolcott Randall D

    2010-09-01

    Full Text Available Abstract Background Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories. Methods Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP, a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities. Results Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics. Conclusions Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.

  17. Tracking fungal community responses to maize plants by DNA- and RNA-based pyrosequencing.

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    Eiko E Kuramae

    Full Text Available We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age in pots associated with four maize cultivars, including two genetically modified (GM cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA. The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most "active" fungi (as recovered via RNA. Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production. Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

  18. Pyrosequencing detects human and animal pathogenic taxa in the grapevine endosphere.

    Science.gov (United States)

    Yousaf, Sohail; Bulgari, Daniela; Bergna, Alessandro; Pancher, Michael; Quaglino, Fabio; Casati, Paola; Campisano, Andrea

    2014-01-01

    Generally, plants are not considered as hosts for human and animal pathogens (HAP). The recent produce-associated outbreaks of food-borne diseases have drawn attention toward significant deficiencies in our understanding of the ecology of HAP, and their potential for interkingdom transfer. To examine the association of microorganisms classified as HAP with plants, we surveyed the presence and distribution of HAP bacterial taxa (henceforth HAPT, for brevity's sake) in the endosphere of grapevine (Vitis vinifera L.) both in the plant stems and leaves. An enrichment protocol was used on leaves to detect taxa with very low abundance in undisturbed tissues. We used pyrosequencing and phylogenetic analyses of the 16S rDNA gene. We identified several HAPT, and focused on four genera (Propionibacterium, Staphylococcus, Clostridium, and Burkholderia). The majority of the bacterial sequences in the genus Propionibacterium, from grapevine leaf and stem, were identified as P. acnes. Clostridia were detected in leaves and stems, but their number was much higher in leaves after enrichment. HAPT were indentified both in leaves and wood of grapevines. This depicts the ability of these taxa to be internalized within plant tissues and maintain their population levels in a variety of environments. Our analysis highlighted the presence of HAPT in the grapevine endosphere and unexpected occurrence of these bacterial taxa in this atypical environment.

  19. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Lisa-Maree Gulino

    Full Text Available Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering. Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales. These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

  20. A Pyrosequencing Investigation of Differences in the Feline Subgingival Microbiota in Health, Gingivitis and Mild Periodontitis.

    Science.gov (United States)

    Harris, Stephen; Croft, Julie; O'Flynn, Ciaran; Deusch, Oliver; Colyer, Alison; Allsopp, Judi; Milella, Lisa; Davis, Ian J

    2015-01-01

    Periodontitis is the most frequently diagnosed health problem in cats yet little is known about the bacterial species important for the disease. The objective of this study was to identify bacterial species associated with health, gingivitis or mild periodontitis (gingivitis or mild periodontitis. Pyrosequencing of the V1-V3 region of the 16S rDNA from these plaque samples generated more than one million reads and identified a total of 267 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all gingival health categories, particularly in health along with Moraxella and Fusobacteria. The Peptostreptococcaceae were the most abundant family in gingivitis and mild periodontitis. Logistic regression analysis identified species from various genera that were significantly associated with health, gingivitis or mild periodontitis. The species identified were very similar to those observed in canine plaque in the corresponding health and disease states. Such similarities were not observed between cat and human at the bacterial species level but with disease progression similarities did emerge at the phylum level. This suggests that interventions targeted at human pathogenic species will not be effective for use in cats but there is more potential for commonalities in interventions for cats and dogs.

  1. Comparative transcriptomic analysis of two closely related ground beetle species with marked genital divergence using pyrosequencing.

    Science.gov (United States)

    Fujimaki, Kotaro; Fujisawa, Tomochika; Yazawa, Shigenobu; Nishimura, Osamu; Sota, Teiji

    2014-09-01

    Ground beetles of the subgenus Ohomopterus (genus Carabus) show marked divergence in species-specific male and female genital morphologies, which contributes to reproductive isolation among species. Characterizing the genetic basis of species-specific genital morphology is essential for understanding their diversification, but genomic information on Ohomopterus is not yet available. We analyzed mRNA extracted from abdominal sections of the last instar larvae and pupae of two sister species, Carabus (Ohomopterus) iwawakianus and C. (O.) uenoi, which show marked differences in genital morphology, to compare transcriptomic profiles using Roche 454 pyrosequencing. We obtained 1,608,572 high-quality reads and assembled them into 176,278 unique sequences, of which 66,049 sequences were combined into 12,662 clusters. Differential expression analyses for sexed pupae suggested that four and five clusters were differentially expressed between species for males and females, respectively. We also identified orthologous sequences of genes involved in genital development in Drosophila, which potentially affect genital development and species-specific genital morphology in Ohomopterus. This study provides the first large transcriptomic data set for a morphologically diversified beetle group, which can facilitate future studies on the genetic basis of species-specific genitalia.

  2. [The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

    Science.gov (United States)

    Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

    2014-11-01

    The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

  3. Multifocal fibrosing thyroiditis and its association with papillary thyroid carcinoma using BRAF pyrosequencing.

    Science.gov (United States)

    Frank, Renee; Baloch, Zubair W; Gentile, Caren; Watt, Christopher D; LiVolsi, Virginia A

    2014-09-01

    Multifocal fibrosing thyroiditis (MFT) is characterized by numerous foci of fibrosis in a stellate configuration with fibroelastotic and fibroblastic centers entrapping epithelial structures. MFT has been proposed as a risk factor for papillary thyroid carcinoma (PTC) development. We attempted to identify whether MFT showed such molecular changes and could possibly be related to PTC. We identified seven cases of PTC with MFT in our institutional pathology database and personal consult service of one of the authors (VAL) for the years 1999 to 2012. Areas of PTC, MFT, and normal tissue were selected for BRAF analysis. Macro-dissection, DNA extraction and PCR amplification, and pyrosequencing were performed to detect BRAF mutations in codon 600. All of the MFT lesions and normal thyroid tissue were negative for BRAF mutations. Of the seven PTCs analyzed, five (71 %) were negative for BRAF mutations, while two cases were positive. In our study, none of the MFT lesions harbored BRAF mutations, whereas 29 % (two of seven) PTCs in the same gland were positive. Hence, in this small study, we found no evidence that the MFT lesion is a direct precursor to PTC. It is likely an incidental bystander in the process and a reflection of the background thyroiditis.

  4. The Prognostic Value of Pyrosequencing-Detected MGMT Promoter Hypermethylation in Newly Diagnosed Patients with Glioblastoma

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    Veronica Villani

    2015-01-01

    Full Text Available O6-methylguanine-DNA-methyltransferase (MGMT has emerged as a relevant predictor of therapeutic response and good prognosis in patients with glioblastoma (GBM. Transcriptionally active MGMT rapidly removes the alkyl adducts, preventing the formation of cross-links and thereby causing resistance to alkylating drugs. Studies with pyrosequencing (PSQ showed that this technique has a higher reproducibility and sensitivity than other techniques. However, the definition of a prognostically relevant threshold for the percentage of MGMT methylation remains one of the most critical issues in the use of PSQ analysis. The aim of this study was to define the cut-off value correlated with good favourable prognostic outcomes. We retrospectively analyzed 51 patients (33 males, 18 females with GBM who underwent surgery or biopsy. The Receiver Operating Characteristics analysis showed that the best possible criteria for PSQ-detected percentage of MGMT methylation that predicted progression-free survival (PFS and overall survival (OS were 19% and 13%, respectively. Patients with ≤19% of PSQ-detected MGMT had a shorter PFS (HR: 0.24, p<0.01; those ones with ≤13% had a shorter OS (HR: 0.33, p<0.05. Our study reinforces the importance of MGMT in the management of GBM patients, but future studies with larger sample sizes are warranted to confirm our findings.

  5. Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

    Science.gov (United States)

    Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y H; Maguire, Anita J; Kienzle, Marco; Klieve, Athol V

    2013-01-01

    Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

  6. Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

    Science.gov (United States)

    Castro-Carrera, T; Toral, P G; Frutos, P; McEwan, N R; Hervás, G; Abecia, L; Pinloche, E; Girdwood, S E; Belenguer, A

    2014-03-01

    Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition

  7. Microplanktonic community structure in a coastal system relative to a Phaeocystis bloom inferred from morphological and tag pyrosequencing methods.

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    Sébastien Monchy

    Full Text Available BACKGROUND: Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC, have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom. METHODOLOGY AND PRINCIPAL FINDINGS: The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae. CONCLUSIONS: Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the

  8. Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

    OpenAIRE

    Oikonomou, Georgios; Machado, Vinicius Silva; Santisteban, Carlos; Schukken, Ynte Hein; Bicalho, Rodrigo Carvalho

    2012-01-01

    Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk sam...

  9. Pyrosequencing Analysis of the Microbial Diversity of Airag, Khoormog and Tarag, Traditional Fermented Dairy Products of Mongolia

    OpenAIRE

    OKI, Kaihei; DUGERSUREN, Jamyan; DEMBEREL, Shirchin; WATANABE, Koichi

    2014-01-01

    Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare’s milk), 5 Khoormog (fermented camel’s milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 ...

  10. Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.

    Science.gov (United States)

    Trama, Jason P; Adelson, Martin E; Mordechai, Eli

    2007-12-01

    Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. To develop a rapid method for identifying patients infected with MCV via swab sampling. Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

  11. De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing

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    Parani Madasamy

    2011-04-01

    Full Text Available Abstract Background Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. Results From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. Conclusion The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil

  12. Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans.

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    Azhahianambi Palavesam

    Full Text Available The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.

  13. Comparative analysis of the intestinal bacterial communities in different species of carp by pyrosequencing.

    Science.gov (United States)

    Li, Tongtong; Long, Meng; Gatesoupe, François-Joël; Zhang, Qianqian; Li, Aihua; Gong, Xiaoning

    2015-01-01

    Gut microbiota is increasingly regarded as an integral component of the host, due to important roles in the modulation of the immune system, the proliferation of the intestinal epithelium and the regulation of the dietary energy intake. Understanding the factors that influence the composition of these microbial communities is essential to health management, and the application to aquatic animals still requires basic investigation. In this study, we compared the bacterial communities harboured in the intestines and in the rearing water of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius cuvieri), and bighead carp (Hypophthalmichthys nobilis), by using 454-pyrosequencing with barcoded primers targeting the V4 to V5 regions of the bacterial 16S rRNA gene. The specimens of the three species were cohabiting in the same pond. Between 6,218 and 10,220 effective sequences were read from each sample, resulting in a total of 110,398 sequences for 13 samples from gut microbiota and pond water. In general, the microbial communities of the three carps were dominated by Fusobacteria, Firmicutes, Proteobacteria and Bacteroidetes, but the abundance of each phylum was significantly different between species. At the genus level, the overwhelming group was Cetobacterium (97.29 ± 0.46 %) in crucian carp, while its abundance averaged c. 40 and 60 % of the sequences read in the other two species. There was higher microbial diversity in the gut of filter-feeding bighead carp than the gut of the two other species, with grazing feeding habits. The composition of intestine microbiota of grass carp and crucian carp shared higher similarity when compared with bighead carp. The principal coordinates analysis (PCoA) with the weighted UniFrac distance and the heatmap analysis suggested that gut microbiota was not a simple reflection of the microbial community in the local habitat but resulted from species-specific selective pressures, possibly dependent on behavioural, immune

  14. Pyrosequencing of bacterial symbionts within Axinella corrugata sponges: diversity and seasonal variability.

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    James R White

    Full Text Available BACKGROUND: Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge "holobiont" system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs. Approximately 65,550 rRNA sequences (24% could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa, and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising >34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. CONCLUSIONS/SIGNIFICANCE: Slight shifts in several bacterial taxa

  15. Cyanobacterial composition and spatial distribution based on pyrosequencing data in the Gurbantunggut Desert, Northwestern China.

    Science.gov (United States)

    Zhang, Bingchang; Li, Renhui; Xiao, Peng; Su, Yangui; Zhang, Yuanming

    2016-03-01

    Cyanobacteria are the primary colonizers and form a dominant component of soil photosynthetic communities in biological soil crusts. They are crucial in improving soil environments, namely accumulating soil carbon and nitrogen. Many classical studies have examined cyanobacterial diversity in desert crusts, but relatively few comprehensive molecular surveys have been conducted. We used 454 pyrosequencing of 16S rRNA to investigate cyanobacterial composition and distribution on regional scales in the Gurbantunggut Desert. The relationship between cyanobacterial distribution and environmental factors was also explored. A total of 24,973 cyanobacteria partial 16S rRNA gene sequences were obtained, and 507OTUs were selected, as most OTUs had very few reads. Among these, 347 OTU sequences were of cyanobacteria origin, belonging to Oscillatoriales, Nostocales, Chroococcales, and uncultured cyanobacterium clone, respectively. Microcoleus vaginatus, Chroococcidiopsis spp. and M. steenstrupii were the dominant species in most areas of the Gurbantunggut Desert. Compared with other desert, the Gurbantunggut Desert differed in the prominence of Chroococcidiopsis spp. and lack of Pseudanabaenales. Species composition and abundance of cyanobacteria also showed distinct variations. Soil texture, precipitation, and nutrients and salt levels affected cyanobacterial distribution. Increased precipitation was helpful in improving cyanobacterial diversity. A higher content of coarse sand promoted the colonization and growth of Oscillatoriales and some phylotypes of Chroococcales. The fine-textured soil with higher nutrients and salts supported more varied populations of cyanobacteria, namely some heterocystous cyanobacteria. The results suggested that the Gurbantunggut Desert was rich in cyanobacteria and that precipitation was a primary regulating factor for cyanobacterial composition on a regional scale. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis

    Directory of Open Access Journals (Sweden)

    Hahn Daniel A

    2009-05-01

    Full Text Available Abstract Background Flesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription. Results We obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (ES. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs. Conclusion Our data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic

  17. Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing.

    Science.gov (United States)

    Li, Ou; Xiao, Rong; Sun, Lihua; Guan, Chenglin; Kong, Dedong; Hu, Xiufang

    2017-01-01

    As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39-38.42%), Burkholderia (2.71-15.98%), Escherichia/Shigella (4.90-25.12%), Pseudomonas (2.68-30.72%) and Sphingomonas (1.83-2.05%) dominated in four planting bases. Pseudomonas (17.94-22.06%), Escherichia/Shigella (6.59-11.59%), Delftia (9.65-22.14%) and Burkholderia (3.12-11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.

  18. Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Ou Li

    Full Text Available As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ to 96.08% (YN, and Delftia (10.39-38.42%, Burkholderia (2.71-15.98%, Escherichia/Shigella (4.90-25.12%, Pseudomonas (2.68-30.72% and Sphingomonas (1.83-2.05% dominated in four planting bases. Pseudomonas (17.94-22.06%, Escherichia/Shigella (6.59-11.59%, Delftia (9.65-22.14% and Burkholderia (3.12-11.05% dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.

  19. Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing

    Science.gov (United States)

    Li, Ou; Sun, Lihua; Guan, Chenglin; Kong, Dedong

    2017-01-01

    As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39–38.42%), Burkholderia (2.71–15.98%), Escherichia/Shigella (4.90–25.12%), Pseudomonas (2.68–30.72%) and Sphingomonas (1.83–2.05%) dominated in four planting bases. Pseudomonas (17.94–22.06%), Escherichia/Shigella (6.59–11.59%), Delftia (9.65–22.14%) and Burkholderia (3.12–11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples. PMID:28931073

  20. Insight into protist diversity in Arctic sea ice and melt-pond aggregate obtained by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Estelle Silvia Kilias

    2014-11-01

    Full Text Available Protists in the central Arctic Ocean are adapted to the harsh environmental conditions of its various habitats. During the Polarstern cruise ARK-XXVI/3 in 2011, at one sea-ice station, large aggregates accumulated at the bottom of the melt ponds. In this study, the protist assemblages of the bottom layer of the sea-ice and melt-pond aggregate were investigated using flow cytometry and 454-pyrosequencing. The objective is to provide a first molecular overview of protist biodiversity in these habitats and to consider the overlaps and/or differences in the community compositions. Results of flow cytometry pointed to a cell size distribution that was dominated by 3–10 µm nanoflagellates. The phylogenetic classification of all sequences was conducted at a high taxonomic level, while a selection of abundant (≥1% of total reads sequences was further classified at a lower level. At a high taxonomic level, both habitats showed very similar community structures, dominated by chrysophytes and chlorophytes. At a lower taxonomic level, dissimilarities in the diversity of both groups were encountered in the abundant biosphere. While sea-ice chlorophytes and chrysophytes were dominated by Chlamydomonas/Chloromonas spp. and Ochromonas spp., the melt-pond aggregate was dominated by Carteria sp., Ochromonas spp. and Dinobryon faculiferum. We suppose that the similarities in richness and community structure are a consequence of melt-pond freshwater seeping through porous sea ice in late summer. Differences in the abundant biosphere nevertheless indicate that environmental conditions in both habitats vary enough to select for different dominant species.

  1. Contrast induced nephropathy

    DEFF Research Database (Denmark)

    Stacul, Fulvio; van der Molen, Aart J; Reimer, Peter

    2011-01-01

    PURPOSE: The Contrast Media Safety Committee (CMSC) of the European Society of Urogenital Radiology (ESUR) has updated its 1999 guidelines on contrast medium-induced nephropathy (CIN). AREAS COVERED: Topics reviewed include the definition of CIN, the choice of contrast medium, the prophylactic me...

  2. Generalized phase contrast:

    DEFF Research Database (Denmark)

    Glückstad, Jesper; Palima, Darwin

    Generalized Phase Contrast elevates the phase contrast technique not only to improve phase imaging but also to cross over and interface with diverse and seemingly disparate fields of contemporary optics and photonics. This book presents a comprehensive introduction to the Generalized Phase Contrast...

  3. Perforations during contrast enema

    International Nuclear Information System (INIS)

    Vogel, H.; Steinkamp, U.; Grabbe, E.; Allgemeines Krankenhaus Ochsenzoll, Hamburg

    1983-01-01

    During contrast enema, perforation into the retroperitoneal space can be differentiated from perforation into the peritoneum and perforation into the intestinal wall associated with formation of barium granulomas or submucosal spreading of the contrast medium. Other special forms are perforation with contrast medium embolism of diverticula; of the processus vermiformis; penetration of contrast medium into fistulous systems and from the operated areas. Risk factors are: balloon catheter, intestinal tubes with a hard tip, preternatural anus, excessive enema pressure, contrast medium additions, preceding manipulations, intestinal diseases, advanced age and delegation of manipulations to assistants and unskilled staff. Children are particularly at risk. (orig.) [de

  4. Contrast induced nephropathy

    DEFF Research Database (Denmark)

    Stacul, Fulvio; van der Molen, Aart J; Reimer, Peter

    2011-01-01

    PURPOSE: The Contrast Media Safety Committee (CMSC) of the European Society of Urogenital Radiology (ESUR) has updated its 1999 guidelines on contrast medium-induced nephropathy (CIN). AREAS COVERED: Topics reviewed include the definition of CIN, the choice of contrast medium, the prophylactic me....../min/1.73 m (2) is CIN risk threshold for intravenous contrast medium. • Hydration with either saline or sodium bicarbonate reduces CIN incidence. • Patients with eGFR = 60 ml/min/1.73 m (2) receiving contrast medium can continue metformin normally....

  5. Molecular markers reveal infestation dynamics of the bed bug (Hemiptera: Cimicidae) within apartment buildings.

    Science.gov (United States)

    Booth, Warren; Saenz, Virna L; Santangelo, Richard G; Wang, Changlu; Schal, Coby; Vargo, Edward L

    2012-05-01

    The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), has experienced an extraordinary global resurgence in recent years, the reasons for which remain poorly understood. Once considered a pest of lower socioeconomic classes, bed bugs are now found extensively across all residential settings, with widespread infestations established in multiapartment buildings. Within such buildings, understanding the population genetic structure and patterns of dispersal may prove critical to the development of effective control strategies. Here, we describe the development of 24 high-resolution microsatellite markers through next generation 454 pyrosequencing and their application to elucidate infestation dynamics within three multistory apartment buildings in the United States. Results reveal contrasting characteristics potentially representative of geographic or locale differences. In Raleigh, NC, an infestation within an apartment building seemed to have started from a single introduction followed by extensive spread. In Jersey City, NJ, two or more introductions followed by spread are evident in two buildings. Populations within single apartments in all buildings were characterized by high levels of relatedness and low levels of diversity, indicative of foundation from small, genetically depauperate propagules. Regardless of the number of unique introductions, genetic data indicate that spread within buildings is extensive, supporting both active and human-mediated dispersal within and between adjacent rooms or apartments spanning multiple floors.

  6. How to misuse echo contrast

    Directory of Open Access Journals (Sweden)

    Missios Anna

    2009-01-01

    Full Text Available Abstract Background Primary intracardiac tumours are rare, there are however several entities that can mimic tumours. Contrast echocardiography has been suggested to aid the differentiation of various suspected masses. We present a case where transthoracic echocardiography completely misdiagnosed a left atrial mass, partly due to use of echo contrast. Case presentation An 80 year-old woman was referred for transthoracic echocardiography because of one-month duration of worsening of dyspnoea. Transthoracic echocardiography displayed a large echodense mass in the left atrium. Intravenous injection of contrast (SonoVue, Bracco Inc., It indicated contrast-enhancement of the structure, suggesting tumour. Transesophageal echocardiography revealed, however, a completely normal finding in the left atrium. Subsequent gastroscopy examination showed a hiatal hernia. Conclusion It is noteworthy that the transthoracic echocardiographic exam completely misdiagnosed what seemed like a left atrial mass, which in part was an effect of the use of echo contrast. This example highlights that liberal use of transoesophageal echocardiography is often warranted if optimal display of cardiac structures is desired.

  7. Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir.

    Directory of Open Access Journals (Sweden)

    Xiomara V Thomas

    Full Text Available BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103 were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.

  8. A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

    Science.gov (United States)

    Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W

    2005-06-01

    The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

  9. Ultra-deep pyrosequencing (UDPS data treatment to study amplicon HCV minor variants.

    Directory of Open Access Journals (Sweden)

    Josep Gregori

    Full Text Available We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn, the mutation frequency (Mf, and the nucleotidic diversity (Pi were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each, in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

  10. Bacterial communities in the rhizosphere of amilaceous maize (Zea mays L. as assessed by pyrosequencing

    Directory of Open Access Journals (Sweden)

    David Correa-Galeote

    2016-07-01

    Full Text Available Maize (Zea mays L. is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere

  11. Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing.

    Science.gov (United States)

    Hong, Pei-Ying; Wheeler, Emily; Cann, Isaac K O; Mackie, Roderick I

    2011-09-01

    Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species--the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E-9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E-14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356-896 OTUs) was >2-fold higher than in the MI (112-567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that

  12. Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

    Science.gov (United States)

    Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

    2009-06-01

    The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

  13. Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

    Science.gov (United States)

    Islam, M. Shahinoor; Zhang, Yanyan; McPhedran, Kerry N.

    2015-01-01

    The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>109 gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O3/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds. PMID:25841014

  14. Next-generation pyrosequencing analysis of microbial biofilm communities on granular activated carbon in treatment of oil sands process-affected water.

    Science.gov (United States)

    Islam, M Shahinoor; Zhang, Yanyan; McPhedran, Kerry N; Liu, Yang; Gamal El-Din, Mohamed

    2015-06-15

    The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10(9) gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O3/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Phase contrast image synthesis

    DEFF Research Database (Denmark)

    Glückstad, J.

    1996-01-01

    A new method is presented for synthesizing arbitrary intensity patterns based on phase contrast imaging. The concept is grounded on an extension of the Zernike phase contrast method into the domain of full range [0; 2 pi] phase modulation. By controlling the average value of the input phase funct...... function and by choosing appropriate phase retardation at the phase contrast filter, a pure phase to intensity imaging is accomplished. The method presented is also directly applicable in dark field image synthesis....

  16. Contrast analysis : A tutorial

    NARCIS (Netherlands)

    Haans, A.

    2018-01-01

    Contrast analysis is a relatively simple but effective statistical method for testing theoretical predictions about differences between group means against the empirical data. Despite its advantages, contrast analysis is hardly used to date, perhaps because it is not implemented in a convenient

  17. 454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature.

    Science.gov (United States)

    Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Rodelas, Belén; Abbas, Ben A; Martinez-Toledo, Maria Victoria; van Loosdrecht, Mark C M; Osorio, F; Gonzalez-Lopez, Jesus

    2015-01-01

    Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44-49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

  18. Pyrosequencing analysis yields comprehensive assessment of microbial communities in pilot-scale two-stage membrane biofilm reactors.

    Science.gov (United States)

    Ontiveros-Valencia, Aura; Tang, Youneng; Zhao, He-Ping; Friese, David; Overstreet, Ryan; Smith, Jennifer; Evans, Patrick; Rittmann, Bruce E; Krajmalnik-Brown, Rosa

    2014-07-01

    We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO3(-)) and perchlorate (ClO4(-)) in contaminated groundwater. The groundwater also contained oxygen (O2) and sulfate (SO4(2-)), which became important electron sinks that affected the NO3(-) and ClO4(-) removal rates. Using pyrosequencing, we elucidated how important phylotypes of each "primary" microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO4(2-) reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the "primary" groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.

  19. 454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

    Directory of Open Access Journals (Sweden)

    Alejandro Gonzalez-Martinez

    2015-01-01

    Full Text Available Identification of anaerobic ammonium oxidizing (anammox bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON and three full-scale bioreactors (anammox, CANON, and DEMON, was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature. The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

  20. Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC

    Directory of Open Access Journals (Sweden)

    Richard Marie-Jeanne

    2011-05-01

    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR mutations, especially in-frame deletions in exon 19 (ΔLRE and a point mutation in exon 21 (L858R predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. Methods We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. Results This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%. In the prospective analysis of 213 samples, 7 (3.3% samples were not analyzed and EGFR mutations were detected in 18 (8.7% patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. Conclusions pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.

  1. Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations

    KAUST Repository

    Arellano, Shawn M.

    2012-10-11

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ13C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge. © 2012 Springer Science+Business Media New York.

  2. Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

    Science.gov (United States)

    Arellano, Shawn M; Lee, On On; Lafi, Feras F; Yang, Jiangke; Wang, Yong; Young, Craig M; Qian, Pei-Yuan

    2013-02-01

    The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

  3. Contrast agents for MRI

    International Nuclear Information System (INIS)

    Bonnemain, B.

    1994-01-01

    Contrast agents MRI (Magnetic Resonance Imaging) have been developed to improve the diagnostic information obtained by this technic. They mainly interact on T1 and T2 parameters and increase consequently normal to abnormal tissues contrast. The paramagnetic agents which mainly act on longitudinal relaxation rate (T1) are gadolinium complexes for which stability is the main parameter to avoid any release of free gadolinium. The superparamagnetic agents that decrease signal intensity by an effect on transversal relaxation rate (T2) are developed for liver, digestive and lymph node imaging. Many area of research are now opened for optimal use of present and future contrast agents in MRI. (author). 28 refs., 4 tabs

  4. Compressive Phase Contrast Tomography

    International Nuclear Information System (INIS)

    Maia, Filipe; MacDowell, Alastair; Marchesini, Stefano; Padmore, Howard A.; Parkinson, Dula Y.; Pien, Jack; Schirotzek, Andre; Yang, Chao

    2010-01-01

    When x-rays penetrate soft matter, their phase changes more rapidly than their amplitude. Interference effects visible with high brightness sources creates higher contrast, edge enhanced images. When the object is piecewise smooth (made of big blocks of a few components), such higher contrast datasets have a sparse solution. We apply basis pursuit solvers to improve SNR, remove ring artifacts, reduce the number of views and radiation dose from phase contrast datasets collected at the Hard X-Ray Micro Tomography Beamline at the Advanced Light Source. We report a GPU code for the most computationally intensive task, the gridding and inverse gridding algorithm (non uniform sampled Fourier transform).

  5. Generalized Phase Contrast

    CERN Document Server

    Glückstad, Jesper

    2009-01-01

    Generalized Phase Contrast elevates the phase contrast technique not only to improve phase imaging but also to cross over and interface with diverse and seemingly disparate fields of contemporary optics and photonics. This book presents a comprehensive introduction to the Generalized Phase Contrast (GPC) method including an overview of the range of current and potential applications of GPC in wavefront sensing and phase imaging, structured laser illumination and image projection, optical trapping and manipulation, and optical encryption and decryption. The GPC method goes further than the restrictive assumptions of conventional Zernike phase contrast analysis and achieves an expanded range of validity beyond weak phase perturbations. The generalized analysis yields design criteria for tuning experimental parameters to achieve optimal performance in terms of accuracy, fidelity and light efficiency. Optimization can address practical issues, such as finding an optimal spatial filter for the chosen application, ...

  6. Mamografia Espectral de Contraste

    OpenAIRE

    Martins, Inês Santiago; Pereira, Inês; Pacheco, Hugo Pisco; Moutinho, Leonor

    2014-01-01

    A mamografia de contraste é uma aplicação recente possível com a mamografia digital directa, que utiliza contraste iodado endovenoso tendo como princípio a neovascularização induzida no cancro da mama, permitindo obter informação morfológica e funcional. Na mamografia espectral de contraste realiza-se uma aquisição simultânea com alta e baixa energia para cada incidência após administração de contraste iodado endovenoso. É depois feita uma imagem recombinada em que são realçadas as áreas que ...

  7. Aspiration of Barium Contrast

    OpenAIRE

    Fuentes Santos, Cristina; Steen, Bárbara

    2014-01-01

    The aspiration of barium contrast is a rare complication that may occur during studies of the digestive tract. Barium is an inert material that can cause anywhere from an asymptomatic mechanical obstruction to serious symptoms of respiratory distress that can result in patient death. We present the case of a 79-year-old male patient in whom we observed the presence of contrast medium residue in the lung parenchyma as an incidental finding during hospitalization. When the patient’s medical fil...

  8. Evaluation of feed grade sodium bisulfate impact on gastrointestinal tract microbiota ecology in broilers via a pyrosequencing platform.

    Science.gov (United States)

    Park, Si Hong; Dowd, Scot E; McReynolds, Jack L; Byrd, James A; Nisbet, David J; Ricke, Steven C

    2015-12-01

    The gastrointestinal microbial community in broiler chickens consists of many different species of bacteria, and the overall microbiota can vary from bird to bird. To control pathogenic bacteria in broilers and improve gut health, numerous potential dietary amendments have been used. In this study, we used a pyrosequencing platform to evaluate the effect of sodium bisulfate on microbiota of the crop, cecum, and ileum of broiler chickens grown over several weeks. The diversity information in each digestive organ sample exhibited considerable variation and was clearly separable, suggesting distinct bacterial populations. Although no apparent microbial clustering occurred between the control and the dietary treatments, we did observe shifts in overall microbiota populations in the crop, ileum, and ceca as well as changes in specific microorganisms such as Bacteroides, Clostridium, and Lactobacillus species that were identified as birds became older. © 2015 Poultry Science Association Inc.

  9. Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes

    DEFF Research Database (Denmark)

    Holmsgaard, Peter Nikolai; Sørensen, Søren Johannes; Hansen, Lars H.

    2013-01-01

    The use of amplicon pyrosequencing makes it possible to produce thousands of sequences of the same gene at relatively low costs. Here we show that it is possible to simultaneously sequence the 16S rRNA gene, IncP-1 trfA gene and mercury reductase gene (merA) as a way for screening the diversity...

  10. 454-Pyrosequencing survey of microbiota in adult Spotted Wing Drosophila (SWD) corroborates a core microbiome and additional symbiotic and entomopathogenic bacterial associates

    Science.gov (United States)

    Complete surveys of insect endosymbionts including species of economic importance have until recently been hampered by a lack of high-throughput genetic assays. We used 454-pyrosequencing of the 16S rRNA gene amplicon of adult spotted wing Drosophila (SWD) Drosophila suzukii (Matsumura) from souther...

  11. Field efficacy of four anthelmintics and confirmation of drug-resistant nematodes by controlled efficacy test and pyrosequencing on a sheep and goat farm in Denmark

    DEFF Research Database (Denmark)

    Pena-Espinoza, Miguel Angel; Thamsborg, Stig M.; Demeler, Janina

    2014-01-01

    and Trichostrongylus colubriformis isolated from adult goats on the farm. Recovered specimens of H. contortus were subjected to pyrosequencing for detection of single nucleotide polymorphisms (SNPs) related to benzimidazole (BZ) resistance. During the FECRT, FECs in untreated lambs dropped significantly by 47%. No FEC...

  12. Are ionic CAT contrast media still justifiable

    International Nuclear Information System (INIS)

    Witt, H.; Trempenau, B.; Dietz, G.

    1984-01-01

    The authors' clinical results revealed no statistically significant differences of tolerance between the two X-ray contrast media 'Ioxitalamat' and 'Ioglicinat'. Side-effects were found in 4.3% of the cases for both contrast media, a rate which is slightly below the one for urography. However, it must not be overlooked that patients exposed to certain risk faktors such as e.g. relative contraindications were as far as possible excluded from the study. (orig./WU) [de

  13. Somatic populations of PGT135-137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics

    Directory of Open Access Journals (Sweden)

    Jiang eZhu

    2012-09-01

    Full Text Available Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use deep sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family-specific primers were used to amplify heavy and light chain-variable domain sequences. 454 pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light chain sequences of ~90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations

  14. Comparative analysis of salivary bacterial microbiome diversity in edentulous infants and their mothers or primary care givers using pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Kimberly D Cephas

    Full Text Available Bacterial contribution to oral disease has been studied in young children, but there is a lack of data addressing the developmental perspective in edentulous infants. Our primary objectives were to use pyrosequencing to phylogenetically characterize the salivary bacterial microbiome of edentulous infants and to make comparisons against their mothers. Saliva samples were collected from 5 edentulous infants (mean age = 4.6±1.2 mo old and their mothers or primary care givers (mean age = 30.8±9.5 y old. Salivary DNA was extracted, used to generate DNA amplicons of the V4-V6 hypervariable region of the bacterial 16S rDNA gene, and subjected to 454-pyrosequencing. On average, over 80,000 sequences per sample were generated. High bacterial diversity was noted in the saliva of adults [1012 operational taxonomical units (OTU at 3% divergence] and infants (578 OTU at 3% divergence. Firmicutes, Proteobacteria, Actinobacteria, and Fusobacteria were predominant bacterial phyla present in all samples. A total of 397 bacterial genera were present in our dataset. Of the 28 genera different (P<0.05 between infants and adults, 27 had a greater prevalence in adults. The exception was Streptococcus, which was the predominant genera in infant saliva (62.2% in infants vs. 20.4% in adults; P<0.05. Veillonella, Neisseria, Rothia, Haemophilus, Gemella, Granulicatella, Leptotrichia, and Fusobacterium were also predominant genera in infant samples, while Haemophilus, Neisseria, Veillonella, Fusobacterium, Oribacterium, Rothia, Treponema, and Actinomyces were predominant in adults. Our data demonstrate that although the adult saliva bacterial microbiome had a greater OTU count than infants, a rich bacterial community exists in the infant oral cavity prior to tooth eruption. Streptococcus, Veillonella, and Neisseria are the predominant bacterial genera present in infants. Further research is required to characterize the development of oral microbiota early in life

  15. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  16. 454 pyrosequencing to describe microbial eukaryotic community composition, diversity and relative abundance: a test for marine haptophytes.

    Directory of Open Access Journals (Sweden)

    Elianne Egge

    Full Text Available Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000-20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.

  17. Aspiration of Barium Contrast

    Directory of Open Access Journals (Sweden)

    Cristina Fuentes Santos

    2014-01-01

    Full Text Available The aspiration of barium contrast is a rare complication that may occur during studies of the digestive tract. Barium is an inert material that can cause anywhere from an asymptomatic mechanical obstruction to serious symptoms of respiratory distress that can result in patient death. We present the case of a 79-year-old male patient in whom we observed the presence of contrast medium residue in the lung parenchyma as an incidental finding during hospitalization. When the patient’s medical file was reviewed, images were found of a barium swallow study that the patient had undergone months earlier, and we were able to observe the exact moment of the aspiration of the contrast material. The patient had been asymptomatic since the test.

  18. Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

    Science.gov (United States)

    Debode, Frederic; Janssen, Eric; Bragard, Claude; Berben, Gilbert

    2017-08-01

    The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

  19. Contrast media: future aspects

    International Nuclear Information System (INIS)

    Weinmann, H.J.; Platzek, J.; Schirmer, H.; Pietsch, H.; Carretero, J.; Harto, J.; Medina, J.; Riefke, B.; Martin, J.

    2005-01-01

    In spite of the dramatic development in CT, there was no major breakthrough in the iodinated contrast media development. New agents based on hybrid between MRI and CT compounds may be a new innovative alternative. This new approach may also open new indications such as radiotherapy. (orig.)

  20. Roentgen contrast medium

    International Nuclear Information System (INIS)

    Tamborski, C.

    1989-01-01

    The patent deals with a roentgen contrast medium containing a perfluorinebrominealkylether of the formula C m F 2m+1 OC n F 2n Br dispersed in water, preferentially in the presence of a non-ionic dispersing agent such as a fluorinated amidoaminoxide. 2 tabs

  1. Iodinated contrast media nephrotoxicity

    International Nuclear Information System (INIS)

    Meyrier, A.

    1994-01-01

    In the late seventies, iodinated contrast agents (ICA) were considered to be a major cause of acute iatrogenic renal failure. Over the last decade new contrast agents have been synthesized, nonionic and less hyperosmolar. The incidence of acute renal failure due to ICAs, varies from 3.7 to 70% of cases according to the series, with an average figure of 10.2%. The pathophysiology of ICA nephrotoxicity was mainly studied in laboratory animal models. Three main factors are involved in an inducing ICA-mediated decrease in glomerular filtration rate: reduction of the renal plasma flow, a direct cytotoxic effect on renal tubular cells and erythrocyte alteration leading to intra-renal sludge. Excluding dysglobulinemias with urinary excretion of immunoglobulin light chains, which represent a special case of maximum nephrotoxicity, 4 main risk factors of renal toxicity have been identified in nondiabetic subjects: previous renal failure with serum creatinine levels greater than 140 μmol per liter, extracellular dehydration, age over 60 and use of high doses of ICA and/or repeated ICA injections before serum creatinine levels return to baseline. Preventive measures for avoiding ICA nephrotoxicity are threefold: maintain or restore adequate hydration with saline infusion, stop NSAID treatment several days before ICA administration, and allow a 5 day interval before repeating contrast media injections. New, nonionic and moderately hyperosmolar contrast agents appear to be much less nephrotoxic than conventional ICAs in laboratory animals and in high-risk patients. It is advisable to select such contrast media for investigating high-risk patients. This approach was recently substantiated in well designed, randomized clinical studies which included more than 2 000 patients. (author)

  2. Pyrosequencing-based assessment of the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions.

    Science.gov (United States)

    Serkebaeva, Yulia M; Kim, Yongkyu; Liesack, Werner; Dedysh, Svetlana N

    2013-01-01

    Northern peatlands play a key role in the global carbon and water budget, but the bacterial diversity in these ecosystems remains poorly described. Here, we compared the bacterial community composition in the surface (0-5 cm depth) and subsurface (45-50 cm) peat layers of an acidic (pH 4.0) Sphagnum-dominated wetland, using pyrosequencing of 16S rRNA genes. The denoised sequences (37,229 reads, average length ∼430 bp) were affiliated with 27 bacterial phyla and corresponded to 1,269 operational taxonomic units (OTUs) determined at 97% sequence identity. Abundant OTUs were affiliated with the Acidobacteria (35.5±2.4% and 39.2±1.2% of all classified sequences in surface and subsurface peat, respectively), Alphaproteobacteria (15.9±1.7% and 25.8±1.4%), Actinobacteria (9.5±2.0% and 10.7±0.5%), Verrucomicrobia (8.5±1.4% and 0.6±0.2%), Planctomycetes (5.8±0.4% and 9.7±0.6%), Deltaproteobacteria (7.1±0.4% and 4.4%±0.3%), and Gammaproteobacteria (6.6±0.4% and 2.1±0.1%). The taxonomic patterns of the abundant OTUs were uniform across all the subsamples taken from each peat layer. In contrast, the taxonomic patterns of rare OTUs were different from those of the abundant OTUs and varied greatly among subsamples, in both surface and subsurface peat. In addition to the bacterial taxa listed above, rare OTUs represented the following groups: Armatimonadetes, Bacteroidetes, Chlamydia, Chloroflexi, Cyanobacteria, Elusimicrobia, Fibrobacteres, Firmicutes, Gemmatimonadetes, Spirochaetes, AD3, WS1, WS4, WS5, WYO, OD1, OP3, BRC1, TM6, TM7, WPS-2, and FCPU426. OTU richness was notably higher in the surface layer (882 OTUs) than in the anoxic subsurface peat (483 OTUs), with only 96 OTUs common to both data sets. Most members of poorly studied phyla, such as the Acidobacteria, Verrucomicrobia, Planctomycetes and the candidate division TM6, showed a clear preference for growth in either oxic or anoxic conditions. Apparently, the bacterial communities in surface and

  3. Interval between injection of contrast material and positive contrast cheliography affects accurate diagnosis of diaphragmatic hernia

    Directory of Open Access Journals (Sweden)

    Ismail A. Sen

    2015-01-01

    Full Text Available The aim of this study was to evaluate the clinical, surgical and diagnostic imaging findings in 11 cats and 3 dogs with suspected acute and chronic traumatic diaphragmatic hernia, and to compare the results of positive contrast cheliography (peritoneography taken immediately and 5 min after the injection of contrast material. Thoracic and abdominal radiography, ultrasonography, and positive contrast cheliography of all animals were performed. Eight cases were considered as acute and six cases were considered chronic. The contrast images taken immediately after the injection of contrast material revealed the contrast material in the thoracic cavity in 8/8 acute trauma patients, but in none of the chronic cases. In 5/6 of these cases contrast material was seen in the thoracal cavity only in additional images taken after 5 min. One patient was diagnosed with FIP and excluded from the study. Twelve cases had complete resolution and one animal died during the early postoperative period. Our results suggest that positive contrast cheliography performed immediately after the injection of contrast material may not reveal chronic cases of diaphragmatic hernia and a second imaging (or imaging after 5 min is indicated in order not to overlook chronic cases.

  4. Mamografia com contraste

    OpenAIRE

    Baptista, Rita; Silva, Carina; Reis, Cláudia

    2016-01-01

    O estudo pretendeu apresentar as indicações clínicas, vantagens e princípios da mamografia com contraste, identificar as evoluyções tecnológicas para a mamografia com contraste e caracterizar as práticas e os desafios dos técnicos de radiologia do Hospital de Santarém (único no país a utilizar esta técnica). O cancro da mama é uma das principais causas de morte nas mulheres, em todo o mundo, mas principalmente nos Estados Unidos da América, Canadá, Europa Ocidental e Austrália. Em Portugal, e...

  5. Current iodinated contrast media

    International Nuclear Information System (INIS)

    Stacul, F.

    2001-01-01

    The number of scientific papers on iodinated contrast media is declining. Indeed, comparative trials between high-osmolality and low-osmolality agents largely showed the higher safety and tolerability of the latter, and this is no longer a matter of discussion. Only financial constraints could prevent a total conversion to low-osmolality agents. Research comparing low-osmolality (nonionic monomers, ionic dimer) and iso-osmolality contrast media (nonionic dimers) are still ongoing. Both classes of nonionic compounds proved safer than the ionic dimer. The relative merits of nonionic monomers and nonionic dimers are a matter for debate, and criteria for a selective use of different agents for different procedures could be discussed. (orig.)

  6. Mathematics revealed

    CERN Document Server

    Berman, Elizabeth

    1979-01-01

    Mathematics Revealed focuses on the principles, processes, operations, and exercises in mathematics.The book first offers information on whole numbers, fractions, and decimals and percents. Discussions focus on measuring length, percent, decimals, numbers as products, addition and subtraction of fractions, mixed numbers and ratios, division of fractions, addition, subtraction, multiplication, and division. The text then examines positive and negative numbers and powers and computation. Topics include division and averages, multiplication, ratios, and measurements, scientific notation and estim

  7. 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides.

    Science.gov (United States)

    Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren

    2009-12-02

    An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects

  8. 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides

    Directory of Open Access Journals (Sweden)

    Jensen Niels

    2009-12-01

    Full Text Available Abstract Background An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Results Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being

  9. Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

    Directory of Open Access Journals (Sweden)

    Sepil Irem

    2012-05-01

    Full Text Available Abstract Background The critical role of Major Histocompatibility Complex (Mhc genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help understanding how parasite-mediated and sexual selection shape and maintain host genetic variation in nature. We believe that study systems like ours can make important contributions to the field of evolutionary biology and emphasize the necessity of integrating long-term field-based studies with detailed genetic analysis to unravel complex evolutionary processes.

  10. Pyrosequencing Reveals Bacterial Communities in Unchlorinated Drinking Water Distribution System: An Integral Study of Bulk Water, Suspended Solids, Loose Deposits, and Pipe Wall Biofilm

    KAUST Repository

    Liu, G.; Bakker, G. L.; Li, S.; Vreeburg, J. H G; Verberk, J. Q J C; Medema, G. J.; Liu, W. T.; Van Dijk, J. C.

    2014-01-01

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected

  11. Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil

    Czech Academy of Sciences Publication Activity Database

    Lladó, Salvador; Covino, Stefano; Solanasa, A. M.; Petruccioli, M.; D´Annibale, A.; Vinas, M.

    2015-01-01

    Roč. 283, č. 1 (2015), s. 35-43 ISSN 0304-3894 Institutional support: RVO:61388971 Keywords : Soil bioremediation * White rot fungi (WRF) * Creosote Subject RIV: EE - Microbiology, Virology Impact factor: 4.836, year: 2015

  12. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed

    2013-01-01

    of the microbial assemblages was different within the snow layers and between snow and freshwater. The highest diversity was seen in snow. In the middle and top snow layers, Proteobacteria, Bacteroidetes and Cyanobacteria dominated, although Actinobacteria and Firmicutes were relatively abundant also. High numbers...... of chloroplasts were also observed. In the deepest snow layer, large percentages of Firmicutes and Fusobacteria were seen. In freshwater, Bacteroidetes, Actinobacteria and Verrucomicrobia were the most abundant phyla while relatively few Proteobacteria and Cyanobacteria were present. Possibly, light intensity...

  13. Contrast Invariant SNR

    DEFF Research Database (Denmark)

    Weiss, Pierre; Escande, Paul; Dong, Yiqiu

    We design an image quality measure independent of local contrast changes, which constitute simple models of illumination changes. Given two images, the algorithm provides the image closest to the first one with the component tree of the second. This problem can be cast as a specific convex progra...... algorithms based on interior point methods. The algorithm has potential applications in change detection, color image processing or image fusion. A Matlab implementation is available at http://www.math.univ-toulouse.fr/_weiss/PageCodes.html....

  14. Pyrosequencing analysis of the microbiota of kusaya gravy obtained from Izu Islands.

    Science.gov (United States)

    Fujii, Tateo; Kyoui, Daisuke; Takahashi, Hajime; Kuda, Takashi; Kimura, Bon; Washizu, Yukio; Emoto, Eiji; Hiramoto, Tadahiro

    2016-12-05

    Kusaya is a salted, dried fish product traditionally produced on the Izu Islands in Japan. Fish are added to kusaya gravy repeatedly and intermittently, and used over several hundred years, which makes unique microbiota and unique flavors. In this study, we performed a metagenomic analysis to compare the composition of the microbiota of kusaya gravy between different islands. Twenty samples obtained from a total of 13 manufacturers on three islands (Hachijojima, Niijima, and Oshima Islands) were analyzed. The statistical analysis revealed that the microbiota in kusaya gravy maintain a stable composition regardless of the production steps, and that the microbiota are characteristic to the particular islands. The bacterial taxa common to all of the samples were not necessarily the dominant ones. On the other hand, the genera Halanaerobium and Tissierella were found to be characteristic to the microbiota of one or two islands. Because these genera are known to be present in the natural environment, it is likely that the bacterial strains peculiar to an island had colonized kusaya gravy for many years. The results of this study revealed an influence of geographical conditions on the microbiota in fermented food. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Field strength and dose dependence of contrast enhancement by gadolinium-based MR contrast agents

    International Nuclear Information System (INIS)

    Rinck, P.A.; Muller, R.N.

    1999-01-01

    The relaxivities r 1 and r 2 of magnetic resonance contrast agents and the T 1 relaxation time values of tissues are strongly field dependent. We present quantitative data and simulations of different gadolinium-based extracellular fluid contrast agents and the modulation of their contrast enhancement by the magnetic field to be able to answer the following questions: How are the dose and field dependences of their contrast enhancement? Is there an interrelationship between dose and field dependence? Should one increase or decrease doses at specific fields? Nuclear magnetic relaxation dispersion data were acquired for the following contrast agents: gadopentetate dimeglumine, gadoterate meglumine, gadodiamide injection, and gadoteridol injection, as well as for several normal and pathological human tissue samples. The magnetic field range stretched from 0.0002 to 4.7 T, including the entire clinical imaging range. The data acquired were then fitted with the appropriate theoretical models. The combination of the diamagnetic relaxation rates (R 1 = 1/T 1 and R 2 = 1/T 2 ) of tissues with the respective paramagnetic contributions of the contrast agents allowed the prediction of image contrast at any magnetic field. The results revealed a nearly identical field and dose-dependent increase of contrast enhancement induced by these contrast agents within a certain dose range. The target tissue concentration (TTC) was an important though nonlinear factor for enhancement. The currently recommended dose of 0.1 mmol/kg body weight seems to be a compromise close to the lower limits of diagnostically sufficient contrast enhancement for clinical imaging at all field strengths. At low field contrast enhancement might be insufficient. Adjustment of dose or concentration, or a new class of contrast agents with optimized relaxivity, would be a valuable contribution to a better diagnostic yield of contrast enhancement at all fields. (orig.)

  16. Transcriptome profiling using pyrosequencing shows genes associated with bast fiber development in ramie (Boehmeria nivea L.).

    Science.gov (United States)

    Chen, Jie; Pei, Zhihua; Dai, Lunjin; Wang, Bo; Liu, Lijun; An, Xia; Peng, Dingxiang

    2014-10-22

    Ramie (Boehmeria nivea L.), popularly known as "China grass", is one of the oldest crops in China and the second most important fiber crop in terms of area sown. Ramie fiber, extracted from the plant bast, is important in the textile industry. However, the molecular mechanism of ramie fiber development remains unknown. A whole sequencing run was performed on the 454 GS FLX + platform using four separately pooled parts of ramie bast. This generated 1,030,057 reads with an average length of 457 bp. Among the 58,369 unigenes (13,386 contigs and 44,983 isotigs) that were generated through de novo assembly, 780 were differentially expressed. As a result, 13 genes that belong to the cellulose synthase gene family (four), the expansin gene family (three) and the xyloglucan endotransglucosylase/hydrolase (XTH) gene family (six) were up-regulated in the top part of the bast, which was in contrast to the other three parts. The identification of these 13 concurrently up-regulated unigenes indicated that the early stage (represented by the top part of the bast) might be important for the molecular regulation of ramie fiber development. Further analysis indicated that four of the 13 unigenes from the expansin (two) and XTH (two) families shared a coincident expression pattern during the whole growth season, which implied they were more relevant to ramie fiber development (fiber quality, etc.) during the different seasons than the other genes. To the best of our knowledge, this study is the first to characterize ramie fiber development at different developmental stages. The identified transcripts will improve our understanding of the molecular mechanisms involved in ramie fiber development. Moreover, the identified differentially expressed genes will accelerate molecular research on ramie fiber growth and the breeding of ramie with better fiber yields and quality.

  17. Comparison between cultivated and total bacterial communities associated with Cucurbita pepo using cultivation-dependent techniques and 454 pyrosequencing.

    Science.gov (United States)

    Eevers, N; Beckers, B; Op de Beeck, M; White, J C; Vangronsveld, J; Weyens, N

    2016-02-01

    Endophytic bacteria often have beneficial effects on their host plants that can be exploited for bioremediation applications but, according to the literature, only 0.001-1% of all endophytic microbes should be cultivable. This study compared the cultivated endophytic communities of the roots and shoots of Cucurbita pepo with the total endophytic communities as determined by cultivation-dependent techniques and 454 pyrosequencing. The ten most abundant taxa of the total communities aligned well with the cultivated taxa; however, the abundance of these taxa in the two communities differed greatly. Enterobacter showed very low presence in the total communities, whereas they were dominantly present in the cultivated communities. Although Rhizobium dominated in total root and shoot communities, it was poorly cultivable and even then only in growth media containing plant extract. Since endophytes likely contribute to plant-growth promotion, cultivated bacterial strains were tested for their plant-growth promoting capabilities, and the results were correlated with their abundance in the total community. Bacillus and Pseudomonas showed promising results when considering cultivability, abundance in the total community and plant-growth promoting capability. This study demonstrated that, although a limited number of bacterial genera were cultivable, current cultivation-dependent techniques may be sufficient for further isolation and inoculation experiments that aim to improve phytoremediation efficiency. Copyright © 2015 Elsevier GmbH. All rights reserved.

  18. Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

    Directory of Open Access Journals (Sweden)

    Edward Alain B. Pajarillo

    2015-04-01

    Full Text Available This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level. Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs.

  19. Bacterial community composition of South China Sea sediments through pyrosequencing-based analysis of 16S rRNA genes.

    Science.gov (United States)

    Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong

    2013-01-01

    Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.

  20. Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing.

    Science.gov (United States)

    Mhuantong, Wuttichai; Wongwilaiwalin, Sarunyou; Laothanachareon, Thanaporn; Eurwilaichitr, Lily; Tangphatsornruang, Sithichoke; Boonchayaanant, Benjaporn; Limpiyakorn, Tawan; Pattaragulwanit, Kobchai; Punmatharith, Thantip; McEvoy, John; Khan, Eakalak; Rachakornkij, Manaskorn; Champreda, Verawat

    2015-01-01

    The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas.

  1. Assessment of bacterial diversity in Hyalomma aegyptium, H. marginatum and H. excavatum ticks through tag-encoded pyrosequencing.

    Science.gov (United States)

    Keskin, Adem; Bursali, Ahmet; Snow, David E; Dowd, Scot E; Tekin, Saban

    2017-12-01

    Ticks are among the most significant human-biting ectoparasites and they play a major role in transmission of many pathogenic agents to humans. In the present study, three species of Hyalomma ticks, Hyalomma aegyptium, H. marginatum and H. excavatum, were examined for the presence of zoonotic bacteria, both male and female ticks alike. Examination of microbial diversity with tag-encoded pyrosequencing indicates that H. marginatum and H. excavatum were more diversity rich than H. aegyptium. Although numerous pathogenic and non-pathogenic bacterial genera were detected, including Acidovorax, Bacillus, Bacteroides, Bdellovibrio, Clostridium, Curvibacter, Escherichia, Flavobacterium, Limnohabitans, Paenibacillus, Ralstonia, Sarcina, Sediminibacterium, Segetibacter Stenotrophomonas and Variovorax, the predominant zoonotic bacteria represented in these ticks were genera Borrelia, Francisella, and Rickettsia. To the authors' knowledge, this work represents the first detection of Yersinia enterocolitica in the tick H. excavatum, raising questions regarding the vector competency of this tick, as well as associations of different disease representations perhaps through previously unforeseen routes of pathogen introduction. Likewise, similar questions are related to the presence of Legionella pneumophila in one H. excavatum sample.

  2. Rapid Development of Microsatellite Markers with 454 Pyrosequencing in a Vulnerable Fish, the Mottled Skate, Raja pulchra

    Science.gov (United States)

    Kang, Jung-Ha; Park, Jung-Youn; Jo, Hyun-Su

    2012-01-01

    The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1–10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy–Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni’s correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species. PMID:22837688

  3. Exploring the variation of oral microbiota in supragingival plaque during and after head-and-neck radiotherapy using pyrosequencing.

    Science.gov (United States)

    Gao, Li; Hu, Yuejian; Wang, Yuxia; Jiang, Wenxin; He, Zhiyan; Zhu, Cailian; Ma, Rui; Huang, Zhengwei

    2015-09-01

    The aim of this article was to study the variation in oral microflora of the subgingival plaque during and after radiotherapy. During and after radiotherapy, microbial samples were collected at seven time points (early stage, medium stage, and later stage of radiotherapy, and 1 month, 3 months, 6 months, and 1 year after radiotherapy) in three subjects for a total of 21 samples. Polymerase chain reaction (PCR) amplification was carried out on the 16S rDNA hypervariable V1-V3 region, and then the PCR products were determined by high-throughput pyrosequencing. The rarefaction curve indicating the richness of the microflora demonstrated that the number of operational taxonomic units (OTUs) was in decline from the early stage of radiotherapy to the time point 1 month after radiotherapy and then trended upward. The Shannon diversity index declined during radiotherapy (ranging from 4.59 to 3.73), and generally rose after radiotherapy, with the lowest value of 3.5 (1 month after radiotherapy) and highest value of 4.75 (6 months after radiotherapy). A total of 120 genera were found; five genera (Actinomyces, Veillonella, Prevotella, Streptococcus, Campylobacter) were found in all subjects across all time points. The richness and diversity of oral ecology decreased with increased radiation dose, and it was gradually restored with time. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Bacterial community composition of South China Sea sediments through pyrosequencing-based analysis of 16S rRNA genes.

    Directory of Open Access Journals (Sweden)

    Daochen Zhu

    Full Text Available BACKGROUND: Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. CONCLUSIONS: This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m.

  5. Rapid development of microsatellite markers with 454 pyrosequencing in a vulnerable fish, the mottled skate, Raja pulchra.

    Science.gov (United States)

    Kang, Jung-Ha; Park, Jung-Youn; Jo, Hyun-Su

    2012-01-01

    The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.

  6. Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

    Science.gov (United States)

    Preusser, Matthias; Berghoff, Anna S.; Manzl, Claudia; Filipits, Martin; Weinhäusel, Andreas; Pulverer, Walter; Dieckmann, Karin; Widhalm, Georg; Wöhrer, Adelheid; Knosp, Engelbert; Marosi, Christine; Hainfellner, Johannes A.

    2014-01-01

    Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing. PMID:24359605

  7. Phase Contrast Imaging

    DEFF Research Database (Denmark)

    1996-01-01

    The invention relates to a method and a system for synthesizing a prescribed intensity pattern based on phase contrast imaging that is not based on the assumption of prior art methods that the pahase shift phi is less than 1 radian. An improved method based on a simple imaging operation...... phasors attain predetermined values for predetermined spatial frequencies, and the phasor value of the specific resolution element of the spatial phase mask corresponds to a distinct intensity level of the image of the resolution element in the intensity pattern, and a spatial phase filter for phase...... shifting of a part of the electromagntic radiation, in combination with an imaging system for generation of the intensity pattern by interference in the image plane of the imaging system between the part of the electromagnetic raidation that has been phase shifted by the phase filter and the remaining part...

  8. Paramagnetic contrast material

    International Nuclear Information System (INIS)

    McNamara, M.T.

    1987-01-01

    Paramagnetic contrast materials have certainly demonstrated clinical utility in a variety of organ systems for improved detection of various neoplastic, inflammatory, infectious, and physiologic abnormalities. Although the more commonly employed extracellular agents, such as Gd-DTPA, have been quite safe and useful, particularly in the CNS, it is almost certain that other substances will achieve more success in various other organs, such as iron oxides in the reticuloendothelial system and persisting extracellular agents in the cardiovascular system. Finally, as MRI technology continues to evolve, producing such exciting new sequences as gradient-echo fast scans, the roles of currently existing and newly discovered paramagnetic pharmaceuticals must be continuously reevaluated both to obtain maximum clinical benefit and to guide the search for newer agents that may further optimize the diagnostic efficacy of MRI

  9. New MR contrast agent

    International Nuclear Information System (INIS)

    Grossman, C.D.; Subramanian, G.; Schneider, R.; Szeverenyi, N.E.; Rosenbaum, A.M.; Gagne, G.; Tillapaugh-Fay, G.; Berlin, R.; Ritter-Hrncirik, C.; Yu, S.

    1990-01-01

    This paper evaluates an MR contrast agent-meglumine tris-(2,6-dicarboxypyridine) gadolinium (III) or gadolinium dipicolinate (Gd-DPC)-produced in-house. Rats were anesthetized with pentobarbital. For renal imaging, bowel motion artifact was minimized with glucagon (0.014 mg/kg, intravenous (IV)). Enhanced images were generated on a 2-T chemical shift imaging system with a 31-cm horizontal bore magnet after IV injection of Gd-DPC (100 μM/kg). Coronal sections of the kidneys and sagittal sections of the brain, 2 mm thick, were made. Six to eight excitations and 128 or 356 phase-encoding steps were used for each image. Control animals were injected with equivalent doses of gadopentetate dimeglumine

  10. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

  11. Protists in the polar regions: comparing occurrence in the Arctic and Southern oceans using pyrosequencing

    Directory of Open Access Journals (Sweden)

    Christian Wolf

    2015-05-01

    Full Text Available In the ongoing discussion of the distribution of protists, whether they are globally distributed or endemic to one or both of the polar regions is the subject of heated debate. In this study, we compared next-generation sequencing data from the Arctic and the Southern oceans to reveal the extent of similarities and dissimilarities between the protist communities in the polar regions. We found a total overlap of operational taxonomic units (OTUs between the two regions of 11.2%. On closer inspection of different taxonomic groups, the overlap ranged between 5.5% (haptophytes and 14.5% (alveolates. Within the different groups, the proportion of OTUs occurring in both regions greatly differed between the polar regions. On the one hand, the overlap between these two regions is remarkable, given the geographical distance between them. On the other hand, one could expect a greater overlap of OTUs between these regions on account of the similar environmental conditions. The overlap suggests a connection between the polar regions for at least certain species or that the evolutionary divergence has been slow, relative to the timescales of isolation. The different proportions of common OTUs among the groups or regions may be a result of different life cycle strategies or environmental adaptations.

  12. Pyrosequencing of Haliotis diversicolor transcriptomes: insights into early developmental molluscan gene expression.

    Directory of Open Access Journals (Sweden)

    Zi-Xia Huang

    Full Text Available BACKGROUND: The abalone Haliotis diversicolor is a good model for study of the settlement and metamorphosis, which are widespread marine ecological phenomena. However, information on the global gene backgrounds and gene expression profiles for the early development of abalones is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, eight non-normalized and multiplex barcode-labeled transcriptomes were sequenced using a 454 GS system to cover the early developmental stages of the abalone H. diversicolor. The assembly generated 35,415 unigenes, of which 7,566 were assigned GO terms. A global gene expression profile containing 636 scaffolds/contigs was constructed and was proven reliable using qPCR evaluation. It indicated that there may be existing dramatic phase transitions. Bioprocesses were proposed, including the 'lock system' in mature eggs, the collagen shells of the trochophore larvae and the development of chambered extracellular matrix (ECM structures within the earliest postlarvae. CONCLUSION: This study globally details the first 454 sequencing data for larval stages of H. diversicolor. A basic analysis of the larval transcriptomes and cluster of the gene expression profile indicates that each stage possesses a batch of specific genes that are indispensable during embryonic development, especially during the two-cell, trochophore and early postlarval stages. These data will provide a fundamental resource for future physiological works on abalones, revealing the mechanisms of settlement and metamorphosis at the molecular level.

  13. Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river.

    Science.gov (United States)

    Bricheux, Geneviève; Morin, Loïc; Le Moal, Gwenaël; Coffe, Gérard; Balestrino, Damien; Charbonnel, Nicolas; Bohatier, Jacques; Forestier, Christiane

    2013-06-01

    Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

  14. Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

    Science.gov (United States)

    Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

    2015-06-25

    Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex

  15. Revealing Rembrandt

    Directory of Open Access Journals (Sweden)

    Andrew J Parker

    2014-04-01

    Full Text Available The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI. Our results emphasised the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt’s portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings.

  16. Comparison of neutral oral contrast versus positive oral contrast medium in abdominal multidetector CT

    International Nuclear Information System (INIS)

    Berther, Ralph; Eckhardt, Boris; Zollikofer, Christoph L.; Patak, Michael A.; Erturk, Sukru M.

    2008-01-01

    To determine whether neutral contrast agents with water-equivalent intraluminal attenuation can improve delineation of the bowel wall and increase overall image quality for a non-selected patient population, a neutral oral contrast agent (3% mannitol) was administered to 100 patients referred for abdominal multidetector row computed tomography (MDCT). Their results were compared with those of 100 patients given a positive oral contrast agent. Qualitative and quantitative measurements were done on different levels of the gastrointestinal tract by three experienced readers. Patients given the neutral oral contrast agent showed significant better qualitative results for bowel distension (P<0.001), homogeneity of the luminal content (P<0.001), delineation of the bowel-wall to the lumen (P<0.001) and to the mesentery (P<0.001) and artifacts (P<0.001), leading to a significant better overall image quality (P<0.001) than patients receiving positive oral contrast medium. The quantitative measurements revealed significant better distension (P<0.001) and wall to lumen delineation (P<0.001) for the patients receiving neutral oral contrast medium. The present results show that the neutral oral contrast agent (mannitol) produced better distension, better homogeneity and better delineation of the bowel wall leading to a higher overall image quality than the positive oral contrast medium in a non-selected patient population. (orig.)

  17. Investigation of ruminal bacterial diversity in dairy cattle fed supplementary monensin alone and in combination with fat, using pyrosequencing analysis.

    Science.gov (United States)

    Kim, M; Eastridge, M L; Yu, Z

    2014-02-01

    The objective of this study was to examine and compare the effects of monensin, both alone and together with dietary fat, on ruminal bacterial communities in dairy cattle fed the following 3 diets: a control diet, the control diet supplemented with monensin, and the control diet supplemented with both monensin and fat. Bacterial communities in the liquid and the adherent fractions of rumen content were analyzed using 454 pyrosequencing analysis of 16S rRNA gene amplicons. Most sequences were assigned to phyla Firmicutes and Bacteroidetes, irrespective of diets and fractions. Prevotella was the most dominant genus, but most sequences could not be classified at the genus level. The proportion of Gram-positive Firmicutes was reduced by 4.5% in response to monensin but increased by 12.8% by combination of monensin and fat, compared with the control diet. Some of the operational taxonomic units in Firmicutes and Bacteroidetes were also affected by monensin or by the combination of monensin with fat. The proportion of numerous bacteria potentially involved in lipolysis and (or) biohydrogenation was increased by both monensin and fat. The Shannon diversity index was decreased in the control diet supplemented with both monensin and fat, compared with the other 2 diet groups. Supplementary fats hinder bacterial attachment to plant particles and then result in decreased bacterial diversity in the rumen. The finding of this study may help in understanding the effect of monensin and fat on ruminant nutrition and the adverse effect of monensin and fat, such as milk fat depression and decreased feed digestibility.

  18. Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

    Directory of Open Access Journals (Sweden)

    Lank Simon M

    2012-08-01

    Full Text Available Abstract Background High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.

  19. Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

    Science.gov (United States)

    Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S

    2016-05-01

    Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  20. Phase Contrast Imaging

    International Nuclear Information System (INIS)

    Menk, Ralf Hendrik

    2008-01-01

    All standard (medical) x-ray imaging technologies, rely primarily on the amplitude properties of the incident radiation, and do not depend on its phase. This is unchanged since the discovery by Roentgen that the intensity of an x-ray beam, as measured by the exposure on a film, was related to the relative transmission properties of an object. However, recently various imaging techniques have emerged which depend on the phase of the x-rays as well as the amplitude. Phase becomes important when the beam is coherent and the imaging system is sensitive to interference phenomena. Significant new advances have been made in coherent optic theory and techniques, which now promise phase information in medical imaging. The development of perfect crystal optics and the increasing availability of synchrotron radiation facilities have contributed to a significant increase in the application of phase based imaging in materials and life sciences. Unique source characteristics such as high intensity, monochromaticity, coherence and high collimating provide an ideal source for advanced imaging. Phase contrast imaging has been applied in both projection and computed tomography modes, and recent applications have been made in the field of medical imaging. Due to the underlying principle of X-ray detection conventional image receptors register only intensities of wave fields and not their phases. During the last decade basically five different methods were developed that translate the phase information into intensity variations. These methods are based on measuring the phase shift φ directly (using interference phenomena), the gradient ∇ φ , or the Laplacian ∇ 2 φ. All three methods can be applied to polychromatic X-ray sources keeping in mind that the native source is synchrotron radiation, featuring monochromatic and reasonable coherent X-ray beams. Due to the vast difference in the coefficients that are driven absorption and phase effects (factor 1,000-10,000 in the energy

  1. Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin

    Science.gov (United States)

    Lowrey, Liam; Woodhams, Douglas C.; Tacchi, Luca

    2015-01-01

    The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (Oncorhynchus mykiss). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases. PMID:26209676

  2. The Value of Contrast Echocardiography

    Directory of Open Access Journals (Sweden)

    Shannon C. Treiber

    2016-01-01

    Full Text Available Purpose: There is much evidence-based research proving the effectiveness of contrast echocardiography, but there are still questions and concerns about its specific uses. This study tested the effectiveness of contrast echocardiography in defining the left ventricular endocardial border. Methods: From 30 patients, a total of 60 echocardiograms –– 30 with and 30 without use of contrast –– were retrospectively reviewed by four blinded cardiologists with advanced training in echocardiography. No single cardiologist reviewed contrast and noncontrast images of the same patient. Each set of 30 echocardiograms was then studied for wall-motion scoring. Visualization of left ventricular wall segments and a global visualization confidence level of interpretation were recorded. Results: Of all wall segments (N = 510, 91% were visualized in echocardiograms with use of contrast, whereas 75% of the walls were visualized in echocardiograms without contrast (P < 0.001. Of 30 examinations, 17 contrast echocardiograms were read with high confidence compared to 6 without contrast use (P = 0.004. The number of walls visualized with contrast was increased in 18 patients (60%, whereas noncontrast echocardiograms yielded more visualized walls in 6 patients (20%, P = 0.002. Conclusions: This study demonstrates that contrast is valuable to echocardiographic imaging. Its use should be supported throughout echocardiography clinics and encouraged in certain patients for whom resting and stress echocardiography results without contrast often prove uninterpretable.

  3. Comparative study of contrast dacryocystogram and nuclear dacryocystogram

    International Nuclear Information System (INIS)

    Chaudhuri, T.K.; Saparoff, G.R.; Dolan, K.D.; Chaudhuri, T.K.

    1975-01-01

    A comparative study was run between conventional radiographic contrast dacryocystogram and radioisotope scan of the lacrimal drainage apparatus (henceforth called ''nuclear dacryocystogram''). A total of 20 contrast dacryocystograms (DCG), 22 irrigations, and 42 nuclear dacryocystograms (DCG) were performed in 21 patients having symptoms of obstruction in the lacrimal drainage system. The study revealed that there was a good correlation between these two diagnostic techniques and nuclear DCG was, perhaps, superior to contrast DCG. (U.S.)

  4. Visibility of minute objects with low contrast

    International Nuclear Information System (INIS)

    Sato, Nobuo

    1982-01-01

    Minimum perceptible contrast was obtained with a penetrameter made of aluminum plates, in which 40 very small holes were perforated. An object can be seen when its contrast exceeds the minimum perceptible contrast. Visual threshold contrast ratio offers minimum perceptible diameter (MPD) which is theoretically obtained from an image contrast corresponding to the technique applied. The MPDs were obtained prior to experiment as a function of system speed, beam quality, exposure range and magnification, which resulted in a reasonable agreement with the experimental results. The visibility of small dot-like objects depends on the granularity as its threshold is governed by the surrounding grainy noise. However it was revealed that MTF was as important as granularity. Dot and lineshaped objects were simulated with small glass beads and nylon fibrils, respectively, in order to make ROC analysis on the different radiographic techniques with a CGR Senograph 500T mammographic unit. The 1.5X magnification technique showed the largest advantage on phantom work basis, but clinical evaluation showed the different weight of the affecting physical factors. The results of ROC analysis were extended to entropy analysis. The visibility in actual mammography depends upon the acutance of images as Xeroradiography proved. (Kako, I.)

  5. Effects of computed tomography contrast medium factors on contrast enhancement

    International Nuclear Information System (INIS)

    Terasawa, Kazuaki; Hatcho, Atsushi; Okuda, Itsuko

    2011-01-01

    The various nonionic iodinated contrast media used in contrast computed tomography (CT) studies differ in terms of their composition, characteristics, and iodine concentration (mgI/ml), as well as the volume injected (ml). Compared with ionic iodinated contrast media, nonionic iodinated contrast media are low-osmolar agents, with different agents having different osmotic pressures. Using a custom-made phantom incorporating a semipermeable membrane, the osmotic flow rate (hounsfield unit (HU)/s) could easily be measured based on the observed increase in CT numbers, and the relationship between the osmotic pressure and the osmotic flow rate could be obtained (r 2 =0.84). In addition, taking the effects of patient size into consideration, the levels of contrast enhancement in the abdominal aorta (AA) and inferior vena cava (IVC) were compared among four types of CT contrast medium. The results showed differences in contrast enhancement in the IVC during the equilibrium phase depending on the type of contrast medium used. It was found that the factors responsible for the differences observed in enhancement in the IVC were the osmotic flow rate and the volume of the blood flow pathways in the circulatory system. It is therefore considered that the reproducibility of contrast enhancement is likely to be reduced in the examination of parenchymal organs, in which scanning must be performed during the equilibrium phase, even if the amount of iodine injected per unit body weight (mgI/kg) is maintained at a specified level. (author)

  6. Analysis of Hepatitis C Virus NS5A Region in Patients with Cirrhosis Using an Ultra-Deep Pyrosequencing Method.

    Science.gov (United States)

    Keskin, Fahriye; Ciftci, Sevgi; Akyuz, Filiz; Abaci, Neslihan; Cakiris, Aris; Akyuz, Umit; Demir, Kadir; Besisik, Fatih; Ustek, Duran; Kaymakoglu, Sabahattin

    2017-09-01

    HCV (Hepatitis C Virus) is genetically more diverse than HBV and HIV (Human Immunodeficiency Virus) and exists as quasispecies within infected individuals. This is due to the lack of efficient proofreading of the viral RNA-dependent RNA polymerase. Consequently, quasispecies emerge depending on the mutation rate of the viral polymerase, which may display a high level of genetic variability in a population. In infected individuals, HCV replicates and circulates as quasispecies composed of a complex mixture of different but closely related genomes that undergoes continuous change due to competitive selection and cooperation between arising mutants. The aim of this study is to investigate mutations in the NS5A region as a whole, including ISDR, PKRBD, IRRDR, and V3 of HCV genotype 1b cirrhosis patients being naive and nonresponders, treated with IFN (interferon) + ribavirin (RBN) by using an ultra-deep pyrosequencing method (UDPS). During the study, five patients (four females, and one male, mean age 59.8 ± 11 years) with HCV related cirrhosis were analyzed. Three patients received IFN + RBN for six months, but two patients did not receive any therapy. HCV-RNA concentrations in patients' sera were determined using a COBAS AMPLICOR HCV MONITOR Test, Version 2.0. Genotyping was performed by using a commercial reverse hybridization method, Line Probe Assay. The quasispecies for the NS5A region were investigated using UDPS. All five patients were HCV genotype 1b (Mean Child-Pugh score 7.2 ± 1.9, 2 pts Child A, 2 pts Child B, and one pt Child C) but only one patient had hepatocellular carcinoma (HCC). A total of 19 different mutations were detected in each of the five patients (ranging from 3 to 6 mutations per patient). In all five patients, several mutations in the ISDR and PKR-BD regions were detected. On the other hand, mutations in the V3 and IRRDR regions were only detected in one patient. UDPS is a new sequencing technology and a very sensitive method in detection

  7. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP.

    Directory of Open Access Journals (Sweden)

    Scot E Dowd

    Full Text Available BACKGROUND: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. METHODS AND FINDINGS: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp. CONCLUSIONS: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP. Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections

  8. Contrast-enhanced endoscopic ultrasonography

    DEFF Research Database (Denmark)

    Reddy, Nischita K; Ioncica, Ana Maria; Saftoiu, Adrian

    2011-01-01

    Contrast agents are increasingly being used to characterize the vasculature in an organ of interest, to better delineate benign from malignant pathology and to aid in staging and directing therapeutic procedures. We review the mechanisms of action of first, second and third generation contrast...... agents and their use in various endoscopic procedures in the gastrointestinal tract. Various applications of contrast-enhanced endoscopic ultrasonography include differentiating benign from malignant mediastinal lymphadenopathy, assessment of depth of invasion of esophageal, gastric and gall bladder...

  9. Properties of conventional contrast media

    International Nuclear Information System (INIS)

    Muetzel, W.

    1981-01-01

    A comprehensive overview is given of the properties of contrast media currently used in computed tomography (CT). The chemical structure of the compounds and the physicochemical properties derived therefrom are described. Emphasis is placed on the importance of the physicochemical properties of contrast media for tolerance and the pharmacokinetic behavior of compounds in the body. An outline is given of the basic ideas governing rational use of contrast media in CT, which result from complex, time-dependent distribution of contrast media in different tissue spaces. (Auth.)

  10. Lexical representation of novel L2 contrasts

    Science.gov (United States)

    Hayes-Harb, Rachel; Masuda, Kyoko

    2005-04-01

    There is much interest among psychologists and linguists in the influence of the native language sound system on the acquisition of second languages (Best, 1995; Flege, 1995). Most studies of second language (L2) speech focus on how learners perceive and produce L2 sounds, but we know of only two that have considered how novel sound contrasts are encoded in learners' lexical representations of L2 words (Pallier et al., 2001; Ota et al., 2002). In this study we investigated how native speakers of English encode Japanese consonant quantity contrasts in their developing Japanese lexicons at different stages of acquisition (Japanese contrasts singleton versus geminate consonants but English does not). Monolingual English speakers, native English speakers learning Japanese for one year, and native speakers of Japanese were taught a set of Japanese nonwords containing singleton and geminate consonants. Subjects then performed memory tasks eliciting perception and production data to determine whether they encoded the Japanese consonant quantity contrast lexically. Overall accuracy in these tasks was a function of Japanese language experience, and acoustic analysis of the production data revealed non-native-like patterns of differentiation of singleton and geminate consonants among the L2 learners of Japanese. Implications for theories of L2 speech are discussed.

  11. The new numbers contrast sensitivity chart for contrast sensitivity measurement

    Directory of Open Access Journals (Sweden)

    Bharkbhum Khambhiphant

    2011-10-01

    Conclusions: These charts show reasonable agreement and can be used interchangeably with the MARS. It is helpful for Thai people who can only read numbers in doing the test. We can use them in routinely contrast sensitivity measurement.

  12. Contrast-enhanced peripheral MRA. Technique and contrast agents

    International Nuclear Information System (INIS)

    Nielsen, Yousef W.; Thomsen, Henrik S.

    2012-01-01

    In the last decade contrast-enhanced magnetic resonance angiography (CE-MRA) has gained wide acceptance as a valuable tool in the diagnostic work-up of patients with peripheral arterial disease. This review presents current concepts in peripheral CE-MRA with emphasis on MRI technique and contrast agents. Peripheral CE-MRA is defined as an MR angiogram of the arteries from the aortic bifurcation to the feet. Advantages of CE-MRA include minimal invasiveness and lack of ionizing radiation. The basic technique employed for peripheral CE-MRA is the bolus-chase method. With this method a paramagnetic MRI contrast agent is injected intravenously and T1-weighted images are acquired in the subsequent arterial first-pass phase. In order to achieve high quality MR angiograms without interfering venous contamination or artifacts, a number of factors need to be taken into account. This includes magnetic field strength of the MRI system, receiver coil configuration, use of parallel imaging, contrast bolus timing technique, and k-space filling strategies. Furthermore, it is possible to optimize peripheral CE-MRA using venous compression techniques, hybrid scan protocols, time-resolved imaging, and steady-state MRA. Gadolinium(Gd)-based contrast agents are used for CE-MRA of the peripheral arteries. Extracellular Gd agents have a pharmacokinetic profile similar to iodinated contrast media. Accordingly, these agents are employed for first-pass MRA. Blood-pool Gd-based agents are characterized by prolonged intravascular stay, due to macromolecular structure or protein binding. These agents can be used for first-pass, as well as steady-state MRA. Some Gd-based contrast agents with low thermodynamic stability have been linked to development of nephrogenic systemic fibrosis in patients with severe renal insufficiency. Using optimized technique and a stable MRI contrast agent, peripheral CE-MRA is a safe procedure with diagnostic accuracy close to that of conventional catheter X

  13. Contrast-enhanced endoscopic ultrasonography

    DEFF Research Database (Denmark)

    Reddy, Nischita K; Ioncică, Ana Maria; Săftoiu, Adrian

    2011-01-01

    Contrast agents are increasingly being used to characterize the vasculature in an organ of interest, to better delineate benign from malignant pathology and to aid in staging and directing therapeutic procedures. We review the mechanisms of action of first, second and third generation contrast...... agents and their use in various endoscopic procedures in the gastrointestinal tract. Various applications of contrast-enhanced endoscopic ultrasonography include differentiating benign from malignant mediastinal lymphadenopathy, assessment of depth of invasion of esophageal, gastric and gall bladder...... cancers and visualization of the portal venous system and esophageal varices. In addition, contrast agents can be used to differentiate pancreatic lesions. The use of color Doppler further increases the ability to diagnose and differentiate various pancreatic malignancies. The sensitivity of power Doppler...

  14. Contrast-enhanced peripheral MRA

    DEFF Research Database (Denmark)

    Nielsen, Yousef W; Thomsen, Henrik S

    2012-01-01

    MRI contrast agent is injected intravenously and T1-weighted images are acquired in the subsequent arterial first-pass phase. In order to achieve high quality MR angiograms without interfering venous contamination or artifacts, a number of factors need to be taken into account. This includes magnetic......-state MRA. Gadolinium(Gd)-based contrast agents are used for CE-MRA of the peripheral arteries. Extracellular Gd agents have a pharmacokinetic profile similar to iodinated contrast media. Accordingly, these agents are employed for first-pass MRA. Blood-pool Gd-based agents are characterized by prolonged...... intravascular stay, due to macromolecular structure or protein binding. These agents can be used for first-pass, as well as steady-state MRA. Some Gd-based contrast agents with low thermodynamic stability have been linked to development of nephrogenic systemic fibrosis in patients with severe renal...

  15. Visual Contrast Sensitivity in Early-Stage Parkinson's Disease.

    Science.gov (United States)

    Ming, Wendy; Palidis, Dimitrios J; Spering, Miriam; McKeown, Martin J

    2016-10-01

    Visual impairments are frequent in Parkinson's disease (PD) and impact normal functioning in daily activities. Visual contrast sensitivity is a powerful nonmotor sign for discriminating PD patients from controls. However, it is usually assessed with static visual stimuli. Here we examined the interaction between perception and eye movements in static and dynamic contrast sensitivity tasks in a cohort of mildly impaired, early-stage PD patients. Patients (n = 13) and healthy age-matched controls (n = 12) viewed stimuli of various spatial frequencies (0-8 cyc/deg) and speeds (0°/s, 10°/s, 30°/s) on a computer monitor. Detection thresholds were determined by asking participants to adjust luminance contrast until they could just barely see the stimulus. Eye position was recorded with a video-based eye tracker. Patients' static contrast sensitivity was impaired in the intermediate spatial-frequency range and this impairment correlated with fixational instability. However, dynamic contrast sensitivity and patients' smooth pursuit were relatively normal. An independent component analysis revealed contrast sensitivity profiles differentiating patients and controls. Our study simultaneously assesses perceptual contrast sensitivity and eye movements in PD, revealing a possible link between fixational instability and perceptual deficits. Spatiotemporal contrast sensitivity profiles may represent an easily measurable metric as a component of a broader combined biometric for nonmotor features observed in PD.

  16. Selective Contrast Adjustment by Poisson Equation

    Directory of Open Access Journals (Sweden)

    Ana-Belen Petro

    2013-09-01

    Full Text Available Poisson Image Editing is a new technique permitting to modify the gradient vector field of an image, and then to recover an image with a gradient approaching this modified gradient field. This amounts to solve a Poisson equation, an operation which can be efficiently performed by Fast Fourier Transform (FFT. This paper describes an algorithm applying this technique, with two different variants. The first variant enhances the contrast by increasing the gradient in the dark regions of the image. This method is well adapted to images with back light or strong shadows, and reveals details in the shadows. The second variant of the same Poisson technique enhances all small gradients in the image, thus also sometimes revealing details and texture.

  17. Contrast Media: Are There Differences in Nephrotoxicity among Contrast Media?

    Science.gov (United States)

    2014-01-01

    Iodinated contrast agents are usually classified based upon their osmolality—high, low, and isosmolar. Iodinated contrast agents are also nephrotoxic in some but not all patients resulting in loss of glomerular filtration rate. Over the past 30 years, nephrotoxicity has been linked to osmolality although the precise mechanism underlying such a link has been elusive. Improvements in our understanding of the pathogenesis of nephrotoxicity and prospective randomized clinical trials have attempted to further explore the relationship between osmolality and nephrotoxicity. In this review, the basis for our current understanding that there are little if any differences in nephrotoxic potential between low and isosmolar contrast media will be detailed using data from clinical studies. PMID:24587997

  18. Impact of enrofloxacin on the human intestinal microbiota revealed by comparative molecular analysis.

    Science.gov (United States)

    Kim, Bong-Soo; Kim, Jong Nam; Yoon, Seok-Hwan; Chun, Jongsik; Cerniglia, Carl E

    2012-06-01

    The indigenous human intestinal microbiota could be disrupted by residues of antibiotics in foods as well as therapeutically administered antibiotics to humans. These disruptions may lead to adverse health outcomes. To observe the possible impact of residues of antibiotics at concentrations below therapeutic levels on human intestinal microbiota, we performed studies using in vitro cultures of fecal suspensions from three individuals with 10 different concentrations (0, 0.1, 0.5, 1, 5, 10, 15, 25, 50 and 150 μg/ml) of the fluoroquinolone, enrofloxacin. The bacterial communities of the control and enrofloxacin dosed fecal samples were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. In addition, changes of functional gene expression were analyzed by a pyrosequencing-based random whole-community mRNA sequencing method. Although each individual had a unique microbial composition, the communities of all individuals were affected by enrofloxacin. The proportions of two phyla, namely, Bacteroidetes and Proteobacteria, were significantly reduced with increasing concentrations of enrofloxacin exposure, while the proportion of Firmicutes increased. Principal Coordinate Analysis (PCoA) using the Fast UniFrac indicated that the community structures of intestinal microbiota were shifted by enrofloxacin. Most of the mRNA transcripts and the anti-microbial drug resistance genes increased with increasing concentrations of enrofloxacin. 16S rRNA gene pyrosequencing of control and enrofloxacin treated fecal suspensions provided valuable information of affected bacterial taxa down to the species level, and the community transcriptomic analyses using mRNA revealed the functional gene expression responses of the changed bacterial communities by enrofloxacin. Published by Elsevier Ltd.

  19. Bacterial tag encoded FLX titanium amplicon pyrosequencing (bTEFAP based assessment of prokaryotic diversity in metagenome of Lonar soda lake, India

    Directory of Open Access Journals (Sweden)

    Pravin Dudhagara

    2015-06-01

    Full Text Available Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP. Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%, Fibrobacter intestinalis (12% and Candidatus Cloacamonas acidaminovorans (11%. Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%, Methanoculleus chikugoensis (12% and Methanolinea tarda (11% were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.

  20. Optimization of biostimulant for bioremediation of contaminated coastal sediment by response surface methodology (RSM) and evaluation of microbial diversity by pyrosequencing.

    Science.gov (United States)

    Subha, Bakthavachallam; Song, Young Chae; Woo, Jung Hui

    2015-09-15

    The present study aims to optimize the slow release biostimulant ball (BSB) for bioremediation of contaminated coastal sediment using response surface methodology (RSM). Different bacterial communities were evaluated using a pyrosequencing-based approach in contaminated coastal sediments. The effects of BSB size (1-5cm), distance (1-10cm) and time (1-4months) on changes in chemical oxygen demand (COD) and volatile solid (VS) reduction were determined. Maximum reductions of COD and VS, 89.7% and 78.8%, respectively, were observed at a 3cm ball size, 5.5cm distance and 4months; these values are the optimum conditions for effective treatment of contaminated coastal sediment. Most of the variance in COD and VS (0.9291 and 0.9369, respectively) was explained in our chosen models. BSB is a promising method for COD and VS reduction and enhancement of SRB diversity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Performances of two biotrickling filters in treating H₂S-containing waste gases and analysis of corresponding bacterial communities by pyrosequencing.

    Science.gov (United States)

    Li, Jianjun; Ye, Guangyun; Sun, Duanfang; Sun, Guoping; Zeng, Xiaowei; Xu, Jian; Liang, Shizhong

    2012-09-01

    Two identical biotrickling filters named BTFa and BTFb were run in parallel to examine their performances in removing hydrogen sulfide. BTFa was filled with ceramic granules, and BTFb was filled with volcanic rocks. The results showed that BTFb was more robust than BTFa under acidic conditions. At empty bed residence times (EBRTs) of 20 and 15 s, the removal efficiency of BTFa was close to 100%. At EBRTs of 10 and 5 s, the removal efficiency of BTFa slightly decreased. The removal efficiencies of BTFa decreased by different degrees at the end of each stage, dropping to 94%, 81%, 60%, and 71%, respectively. However, the H(2)S removal efficiency in BTFb consistently reached 99% throughout the experiment. Pyrosequencing analyses indicated that members of Thiomonas dominated in both BTFs, but the relative abundance of Acidithiobacillus was higher in BTFb than in BTFa.

  2. Multiscale image contrast amplification (MUSICA)

    Science.gov (United States)

    Vuylsteke, Pieter; Schoeters, Emile P.

    1994-05-01

    This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

  3. Contrast enhancement CT by iopamidol

    International Nuclear Information System (INIS)

    Mori, Masaki; Makita, Nobue; Yanai, Kyoko

    1984-01-01

    In order to evaluate the contrast enhancement effect and safety of iopamidol (IOP) in CT examination, IOP was compared with angiographin (AG). In the liver and abdominal aorta, peak CT values were obtained earlier and were higher in the group with AG than in the group with IOP. However, CT values in the group with IOP decreased a little more slowly than those in the group with AG. There was no significant difference in the effect on contrast enhancement between the groups. Intravenous injection of IOP caused lower degree of burning sensation than that of AG, and some of the patients with IOP did not feel burning sensation at all. Changes in clinical laboratory values were slight before and after intravenous injection of IOP. These results suggest that IOP is satisfactory in terms of safety and effect on contrast enhancement in CT examination. (Namekawa, K.)

  4. Contrast-guided image interpolation.

    Science.gov (United States)

    Wei, Zhe; Ma, Kai-Kuang

    2013-11-01

    In this paper a contrast-guided image interpolation method is proposed that incorporates contrast information into the image interpolation process. Given the image under interpolation, four binary contrast-guided decision maps (CDMs) are generated and used to guide the interpolation filtering through two sequential stages: 1) the 45(°) and 135(°) CDMs for interpolating the diagonal pixels and 2) the 0(°) and 90(°) CDMs for interpolating the row and column pixels. After applying edge detection to the input image, the generation of a CDM lies in evaluating those nearby non-edge pixels of each detected edge for re-classifying them possibly as edge pixels. This decision is realized by solving two generalized diffusion equations over the computed directional variation (DV) fields using a derived numerical approach to diffuse or spread the contrast boundaries or edges, respectively. The amount of diffusion or spreading is proportional to the amount of local contrast measured at each detected edge. The diffused DV fields are then thresholded for yielding the binary CDMs, respectively. Therefore, the decision bands with variable widths will be created on each CDM. The two CDMs generated in each stage will be exploited as the guidance maps to conduct the interpolation process: for each declared edge pixel on the CDM, a 1-D directional filtering will be applied to estimate its associated to-be-interpolated pixel along the direction as indicated by the respective CDM; otherwise, a 2-D directionless or isotropic filtering will be used instead to estimate the associated missing pixels for each declared non-edge pixel. Extensive simulation results have clearly shown that the proposed contrast-guided image interpolation is superior to other state-of-the-art edge-guided image interpolation methods. In addition, the computational complexity is relatively low when compared with existing methods; hence, it is fairly attractive for real-time image applications.

  5. Iodinated contrast media nephrotoxicity. Nephrotoxicite des produits de contraste iodes

    Energy Technology Data Exchange (ETDEWEB)

    Meyrier, A. (Hopital Avicenne, 93 - Bobigny (France))

    1994-01-01

    In the late seventies, iodinated contrast agents (ICA) were considered to be a major cause of acute iatrogenic renal failure. Over the last decade new contrast agents have been synthesized, nonionic and less hyperosmolar. The incidence of acute renal failure due to ICAs, varies from 3.7 to 70% of cases according to the series, with an average figure of 10.2%. The pathophysiology of ICA nephrotoxicity was mainly studied in laboratory animal models. Three main factors are involved in an inducing ICA-mediated decrease in glomerular filtration rate: reduction of the renal plasma flow, a direct cytotoxic effect on renal tubular cells and erythrocyte alteration leading to intra-renal sludge. Excluding dysglobulinemias with urinary excretion of immunoglobulin light chains, which represent a special case of maximum nephrotoxicity, 4 main risk factors of renal toxicity have been identified in nondiabetic subjects: previous renal failure with serum creatinine levels greater than 140 [mu]mol per liter, extracellular dehydration, age over 60 and use of high doses of ICA and/or repeated ICA injections before serum creatinine levels return to baseline. Preventive measures for avoiding ICA nephrotoxicity are threefold: maintain or restore adequate hydration with saline infusion, stop NSAID treatment several days before ICA administration, and allow a 5 day interval before repeating contrast media injections. New, nonionic and moderately hyperosmolar contrast agents appear to be much less nephrotoxic than conventional ICAs in laboratory animals and in high-risk patients. It is advisable to select such contrast media for investigating high-risk patients. This approach was recently substantiated in well designed, randomized clinical studies which included more than 2 000 patients. (author).

  6. Quantitative contrast-enhanced mammography for contrast medium kinetics studies

    Science.gov (United States)

    Arvanitis, C. D.; Speller, R.

    2009-10-01

    Quantitative contrast-enhanced mammography, based on a dual-energy approach, aims to extract quantitative and temporal information of the tumour enhancement after administration of iodinated vascular contrast media. Simulations using analytical expressions and optimization of critical parameters essential for the development of quantitative contrast-enhanced mammography are presented. The procedure has been experimentally evaluated using a tissue-equivalent phantom and an amorphous silicon active matrix flat panel imager. The x-ray beams were produced by a tungsten target tube and spectrally shaped using readily available materials. Measurement of iodine projected thickness in mg cm-2 has been performed. The effect of beam hardening does not introduce nonlinearities in the measurement of iodine projected thickness for values of thicknesses found in clinical investigations. However, scattered radiation introduces significant deviations from slope equal to unity when compared with the actual iodine projected thickness. Scatter correction before the analysis of the dual-energy images provides accurate iodine projected thickness measurements. At 10% of the exposure used in clinical mammography, signal-to-noise ratios in excess of 5 were achieved for iodine projected thicknesses less than 3 mg cm-2 within a 4 cm thick phantom. For the extraction of temporal information, a limited number of low-dose images were used with the phantom incorporating a flow of iodinated contrast medium. The results suggest that spatial and temporal information of iodinated contrast media can be used to indirectly measure the tumour microvessel density and determine its uptake and washout from breast tumours. The proposed method can significantly improve tumour detection in dense breasts. Its application to perform in situ x-ray biopsy and assessment of the oncolytic effect of anticancer agents is foreseeable.

  7. Intensive care unit environmental surfaces are contaminated by multidrug-resistant bacteria in biofilms: combined results of conventional culture, pyrosequencing, scanning electron microscopy, and confocal laser microscopy.

    Science.gov (United States)

    Hu, H; Johani, K; Gosbell, I B; Jacombs, A S W; Almatroudi, A; Whiteley, G S; Deva, A K; Jensen, S; Vickery, K

    2015-09-01

    Hospital-associated infections cause considerable morbidity and mortality, and are expensive to treat. Organisms causing these infections can be sourced from the inanimate environment around a patient. Could the difficulty in eradicating these organisms from the environment be because they reside in dry surface biofilms? The intensive care unit (ICU) of a tertiary referral hospital was decommissioned and the opportunity to destructively sample clinical surfaces was taken in order to investigate whether multidrug-resistant organisms (MDROs) had survived the decommissioning process and whether they were present in biofilms. The ICU had two 'terminal cleans' with 500 ppm free chlorine solution; items from bedding, surrounds, and furnishings were then sampled with cutting implements. Sections were sonicated in tryptone soya broth and inoculated on to chromogenic plates to demonstrate MDROs, which were confirmed with the Vitek2 system. Genomic DNA was extracted directly from ICU samples, and subjected to polymerase chain reaction (PCR) for femA to detect Staphylococcus aureus and the microbiome by bacterial tag-encoded FLX amplicon pyrosequencing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed on environmental samples. Multidrug-resistant bacteria were cultured from 52% (23/44) of samples cultured. S. aureus PCR was positive in 50%. Biofilm was demonstrated in 93% (41/44) of samples by CLSM and/or SEM. Pyrosequencing demonstrated that the biofilms were polymicrobial and contained species that had multidrug-resistant strains. Dry surface biofilms containing MDROs are found on ICU surfaces despite terminal cleaning with chlorine solution. How these arise and how they might be removed requires further study. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Microbial community diversities and taxa abundances in soils along a seven-year gradient of potato monoculture using high throughput pyrosequencing approach.

    Directory of Open Access Journals (Sweden)

    Xing Liu

    Full Text Available BACKGROUND: Previous studies have focused on linking soil community structure, diversity, or specific taxa to disturbances. Relatively little attention has been directed to crop monoculture soils, particularly potato monoculture. Information about microbial community changes over time between monoculture and non-monoculture treatments is lacking. Furthermore, few studies have examined microbial communities in potato monoculture soils using a high throughput pyrosequencing approach. METHODOLOGY/PRINCIPAL FINDINGS: Soils along a seven-year gradient of potato monoculture were collected and microbial communities were characterized using high throughput pyrosequencing approach. Principal findings are as follows. First, diversity (H(Shannon and richness (S(Chao1 indices of bacterial community, but not of fungal community, were linearly decreased over time and corresponded to a decline of soil sustainability represented by yield decline and disease incidence increase. Second, Fusarium, the only soilborne pathogen-associated fungal genus substantially detected, was linearly increased over time in abundance and was closely associated with yield decline. Third, Fusarium abundance was negatively correlated with soil organic matter (OM and total nitrogen (TN but positively with electrical conductivity (EC. Fourth, Fusarium was correlated in abundances with 6 bacterial taxa over time. CONCLUSIONS: Soil bacterial and fungal communities exhibited differential responses to the potato monoculture. The overall soil bacterial communities were shaped by potato monoculture. Fusarium was the only soilborne pathogen-associated genus associated with disease incidence increase and yield decline. The changes of soil OM, TN and EC were responsible for Fusarium enrichment, in addition to selections by the monoculture crop. Acidobacteria and Nitrospirae were linearly decreased over time in abundance, corresponding to the decrease of OM, suggesting their similar

  9. The contrasting responses of soil microorganisms in two rice cultivars to elevated ground-level ozone

    International Nuclear Information System (INIS)

    Feng, Youzhi; Yu, Yongjie; Tang, Haoye; Zu, Qianhui; Zhu, Jianguo; Lin, Xiangui

    2015-01-01

    Although elevated ground-level O 3 has a species–specific impact on plant growth, the differences in soil biota responses to O 3 pollution among rice cultivars are rarely reported. Using O 3 Free-Air Concentration Enrichment, the responses of the rhizospheric bacterial communities in the O 3 -tolerant (YD6) and the O 3 -sensitive (IIY084) rice cultivars to O 3 pollution and their differences were assessed by pyrosequencing at rice tillering and anthesis stages. Elevated ground-level O 3 negatively influenced the bacterial community in cultivar YD6 at both rice growth stages by decreasing the bacterial phylogenetic diversities and response ratios. In contrast, in cultivar IIY084, the bacterial community responded positively at the rice tillering stage under O 3 pollution. However, several keystone bacterial guilds were consistently negatively affected by O 3 pollution in two rice cultivars. These findings indicate that continuously O 3 pollution would negatively influence rice agroecosystem and the crop cultivar is important in determining the soil biota responses to elevated O 3 . - Highlights: • We investigated the soil biota in two rice cultivars in presence of elevated O 3 . • The contrasting responses of soil biota were found between two rice cultivars. • Some keystone bacterial guilds were consistently negatively affected by O 3 pollution. • The crop cultivar is important in determining soil biota responses to elevated O 3 . - The crop cultivar is important in determining the soil biota responses to elevated O 3

  10. Ultrasound Contrast Agent Microbubble Dynamics

    NARCIS (Netherlands)

    Overvelde, M.L.J.; Vos, Henk; de Jong, N.; Versluis, Michel; Paradossi, Gaio; Pellegretti, Paolo; Trucco, Andrea

    2010-01-01

    Ultrasound contrast agents are traditionally used in ultrasound-assisted organ perfusion imaging. Recently the use of coated microbubbles has been proposed for molecular imaging applications where the bubbles are covered with a layer of targeting ligands to bind specifically to their target cells.

  11. Color contrasting in radioscopy systems

    International Nuclear Information System (INIS)

    Lopaev, V.P.; Pavlov, S.V.; Nazarenko, V.G.

    1979-01-01

    Transformation principles for achromatic radioscopy control systems to color ones have been considered. Described is the developed ''Gamma 1'' roentgen-TV facility with color contrasting, which is based on the principle of analog conversion of brightness signal to a hue. By means of color channels amplifiers realized are the special amplitude characteristics, permitting in comparison with the common method of analogous transformation to obtain the greater number of hues within the identical range of brightnesses of image under investigation due to introducing purple colors. The investigation of amplitude resolution capability of color contrasting device has shown, that in the case of color contrasting of image the amplitude resolution is 1.7-1.8 time higher than in the case of achromatic one. Defectoscopic sensitivity during the testing of 5-20 mm thick steel products in the process of experimental-production tests turned out to be 1.1-1.3 time higher when using color contrasting of radioscopic image. Realization simplicity, high resolution, noise stability and wide functional possibilities of the facility show the prospects for its using during the quality control of welded joints in products of power engineering

  12. Quantitative imaging by pixel-based contrast-enhanced ultrasound reveals a linear relationship between synovial vascular perfusion and the recruitment of pathogenic IL-17A-F+IL-23+ CD161+ CD4+ T helper cells in psoriatic arthritis joints.

    Science.gov (United States)

    Fiocco, Ugo; Stramare, Roberto; Martini, Veronica; Coran, Alessandro; Caso, Francesco; Costa, Luisa; Felicetti, Mara; Rizzo, Gaia; Tonietto, Matteo; Scanu, Anna; Oliviero, Francesca; Raffeiner, Bernd; Vezzù, Maristella; Lunardi, Francesca; Scarpa, Raffaele; Sacerdoti, David; Rubaltelli, Leopoldo; Punzi, Leonardo; Doria, Andrea; Grisan, Enrico

    2017-02-01

    To develop quantitative imaging biomarkers of synovial tissue perfusion by pixel-based contrast-enhanced ultrasound (CEUS), we studied the relationship between CEUS synovial vascular perfusion and the frequencies of pathogenic T helper (Th)-17 cells in psoriatic arthritis (PsA) joints. Eight consecutive patients with PsA were enrolled in this study. Gray scale CEUS evaluation was performed on the same joint immediately after joint aspiration, by automatic assessment perfusion data, using a new quantification approach of pixel-based analysis and the gamma-variate model. The set of perfusional parameters considered by the time intensity curve includes the maximum value (peak) of the signal intensity curve, the blood volume index or area under the curve, (BVI, AUC) and the contrast mean transit time (MTT). The direct ex vivo analysis of the frequencies of SF IL17A-F + CD161 + IL23 + CD4 + T cells subsets were quantified by fluorescence-activated cell sorter (FACS). In cross-sectional analyses, when tested for multiple comparison setting, a false discovery rate at 10%, a common pattern of correlations between CEUS Peak, AUC (BVI) and MTT parameters with the IL17A-F + IL23 + - IL17A-F + CD161 + - and IL17A-F + CD161 + IL23 + CD4 + T cells subsets, as well as lack of correlation between both peak and AUC values and both CD4 + T and CD4 + IL23 + T cells, was observed. The pixel-based CEUS assessment is a truly measure synovial inflammation, as a useful tool to develop quantitative imaging biomarker for monitoring target therapeutics in PsA.

  13. Contrast-enhanced MR angiography in Leriche's syndrome

    International Nuclear Information System (INIS)

    Link, J.; Steffens, J.C.; Brossmann, J.; Heller, M.; Loose, R.

    1998-01-01

    Purpose: To determine the usefulness of contrast-enhanced MR angiography for the diagnosis of Leriche's syndrome. Material and methods: Leriche's syndrome was seen in 7 patients via DSA. In [dition, contrast-enhanced MR angiography was performed (T R 7.8 ms/T E 2.1 ms, flip angle 30 , slab thickness 116 mm, slice thickness 1.82 mm, 64 partitions, FOV 500x438 mm, matrix 224x512). Results: Diagnosis of Leriche's syndrome was possible by contrast-enhanced MR angiography in each case. Visualisation of the femoral arteries was not possible in two patients by intraarterial DSA, in three other patients there was an insufficient contrast in the femoral arteries with DSA. Contrast-enhanced MR angiography revealed good visualisation of the femoral arteries in these patients. In [dition, contrast-enhanced MR angiography allowed complete visualisation of the patent lower limb arteries. In intraarterial DSA visualisation of the lower limb arteries was achieved reached in only one patient, but was incomplete. Conclusion: Contrast-enhanced MR angiography yielded the correct diagnosis of Leriche's syndrome in all 7 patients. Contrast-enhanced MR angiography was superior to DSA in the assessment of the distal run-off vessels in five of seven patients. (orig.) [de

  14. Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

    KAUST Repository

    Prest, Emmanuelle I E C; El Chakhtoura, Joline; Hammes, Frederik A.; Saikaly, Pascal; van Loosdrecht, Mark C.M.; Vrouwenvelder, Johannes S.

    2014-01-01

    concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two

  15. Ionic versus nonionic contrast media

    International Nuclear Information System (INIS)

    Zylak, C.J.; Gafni, A.

    1988-01-01

    The efficacy and effectiveness of the nonionic contrast media have been established. Widespread usage has been hampered because of the approximate tenfold increase in cost compared with the ionic media. An economic evaluation considering costs and consequences of both interventions (ionic vs nonionic contrast media) was performed; it is a cost effectiveness (CEA) and a cost-benefit analysis (CBA) for the Canadian experience. The results of the CEA demonstrate a value per life-year saved within an acceptable range when compared with value for quality-adjusted life years for programs such as treatment of severe (diastolic≥ 105 mm Hg) and mild (diastolic 95-104 mm Hg) hypertension in men aged 40. The CBA showed a net cost to society when benefits were measured as future treatment costs saved plus productivity gained. However, if people are willing to pay a small amount for the comfort of the new intervention, this will result in a break-even situation

  16. Contrast-enhanced MR angiography

    International Nuclear Information System (INIS)

    Bosmans, H.; Marchal, G.

    1996-01-01

    Despite many optimizations, the current limitations of plain MR angiography include: Saturation that impairs the visualization of veins and arteries with slow flow and spin-dephasing signal voids in locations with turbulent flow. Recently, the use of contrast agents has been proposed to cope with these remaining problems. Because of induced shortening of the T1 of the blood, saturation in the blood vessels is overcome. As a result, arteries and veins are visualized with the same signal intensity, which makes the technique less flow-dependent. In combination with short T1-weighted acquisitions, today CE MRA can be obtained while the patient is holding his breath. This last approach is most promising for abdominal applications since the respiratory motion can be frozen. As these acquisitions also use very short echo times, spin dephasing can be reduced. In conclusion, the use of contrast agents has greatly increased the clinical usefulness of MR angiography. (orig.) [de

  17. Minimum resolvable power contrast model

    Science.gov (United States)

    Qian, Shuai; Wang, Xia; Zhou, Jingjing

    2018-01-01

    Signal-to-noise ratio and MTF are important indexs to evaluate the performance of optical systems. However,whether they are used alone or joint assessment cannot intuitively describe the overall performance of the system. Therefore, an index is proposed to reflect the comprehensive system performance-Minimum Resolvable Radiation Performance Contrast (MRP) model. MRP is an evaluation model without human eyes. It starts from the radiance of the target and the background, transforms the target and background into the equivalent strips,and considers attenuation of the atmosphere, the optical imaging system, and the detector. Combining with the signal-to-noise ratio and the MTF, the Minimum Resolvable Radiation Performance Contrast is obtained. Finally the detection probability model of MRP is given.

  18. Effects of theophyline on contrast

    OpenAIRE

    A.R. Fatahiyan; B. Baqerii; A. Mohseni; A. Makhlouq

    2006-01-01

    Background and purpose: Contrast-induced nephropathy (CN) is one of the most common causes of iatrogenic acute renal failure. In fact CN is the third leading cause of new ARF in hospitalized patients. Radiocontrast-associated ARF is a significant problem in patients with cardiovascular disease. The risk factors for cardiovascular disease also predispose these patients to an increased risk of renal failure. Various strategies have been suggested for preventing CN. Since adenosine may play a ro...

  19. Contrast detail phantom for SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Cabrejas, M.L. de; Arashiro, J G; Giannone, C. [Comision Nacional de Energia Atomica, Buenos Aires (Argentina); Camuyrano, M; Nohara, G [Universidad de Buenos Aires, Buenos Aires (Argentina). Facultad Ciencias Exactas

    1996-06-01

    A new low variable contrast phantom for single photon emission computed tomography (SPECT) was constructed, tested and compared with other existing phantoms. It contains simulated cylindrical lesions of four different diameters (D{sub i}), embedded in a cylindrical scattering medium and a uniform section to evaluate tomographic uniformity. The concentration of tracer in the simulated lesions and the scattering medium (background) can be varied to simulate hot and cold lesions. Different applications of the phantom were tested, including determination of the minimum object contrast (OCm) necessary to detect lesions as a function of lesion size, lesion type (hot or cold) and acquisition and processing protocols by visual inspection. This parameter allows categorization of instruments comparing an `image quality index` (IQI). Preliminary comparison with the Britten contrast processing method showed that the detectable OCm was of the same order of magnitude, but the presented device seems more suitable for training and intercomparison purposes. The constructed phantom, of simple design, has proved to be useful for acquisition and processing condition evaluation, OCm estimation and external quality control. (author). 11 refs, 4 figs.

  20. Advancing High Contrast Adaptive Optics

    Science.gov (United States)

    Ammons, M.; Poyneer, L.; GPI Team

    2014-09-01

    A long-standing challenge has been to directly image faint extrasolar planets adjacent to their host suns, which may be ~1-10 million times brighter than the planet. Several extreme AO systems designed for high-contrast observations have been tested at this point, including SPHERE, Magellan AO, PALM-3000, Project 1640, NICI, and the Gemini Planet Imager (GPI, Macintosh et al. 2014). The GPI is the world's most advanced high-contrast adaptive optics system on an 8-meter telescope for detecting and characterizing planets outside of our solar system. GPI will detect a previously unstudied population of young analogs to the giant planets of our solar system and help determine how planetary systems form. GPI employs a 44x44 woofer-tweeter adaptive optics system with a Shack-Hartmann wavefront sensor operating at 1 kHz. The controller uses Fourier-based reconstruction and modal gains optimized from system telemetry (Poyneer et al. 2005, 2007). GPI has an apodized Lyot coronal graph to suppress diffraction and a near-infrared integral field spectrograph for obtaining planetary spectra. This paper discusses current performance limitations and presents the necessary instrumental modifications and sensitivity calculations for scenarios related to high-contrast observations of non-sidereal targets.

  1. Electrofluidic systems for contrast management

    Science.gov (United States)

    Rebello, Keith J.; Maranchi, Jeffrey P.; Tiffany, Jason E.; Brown, Christopher Y.; Maisano, Adam J.; Hagedon, Matthew A.; Heikenfeld, Jason C.

    2012-06-01

    Operating in dynamic lighting conditions and in greatly varying backgrounds is challenging. Current paints and state-ofthe- art passive adaptive coatings (e.g. photochromics) are not suitable for multi- environment situations. A semi-active, low power, skin is needed that can adapt its reflective properties based on the background environment to minimize contrast through the development and incorporation of suitable pigment materials. Electrofluidic skins are a reflective display technology for electronic ink and paper applications. The technology is similar to that in E Ink but makes use of MEMS based microfluidic structures, instead of simple black and white ink microcapsules dispersed in clear oil. Electrofluidic skin's low power operation and fast switching speeds (~20 ms) are an improvement over current state-ofthe- art contrast management technologies. We report on a microfluidic display which utilizes diffuse pigment dispersion inks to change the contrast of the underlying substrate from 5.8% to 100%. Voltage is applied and an electromechanical pressure is used to pull a pigment dispersion based ink from a hydrophobic coated reservoir into a hydrophobic coated surface channel. When no voltage is applied, the Young-Laplace pressure pushes the pigment dispersion ink back down into the reservoir. This allows the pixel to switch from the on and off state by balancing the two pressures. Taking a systems engineering approach from the beginning of development has enabled the technology to be integrated into larger systems.

  2. Attention enhances contrast appearance via increased input baseline of neural responses.

    Science.gov (United States)

    Cutrone, Elizabeth K; Heeger, David J; Carrasco, Marisa

    2014-12-30

    Covert spatial attention increases the perceived contrast of stimuli at attended locations, presumably via enhancement of visual neural responses. However, the relation between perceived contrast and the underlying neural responses has not been characterized. In this study, we systematically varied stimulus contrast, using a two-alternative, forced-choice comparison task to probe the effect of attention on appearance across the contrast range. We modeled performance in the task as a function of underlying neural contrast-response functions. Fitting this model to the observed data revealed that an increased input baseline in the neural responses accounted for the enhancement of apparent contrast with spatial attention. © 2014 ARVO.

  3. Contrasts and synergies in different biofuel reports.

    Science.gov (United States)

    Michalopoulos, A; Landeweerd, L; Van der Werf-Kulichova, Z; Puylaert, P G B; Osseweijer, P

    2011-04-06

    The societal debate on biofuels is characterised by increased complexity. This can hinder the effective governance of the field. This paper attempts a quantitative bird's eye meta-analysis of this complexity by mapping different stakeholder perspectives and expected outcomes as seen in the secondary literature on biofuels, along the lines of the People-Planet-Profit framework. Our analysis illustrates the tension between stated and actual drivers of large scale biofuel development, especially for first generation biofuels. Although environmental (Planet) aspects have dominated the biofuel debate, their overall assessment is mostly negative with regard to first generation biofuels. By contrast, economic (Profit) aspects are the only ones that are assessed positively with regard to first generation biofuels. Furthermore, positive and negative assessments of biofuel development are strongly influenced by the differences in focus between different stakeholder clusters. Stakeholders who appear generally supportive to biofuel development (industry) focus relatively more on aspects that are generally assessed as positive (Profit). By contrast, non-supportive stakeholders (NGO's) tend to focus mainly on aspects that are generally assessed as negative (Planet). Moreover, our analysis of reference lists revealed few citations of primary scientific data, and also that intergovernmental organizations produce the most influential publications in the debate. The surprising lack of listed references to scientific (primary) data reveals a need to assess in which arena the transition of scientific data towards secondary publications takes place, and how one can measure its quality. This work should be understood as a first effort to take some control over a complex and contradictory number of publications, and to allow the effective governance of the field through the identification of areas of overlapping consensus and persisting controversy, without reverting to claims on

  4. Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

    Science.gov (United States)

    Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

    2016-01-01

    Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

  5. Phase contrast STEM for thin samples: Integrated differential phase contrast.

    Science.gov (United States)

    Lazić, Ivan; Bosch, Eric G T; Lazar, Sorin

    2016-01-01

    It has been known since the 1970s that the movement of the center of mass (COM) of a convergent beam electron diffraction (CBED) pattern is linearly related to the (projected) electrical field in the sample. We re-derive a contrast transfer function (CTF) for a scanning transmission electron microscopy (STEM) imaging technique based on this movement from the point of view of image formation and continue by performing a two-dimensional integration on the two images based on the two components of the COM movement. The resulting integrated COM (iCOM) STEM technique yields a scalar image that is linear in the phase shift caused by the sample and therefore also in the local (projected) electrostatic potential field of a thin sample. We confirm that the differential phase contrast (DPC) STEM technique using a segmented detector with 4 quadrants (4Q) yields a good approximation for the COM movement. Performing a two-dimensional integration, just as for the COM, we obtain an integrated DPC (iDPC) image which is approximately linear in the phase of the sample. Beside deriving the CTFs of iCOM and iDPC, we clearly point out the objects of the two corresponding imaging techniques, and highlight the differences to objects corresponding to COM-, DPC-, and (HA) ADF-STEM. The theory is validated with simulations and we present first experimental results of the iDPC-STEM technique showing its capability for imaging both light and heavy elements with atomic resolution and a good signal to noise ratio (SNR). Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Contrast enhanced MRA: do contrast agents with a higher T1 relaxitivity improve the visualization of carotid artery stenoses?

    International Nuclear Information System (INIS)

    Friese, S.; Krapf, H.; Skalej, M.; Kueker, W.; Fetter, M.; Vonthein, R.

    2001-01-01

    CE-MRA is a powerful tool for the non-invasive evaluation of carotid artery occlusive disease. However, due to certain drawbacks, it has not completely replaced DSA. The purpose of this study was to evaluate if Gd-BOPTA, a contrast agent with high T 1 relaxivity, can increase the diagnostic accuracy of CE-MRA. Material and Methods: The CE-MRA examinations of 54 consecutive patients were evaluated by two experienced radiologists, independently. The examinations of 27 patients were contrasted either with 20 ml Gd-BOPTA or with 20 ml Gd-DTPA. The reviewers were blinded to the contrast agent chosen and to the ultrasound results. They rated the overall image quality and the degree of the ICA stenoses. Results: For the estimation of the degree of the ICA stenoses there was a high interrater validity. In comparison to the ultrasound findings, 6 of 50 high-degree stenoses were underestimated as moderate stenoses. In one of seven sonographically occluded vessels, MRA revealed residual patency in the vessel lumen. It was not possible to identify the contrast agent that was taken for a study. Subjective estimation of the image quality (arterial contrast of the ICA, contrast of the other vessels, and general impression) did not significantly change with the contrast agent employed. Conclusion: The diagnostic accuracy of CE-MRA for the evaluation of internal carotid artery stenoses is not improved by Gd-BOPTA if identical volumina of contrast media are applied. The potential of this contrast agent can be the reduction of the amount of contrast without loss of diagnostic information. Further studies are necessary. (orig.) [de

  7. The microbiome of Brazilian mangrove sediments as revealed by metagenomics.

    Directory of Open Access Journals (Sweden)

    Fernando Dini Andreote

    Full Text Available Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04 in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.

  8. The microbiome of Brazilian mangrove sediments as revealed by metagenomics.

    Science.gov (United States)

    Andreote, Fernando Dini; Jiménez, Diego Javier; Chaves, Diego; Dias, Armando Cavalcante Franco; Luvizotto, Danice Mazzer; Dini-Andreote, Francisco; Fasanella, Cristiane Cipola; Lopez, Maryeimy Varon; Baena, Sandra; Taketani, Rodrigo Gouvêa; de Melo, Itamar Soares

    2012-01-01

    Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2)S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.

  9. Radiographic contrast media, function and future reassessed

    International Nuclear Information System (INIS)

    Sovak, M.

    1988-01-01

    A brief review is given of the use of radiographic contrast media in cholecysto- and cholangiography, central nervous system imaging, urography and angiography. Other aspects briefly discussed are the use of large i.v. doses of contrast media for CT contrast enhancement and the design theory for improved water soluble contrast media. (U.K.)

  10. Ultrasound contrast agents: an overview.

    Science.gov (United States)

    Cosgrove, David

    2006-12-01

    With the introduction of microbubble contrast agents, diagnostic ultrasound has entered a new era that allows the dynamic detection of tissue flow of both the macro and microvasculature. Underpinning this development is the fact that gases are compressible, and thus the microbubbles expand and contract in the alternating pressure waves of the ultrasound beam, while tissue is almost incompressible. Special software using multiple pulse sequences separates these signals from those of tissue and displays them as an overlay or on a split screen. This can be done at low acoustic pressures (MIdeveloped for myocardial perfusion. In radiology, the most important application is the liver, especially for focal disease. The approach parallels that of dynamic CT or MRI but ultrasound has the advantages of high spatial and temporal resolution. Thus, small lesions that can be indeterminate on CT can often be studied with ultrasound, and situations where the flow is very rapid (e.g., focal nodular hyperplasia where the first few seconds of arterial perfusion may be critical to making the diagnosis) are readily studied. Microbubbles linger in the extensive sinusoidal space of normal liver for several minutes whereas they wash out rapidly from metastases, which have a low vascular volume and thus appear as filling defects. The method has been shown to be as sensitive as three-phase CT. Microbubbles have clinical uses in many other applications where knowledge of the microcirculation is important (the macrocirculation can usually be assessed adequately using conventional Doppler though there are a few important situations where the signal boost given by microbubbles is useful, e.g., transcranial Doppler for evaluating vasospasm after subarachnoid haemorrhage). An important situation where demonstrating tissue devitalisation is important is in interstitial ablation of focal liver lesions: using microbubble contrast agents at the end of a procedure allows immediate evaluation of the

  11. High throughput pyrosequencing technology for molecular differential detection of Babesia vogeli, Hepatozoon canis, Ehrlichia canis and Anaplasma platys in canine blood samples.

    Science.gov (United States)

    Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Kongklieng, Amornmas; Tantrawatpan, Chairat; Boonmars, Thidarut; Lulitanond, Viraphong; Taweethavonsawat, Piyanan; Chungpivat, Sudchit; Maleewong, Wanchai

    2014-06-01

    Canine babesiosis, hepatozoonosis, ehrlichiosis, and anaplasmosis are tick-borne diseases caused by different hemopathogens. These diseases are causes of morbidity and mortality in dogs. The classic method for parasite detection and differentiation is based on microscopic observation of blood smears. The limitations of the microscopic method are that its performance requires a specially qualified person with professional competence, and it is ineffective in differentiating closely related species. This study applied PCR amplification with high throughput pyrosequencing for molecular differential detection of the following 4 hemoparasites common to tropical areas in dog blood samples: Babesia vogeli, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. PCR was initially used to amplify specific target regions of the ribosomal RNA genes of each parasite using 2 primer pairs that included 18S rRNA for protozoa (B. vogeli and H. canis) and 16S rRNA for rickettsia (E. canis and A. platys). Babesia vogeli and H. canis were discriminated using 9 nucleotide positions out of 30 base pairs, whereas E. canis and A. platys were differentiated using 15 nucleotide positions out of 34 base pairs that were determined from regions adjacent to 3' ends of the sequencing primers. This method provides a challenging alternative for a rapid diagnosis and surveillance of these tick-borne diseases in canines. Copyright © 2014 Elsevier GmbH. All rights reserved.

  12. Characterization of the Drug Resistance Profiles of Patients Infected with CRF07_BC Using Phenotypic Assay and Ultra-Deep Pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Szu-Wei Huang

    Full Text Available The usefulness of ultra-deep pyrosequencing (UDPS for the diagnosis of HIV-1 drug resistance (DR remains to be determined. Previously, we reported an explosive outbreak of HIV-1 circulating recombinant form (CRF 07_BC among injection drug users (IDUs in Taiwan in 2004. The goal of this study was to characterize the DR of CRF07_BC strains using different assays including UDPS. Seven CRF07_BC isolates including 4 from early epidemic (collected in 2004-2005 and 3 from late epidemic (collected in 2008 were obtained from treatment-naïve patient's peripheral blood mononuclear cells. Viral RNA was extracted directly from patient's plasma or from cultural supernatant and the pol sequences were determined using RT-PCR sequencing or UDPS. For comparison, phenotypic drug susceptibility assay using MAGIC-5 cells (in-house phenotypic assay and Antivirogram were performed. In-house phenotypic assay showed that all the early epidemic and none of the late epidemic CRF07_BC isolates were resistant to most protease inhibitors (PIs (4.4-47.3 fold. Neither genotypic assay nor Antivirogram detected any DR mutations. UDPS showed that early epidemic isolates contained 0.01-0.08% of PI DR major mutations. Furthermore, the combinations of major and accessory PI DR mutations significantly correlated with the phenotypic DR. The in-house phenotypic assay is superior to other conventional phenotypic assays in the detection of DR variants with a frequency as low as 0.01%.

  13. The Analysis of a Microbial Community in the UV/O3-Anaerobic/Aerobic Integrated Process for Petrochemical Nanofiltration Concentrate (NFC Treatment by 454-Pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Chao Wei

    Full Text Available In this study, high-throughput pyrosequencing was applied on the analysis of the microbial community of activated sludge and biofilm in a lab-scale UV/O3- anaerobic/aerobic (A/O integrated process for the treatment of petrochemical nanofiltration concentrate (NFC wastewater. NFC is a type of saline wastewater with low biodegradability. From the anaerobic activated sludge (Sample A and aerobic biofilm (Sample O, 59,748 and 51,231 valid sequence reads were obtained, respectively. The dominant phylotypes related to the metabolism of organic compounds, polycyclic aromatic hydrocarbon (PAH biodegradation, assimilation of carbon from benzene, and the biodegradation of nitrogenous organic compounds were detected as genus Clostridium, genera Pseudomonas and Stenotrophomonas, class Betaproteobacteria, and genus Hyphomicrobium. Furthermore, the nitrite-oxidising bacteria Nitrospira, nitrite-reducing and sulphate-oxidising bacteria (NR-SRB Thioalkalivibrio were also detected. In the last twenty operational days, the total Chemical Oxygen Demand (COD and Total Organic Carbon (TOC removal efficiencies on average were 64.93% and 62.06%, respectively. The removal efficiencies of ammonia nitrogen and Total Nitrogen (TN on average were 90.51% and 75.11% during the entire treatment process.

  14. Real-time monitoring of methane oxidation in a simulated landfill cover soil and MiSeq pyrosequencing analysis of the related bacterial community structure.

    Science.gov (United States)

    Xing, Zhilin; Zhao, Tiantao; Gao, Yanhui; He, Zhi; Zhang, Lijie; Peng, Xuya; Song, Liyan

    2017-10-01

    Real-time CH 4 oxidation in a landfill cover soil was studied using automated gas sampling that determined biogas (CH 4 and CO 2 ) and O 2 concentrations at various depths in a simulated landfill cover soil (SLCS) column reactor. The real-time monitoring system obtained more than 10,000 biogas (CH 4 and CO 2 ) and O 2 data points covering 32 steady states of CH 4 oxidation with 32 different CH 4 fluxes (0.2-125mol·m -2 ·d -1 ). The kinetics of CH 4 oxidation at different depths (0-20cm, 20-40cm, and 40-60cm) of SLCS were well fit by a CH 4 -O 2 dual-substrate model based on 32 values (averaged, n=5-15) of equilibrated CH 4 concentrations. The quality of the fit (R 2 ranged from 0.90 to 0.96) was higher than those reported in previous studies, which suggests that real time monitoring is beneficial for CH 4 oxidation simulations. MiSeq pyrosequencing indicated that CH 4 flux events changed the bacterial community structure (e.g., increased the abundance of Bacteroidetes and Methanotrophs) and resulted in a relative increase in the amount of type I methanotrophs (Methylobacter and Methylococcales) and a decrease in the amount of type II methanotrophs (Methylocystis). Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Pyrosequencing analysis of free-living and attached bacterial communities in Meiliang Bay, Lake Taihu, a large eutrophic shallow lake in China.

    Science.gov (United States)

    Tang, Xiangming; Li, Linlin; Shao, Keqiang; Wang, Boweng; Cai, Xianlei; Zhang, Lei; Chao, Jianying; Gao, Guang

    2015-01-01

    To elucidate the relationship between particle-attached (PA, ≥ 5.0 μm) and free-living (FL, 0.2-5.0 μm) bacterial communities, samplings were collected seasonally from November 2011 to August 2012 in Meiliang Bay, Lake Taihu, China. We used 454 pyrosequencing of 16S rRNA genes to study bacterial diversity and structure of PA and FL communities. The analysis rendered 37,985 highly qualified reads, subsequently assigned to 1755 operational taxonomic units (97% similarity) for the 8 samples. Although 27 high-level taxonomic groups were obtained, the 3 dominant phyla (Proteobacteria, Actinobacteria, and Bacteroidetes) comprised about 75.9% and 82.4% of the PA and FL fractions, respectively. Overall, we found no significant differences between community types, as indicated by ANOSIM R statistics (R = 0.063, P > 0.05) and the Parsimony test (P = 0.222). Dynamics of bacterial communities were correlated with changes in concentrations of total suspended solids (TSS) and total phosphorus (TP). In summer, a significant taxonomic overlap in the 2 size fractions was observed when Cyanobacteria, a major contributor of TSS and TP, dominated in the water, highlighting the potential rapid exchange between PA and FL bacterial populations in large shallow eutrophic lakes.

  16. Composition of Groundwater Bacterial Communities before and after Air Surging in a Groundwater Heat Pump System According to a Pyrosequencing Assay

    Directory of Open Access Journals (Sweden)

    Heejung Kim

    2017-11-01

    Full Text Available The geothermal energy of groundwater has aroused increasing interest as a solution to climate change. The groundwater heat pumps (GWHP system using groundwater is the most environmentally friendly system to date and has been examined in several studies. However, biological clogging by microorganisms negatively affects the thermal efficiency of the GWHP system. In this study, we employed air surging, the most popular among well management methods, and pyrosequencing to analyze the genetic diversity in bacteria before and after air surging in a geothermal well. Furthermore, the diversity of dominant bacterial genera and those related to clogging were evaluated. The bacterial diversity of the groundwater well increased after air surging. Nevertheless, the proportion of bacterial genera thought to be related to microbiological clogging decreased. In cooling and heating systems based on the geothermal energy of groundwater, the wells should be maintained regularly by air surging to reduce efficiency problems caused by microbiological clogging and to prevent secondary damage to human health, e.g., pneumonia due to human pathogenic bacteria including Pseudomonas aeruginosa and Acinetobacter.

  17. The Analysis of a Microbial Community in the UV/O3-Anaerobic/Aerobic Integrated Process for Petrochemical Nanofiltration Concentrate (NFC) Treatment by 454-Pyrosequencing

    Science.gov (United States)

    Wei, Chao; He, Wenjie; Wei, Li; Li, Chunying; Ma, Jun

    2015-01-01

    In this study, high-throughput pyrosequencing was applied on the analysis of the microbial community of activated sludge and biofilm in a lab-scale UV/O3- anaerobic/aerobic (A/O) integrated process for the treatment of petrochemical nanofiltration concentrate (NFC) wastewater. NFC is a type of saline wastewater with low biodegradability. From the anaerobic activated sludge (Sample A) and aerobic biofilm (Sample O), 59,748 and 51,231 valid sequence reads were obtained, respectively. The dominant phylotypes related to the metabolism of organic compounds, polycyclic aromatic hydrocarbon (PAH) biodegradation, assimilation of carbon from benzene, and the biodegradation of nitrogenous organic compounds were detected as genus Clostridium, genera Pseudomonas and Stenotrophomonas, class Betaproteobacteria, and genus Hyphomicrobium. Furthermore, the nitrite-oxidising bacteria Nitrospira, nitrite-reducing and sulphate-oxidising bacteria (NR-SRB) Thioalkalivibrio were also detected. In the last twenty operational days, the total Chemical Oxygen Demand (COD) and Total Organic Carbon (TOC) removal efficiencies on average were 64.93% and 62.06%, respectively. The removal efficiencies of ammonia nitrogen and Total Nitrogen (TN) on average were 90.51% and 75.11% during the entire treatment process. PMID:26461260

  18. Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and Sanger sequencing: Mafa-class I polymorphism.

    Science.gov (United States)

    Shiina, Takashi; Yamada, Yukiho; Aarnink, Alice; Suzuki, Shingo; Masuya, Anri; Ito, Sayaka; Ido, Daisuke; Yamanaka, Hisashi; Iwatani, Chizuru; Tsuchiya, Hideaki; Ishigaki, Hirohito; Itoh, Yasushi; Ogasawara, Kazumasa; Kulski, Jerzy K; Blancher, Antoine

    2015-10-01

    Although the low polymorphism of the major histocompatibility complex (MHC) transplantation genes in the Filipino cynomolgus macaque (Macaca fascicularis) is expected to have important implications in the selection and breeding of animals for medical research, detailed polymorphism information is still lacking for many of the duplicated class I genes. To better elucidate the degree and types of MHC polymorphisms and haplotypes in the Filipino macaque population, we genotyped 127 unrelated animals by the Sanger sequencing method and high-resolution pyrosequencing and identified 112 different alleles, 28 at cynomolgus macaque MHC (Mafa)-A, 54 at Mafa-B, 12 at Mafa-I, 11 at Mafa-E, and seven at Mafa-F alleles, of which 56 were newly described. Of them, the newly discovered Mafa-A8*01:01 lineage allele had low nucleotide similarities (Filipino macaque population would identify these and other high-frequency Mafa-class I haplotypes that could be used as MHC control animals for the benefit of biomedical research.

  19. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

    Directory of Open Access Journals (Sweden)

    Paula González-Alonso

    2015-08-01

    Full Text Available Mutations in Human Epidermal Growth Factor Receptors (HER are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS, alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC, ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  20. Clinical applications of contrast echocardiography

    International Nuclear Information System (INIS)

    Jorge, Leon Galindo

    2005-01-01

    The echocardiography is the technique more used for the diagnosis and pursuit of the cardiovascular illnesses; therefore, their diagnostic precision has acquired a vital importance in the handling of the patients with cardiovascular pathologies. However, with relative frequency, the diagnostic capacity of the echocardiography exam is diminished by limitations of the acoustic window, mainly in-patient with obesity, lung illnesses and alterations of the thoracic wall. This can be obviated with the use of the intra-esophagus echocardiography, although this it is a procedure semi-invasive and not very practical of carrying out in all the patients with problems of acoustic window. In this article the clinical applications are revised more common of the contrast echocardiography

  1. Ultrasound contrast agents: An overview

    International Nuclear Information System (INIS)

    Cosgrove, David

    2006-01-01

    With the introduction of microbubble contrast agents, diagnostic ultrasound has entered a new era that allows the dynamic detection of tissue flow of both the macro and microvasculature. Underpinning this development is the fact that gases are compressible, and thus the microbubbles expand and contract in the alternating pressure waves of the ultrasound beam, while tissue is almost incompressible. Special software using multiple pulse sequences separates these signals from those of tissue and displays them as an overlay or on a split screen. This can be done at low acoustic pressures (MI < 0.3) so that the microbubbles are not destroyed and scanning can continue in real time. The clinical roles of contrast enhanced ultrasound scanning are expanding rapidly. They are established in echocardiography to improve endocardial border detection and are being developed for myocardial perfusion. In radiology, the most important application is the liver, especially for focal disease. The approach parallels that of dynamic CT or MRI but ultrasound has the advantages of high spatial and temporal resolution. Thus, small lesions that can be indeterminate on CT can often be studied with ultrasound, and situations where the flow is very rapid (e.g., focal nodular hyperplasia where the first few seconds of arterial perfusion may be critical to making the diagnosis) are readily studied. Microbubbles linger in the extensive sinusoidal space of normal liver for several minutes whereas they wash out rapidly from metastases, which have a low vascular volume and thus appear as filling defects. The method has been shown to be as sensitive as three-phase CT. Microbubbles have clinical uses in many other applications where knowledge of the microcirculation is important (the macrocirculation can usually be assessed adequately using conventional Doppler though there are a few important situations where the signal boost given by microbubbles is useful, e.g., transcranial Doppler for evaluating

  2. Phase contrast STEM for thin samples: Integrated differential phase contrast

    International Nuclear Information System (INIS)

    Lazić, Ivan; Bosch, Eric G.T.; Lazar, Sorin

    2016-01-01

    It has been known since the 1970s that the movement of the center of mass (COM) of a convergent beam electron diffraction (CBED) pattern is linearly related to the (projected) electrical field in the sample. We re-derive a contrast transfer function (CTF) for a scanning transmission electron microscopy (STEM) imaging technique based on this movement from the point of view of image formation and continue by performing a two-dimensional integration on the two images based on the two components of the COM movement. The resulting integrated COM (iCOM) STEM technique yields a scalar image that is linear in the phase shift caused by the sample and therefore also in the local (projected) electrostatic potential field of a thin sample. We confirm that the differential phase contrast (DPC) STEM technique using a segmented detector with 4 quadrants (4Q) yields a good approximation for the COM movement. Performing a two-dimensional integration, just as for the COM, we obtain an integrated DPC (iDPC) image which is approximately linear in the phase of the sample. Beside deriving the CTFs of iCOM and iDPC, we clearly point out the objects of the two corresponding imaging techniques, and highlight the differences to objects corresponding to COM-, DPC-, and (HA) ADF-STEM. The theory is validated with simulations and we present first experimental results of the iDPC-STEM technique showing its capability for imaging both light and heavy elements with atomic resolution and a good signal to noise ratio (SNR). - Highlights: • First DPC-based atomic resolution images of potential and charge density are obtained. • This is enabled by integration and differentiation of 2D DPC signals, respectively. • Integrated DPC (iDPC) based on 4 quadrant imaging is compared to iCOM imaging. • Noise analysis and comparison with standard STEM imaging modes is provided. • iDPC allows direct imaging of light (C, N, O …) and heavy (Ga, Au …) atoms together.

  3. Phase contrast STEM for thin samples: Integrated differential phase contrast

    Energy Technology Data Exchange (ETDEWEB)

    Lazić, Ivan, E-mail: ivan.lazic@fei.com; Bosch, Eric G.T.; Lazar, Sorin

    2016-01-15

    It has been known since the 1970s that the movement of the center of mass (COM) of a convergent beam electron diffraction (CBED) pattern is linearly related to the (projected) electrical field in the sample. We re-derive a contrast transfer function (CTF) for a scanning transmission electron microscopy (STEM) imaging technique based on this movement from the point of view of image formation and continue by performing a two-dimensional integration on the two images based on the two components of the COM movement. The resulting integrated COM (iCOM) STEM technique yields a scalar image that is linear in the phase shift caused by the sample and therefore also in the local (projected) electrostatic potential field of a thin sample. We confirm that the differential phase contrast (DPC) STEM technique using a segmented detector with 4 quadrants (4Q) yields a good approximation for the COM movement. Performing a two-dimensional integration, just as for the COM, we obtain an integrated DPC (iDPC) image which is approximately linear in the phase of the sample. Beside deriving the CTFs of iCOM and iDPC, we clearly point out the objects of the two corresponding imaging techniques, and highlight the differences to objects corresponding to COM-, DPC-, and (HA) ADF-STEM. The theory is validated with simulations and we present first experimental results of the iDPC-STEM technique showing its capability for imaging both light and heavy elements with atomic resolution and a good signal to noise ratio (SNR). - Highlights: • First DPC-based atomic resolution images of potential and charge density are obtained. • This is enabled by integration and differentiation of 2D DPC signals, respectively. • Integrated DPC (iDPC) based on 4 quadrant imaging is compared to iCOM imaging. • Noise analysis and comparison with standard STEM imaging modes is provided. • iDPC allows direct imaging of light (C, N, O …) and heavy (Ga, Au …) atoms together.

  4. Molecular Techniques Revealed Highly Diverse Microbial Communities in Natural Marine Biofilms on Polystyrene Dishes for Invertebrate Larval Settlement

    KAUST Repository

    Lee, On On

    2014-01-09

    Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups. © 2014 Springer Science+Business Media New York.

  5. Multiscale differential phase contrast analysis with a unitary detector

    KAUST Repository

    Lopatin, Sergei; Ivanov, Yurii P.; Kosel, Jü rgen; Chuvilin, Andrey

    2015-01-01

    A new approach to generate differential phase contrast (DPC) images for the visualization and quantification of local magnetic fields in a wide range of modern nano materials is reported. In contrast to conventional DPC methods our technique utilizes the idea of a unitary detector under bright field conditions, making it immediately usable by a majority of modern transmission electron microscopes. The approach is put on test to characterize the local magnetization of cylindrical nanowires and their 3D ordered arrays, revealing high sensitivity of our method in a combination with nanometer-scale spatial resolution.

  6. Temporal adaptation enhances efficient contrast gain control on natural images.

    Directory of Open Access Journals (Sweden)

    Fabian Sinz

    Full Text Available Divisive normalization in primary visual cortex has been linked to adaptation to natural image statistics in accordance to Barlow's redundancy reduction hypothesis. Using recent advances in natural image modeling, we show that the previously studied static model of divisive normalization is rather inefficient in reducing local contrast correlations, but that a simple temporal contrast adaptation mechanism of the half-saturation constant can substantially increase its efficiency. Our findings reveal the experimentally observed temporal dynamics of divisive normalization to be critical for redundancy reduction.

  7. Multiscale differential phase contrast analysis with a unitary detector

    KAUST Repository

    Lopatin, Sergei

    2015-12-30

    A new approach to generate differential phase contrast (DPC) images for the visualization and quantification of local magnetic fields in a wide range of modern nano materials is reported. In contrast to conventional DPC methods our technique utilizes the idea of a unitary detector under bright field conditions, making it immediately usable by a majority of modern transmission electron microscopes. The approach is put on test to characterize the local magnetization of cylindrical nanowires and their 3D ordered arrays, revealing high sensitivity of our method in a combination with nanometer-scale spatial resolution.

  8. Evaluation of contrast in duplicated radiographs

    International Nuclear Information System (INIS)

    Thunthy, K.H.; Weinberg, R.

    1982-01-01

    This investigation evaluated changes in the contrast of duplicated radiographs made at different ultraviolet light exposures. Increasing ultraviolet light exposure had different effects on the duplicates of originals of different background densities. When correctly exposed, a duplicate radiograph enhanced contrast. When originals had the same contrast but different background densities, their duplicates did not have the same contrast. It was not possible to duplicate accurately all the different contrasts measured on an original. It was possible, however, to produce duplicates with all contrasts greater than those of the original

  9. Binocular contrast discrimination needs monocular multiplicative noise

    Science.gov (United States)

    Ding, Jian; Levi, Dennis M.

    2016-01-01

    The effects of signal and noise on contrast discrimination are difficult to separate because of a singularity in the signal-detection-theory model of two-alternative forced-choice contrast discrimination (Katkov, Tsodyks, & Sagi, 2006). In this article, we show that it is possible to eliminate the singularity by combining that model with a binocular combination model to fit monocular, dichoptic, and binocular contrast discrimination. We performed three experiments using identical stimuli to measure the perceived phase, perceived contrast, and contrast discrimination of a cyclopean sine wave. In the absence of a fixation point, we found a binocular advantage in contrast discrimination both at low contrasts (discrimination mechanisms: a nonlinear contrast transducer and multiplicative noise (MN). A binocular combination model (the DSKL model; Ding, Klein, & Levi, 2013b) was first fitted to both the perceived-phase and the perceived-contrast data sets, then combined with either the nonlinear contrast transducer or the MN mechanism to fit the contrast-discrimination data. We found that the best model combined the DSKL model with early MN. Model simulations showed that, after going through interocular suppression, the uncorrelated noise in the two eyes became anticorrelated, resulting in less binocular noise and therefore a binocular advantage in the discrimination task. Combining a nonlinear contrast transducer or MN with a binocular combination model (DSKL) provides a powerful method for evaluating the two putative contrast-discrimination mechanisms. PMID:26982370

  10. Brute force absorption contrast microtomography

    Science.gov (United States)

    Davis, Graham R.; Mills, David

    2014-09-01

    In laboratory X-ray microtomography (XMT) systems, the signal-to-noise ratio (SNR) is typically determined by the X-ray exposure due to the low flux associated with microfocus X-ray tubes. As the exposure time is increased, the SNR improves up to a point where other sources of variability dominate, such as differences in the sensitivities of adjacent X-ray detector elements. Linear time-delay integration (TDI) readout averages out detector sensitivities on the critical horizontal direction and equiangular TDI also averages out the X-ray field. This allows the SNR to be increased further with increasing exposure. This has been used in dentistry to great effect, allowing subtle variations in dentine mineralisation to be visualised in 3 dimensions. It has also been used to detect ink in ancient parchments that are too damaged to physically unroll. If sufficient contrast between the ink and parchment exists, it is possible to virtually unroll the tomographic image of the scroll in order that the text can be read. Following on from this work, a feasibility test was carried out to determine if it might be possible to recover images from decaying film reels. A successful attempt was made to re-create a short film sequence from a rolled length of 16mm film using XMT. However, the "brute force" method of scaling this up to allow an entire film reel to be imaged presents a significant challenge.

  11. Integral equations with contrasting kernels

    Directory of Open Access Journals (Sweden)

    Theodore Burton

    2008-01-01

    Full Text Available In this paper we study integral equations of the form $x(t=a(t-\\int^t_0 C(t,sx(sds$ with sharply contrasting kernels typified by $C^*(t,s=\\ln (e+(t-s$ and $D^*(t,s=[1+(t-s]^{-1}$. The kernel assigns a weight to $x(s$ and these kernels have exactly opposite effects of weighting. Each type is well represented in the literature. Our first project is to show that for $a\\in L^2[0,\\infty$, then solutions are largely indistinguishable regardless of which kernel is used. This is a surprise and it leads us to study the essential differences. In fact, those differences become large as the magnitude of $a(t$ increases. The form of the kernel alone projects necessary conditions concerning the magnitude of $a(t$ which could result in bounded solutions. Thus, the next project is to determine how close we can come to proving that the necessary conditions are also sufficient. The third project is to show that solutions will be bounded for given conditions on $C$ regardless of whether $a$ is chosen large or small; this is important in real-world problems since we would like to have $a(t$ as the sum of a bounded, but badly behaved function, and a large well behaved function.

  12. Renal streaky artifact during contrast-enhanced abdominal and pelvic CT: Comparison of high versus low osmolality contrast media

    International Nuclear Information System (INIS)

    Kim, Dae Hong; Kim, Jong Chul; Lee, Chung Keun; Shin, Kyoung Suk

    1994-01-01

    Introduction of low osmolality contrast agent(LOCA) has allowed safer, more comfortable contrast-enhanced CT examination, but there has been significant increase in image degradation when evaluating the kidneys due to streaky artifact. The authors reviewed findings of contrast- enhanced abdominal and pelvic computed tomography(CT) to know the difference of renal streaky artifact between a high osmolality contrast agent (HOCA) and LOCA. This study included two hundred contrast-enhanced CT in 200 patients, 100 performed with HOCA(meglumine ioglicate, 150 ml) and 100 performed with LOCA (iopromide,150 ml). The severity of renal streaky artifact was compared between HOCA and LOCA groups. Of the scans performed with HOCA, 40 had no artifact, 52 had grade I artifact, 6 had grade II artifact, and 2 had grade III artifact. Of the scans preformed with LOCA, 23 had no artifact, 44 had grade I artifact, 29 had grade II artifact, and 4 had grade III artifact. There was significant difference in the degree of the streaky artifact depending upon the osmolality of the contrast media used(by χ 2 -test, P=.0001). The results of this study revealed a statistically significant increased incidence of artifacts and distortions of renal image with LOCA when compared with HOCA

  13. Contrast enhanced CT of spinal cord angioma

    International Nuclear Information System (INIS)

    Nakamura, Takahiko; Ebitani, Tsutomu; Honma, Takao; Sofue, Muroto; Nakamura, Shigeru

    1982-01-01

    Contrast enhanced CT on 6 patients with spinal cord angioma showed enhancement in 2 of them. The conditions to produce contrast enhancement were the window width of 100 - 200, and the window level of 0 - 50. In spinal cord angioma, contrast enhanced CT is presently only an adjunct to angiography and myelography. Nevertheless, contrast enhanced CT is useful in the screening test for spinal cord angioma, in the patients who are nonindicated to angiography, and in the postoperative follow-up. (Ueda, J.)

  14. Contrast reaction training in US radiology residencies: a COARDRI study.

    Science.gov (United States)

    LeBedis, Christina A; Rosenkrantz, Andrew B; Otero, Hansel J; Decker, Summer J; Ward, Robert J

    To perform a survey-based assessment of current contrast reaction training in US diagnostic radiology residency programs. An electronic survey was distributed to radiology residency program directors from 9/2015-11/2015. 25.7% of programs responded. 95.7% of those who responded provide contrast reaction management training. 89.4% provide didactic lectures (occurring yearly in 71.4%). 37.8% provide hands-on simulation training (occurring yearly in 82.3%; attended by both faculty and trainees in 52.9%). Wide variability in contrast reaction education in US diagnostic radiology residency programs reveals an opportunity to develop and implement a national curriculum. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Using Contrastive Rhetoric in the ESL Classroom

    Science.gov (United States)

    Quinn, Janet M.

    2012-01-01

    Contrastive rhetoric studies the writing of second language learners to understand how it is affected by their first language and culture. The field of contrastive rhetoric is as multidimensional as second language writing is complex. It draws on the work of contrastive analysis, anthropology, linguistics, pedagogy, culture studies, translation…

  16. Contrast media are helpful in computerized tomography

    International Nuclear Information System (INIS)

    Anon.

    1987-01-01

    Contrast media very often can decisively improve the image contrasts, and thus contribute to making the differential diagnosis more specific. This survey of a symposium on the current status of CT refers particularly to the lectures that discussed the use of contrast media for neuroradiology, and the risks involved. (orig.) [de

  17. Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.

    Science.gov (United States)

    Boyer, Stephane; Brown, Samuel D J; Collins, Rupert A; Cruickshank, Robert H; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D

    2012-01-01

    DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.

  18. Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.

    Directory of Open Access Journals (Sweden)

    Stephane Boyer

    Full Text Available DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI is over 600 base pairs (bp, amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential 'mini-barcodes' for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R. This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey DNA from 46 landsnail (predator faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1 when dealing with degraded DNA for which only small fragments can be amplified, (2 for cases where no consensus has yet been reached on the appropriate barcode gene, or (3 to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.

  19. Algorithms for contrast enhancement of electronic portal images

    International Nuclear Information System (INIS)

    Díez, S.; Sánchez, S.

    2015-01-01

    An implementation of two new automatized image processing algorithms for contrast enhancement of portal images is presented as suitable tools which facilitate the setup verification and visualization of patients during radiotherapy treatments. In the first algorithm, called Automatic Segmentation and Histogram Stretching (ASHS), the portal image is automatically segmented in two sub-images delimited by the conformed treatment beam: one image consisting of the imaged patient obtained directly from the radiation treatment field, and the second one is composed of the imaged patient outside it. By segmenting the original image, a histogram stretching can be independently performed and improved in both regions. The second algorithm involves a two-step process. In the first step, a Normalization to Local Mean (NLM), an inverse restoration filter is applied by dividing pixel by pixel a portal image by its blurred version. In the second step, named Lineally Combined Local Histogram Equalization (LCLHE), the contrast of the original image is strongly improved by a Local Contrast Enhancement (LCE) algorithm, revealing the anatomical structures of patients. The output image is lineally combined with a portal image of the patient. Finally the output images of the previous algorithms (NLM and LCLHE) are lineally combined, once again, in order to obtain a contrast enhanced image. These two algorithms have been tested on several portal images with great results. - Highlights: • Two Algorithms are implemented to improve the contrast of Electronic Portal Images. • The multi-leaf and conformed beam are automatically segmented into Portal Images. • Hidden anatomical and bony structures in portal images are revealed. • The task related to the patient setup verification is facilitated by the contrast enhancement then achieved.

  20. Single and double contrast arthrography in lesions of the glenohumeral joint

    International Nuclear Information System (INIS)

    Ahovuo, J.

    1984-01-01

    One hundred and forty-five arthrograms from single and double contrast arthrography were compared with respect to their accuracy in revealing lesions of the glenohumeral joint. Total and partial rotator cuff tears were accurately delineated by both single and double contrast arthrography. The size of the tear did not correlate with surgical findings with either technique. The volume of the injected contrast medium was larger in full-thickness rotator cuff tears than in normal arthrograms (p 2 test). No difference in filling frequency of the sheath could be observed between single and double contrast arthrography. This study did not reveal any major advantages of double contrast arthrography over single contrast examinations in lesions of the rotator cuff and those of the tendon of the long head of the biceps brachii. (orig.)

  1. Effect of intensifying screens to subject contrast

    International Nuclear Information System (INIS)

    Lee, In Ja; Huh, Joon; Kang, Hong Seok; Yoon, Chul Ho

    1990-01-01

    It is an experimental report about object contrast according to the Kinds of screen and obtained results as follows ; 1. In the same object radiography, use acquired the difference of the object contrast between LT-II(CaWO 4 system) and G4(Gd system) screens. 2. object contrast more decreased in Gd system screen than CaWO 4 and object contrast controlled by X-ray tube voltage. 3. The difference of object contrast seen clearly in 80 kVp

  2. Contrast Enhanced US in the Abdomen

    International Nuclear Information System (INIS)

    Chung, Yong Eun; Kim, Ki Whang

    2012-01-01

    Contrast enhanced ultrasound, which was introduced in 1996, has been widely used in Europe and Eastern Asia. Ultrasound contrast agent can be classified as first generation and second generation, depending on the gas within the microbubble. With the first generation contrast agent, the high MI technique was used, and only intermittent scanning was possible due to destruction of the microbubble during scanning. Use of the second generation contrast agent with the low MI technique makes continuous scanning possible. Contrast enhanced US can be used in detection and differentiation of focal liver lesions. It is also helpful for monitoring of radiofrequency ablation and for targeting of US guided biopsy. Currently, because morphologic criteria alone may not reflect the response of the tumor to treatment, new criteria are needed for treatment evaluation after administration of anti-angiogenic agents. Contrast enhanced US could provide quantitative markers for evaluation of the response to treatment via use of dynamic contrast enhanced US. Due to cost-effectiveness, contrast enhanced US is not yet widely used in Korea; however, considering recent issues regarding contrast agent related adverse reaction, such as contrast induced nephropathy and nephrogenic systemic fibrosis, and radiation exposure, contrast enhanced US might be more widely used in Korea, as an alternative imaging modality in the future.

  3. MRI and CT contrast media extravasation

    Science.gov (United States)

    Heshmatzadeh Behzadi, Ashkan; Farooq, Zerwa; Newhouse, Jeffery H.; Prince, Martin R.

    2018-01-01

    Abstract Background: This systematic review combines data from multiple papers on contrast media extravasation to identify factors contributing to increased extravasation risk. Methods: Data were extracted from 17 papers reporting 2191 extravasations in 1,104,872 patients (0.2%) undergoing computed tomography (CT) or magnetic resonance imaging (MRI). Results: Extravasation rates were 0.045% for gadolinium-based contrast agents (GBCA) and nearly 6-fold higher, 0.26% for iodinated contrast agents. Factors associated with increased contrast media extravasations included: older age, female gender, using an existing intravenous (IV) instead of placing a new IV in radiology, in-patient status, use of automated power injection, high injection rates, catheter location, and failing to warm up the more viscous contrast media to body temperature. Conclusion: Contrast media extravasation is infrequent but nearly 6 times less frequent with GBCA for MRI compared with iodinated contrast used in CT. PMID:29489663

  4. Contrasting styles of deep-marine pyroclastic eruptions revealed from Axial Seamount push core records

    Science.gov (United States)

    Portner, Ryan A.; Clague, David A.; Helo, Christoph; Dreyer, Brian M.; Paduan, Jennifer B.

    2015-08-01

    A comprehensive understanding of explosive basaltic eruption processes in the deep-sea relies upon detailed analysis and comparison of the variety of volcaniclastic lithologies on the seafloor, which has been challenged by insufficient sample recovery. A dedicated ROV-based sampling approach using long push cores offers an unparalleled opportunity to fully characterize the diversity of unconsolidated volcaniclastic lithofacies on a recently active seamount. Lithofacies from Axial Seamount record two styles of pyroclastic eruptions, strombolian and phreatomagmatic, at 1.5 km water depth. Strombolian eruptions are represented by abundant fluidal and highly vesicular (up to 50%) vitriclasts within limu o Pele lapilli tuff and tuffaceous mud lithofacies. Lapilli-ash grain size, normal grading, good sorting, rip-up clasts and homogeneous glass geochemistry characterize individual limu o Pele lapilli tuff beds, and imply proximal deposition from a turbidity flow associated with a single eruption (i.e. event bed). Limu o Pele lapilli tuff beds are interbedded with poorly sorted, chemically heterogeneous and bioturbated tuffaceous mud units that preserve reworking and biologic habitation of more distal pyroclastic fallout and dilute turbidity flows. The phreatomagmatic eruption style is preserved by hydrothermal mineral-bearing muddy tuff that exhibits characteristics distinct from lapilli ash and tuffaceous mud lithofacies. Hydrothermal muddy tuff lithofacies are well-sorted and fine-grained with notable components of non-fluidal basaltic ash (∼45%), fluidal ash (∼30%) and accessory lithics (∼25%). Heterogeneous geochemistry of ash shards implies that juvenile components are minimal. The abundance, mineralogy and texture of lithic components (Fe-Mg clays, pyrite, epidote, actinolite, altered glass, basalt/diabase, hydrothermal breccia and agglutinate), and very fine-grain size of basaltic ash, are consistent with phreatomagmatic eruption deposits. A lack of bioturbation or other interbedded lithofacies, and presence of normal grading suggests prolonged eruption activity and deposition via turbidity flows or suspension fallout. The proximity of ancient hydrothermal muddy tuff lithofacies and active hydrothermal vents to caldera walls suggest that phreatomagmatic activity was linked to shallow circulation of fluids along caldera ring-faults rooted to underlying magma conduits and shallow reservoirs. This study provides evidence for two distinctly different pyroclastic eruption styles and provides a framework to further develop existing models of deep-sea explosive volcanism.

  5. Comment on "Satellites reveal contrasting responses of regional climate to the widespread greening of Earth".

    Science.gov (United States)

    Li, Yue; Zeng, Zhenzhong; Huang, Ling; Lian, Xu; Piao, Shilong

    2018-06-15

    Forzieri et al (Reports, 16 June 2017, p. 1180) used satellite data to show that boreal greening caused regional warming. We show that this positive sensitivity of temperature to the greening can be derived from the positive response of vegetation to boreal warming, which indicates that results from a statistical regression with satellite data should be carefully interpreted. Copyright © 2018, American Association for the Advancement of Science.

  6. Drought stress tolerance strategies revealed by RNA-Seq in two sorghum genotypes with contrasting WUE

    NARCIS (Netherlands)

    Fracasso, Alessandra; Trindade, Luisa M.; Amaducci, Stefano

    2016-01-01

    Background: Drought stress is the major environmental stress that affects plant growth and productivity. It triggers a wide range of responses detectable at molecular, biochemical and physiological levels. At the molecular level the response to drought stress results in the differential

  7. Multi-decadal analysis reveals contrasting patterns of resilience and decline in coral assemblages

    Science.gov (United States)

    Tanner, Jason E.

    2017-12-01

    A 50-yr study of coral dynamics at Heron Island, on Australia's Great Barrier Reef, shows that community change on a single reef is highly variable and that while some areas of the reef are in decline, others are recovering well 40 yr after a major cyclonic disturbance that eliminated all corals in the study plot. At one site, the genus-level composition in 2012 was identical to that before the cyclone, although it took around 30 yr to recover, and there were still differences at the species level. The colony size structure of some species at this site, notably the dominant Acropora digitifera, which had over 40% cover in 2012, also recovered after 30 yr, although sub-dominant species still lacked large colonies even after 40 yr. Given the small scale of the individual study plots (1 m2), this shows an unexpected degree of determinism in assemblage structure. At a second site, however, both composition and size structure changed dramatically over the last 40 yr of the study as both external and internal factors altered local environmental conditions. At both sites, major changes in composition appear to be related to drying out of the reef crest due to changes in flow regimes and/or natural accretion. At the site that has recovered, erosion has reversed this drying out, whereas no such erosion has occurred at the second site. If such erosion occurs, or sea levels increase due to global warming, then the second site may also prove to be resilient over decadal time scales.

  8. Puffins reveal contrasting relationships between forage fish and ocean climate in the North Pacific

    Science.gov (United States)

    Sydeman, William J.; Piatt, John F.; Thompson, Sarah Ann; Garcia-Reyes, Marisol; Hatch, Scott A.; Arimitsu, Mayumi L.; Slater, Leslie; Williams, Jeffrey C.; Rojek, Nora A.; Zador, Stephani G.; Renner, Heather M.

    2017-01-01

    Long-term studies of predator food habits (i.e., ‘predator-based sampling’) are useful for identifying patterns of spatial and temporal variability of forage nekton in marine ecosystems. We investigated temporal changes in forage fish availability and relationships to ocean climate by analyzing diet composition of three puffin species (horned puffin Fratercula corniculata, tufted puffin Fratercula cirrhata, and rhinoceros auklet Cerorhinca monocerata) from five sites in the North Pacific from 1978–2012. Dominant forage species included squids and hexagrammids in the western Aleutians, gadids and Pacific sand lance (Ammodytes personatus) in the eastern Aleutians and western Gulf of Alaska (GoA), and sand lance and capelin (Mallotus villosus) in the northern and eastern GoA. Interannual fluctuations in forage availability dominated variability in the western Aleutians, whereas lower-frequency shifts in forage fish availability dominated elsewhere. We produced regional multivariate indicators of sand lance, capelin, and age-0 gadid availability by combining data across species and sites using Principal Component Analysis, and related these indices to environmental factors including sea level pressure (SPL), winds, and sea surface temperature (SST). There was coherence in the availability of sand lance and capelin across the study area. Sand lance availability increased linearly with environmental conditions leading to warmer ocean temperatures, whereas capelin availability increased in a non-linear manner when environmental changes led to lower ocean temperatures. Long-term studies of puffin diet composition appear to be a promising tool for understanding the availability of these difficult-to-survey forage nekton in remote regions of the North Pacific.

  9. Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis?

    Science.gov (United States)

    Gaberel, Thomas; Gakuba, Clement; Goulay, Romain; Martinez De Lizarrondo, Sara; Hanouz, Jean-Luc; Emery, Evelyne; Touze, Emmanuel; Vivien, Denis; Gauberti, Maxime

    2014-10-01

    The aim of the present study was to investigate the impact of different stroke subtypes on the glymphatic system using MRI. We first improved and characterized an in vivo protocol to measure the perfusion of the glymphatic system using MRI after minimally invasive injection of a gadolinium chelate within the cisterna magna. Then, the integrity of the glymphatic system was evaluated in 4 stroke models in mice including subarachnoid hemorrhage (SAH), intracerebral hemorrhage, carotid ligature, and embolic ischemic stroke. We were able to reliably evaluate the glymphatic system function using MRI. Moreover, we provided evidence that the glymphatic system was severely impaired after SAH and in the acute phase of ischemic stroke, but was not altered after carotid ligature or in case of intracerebral hemorrhage. Notably, this alteration in glymphatic perfusion reduced brain clearance rate of low-molecular-weight compounds. Interestingly, glymphatic perfusion after SAH can be improved by intracerebroventricular injection of tissue-type plasminogen activator. Moreover, spontaneous arterial recanalization was associated with restoration of the glymphatic function after embolic ischemic stroke. SAH and acute ischemic stroke significantly impair the glymphatic system perfusion. In these contexts, injection of tissue-type plasminogen activator either intracerebroventricularly to clear perivascular spaces (for SAH) or intravenously to restore arterial patency (for ischemic stroke) may improve glymphatic function. © 2014 American Heart Association, Inc.

  10. Comparative phylogeography of three trematomid fishes reveals contrasting genetic structure patterns in benthic and pelagic species

    Czech Academy of Sciences Publication Activity Database

    Van de Putte, A. P.; Janko, Karel; Kašparová, Eva; Maes, G. E.; Rock, J.; Koubbi, P.; Volckaert, F. A. M.; Choleva, Lukáš; Fraser, K. P. P.; Smykla, J.; Van Houdt, J. K. J.; Marschall, C.

    2012-01-01

    Roč. 8, č. 1 (2012), s. 23-34 ISSN 1874-7787 R&D Projects: GA AV ČR KJB600450903 Institutional research plan: CEZ:AV0Z50450515 Keywords : microsatellite markers * mitochondrial * larval duration Subject RIV: EG - Zoology Impact factor: 1.339, year: 2012

  11. Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Ayaka Yamamuro

    Full Text Available Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2 (based on the projected area of the anode. In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

  12. Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

    Science.gov (United States)

    Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

    2014-01-01

    Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2) (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

  13. Bacterial Pathogens and Community Composition in Advanced Sewage Treatment Systems Revealed by Metagenomics Analysis Based on High-Throughput Sequencing

    Science.gov (United States)

    Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

    2015-01-01

    This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence. PMID:25938416

  14. Flash Infrared Thermography Contrast Data Analysis Technique

    Science.gov (United States)

    Koshti, Ajay

    2014-01-01

    This paper provides information on an IR Contrast technique that involves extracting normalized contrast versus time evolutions from the flash thermography inspection infrared video data. The analysis calculates thermal measurement features from the contrast evolution. In addition, simulation of the contrast evolution is achieved through calibration on measured contrast evolutions from many flat-bottom holes in the subject material. The measurement features and the contrast simulation are used to evaluate flash thermography data in order to characterize delamination-like anomalies. The thermal measurement features relate to the anomaly characteristics. The contrast evolution simulation is matched to the measured contrast evolution over an anomaly to provide an assessment of the anomaly depth and width which correspond to the depth and diameter of the equivalent flat-bottom hole (EFBH) similar to that used as input to the simulation. A similar analysis, in terms of diameter and depth of an equivalent uniform gap (EUG) providing a best match with the measured contrast evolution, is also provided. An edge detection technique called the half-max is used to measure width and length of the anomaly. Results of the half-max width and the EFBH/EUG diameter are compared to evaluate the anomaly. The information provided here is geared towards explaining the IR Contrast technique. Results from a limited amount of validation data on reinforced carbon-carbon (RCC) hardware are included in this paper.

  15. Contrast media. Safety issues and ESUR guidelines

    Energy Technology Data Exchange (ETDEWEB)

    Thomsen, H.S. (ed.) [Copenhagen Univ. Hospital, Herlev (Denmark). Dept. of Diagnostic Radiology 54E2

    2006-07-01

    In 1994 the European Society of Urogenital Radiology (ESUR) set up a committee to consider the safety of the contrast media used in radiology departments. Since then, the committee has questioned members, reviewed the literature, proposed guidelines, and discussed these proposals with participants at the annual symposia on urogenital radiology. This book represents the end result of this hard work. It contains all of the agreed guidelines, updated when necessary, and thereby comprehensively covers the many different safety issues relating to the diverse contrast media: barium contrast media, iodinated contrast media, MR contrast media (both gadolinium-based extracellular and organ-specific) and ultrasound contrast media. The prevention and treatment of both acute and delayed non-renal adverse reactions as well as the renal adverse reactions are covered in detail. The inclusion of all the ESUR guidelines within one book will offer an invaluable, unique and unparalleled resource. (orig.)

  16. Contrast media. Safety issues and ESUR guidelines

    International Nuclear Information System (INIS)

    Thomsen, H.S.

    2006-01-01

    In 1994 the European Society of Urogenital Radiology (ESUR) set up a committee to consider the safety of the contrast media used in radiology departments. Since then, the committee has questioned members, reviewed the literature, proposed guidelines, and discussed these proposals with participants at the annual symposia on urogenital radiology. This book represents the end result of this hard work. It contains all of the agreed guidelines, updated when necessary, and thereby comprehensively covers the many different safety issues relating to the diverse contrast media: barium contrast media, iodinated contrast media, MR contrast media (both gadolinium-based extracellular and organ-specific) and ultrasound contrast media. The prevention and treatment of both acute and delayed non-renal adverse reactions as well as the renal adverse reactions are covered in detail. The inclusion of all the ESUR guidelines within one book will offer an invaluable, unique and unparalleled resource. (orig.)

  17. Contrast-enhanced harmonic endoscopic ultrasound

    DEFF Research Database (Denmark)

    Săftoiu, A; Dietrich, C F; Vilmann, P

    2012-01-01

    Second-generation intravenous blood-pool ultrasound contrast agents are increasingly used in endoscopic ultrasound (EUS) for characterization of microvascularization, differential diagnosis of benign and malignant focal lesions, and improving staging and guidance of therapeutic procedures. Although...... initially used as Doppler signal enhancers, second-generation microbubble contrast agents are now used with specific contrast harmonic imaging techniques, which benefit from the highly nonlinear behavior of the microbubbles. Contrast-specific modes based on multi-pulse technology are used to perform...... contrast-enhanced harmonic EUS based on a very low mechanical index (0.08 - 0.12). Quantification techniques based on dynamic contrast-enhanced ultrasound have been recommended for perfusion imaging and monitoring of anti-angiogenic treatment, mainly based on time-intensity curve analysis. Most...

  18. Multi-step contrast sensitivity gauge

    Science.gov (United States)

    Quintana, Enrico C; Thompson, Kyle R; Moore, David G; Heister, Jack D; Poland, Richard W; Ellegood, John P; Hodges, George K; Prindville, James E

    2014-10-14

    An X-ray contrast sensitivity gauge is described herein. The contrast sensitivity gauge comprises a plurality of steps of varying thicknesses. Each step in the gauge includes a plurality of recesses of differing depths, wherein the depths are a function of the thickness of their respective step. An X-ray image of the gauge is analyzed to determine a contrast-to-noise ratio of a detector employed to generate the image.

  19. Contrast settling in cerebral aneurysm angiography

    International Nuclear Information System (INIS)

    Wang Zhijie; Hoffmann, Kenneth R; Guterman, Lee R; Wang Zhou; Rudin, Stephen; Meng Hui

    2005-01-01

    During angiography, blood flow is visualized with a radiopaque contrast agent, which is denser than blood. In complex vasculature, such as cerebral saccular aneurysms, the density difference may produce an appreciable gravity effect, where the contrast material separates from blood and settles along the gravity direction. Although contrast settling has been occasionally reported before, the fluid mechanics behind it have not been explored. Furthermore, the severity of contrast settling in cerebral aneurysms varies significantly from case to case. Therefore, a better understanding of the physical principles behind this phenomenon is needed to evaluate contrast settling in clinical angiography. In this study, flow in two identical groups of sidewall aneurysm models with varying parent-vessel curvature was examined by angiography. Intravascular stents were deployed into one group of the models. To detect contrast settling, we used lateral view angiography. Time-intensity curves were analysed from the angiographic data, and a computational fluid dynamic analysis was conducted. Results showed that contrast settling was strongly related to the local flow dynamics. We used the Froude number, a ratio of flow inertia to gravity force, to characterize the significance of gravity force. An aneurysm with a larger vessel curvature experienced higher flow, which resulted in a larger Froude number and, thus, less gravitational settling. Addition of a stent reduced the aneurysmal flow, thereby increasing the contrast settling. We found that contrast settling resulted in an elevated washout tail in the time-intensity curve. However, this signature is not unique to contrast settling. To determine whether contrast settling is present, a lateral view should be obtained in addition to the anteroposterior (AP) view routinely used clinically so as to rule out contrast settling and hence to enable a valid time-intensity curve analysis of blood flow in the aneurysm

  20. Identification of IL-28B Genotype Modification in Hepatocytes after Living Donor Liver Transplantation by Laser Capture Microdissection and Pyrosequencing Analysis

    Directory of Open Access Journals (Sweden)

    King-Wah Chiu

    2018-01-01

    Full Text Available The aim of this study is to elucidate the biogenetic modification of donor and recipient interleukin-28B (IL-28B genotypes in liver graft biopsies after living donor liver transplantation (LDLT for chronic hepatitis C virus- (HCV- related, end-stage liver disease. Fifty liver graft biopsies were collected from recipients during LDLT treatment for HCV-related, end-stage liver disease. DNA was extracted from all 50 liver tissues, and the IL-28B single-nucleotide polymorphisms (SNPs rs8099917 and rs12979860 were studied for allelic discrimination by real-time PCR analysis. Blood samples were obtained from donors and recipients on postoperative day 0 (POD0, POD7, and POD30. We randomly selected five liver biopsies and isolated the hepatocytes by laser capture microdissection (LCM to evaluate genotype modifications resulting from LDLT. After LDLT, the IL-28B SNP rs8099917 was identified not only in the liver graft biopsies and donors’ sera (TT = 41 : 43; GT = 9 : 5; GG = 0 : 2, but also in liver graft biopsies and recipients’ sera on POD0 (TT = 41 : 44; GT = 9 : 4; GG = 0 : 2, POD7 (TT = 41 : 30; GT = 9 : 18; GG = 0 : 2, and POD30 (TT = 41 : 29; GT = 9 : 19; GG = 0 : 2. A significant difference was observed between the rs8099917 allele frequencies of liver graft biopsies and recipients’ sera on POD30 (p=0.039. In addition, a significant difference was also noted between the rs12979860 allele frequencies of liver graft biopsies and donors’ sera (CT = 49 : 39; TT = 1 : 10 (p=0.012 and of liver graft biopsies and recipients’ sera on POD0 (CT = 49 : 39; TT = 1 : 11 (p=0.002, POD7 (CT = 49 : 42; TT = 1 : 8 (p=0.016, and POD30 (CT = 49 : 41; TT = 1 : 9 (p=0.008. This phenomenon was confirmed by pyrosequencing of hepatocytes isolated by LCM. Following LDLT, the TT-to-GT IL-28B genotype modification predominated in rs8099917, and the CC-to-CT modification predominated

  1. Transcriptomic analysis of grain amaranth (Amaranthus hypochondriacus using 454 pyrosequencing: comparison with A. tuberculatus, expression profiling in stems and in response to biotic and abiotic stress

    Directory of Open Access Journals (Sweden)

    Vargas-Ortiz Erandi

    2011-07-01

    Full Text Available Abstract Background Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs, transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scale transcriptome analysis was performed to generate stem- and (abiotic stress-responsive gene expression profiles in grain amaranth. Results A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs. The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing

  2. Contrast media on abdominal computed tomography

    International Nuclear Information System (INIS)

    Skalpe, I.O.; Oestensen, H.M.

    1984-01-01

    Abdominal computed tomography was performed in 55 patients before and after intravenous injection of 60 ml of a non-ionic (iohexol) or an ionic (metrizoate) contrast medium. The adverse effects were recorded and a series of measurements of attenuation values before and after the contrast medium injection was performed in the aorta and in hepatic and renal parenchyma. Only minor adverse effects were seen with both contrast media, but iohexol was clearly better tolerated than metrizoate. No difference in the enhancement properties was found between the two contrast media. (orig.)

  3. An Improved Image Contrast Assessment Method

    Directory of Open Access Journals (Sweden)

    Yuanyuan Fan

    2013-07-01

    Full Text Available Contrast is an important factor affecting the image quality. In order to overcome the problems of local band-limited contrast, a novel image contrast assessment method based on the property of HVS is proposed. Firstly, the image by low-pass filter is performed fast wavelet decomposition. Secondly, all levels of band-pass filtered image and its corresponding low-pass filtered image are obtained by processing wavelet coefficients. Thirdly, local band-limited contrast is calculated, and the local band-limited contrast entropy is calculated according to the definition of entropy, Finally, the contrast entropy of image is obtained by averaging the local band-limited contrast entropy weighed using CSF coefficient. The experiment results show that the best contrast image can be accurately identified in the sequence images obtained by adjusting the exposure time and stretching gray respectively, the assessment results accord with human visual characteristics and make up the lack of local band-limited contrast.

  4. Contrast enhanced ultrasound in liver imaging

    International Nuclear Information System (INIS)

    Nielsen, Michael Bachmann; Bang, Nanna

    2004-01-01

    Ultrasound contrast agents were originally introduced to enhance the Doppler signals when detecting vessels with low velocity flow or when imaging conditions were sub-optimal. Contrast agents showed additional properties, it was discovered that a parenchymal enhancement phase in the liver followed the enhancement of the blood pool. Contrast agents have made ultrasound scanning more accurate in detection and characterization of focal hepatic lesions and the sensitivity is now comparable with CT and MRI scanning. Further, analysis of the transit time of contrast agent through the liver seems to give information on possible hepatic involvement, not only from focal lesions but also from diffuse benign parenchymal disease. The first ultrasound contrast agents were easily destroyed by the energy from the sound waves but newer agents have proved to last for longer time and hereby enable real-time scanning and make contrast enhancement suitable for interventional procedures such as biopsies and tissue ablation. Also, in monitoring the effect of tumour treatment contrast agents have been useful. A brief overview is given on some possible applications and on different techniques using ultrasound contrast agents in liver imaging. At present, the use of an ultrasound contrast agent that allows real-time scanning with low mechanical index is to be preferred

  5. Polymeric nanoparticles as OCT contrast agents

    Energy Technology Data Exchange (ETDEWEB)

    Al Rawashdeh, Wa' el [RWTH Aachen University, Experimental Molecular Imaging (Germany); Kray, Stefan [RWTH Aachen University, Institute for Semiconductor Electronics (Germany); Pich, Andrij; Pargen, Sascha; Balaceanu, Andreea [RWTH Aachen University, Interactive Material Research (DWI) (Germany); Lenz, Markus; Spoeler, Felix [RWTH Aachen University, Institute for Semiconductor Electronics (Germany); Kiessling, Fabian, E-mail: fkiessling@ukaachen.de; Lederle, Wiltrud [RWTH Aachen University, Experimental Molecular Imaging (Germany)

    2012-12-15

    In this study, the optical properties of two nano-sized polymer colloids in optical coherence tomography (OCT) were compared in vitro with respect to their potential use as contrast agents. We used two types of particles: compact hydrophobic spherical polystyrene (PS) particles and soft water-swollen nanogel (NG) particles both with grafted hydrophilic shell, both prepared at two different sizes (PS at 300 and 150 nm, NG at 300 and 200 nm). The OCT backscattering signals of the particles in a vessel-mimicking highly scattering agar/TiO{sub 2} phantom were compared on either number of particles or weight percent. Larger particles and higher concentrations produced higher OCT contrast. At each concentration tested, a markedly higher contrast was achieved by PS particles than NG particles. PS particles generated a markedly higher OCT contrast than the phantom at concentrations of at least 1 Multiplication-Sign 10{sup 10} or 0.1 % for PS 300 nm and at least 3 Multiplication-Sign 10{sup 11} particles/mL or 0.4 % for PS 150 nm. The contrast generated by NG 300 nm was above the phantom contrast at concentrations of at least 3 Multiplication-Sign 10{sup 11} particles/mL or 1 %, whereas NG 200 nm only at 4 %. At any given weight percent, the differences in OCT contrast between differently sized particles were much less evident than in the comparison based on particle number. PS 300 nm generated also a good contrast ex vivo on chicken muscle tissue. These results strongly suggest that PS spheres have strong potential as intravascular OCT contrast agent, while NG particles need further contrast enhancer for being used as OCT contrast agent.

  6. Motion contrast using optical coherence tomography

    Science.gov (United States)

    Fingler, Jeffrey Paul

    Diagnosis of ophthalmic diseases like age-related macular degeneration is very important for treatment of the disease as well as the development of future treatments. Optical coherence tomography (OCT) is an optical interference technique which can measure the three-dimensional structural information of the reflecting layers within a sample. In retinal imaging, OCT is used as the primary diagnostic tool for structural abnormalities such as retinal holes and detachments. The contrast within the images of this technique is based upon reflectivity changes from different regions of the retina. This thesis demonstrates the developments of methods used to produce additional contrast to the structural OCT images based on the tiny fluctuations of motion experienced by the mobile scatterers within a sample. Motion contrast was observed for motions smaller than 50 nm in images of a variety of samples. Initial contrast method demonstrations used Brownian motion differences to separate regions of a mobile Intralipid solution from a static agarose gel, chosen in concentration to minimize reflectivity contrast. Zebrafish embryos in the range of 3-4 days post fertilization were imaged using several motion contrast methods to determine the capabilities of identifying regions of vascular flow. Vasculature identification was demonstrated in zebrafish for blood vessels of all orientations as small as 10 microns in diameter. Mouse retinal imaging utilized the same motion contrast methods to determine the contrast capabilities for motions associated with vasculature within the retina. Improved contrast imaging techniques demonstrated comparable images to fluorescein angiography, the gold standard of retinal vascular imaging. Future studies can improve the demonstrated contrast analysis techniques and apply them towards human retinal motion contrast imaging for ophthalmic diagnostic purposes.

  7. CHANGE DETECTION VIA SELECTIVE GUIDED CONTRASTING FILTERS

    Directory of Open Access Journals (Sweden)

    Y. V. Vizilter

    2017-05-01

    Full Text Available Change detection scheme based on guided contrasting was previously proposed. Guided contrasting filter takes two images (test and sample as input and forms the output as filtered version of test image. Such filter preserves the similar details and smooths the non-similar details of test image with respect to sample image. Due to this the difference between test image and its filtered version (difference map could be a basis for robust change detection. Guided contrasting is performed in two steps: at the first step some smoothing operator (SO is applied for elimination of test image details; at the second step all matched details are restored with local contrast proportional to the value of some local similarity coefficient (LSC. The guided contrasting filter was proposed based on local average smoothing as SO and local linear correlation as LSC. In this paper we propose and implement new set of selective guided contrasting filters based on different combinations of various SO and thresholded LSC. Linear average and Gaussian smoothing, nonlinear median filtering, morphological opening and closing are considered as SO. Local linear correlation coefficient, morphological correlation coefficient (MCC, mutual information, mean square MCC and geometrical correlation coefficients are applied as LSC. Thresholding of LSC allows operating with non-normalized LSC and enhancing the selective properties of guided contrasting filters: details are either totally recovered or not recovered at all after the smoothing. These different guided contrasting filters are tested as a part of previously proposed change detection pipeline, which contains following stages: guided contrasting filtering on image pyramid, calculation of difference map, binarization, extraction of change proposals and testing change proposals using local MCC. Experiments on real and simulated image bases demonstrate the applicability of all proposed selective guided contrasting filters. All

  8. High-contrast MacNeille-PBS-based LCOS projection systems

    Science.gov (United States)

    Chen, Jianmin; Robinson, Michael G.; Sharp, Gary D.

    2005-04-01

    Contrast limits are investigated for MacNeille PBS based LCOS projection systems that use retarder stack filters (RSF). The two contributing factors are considered separately; namely the color management system and the panel port. To enhance performance of the former, skew ray compensated RSFs are introduced. For the latter, a general methodology is presented to optimize contrast by compensating the LCOS panel. It is shown that the orientation of the LCOS panel and compensator, relative to the MacNeille PBS, is critical. The significant impact of AR coating performance on system contrast is also revealed. A high contrast architecture will be presented by way of example.

  9. Next generation sequencing reveals the hidden diversity of zooplankton assemblages.

    Directory of Open Access Journals (Sweden)

    Penelope K Lindeque

    Full Text Available BACKGROUND: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. METHODOLOGY/PRINCIPLE FINDINGS: Plankton net hauls (200 µm were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. CONCLUSIONS: Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may

  10. Comparative evaluation of positive contrast and double contrast gastrography in dogs

    International Nuclear Information System (INIS)

    Dileepkumar, K.M.; Rajankutty, K.; Sarada, Amma T.; Devanand, C.B.; Vijayan, N.

    2012-01-01

    A contrast radiography of stomach with oral barium sulphate suspension 25% (5 mL/kg b.wt positive contrast) and double contrast with oral barium sulphate 25% (3 mL/kg b.wt) followed by air (2 to 10 mL/kg b.wt, negative contrast) was done on six dogs to study the affections of stomach. Contrast radiography using barium sulphate alone was found satisfactory to identify most of the lesions of the stomach. Double contrast radiography using barium sulphate and air, required sedation to control the animals for proper administration. For the diagnosis of mucosal lesions, double contrast radiography was better than barium sulphate alone. Key words: Barium, Contrast radiography, Dog, Double contrast, Stomach

  11. Contrast-enhanced MR imaging monitoring of acute tumor response to chemotherapy

    International Nuclear Information System (INIS)

    Ranney, D.F.; Cohen, J.M.; Antich, P.P.; Endman, W.A.; Kulkarni, P.; Weinreb, J.C.; Giovanella, B.

    1987-01-01

    Treatment responses of human malignant melanomas were monitored at millimeter resolution in athymic mice by injecting a new polymeric contrast agent, Gd-DTPA-dextran (0.1 mmol Gd/kg, intravenously). Proton MR imaging (0.35 T, spin-echo, repetition time = 0.5 second, echo time = 50 msec) was performed 30 hours after administering diphtheria toxin. Pre-contrast medium images revealed only homogeneous intermediate-intensity tumor masses. Post-contrast medium images of untreated (viable) tumors demonstrated 32% enhancement throughout the entire mass. Post-contrast medium images of toxin-treated tumors revealed marked enhancement (65%) of the histologically viable outer rims, lesser enhancement (38%) of heavily damaged subregions, and no enhancement of dead tumor. These acute, contrast medium-enhanced MR images accurately identified tumor subregions that survived for longer than one week

  12. Contrasts agents in magnetic resonance imaging

    International Nuclear Information System (INIS)

    Bonnet, P.A.; Fernandez, J.P.; Milhavet, J.C.; Chapat, J.P.; Almes, C.; Bruel, J.M.; Rouanet, J.P.; Lamarque, J.L.

    1984-01-01

    Changing different parameters involved in imaging procedures, paramagnetic substances provide contrast enhancement in MRI. Contrast agents presently studied in animals and clinical trials, are either salts or complexes of mineral ions either nitroxide stable free radicals. Their development should extend the possibilities of tissular characterization and fonctional or metabolic evaluation of the MRI [fr

  13. Parametric based morphological transformation for contrast ...

    Indian Academy of Sciences (India)

    for contrast enhancement of color images in poor-lighting ... high and low mean brightness value, there is a significant change in the view of the enhanced ... The two most fundamental morphological operations dilation and erosion are defined by ..... Menotti David 2007 Multi-histogram equalization methods for contrast ...

  14. Discrimination of Arabic Contrasts by American Learners

    Science.gov (United States)

    Al Mahmoud, Mahmoud S.

    2013-01-01

    This article reports on second language perception of non-native contrasts. The study specifically tests the perceptual assimilation model (PAM) by examining American learners' ability to discriminate Arabic contrasts. Twenty two native American speakers enrolled in a university level Arabic language program took part in a forced choice AXB…

  15. Comparison and Contrast in Perceptual Categorization

    Science.gov (United States)

    Hampton, James A.; Estes, Zachary; Simmons, Claire L.

    2005-01-01

    People categorized pairs of perceptual stimuli that varied in both category membership and pairwise similarity. Experiments 1 and 2 showed categorization of 1 color of a pair to be reliably contrasted from that of the other. This similarity-based contrast effect occurred only when the context stimulus was relevant for the categorization of the…

  16. Contrast sensitivity abnormalities in deaf individuals

    Directory of Open Access Journals (Sweden)

    Masoud Khorrami-Nejad

    2018-01-01

    Conclusion: Hearing impaired boys are at a greater risk for contrast sensitivity abnormalities than boys with normal hearing. The larger frequency of contrast sensitivity abnormalities in high spatial frequencies than in other frequencies may demonstrate greater defects in the central visual system compared with the periphery in individuals with hearing loss.

  17. Influence of radiographic contrast media on phagocytosis

    International Nuclear Information System (INIS)

    Rasmussen, F.; Georgsen, J.; Grunnet, N.; Aalborg Sygehus

    1988-01-01

    To evaluate the influence of radiographic contrast media (CM) on human polymorphonuclear leucocytes (PML), the ability of these cells to ingest latex particles after in vitro exposure to five different radiographic contrast media was investigated. All CM inhibited the phagocytic properties of PML. The inhibition was dose dependent. The inhibitory effect was partly due to hyperosmolality but CM specific inhibition was also evident. (orig.)

  18. Modeling of ultrasound propagation through contrast agents

    NARCIS (Netherlands)

    Grootens, J.J.F.A.H.; Mischi, M.; Böhmer, M.; Korsten, H.; Aarts, R.M.; Vander Sloten, Jos; Verdonck, Pascal; Nyssen, Marc

    2008-01-01

    In the past years many advances have been made in the detection of ultrasound contrast agents (UCA) by exploiting their nonlinear behavior. However, little attention has been paid to the nonlinear distortion of ultrasound (US) waves propagating through contrast media. The aim of this study is to

  19. Adverse reactions to intravascularly administered contrast media

    International Nuclear Information System (INIS)

    Olin, T.

    1986-01-01

    A hypothesis is formulated about the mechanisms causing adverse reactions to contrast media. Contrast media act in two ways. They stimulate the mast cells to release histamine and leukotrienes, and they inhibit the enzymes which otherwise degrade leukotrienes. Thus individuals, especially those with a history of allergy, are easily exposed to undue amounts of leukotrienes and these are responsible for the adverse reactions. (orig.)

  20. Lesion Contrast Enhancement in Medical Ultrasound Imaging

    DEFF Research Database (Denmark)

    Stetson, Paul F.; Sommer, F.G.; Macovski, A.

    1997-01-01

    Methods for improving the contrast-to-noise ratio (CNR) of low-contrast lesions in medical ultrasound imaging are described. Differences in the frequency spectra and amplitude distributions of the lesion and its surroundings can be used to increase the CNR of the lesion relative to the background...

  1. Microbial Diversity of Browning Peninsula, Eastern Antarctica Revealed Using Molecular and Cultivation Methods.

    Science.gov (United States)

    Pudasaini, Sarita; Wilson, John; Ji, Mukan; van Dorst, Josie; Snape, Ian; Palmer, Anne S; Burns, Brendan P; Ferrari, Belinda C

    2017-01-01

    Browning Peninsula is an ice-free polar desert situated in the Windmill Islands, Eastern Antarctica. The entire site is described as a barren landscape, comprised of frost boils with soils dominated by microbial life. In this study, we explored the microbial diversity and edaphic drivers of community structure across this site using traditional cultivation methods, a novel approach the soil substrate membrane system (SSMS), and culture-independent 454-tag pyrosequencing. The measured soil environmental and microphysical factors of chlorine, phosphate, aspect and elevation were found to be significant drivers of the bacterial community, while none of the soil parameters analyzed were significantly correlated to the fungal community. Overall, Browning Peninsula soil harbored a distinctive microbial community in comparison to other Antarctic soils comprised of a unique bacterial diversity and extremely limited fungal diversity. Tag pyrosequencing data revealed the bacterial community to be dominated by Actinobacteria (36%), followed by Chloroflexi (18%), Cyanobacteria (14%), and Proteobacteria (10%). For fungi, Ascomycota (97%) dominated the soil microbiome, followed by Basidiomycota. As expected the diversity recovered from culture-based techniques was lower than that detected using tag sequencing. However, in the SSMS enrichments, that mimic the natural conditions for cultivating oligophilic "k-selected" bacteria, a larger proportion of rare bacterial taxa (15%), such as Blastococcus, Devosia, Herbaspirillum, Propionibacterium and Methylocella and fungal (11%) taxa, such as Nigrospora, Exophiala, Hortaea , and Penidiella were recovered at the genus level. At phylum level, a comparison of OTU's showed that the SSMS shared 21% of Acidobacteria, 11% of Actinobacteria and 10% of Proteobacteria OTU's with soil. For fungi, the shared OTUs was 4% (Basidiomycota) and <0.5% (Ascomycota). This was the first known attempt to culture microfungi using the SSMS which resulted in

  2. Contrast configuration influences grouping in apparent motion.

    Science.gov (United States)

    Ma-Wyatt, Anna; Clifford, Colin W G; Wenderoth, Peter

    2005-01-01

    We investigated whether the same principles that influence grouping in static displays also influence grouping in apparent motion. Using the Ternus display, we found that the proportion of group motion reports was influenced by changes in contrast configuration. Subjects made judgments of completion of these same configurations in a static display. Generally, contrast configurations that induced a high proportion of group motion responses were judged as more 'complete' in static displays. Using a stereo display, we then tested whether stereo information and T-junction information were critical for this increase in group motion. Perceived grouping was consistently higher for same contrast polarity configurations than for opposite contrast polarity configurations, regardless of the presence of stereo information or explicit T-junctions. Thus, while grouping in static and moving displays showed a similar dependence on contrast configuration, motion grouping showed little dependence on stereo or T-junction information.

  3. Contrast Agent in Magnetic Resonance Imaging

    DEFF Research Database (Denmark)

    Vu-Quang, Hieu

    2015-01-01

    Nanoparticles have been employed as contrast agent in magnetic resonance imaging (MRI) in order to improve sensitivity and accuracy in diagnosis. In addition, these contrast agents are potentially combined with other therapeutic compounds or near infrared bio-imaging (NIR) fluorophores to obtain...... theranostic or dual imaging purposes, respectively. There were two main types of MRI contrast agent that were synthesized during this PhD project including fluorine containing nanoparticles and magnetic nanoparticles. In regard of fluorine containing nanoparticles, there were two types contrast agent...... cancer cells for cancer diagnosis in MRI. F127-Folate coated SPION were stable in various types of suspension medium for over six months. They could specifically target folate receptor of cancer cells in vitro and in vivo thus enhancing the contrast in MRI T2/T2* weighted images. These are preliminary...

  4. Application of phase contrast imaging to mammography

    International Nuclear Information System (INIS)

    Tohyama, Keiko; Yamada, Katsuhiko; Katafuchi, Tetsuro; Matsuo, Satoru; Morishita, Junji

    2005-01-01

    Phase contrast images were obtained experimentally by using a customized mammography unit with a nominal focal spot size of 100 μm and variable source-to-image distances of up to 1.5 m. The purpose of this study was to examine the applicability and potential usefulness of phase contrast imaging for mammography. A mammography phantom (ACR156 RMI phantom) was imaged, and its visibility was examined. The optical density of the phantom images was adjusted to approximately 1.3 for both the contact and phase contrast images. Forty-one observers (18 medical doctors and 23 radiological technologists) participated in visual evaluation of the images. Results showed that, in comparison with the images of contact mammography, the phantom images of phase contrast imaging demonstrated statistically significantly superior visibility for fibers, clustered micro-calcifications, and masses. Therefore, phase contrast imaging obtained by using the customized mammography unit would be useful for improving diagnostic accuracy in mammography. (author)

  5. Orientation tuning of contrast masking caused by motion streaks.

    Science.gov (United States)

    Apthorp, Deborah; Cass, John; Alais, David

    2010-08-01

    We investigated whether the oriented trails of blur left by fast-moving dots (i.e., "motion streaks") effectively mask grating targets. Using a classic overlay masking paradigm, we varied mask contrast and target orientation to reveal underlying tuning. Fast-moving Gaussian blob arrays elevated thresholds for detection of static gratings, both monoptically and dichoptically. Monoptic masking at high mask (i.e., streak) contrasts is tuned for orientation and exhibits a similar bandwidth to masking functions obtained with grating stimuli (∼30 degrees). Dichoptic masking fails to show reliable orientation-tuned masking, but dichoptic masks at very low contrast produce a narrowly tuned facilitation (∼17 degrees). For iso-oriented streak masks and grating targets, we also explored masking as a function of mask contrast. Interestingly, dichoptic masking shows a classic "dipper"-like TVC function, whereas monoptic masking shows no dip and a steeper "handle". There is a very strong unoriented component to the masking, which we attribute to transiently biased temporal frequency masking. Fourier analysis of "motion streak" images shows interesting differences between dichoptic and monoptic functions and the information in the stimulus. Our data add weight to the growing body of evidence that the oriented blur of motion streaks contributes to the processing of fast motion signals.

  6. Moho Density Contrast in Central Eurasia from GOCE Gravity Gradients

    Directory of Open Access Journals (Sweden)

    Mehdi Eshagh

    2016-05-01

    Full Text Available Seismic data are primarily used in studies of the Earth’s inner structure. Since large parts of the world are not yet sufficiently covered by seismic surveys, products from the Earth’s satellite observation systems have more often been used for this purpose in recent years. In this study we use the gravity-gradient data derived from the Gravity field and steady-state Ocean Circulation Explorer (GOCE, the elevation data from the Shuttle Radar Topography Mission (SRTM and other global datasets to determine the Moho density contrast at the study area which comprises most of the Eurasian plate (including parts of surrounding continental and oceanic tectonic plates. A regional Moho recovery is realized by solving the Vening Meinesz-Moritz’s (VMM inverse problem of isostasy and a seismic crustal model is applied to constrain the gravimetric solution. Our results reveal that the Moho density contrast reaches minima along the mid-oceanic rift zones and maxima under the continental crust. This spatial pattern closely agrees with that seen in the CRUST1.0 seismic crustal model as well as in the KTH1.0 gravimetric-seismic Moho model. However, these results differ considerably from some previously published gravimetric studies. In particular, we demonstrate that there is no significant spatial correlation between the Moho density contrast and Moho deepening under major orogens of Himalaya and Tibet. In fact, the Moho density contrast under most of the continental crustal structure is typically much more uniform.

  7. Adhesive capsulitis: contrast-enhansed shoulder MRI findings

    International Nuclear Information System (INIS)

    Gokalp, Gokhan; Yildirim, Nalan; Yazici, Zeynep; Algin, Oktay

    2011-01-01

    Full text: Evaluation of contrast-enhanced magnetic resonance imaging (CE-MRI) findings in cases clinically diagnosed as adhesive capsulitis (AC). CE-MRI images of 12 cases diagnosed as AC (13 shoulder joints) and nine control cases were retrospectively evaluated. AC diagnosis was establlished based on the history and clinical symptoms. MR signal intensity changes in the axillary pouch, rotator interval, biceps anchor and anterior posterior capsules were analysed with regard to the presence of abnormal soft tissue and contrast enhancement. Capsular and synovial thickening were measured in the axillary recess and rotator interval on coronal oblique CE T1-weighted images. Patient and control groups were compared by Fisher's exact and McNemar tests in terms of signal intensity changes and contrast enhancement in the described areas. Results: Comparison of the group with AC and the control group regarding intensity changes showed a statistically significant difference in the axillary pouch (P 0.05). Comparison of AC and control groups in terms of contrast enhancement revealed statistically significant differences in the axillary pouch, rotator interval, biceps anchor and anterior-posterior capsules (P < 0.001). A significant difference was determined between the AC and control groups with regard to thickening in axillary pouch and rotator interval (P < 0.001). CE studies are useful for diagnosis of AC as it demonstrates thickening of specific soft-tissue areas like joint capsule and synovium.

  8. Contrasting effects of fresh and fermented kimchi consumption on gut microbiota composition and gene expression related to metabolic syndrome in obese Korean women.

    Science.gov (United States)

    Han, Kyungsun; Bose, Shambhunath; Wang, Jing-hua; Kim, Bong-Soo; Kim, Mi Jeong; Kim, Eun-Jung; Kim, Hojun

    2015-05-01

    Accumulating evidence suggests relationship of compositional changes of gut microbiota with onset of metabolic disorders and obesity. Kimchi, a traditional Korean side dish, is known for its beneficial impact on metabolic parameters and anti-obesity effects. The current study was designed to evaluate the association between gut microbiota and human genome after kimchi intervention in an effort to understand the molecular mechanism(s) underlying the antiobesity impact of kimchi. Twenty-four obese women were randomly assigned to either fresh or fermented kimchi group for eight weeks of kimchi intervention. Pyrosequencing of fecal microbiota and microarray analyses of blood samples revealed that fresh and fermented kimchi interventions exerted differential effects on the obesity-related clinical parameters. Correlations of these effects with changes in blood gene expression and gut microbial population were more evident in the fermented kimchi group than the fresh kimchi group. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends.

    Science.gov (United States)